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A Preclinical Study of Flavopiridol in the Treatment of Acute Lymphoblastic Leukemia

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PAGE 1

1 A PRECLINICAL STUDY OF FLAVOPIRIDO L IN THE TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA By KELLY MARIE JACKMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Kelly Marie Jackman

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3 To those whose lives have been touched by can cer; especially women who have lost their fathers. Even though our loved ones have moved on, a small part of them is still here in us.

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4 ACKNOWLEDGMENTS I would first like to thank my me ntor, Dr. Hunger. I appreciate the time that he has given to assist me in my writing and scientific developm ent. Because of his patient guidance and honesty, my ability to communicate in a more sophisticated and organized manner has matured tremendously. My professional demeanor has also changed a great deal during my graduate career; part of which I owe to the example set by Dr. Hunger. I w ill always be grateful for his mentorship. I would also like to thank the members of my committee, Drs. Rowe, Kilberg, and Fletcher for their input into this work and their contribut ion to making sure that it progressed in a timely fashion. My appreciation also goes to the past a nd present members of the Hunger laboratory. Through discussions of various t opics, both professional and non-scientific, Dr. Victor Prima has helped me to learn how to articulate and defe nd my ideas; skills which are integral to the graduate experience. Dr. Mi Zhou was and still is a wonderful friend and an important source of personal support. Both of these individuals ha ve taught me so much about the cultures of Ukraine and China, respectively, which has made my time in the lab a truly unique experience. I would also like to thank Carole Frye for her valuable advice a nd technical assistance with my experiments. My thanks also go to Amanda Ri ce, who became a great friend in the short time that she worked in the lab. Without the tireless help of th e individuals in the Flow Cy tometry Core Lab, this work would not have been possible. I would like to thank Neil Bens on, Bhavna Bhardwaj, and Steve McClellan for assisting me with my experiments. Bhavna and Steve were willing spirits as they performed most of the raw data analyses contai ned in this dissertation, for which I was always

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5 grateful. I would also like to th ank Linda Young in the Department of Statistics for patiently helping me through all of the statistics required to properly analyze my data. Other members of the College of Medicine that I wish to thank are Judy Adams, my graduate secretary, as well as Cathy Hymon, secretary for Pediatric Hematology/Oncology. Without these ladies I would not have been able to navigate the huge system that is UF. I would also like to remember my fello w students and members of GSO. Finally, and most importantly, I would like to thank my mom. She ha s been there through everything; so many events that it becomes diff icult to list them all. She has held my hand literally and figuratively when it was time for many of the special people in my life, and hers, to leave us. She has been there through the frustra tions and triumphs of my life and academic career and has always supported me. My mom is truly my best friend and I know that without her I would not have gotten as far as I have. I hope th at my achievement brings to her a sense of satisfaction that a small part of the plan that she and my father set in to motion many years ago continues on.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 BACKGROUND....................................................................................................................14 General Treatment of Acute Ly mphoblastic Leukemia (ALL)..............................................14 Relapsed ALL: The Clinical Problem....................................................................................15 Important Regulators of Cell Cycle........................................................................................16 Cell Cycle Regulators in Cancer and ALL.............................................................................17 Flavopiridol (FP).............................................................................................................. ......20 In Vitro Testing of Flavopiridol in Combina tion with Other Agents: Sequence of Administration and Synergy...............................................................................................22 Efficacy of Flavopiridol in Clinical Trials..............................................................................23 Biological Correlates of Clinical Activity..............................................................................26 Clinical Trials of Paclitaxel (PAC) a nd Combining Flavopiridol with Paclitaxel.................28 Project Rationale.............................................................................................................. .......29 2 FLAVOPIRIDOL DISPLAYS PRECLI NICAL ACTIVITY IN ACUTE LYMPHOBLASTIC LEUKEMIA.........................................................................................36 Introduction................................................................................................................... ..........36 Methods........................................................................................................................ ..........37 In Vitro Drug Sensitivity Testing....................................................................................37 Western Blot Analyses....................................................................................................38 Measurement of Cell Death.............................................................................................39 Cell Cycle Analysis.........................................................................................................40 Results........................................................................................................................ .............40 ALL Cell Lines Used for in Vitro Testing Lack p16 Protein Expression.......................40 In Vitro Drug Sensitivity in ALL Cell Lines...................................................................41 FP Induces Apoptosis in ALL Cell Lines........................................................................41 FP Induces Cell Cycle Arrest in ALL Cell Lines which Correlates with Effects on pp-Rb Protein Expression............................................................................................42 Apoptotic Effects of FP in Human Serum.......................................................................43 Discussion..................................................................................................................... ..........44

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7 3 PRECLINICAL STUDIES OF FLAVOPIR IDOL COMBINED WITH PACLITAXEL IN ACUTE LYMPHOBLASTIC LEUKEMIA.....................................................................52 Introduction................................................................................................................... ..........52 Methods........................................................................................................................ ..........53 Materials...................................................................................................................... ....53 Single Agent in Vitro Sensitivity Assays........................................................................53 Drug Combination Studies..............................................................................................54 Treatment Sequence........................................................................................................54 Statistical Analysis..........................................................................................................55 Results........................................................................................................................ .............55 Single Agent FP Treatment.............................................................................................55 Single Agent PAC Treatment..........................................................................................56 Combination Treatment with FP and PAC......................................................................56 Determination of Optimal Schedule for PAC+FP...........................................................57 Activity of PAC in Human Serum...................................................................................57 Combination Studies in Human Serum...........................................................................58 Discussion..................................................................................................................... ..........58 4 CONCLUSIONS AND DISCUSSION..................................................................................67 FP Single Agent Studies........................................................................................................ .67 Establishing an in Vitro Treatment Model of ALL.........................................................67 Drug Sensitivity Testing via Cell Proliferation Assays...................................................67 The Mechanism of Cell Death Induced by FP in ALL Cell Lines..................................68 FP Activity in Human Serum..........................................................................................69 PAC+FP Combination Studies...............................................................................................70 Note about Statistical Analysis........................................................................................70 Enhancement of PAC Activity by FP..............................................................................71 Methods of Determining Synergy...................................................................................72 FP Combined with PAC..................................................................................................75 Sequence Dependent Enhancement.................................................................................77 Drug Sensitivity in Human Serum..................................................................................78 Placing Perspective on this Project.........................................................................................79 Potential Side Effects of Single Agent and Combination Therapy.................................79 Where Does FP Fit into the Treatment Scheme of ALL?...............................................79 Future Directions.............................................................................................................80 LIST OF REFERENCES............................................................................................................. ..96 BIOGRAPHICAL SKETCH.......................................................................................................108

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8 LIST OF TABLES Table page 4-1. Mixed model analysis for FP treatment duration in Nalm-6.................................................87 4-2. Mixed model analysis for FP treatment duration in RCH-ACV...........................................87 4-3. Differences between treatment durati on at a given FP concentration in Nalm-6..................87 4-4. Differences between treatment durati on at a given FP concentration in RCH-ACV............87 4-5. Mixed model analysis of PAC single agent treatment in Nalm-6.........................................88 4-6. Mixed model analysis of PAC single agent treatment in RCH-ACV...................................88 4-7. Differences in cell death based on incuba tion time after 6 or 24 hours PAC treatment in Nalm-6......................................................................................................................... ......89 4-8. Differences in cell death based on incuba tion time after 6 or 24 hours PAC treatment in RCH-ACV........................................................................................................................ ..90 4-9. Combination Index (CI) valu es for drug combination studies using a variety of ratios.......90 4-10. Mixed model analysis of Nalm -6 and RCH-ACV combination data..................................90 4-11. Mixed model analysis of Molt-4 and Jurkat combination data...........................................91 4-12. Significant differences in treatment fo r a given cell line and drug concentration...............91 4-13. Significant differences in treatment fo r a given cell line and drug concentration...............91 4-14. Combination Index (CI) valu es for drug combination studies............................................92 4-15. One-Way Analysis of Variance of treatment sequence in Nalm-6.....................................92 4-16. Weighted One-Way Analysis of Va riance of treatment sequence in RCH-ACV...............92 4-17. Significant differences between standa rd treatment sequence, reverse treatment sequence, and single agent controls in Nalm-6..................................................................92 4-18. Significant differences between standa rd treatment sequence, reverse treatment sequence, and single agent controls in RCH-ACV............................................................93 4-19. Mixed model analysis for comparison of cell death induced by PAC in FBS vs. HS........93 4-20. Comparison of cell death induced by PAC in FBS vs. HS..................................................94 4-21. Mixed model analysis of cell viability FBS vs. HS.............................................................94

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9 4-22. Mixed model analysis of combination studies in human serum..........................................94 4-23. Significant differences in treatment for combination studies in human serum...................95

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10 LIST OF FIGURES Figure page 1-1. Treatment of Childhood Acute Lymphoblastic Leukemia (ALL).........................................32 1-2. Cyclin dependent kinase (CDK) inhi bitors function in the transition from G1 (Gap 1) to S (DNA synthesis) phase of the cell cycle.........................................................................33 1-3. p16 works in concert with pRb to regulate the G1-S transition.............................................34 1-4. Flavopiridol is a pan-CDK inhibitor......................................................................................35 2-1. Fifty percent inhibitory concentration (IC50) determinations via WST-1 in cell lines that lack p16 protein expression...............................................................................................47 2-2. Flavopiridol induces apoptosis in ALL ce ll lines in a concentration dependent manner......48 2-3. Flavopiridol induces G1-S and G2-M (Gap 2-mitotic) arrest in RCH-ACV with reduced phosphorylation of pRb ....................................................................................................49 2-4. Flavopiridol induces transient G1-S arrest in Nalm-6. ........................................................50 2-5. Efficacy of FP in human serum............................................................................................ .51 3-1. Experimental design for PAC single agent treatment...........................................................60 3-2. Cell death induced by tr eatment with FP or PAC in Nalm-6 and RCH-ACV. ...................61 3-3. Flavopiridol enhances the e fficacy of PAC in ALL cell lines...............................................62 3-4. PAC FP is a more efficacious treatment sequence than FP PAC or concurrent exposure in Nalm-6............................................................................................................63 3-5. PAC FP is a more efficacious treatment sequence than FP PAC or concurrent exposure in RCH-ACV......................................................................................................64 3-6. Efficacy of PAC in Nalm-6 in the presence of human serum...............................................65 3-7. Flavopiridol enhances the effi cacy of PAC in human serum.................................................66 4-1. Growth curves used to establish ce ll concentration for proliferation assays.........................82 4-2. Representative dose-response curves generated from cell proliferation assays....................83 4-3. Illustration of isobologram anal ysis of combined drug effects.............................................84 4-4. Preliminary combination data at a variety of ratios in Nalm-6.............................................85

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11 4-5. Preliminary combination data at a variety of ratios in the pres ence of human serum...........86

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12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy A PRECLINICAL STUDY OF FLAVOPIRIDO L IN THE TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA By Kelly Marie Jackman May 2007 Chair: Stephen P. Hunger Major: Medical Sciences--Physiology and Pharmacology Approximately 80% of children with acute ly mphoblastic leukemia (ALL) will be cured; however, it is essential to study nove l agents and new combinations of existing therapies for their potential use in relapsed patient s. Loss of p16 function might play a part in the progression of ALL, which makes this pathway an interesting ta rget for novel therapeuti cs. I have chosen to study flavopiridol (FP), a semi-s ynthetic flavonoid that targets the p16 pathway. FP acts as a pan-cyclin dependent kinase inhibitor with the ab ility to induce apoptosis and cell cycle arrest in human cancer cells. My studies have shown that at a concentration approximately equal to the IC50, FP induces a transient G1-S arrest and a low percentage of apoptosis in ALL cell lines. At approximately twice the IC50, FP induces a sustained G1-S and G2-M arrest with a high percentage of apoptosis. My work has al so shown that FP treatment decreases the phosphorylation of retinoblastoma protein on sp ecific serine residues; an indication of a reduction in endogenous CDK activity. Further, despit e a high level of binding by FP to proteins present human serum and subsequent reduction in its in vitro activity reported by others, I show that there is not a substantial difference in FP activity in the presence of human serum when compared to fetal bovine serum.

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13 Based on disappointing results from early clinic al studies of FP by others, I chose to test FP in combination with paclitaxel. PAC has a mechanism of action which is complementary to that of FP. PAC enhances the activity of CD K 1, inhibits microtubule depolymerization, and induces G2-M arrest. Others have reported that FP enhances the efficacy of PAC in a sequencedependent manner in cell types other than ALL. My results show that FP enhances the efficacy of PAC in ALL cell lines and that this e nhancement is dependent on the sequence of administration. In this study I established optimal times of exposure for each of the agents when used in combination and confirmed that the e nhancement of PAC activit y by FP is present both in fetal bovine serum and human serum.

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14 CHAPTER 1 BACKGROUND General Treatment of Acute Ly mphoblastic Leukemia (ALL) Acute lymphoblastic leukemia (ALL) is th e most common form of childhood cancer, accounting for approximately 30% of pediatric malignancies (1). With current multi-agent chemotherapy regimens, approximately 80% of pa tients are cured of their disease; however, relapse remains a significant clinical problem (2 ). Treatment of children with ALL consists of three phases: induction, c onsolidation and maintenance (Figure 1-1) (2, 3). The total length of treatment lasts 2-3 yrs. The purpose of the first phase of treatment, which lasts approximately one month, is to induce a complete remission or an absence of morphologically detectable leukemic blast cells in the blood or bone marrow. This is successfully achieved in 99% of patients with three or four drugs (2, 3). The consolidation phase of therapy lasts 4-8 months and is designed to reduce the number of remaining leukemic blast cells using the agents listed in Figure 1-1 (3). Maintenance therapy (1.5 to 2.5 years) consists of methotrexate and 6mercaptopurine; in addition to vincristine and either pred nisone or dexamethasone. Patients with recurrent ALL receive more in tensive therapy involving any or all of the agents previously outlined in other phases with other active agents such as ifosfamide, etoposide or teniposide often added. Stem ce ll transplant is also frequen tly performed for patients whose disease recurs during treatment or within 6 months of completing therapy. In the case of relapse outside of the bone marrow, such as leukemic bl asts found in the central nervous system or testes, radiation can be administ ered at that site if it has no t been previously administered. There are agents which are currently used on an experimental basis in children with ALL. These include newer cytotoxic agents such as clofarabine, a nucleoside analog, agents which target tyrosine kinases and those that target hist one deacetylases (2). Ima tinib mesylate (Gleevec)

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15 targets the tyrosine kinase formed by the BCR-AB L fusion protein resulti ng from a translocation between chromosome number 9 and chromosome number 22 (Philadelphia chromosome) as well as other tyrosine kinases. Use of this ag ent has induced remissions in BCR-ABL positive ALL (4-6). Other therapies under investigation incl ude the use of RNA inte rference technology, gene therapy and immunotherapy (2). Relapsed ALL: The Clinical Problem Relapse can occur in a number of sites, in cluding but not limited to the bone marrow, central nervous system, and testes. Survival ra tes after bone marrow relapse range from 5% to 57% and are especially poor for those with a relaps e within 36 months of initial diagnosis (7-11). Increased dosage, the use of other existing chem otherapy agents not typically used in primary treatment (etoposide, ifosfamide and others) and widespread us e of stem cell transplantation have not significantly improved outcome for these patients. In addition, while complete remission rates for children who relapse more than 3 years after diagnosis are similar to those seen at initial diagnosis (>95%), patients who relapse less than 3 years after diagnosis often fail to attain a second remission (12). Thus, there is a need to develop novel agents and/or new combinations of existing agents in order to improve the outcome of relapsed pedi atric ALL patients. Many new agents have been developed that have novel modes of action. Some of these include the cy clin-dependent kinase inhibitors (CDKIs), examples being flavopiridol and UCN-01 (13, 14). Other drugs which are typically used in other types of cancers, such as the microt ubule depolymerization inhibitors paclitaxel and docetaxel, have b een used experimentally in ALL with mixed results (15-17). It becomes essential to study the biology that make s relapsed ALL different from ALL at initial diagnosis so that priority can be given to the study of the most promising agents.

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16 Important Regulators of Cell Cycle Gene expression profiling reveals that several key pathways are altered at the time of ALL relapse vs. at initial diagnosis including cell cycle regulation, DNA repair and apoptosis (18). This project has focused on preclinic al testing of agents that targ et the aberrations in cell cycle regulation present in relapsed ALL. Regulatory proteins can be broadl y separated into those which regulate the transition from G1 (Gap 1) to S (DNA synthe sis) phase and from G2 (Gap 2) to M (mitosis) phase. CDK 2, CDK 4, and CDK 6 regulate the transition from G1 to S and CDK 1 (cdc2) regulates the transition from G2 to M (Figure 1-2) (19). Most molecules of interest in this project function in th e restriction point from G1 to S or the point at which the cell is committed to divide with or without the presence of growth factors (20), as beyond this point the cell is less likely to respond to external stimuli such as a drug. CDK 4 and CDK 6 become functional after cyclin D1, cyclin D2, or cycl in D3 binding (19). These kinases phosphorylate retinoblastoma protein (pRb) at specific serine a nd/or threonine residues. This phosphorylation is normally prevented by p16 (cyclin-dependent kina se 4 inhibitor A; INK4A), which binds to CDK 4 and CDK 6 in the place of the cyclin (21). p16 is part of the INK4 family of proteins, including p15 (INK4B), p18(INK4C), and p19(INK4 D), which work to inhibit CDK 4 and CDK 6, along with members of the CIP/KIP family, in cluding p27 (cyclin depende nt kinase inhibitor 1B; KIP1), p57 (cyclin dependent kinase inhibitor 1C; KIP2) a nd p21 (cyclin dependent kinase inhibitor 1A; WAF1/CIP1) (Figure 1-2) (22). When p16 is present, the hypophosphorylated form of pRb acts as a tumor suppressor by binding to E2F transcription factor making E2F unable to bind to DP-1 and 2 (Figure 1-3). These molecules function as transcription factors that act in DNA synthesis and nucleotide metabolism (22). p16 has been a major interest in this proj ect; however, other molecules such as p15, p21, and p27 have been studied by the Hunger lab an d others for their po ssible roles in the

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17 progression of ALL (see below). p15 shares gr eat homology with p16, is found within 25kb of the p16 gene on chromosome 9 (23), and acts as a TGF(transforming growth factor) induced inhibitor of CDK 4 and CDK 6 (24). p21 and p27 regulate not only CDK 4 and CDK 6, but also CDK 2, which functions in concert with CDK 4 and CDK 6 to phosphorylate pRb (19). It is CDK 2 that actually completes the hyperphosphorylation of pR b. This inhibits the tumor suppressive nature of pRb and allows progression of the cell cycle through S-phase. p21 is activated by the p53 transc ription factor (22). P53 is regu lated by MDM-2 (mouse double minute 2; HDM-2 in humans) which inactivates the transcriptional activity of p53, flags it for ubiquitylation, and ensures its tr ansport from the nucleus into the cytoplasm. An alternate reading frame of the p16 locus produces p14 (alternate read ing frame; ARF) which acts as a tumor suppressor by preventing the p53 suppression activity of MDM-2. Cell Cycle Regulators in Cancer and ALL Deletion of p16 is the most common form of gene tic alteration in can cer among cell cycle regulators (25). Studies have shown that greater than 30% of ALL cases have p15 and p16 deletions, with that percentage increasing to greater than 50% in Tcell ALL and remaining greater than 20% in B-precursor ALL (26). It has been found by th e Hunger lab and others that a substantial number of patients develop p15 and/or p16 deletions in the bone marrow between the time of initial diagnosis and relapse of ALL (27, 28). The p15 promoter has also been studied and has been found to undergo methylation be tween diagnosis and relapse, much more commonly than the p16 promoter (29-31). This p15 methylation takes place in CpG islands at the 5 end of the gene, which results in loss of tr anscription in the prom oter region (32). A study from the Hunger laboratory used 18 matched speci men pairs from children with ALL at initial diagnosis and first rela pse to determine if p15/p16 deletions or hypermethylation of the p15

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18 promoter occurred between diagnosis and relapse ( 27). Results showed that out of 14 pairs that were germline at diagnosis, three de veloped homozygous deletions of both p15 and p16 and two developed homozygous p16 deletions and retained germline p15 status between the time of initial diagnosis and relapse. p15 promoter hypermethylation deve loped in two patients between diagnosis and relapse. Out of the eigh teen total cases, seven had homozygous p15 deletions, nine had homozygous p16 deletions, and two of eight cases tested had p15 promoter hypermethylation at relapse. Similar findings have been reported by Carter, et al., showing that out of a group of 25 pediatric ALL patients, at diagnosis 32% and 20% had homozygous and hemizygous p16 (exon 2) deletions respectively ( 28). The incidence of homozygous p16 deletion at relapse increased to 64%, illustra ting the potential importance of the loss p16 in the progression of ALL. The prevalence of p15 and p16 alterations is much hi gher than the level of p21 and p27 alterations found in ALL (33, 34). Both p21 and p27 function to inhibit CDK 2 and CDK 4 (3537). p21 is regulated by p53 in order to control cell growth (38). Hayette, et al. performed a study of alterations of molecules which inhibit CDKs in leukemia, using bone marrow or peripheral blood from 121 newly diagnosed ALL cases, 85 ne wly diagnosed acute myeloid leukemia cases, and 42 newly diagnosed B-cell chronic lymphocytic leukemia cases (34). Via Southern blot this group found that p16 was inactivated in 25 of 38 T-cell A LL cases and 28 of 83 B-lineage ALLs. After testing 40 ALL samples with a p16 aberration, it was found th at 22 cases (55%) had biallelic p15 deletions and 11 cases (28%) had monoa llelic deletions. All cases with a p15 deletion also had an anomaly in the p16 gene There were no alterations found in p21 and monoallelic deletion of p27 was present in 4 of 85 acute myeloid leukemia cases tested. These data show that p21 and p27 alterations are much less prevalen t in leukemia than deletions of p15

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19 and p16 Another study by Kawamura, et al. further illustrates this point by analyzing 71 primary T-ALL samples and 18 T-ALL ce ll lines for alterations in p15, p16, p21, p53 and RAS via polymerase chain reaction-single strand conforma tion polymorphism analysis (33). They found that none had alterations in p21 In contrast, 18 of 47 (38%) newly diagnosed patients had p16 alterations and 7 of 14 (50%) patients had p16 alterations at relapse. Gene deletion is not the only cause of lo ss of functional p16. Many samples have been found to have an intact p16 gene, but no protein expression. An interesting study by Nakamura, et al. notes that when p16 expression was i nvestigated in childhood ALL samples via Western blot, 18 of 22 samples with an intact p16 gene did not express p16 pr otein; however, protein expression was able to be induced after treatment with a demethylating agent, indicating that the loss of p16 protein expression was due to gene hypermethylation (39). Others have reported similar results in T-cell ALL and/or AML (4042). A separate study of pediatric T-cell ALL patients reported that only 9 of the 45 samples with intact p16 expressed p16 protein (43). This study found that p16 was altered at the DNA, RNA or protein level in 115 of the 124 (93%) samples tested and concluded that alteration in both p16 and p15 we re essential to the progression of T-cell ALL. Most recently a study of adults with untreated ALL found that not one of the samples tested (n= 91) expressed p16 protein (44). A study performed by Carter, et al. on 45 patie nt samples via quantit ative PCR techniques found that ALL patients with a hemizygous deletion of p16 at diagnosis were 6.5 times more likely (P=0.00687) to relapse and th ose with a homozygous deletion had an even higher risk ratio of 11.5 (P=0.000539) (45). In contrast to the findings of Carter et al., Einsiedel et al. found that there was no association between p16 deletions and event free survival in ALL (46). p15 and p16 status could be correlated to two major pr ognostic indicators: T-cell immunophenotype and first

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20 remission duration. This study did not assess for he mizygous deletions, as Carter did, because of theoretic and methodologic considerati ons. Another group compared wildtype p16 to hemizygous deletions and found no difference in potential for event free survival (47). Given this information it becomes apparent that despite the fact that its prognostic value is still somewhat controversial, p16 alterations occur commonly in relapsed ALL and are often acquired during disease pr ogression. Deletion of p16 and hypermethylation of the p15 promoter region occur much more frequently in ALL th an alterations in other cell cycle regulatory molecules such as p21 or p27. Therefore the p15/p16 pathway is an attractive target for therapeutic intervention in re lapsed ALL. Several agents exist that modul ate cell cycle progression and are logical candidate s to test in relapsed ALL. One such agent is flavopiridol, a description of which follows. Flavopiridol Flavopiridol (FP) is a semisynt hetic flavonoid derived from roh itukine, an alkaloid isolated from a plant indigenous to Indi a (48). Flavopiridol has a variety of mechanisms of action; however, most relevant to my studies is the abi lity of FP to decrease th e activity of CDKs and induce cell cycle arrest (Figure 1-4). Cell cycle re gulatory elements such as the CDK inhibitors p15 and p16 are altered in ALL between diagnosis and relapse, indicating that this loss of checkpoint control in the cell cycle could be a critical factor in the progression of the disease and an attractive target for novel therapeutic agents FP competitively binds to the ATP binding cleft of the CDK (14) and is capable of reducing the activity of CDK 1, CDK 2, CDK 4, CDK 6, and CDK 7 with IC50 values in the range of 20-400 nM (49) FP also reduces the activity of CDK 9 (50-52). FP induces cell cycle arrest at the G1-S phase border as well as during G2-M. Inhibition of CDK 2 and CDK 4 has been correlated to G1 arrest in MCF-7 breas t carcinoma cells (14).

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21 Another group had similar findings using MDA-468 breast carcinoma cells that were synchronized either in G1 phase with aphidicolin or synchr onized in M phase with nocodazole (53). MDA-468 cells treated with 200 nM FP after release from aphidicolin G1 block arrested in G2-M after 24 hours. Cultures released from noc odazole M phase block and treated with 200 nM FP showed a G1-S arrest when compared to contro l cultures not treated with FP. The ability of FP to induce cell death has been tested in vitro in a variety of cancer cell types, including adult leukemia. An early study in a variety of solid tumor cell types and HL-60 leukemia cells found that FP was cytotoxic as measured by trypan blue exclusion and colony formation assays (54). Previous studies had only shown that FP was cytostatic (53). The former study also found that 90% cell death was induced 72 hours following a 24 hour exposure to 250300 nM FP compared to 50% cell death induc ed immediately following the 24 hour drug exposure, thus showing that more time was need ed to achieve a maximum cell death response. This group also showed that both logarithmically growing and cytostatic cell lines were affected by FP treatment. Similar results have been found by others testing non-small cell lung carcinoma cell lines (55). It was found that se ven different cell lines were sens itive to FP at concentrations ranging from 100-500 nM; regardless of whether the cell lines were in logarithmic growth phase or cytostatic. This group also show ed that cell cycle arrest preced ed cell death in most cases and that maximal cell death occurred 72 hours post-tr eatment with concentrations of FP 500 nM or below. These data illustrate the cytotoxic acti on of FP during a prolonged exposure. This activity combined with the ability of FP to inhibit CDK ac tivity and induce cell cycle arrest contribute to this projects focus on testing the efficacy of FP as a potential treatment for ALL.

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22 In Vitro Testing of Flavopiridol in Combin ation with Other Agents: Sequence of Administration and Synergy It has been found that administering FP with traditional antineoplastic agents can improve the efficacy of those agents and that in some cases this interacti on is synergistic. The enhancement of a traditional agent by FP has b een shown to be dependent on the sequence in which the drugs are given, such as the enhan cement of paclitaxel (PAC) activity by FP (56). Paclitaxel (trade name Taxol) prevents microt ubule depolymerization ( 57) and induces G2-M phase arrest (58). An example of the enhancem ent of PAC activity by FP can be found in work by Motwani, et al. in which MKN-74 human gast ric carcinoma cells a nd MCF-7 human breast carcinoma cells were exposed to PAC, FP, or bot h agents either sequentially or simultaneously (56). When MKN-74 cells were exposed to PA C and FP for 24 hours, the level of apoptosis increased from 3 +/1% with FP alone to 8 +/1%. A significant increase was then seen when the drugs were used sequentially. MKN-74 cells were exposed to PAC for 18 hours followed by FP for 24 hours and the level of apoptosis was 40 +/2%; however, when using FP followed by PAC the level was 8 +/1%, which was not signif icantly different from the amount of apoptosis found after exposing the cells to FP for 24 hour s followed by no drug for 18 hours. Caspase-3, the final activator of the apoptotic cascade wa s activated when MKN-74 and MCF-7 were treated with PAC followed by FP. Without FP, PAC only mi nimally activated caspase-3. If the sequence of administration was reverse d, FP inhibited the function of PAC by preventing mitosis and CDK 1 activity. Similar results in regard to cyto toxicity have been achieved when using FP in conjunction with docetaxel in vitro and in xenograft tumor models (59). When testing eight agents against a human non-small cell lung carcinoma cell line (A549), Bible and Kaufmann found that seven of the eigh t agents had synergy with FP that was sequence specific (60). These authors extensively studied the possibility that treatment with PAC and FP

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23 could show sequence dependent synergy. Their finding that the effects of PAC were more pronounced when administered be fore FP treatment as opposed to after or concomitantly was particularly intriguing. A marked decrease in clonigenic cell survival over PAC alone and FP alone was seen when PAC treatment was follo wed by FP. Synergy was assessed through the use of combination index or CI. A CI of 1.0 indicates that the rela tionship between the drugs being studied is nearly additive, wh ile a CI of <1.0 indicates syne rgy and a CI of >1.0 indicates antagonism (61). At the concentration at wh ich cell proliferation was inhibited by 75% (IC75) and 95% (IC95), combination indices of 0.49 +/0.21 and 0.20 +/0.14 were found, respectively, indicating synergy if PAC was given before FP in the treatment sequence (60). Antagonism was found if PAC followed FP. Others have tested many agents in conjunc tion with FP in myeloid leukemia cell lines. These agents have included phorbol 12-myrist ate 13-acetate (PMA), imatinib mesylate (Gleevec), bryostatin 1, bortezomib (Velcade) and suberoylanilide hydroxamic acid (SAHA) (62-66). All have shown promising results for the ability of FP to enhance the activity of other agents. Efficacy of Flavopiridol in Clinical Trials Based on its action as a CDK inhi bitor and promising preclinical activity, FP was tested in phase I human trials. Studies designed to obtain clinical pharmacology data after giving FP as a 72 hour infusion readily achieved plasma concentrat ions that were comparable to that found to be effective in vitro (67, 68). However, most clinical tria ls involving cancer pa tients gave FP as a 72 hour infusion every 2-3 weeks and found that it had limited efficacy as a single agent (69-75). One of these trials found that FP had antitumor activity in certain patients with renal, prostate, and colon cancer, and non-Hodgkins lymphoma (69). The two maximum tolerated doses (MTDs) found in this phase I study gave peak pl asma concentrations of 271 nM and 344 nM, the

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24 second after antidiarrheal prophylax is. The concentrations of FP needed to inhibit cyclin dependent kinase function (200 to 400 nM) were safely achieved in this study. Despite both in vitro and in vivo data showing that FP was cytosta tic and cytotoxic in non-small cell lung carcinoma cells, Shapiro and colleagues stopped a phase II study after only 20 of 45 patients projected to be in the study were treated, as no responses were obs erved in these individuals (72). This study also noted that a mean steadystate plasma concentration of 200.9nM was achieved, which was well within the FP con centration range found to be effective in vitro Questions regarding dose and le ngth of treatment have b een the main focus of many clinical trials involving FP. In addition to the traditional 72 hour infusion schedule, FP has also been tested as a 24 hour continuous infusi on given every two weeks and a 1 hour bolus administered over a range of schedules. Flinn, et al. found that FP had no clinical activity in patients with fludarabine refrac tory chronic lymphocytic leukem ia (CLL) when given as a 24 hour infusion (76). A study from the same group compared FP activity in CLL when the agent was administered as a 72 hour infusion to a 1 hour bolus and found that the 72 hour schedule did not result in any patient responses ; however, the bolus dose did resu lt in slight clinical activity (75). A separate phase I study us ing FP as a single agent in patie nts with advanced neoplasms tested FP at varying 1 hour infusion doses over 5-days, 3-days and 1-day every 3 weeks (77). During the trial, median peak total concentrations at the MT D of 1.7 M (range 1.3 to 4.2 M) for 5-day administration, 3.2 M (range 1.7 to 4.8 M) for 3-day administration, and 3.9 M (range 1.8 to 5.1 M) for 1-day administration were found. Twelve of the 55 patients studied had stable disease for greater than or equal to thr ee months with a median duration of six months (range, three to eleven months). A similar st udy was conducted by the Nati onal Cancer Institute of Canada using FP as a bolus infusion over 3 days in patients with untreated or relapsed mantle-

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25 cell lymphoma (78). No complete responses we re observed; however, 11% of patients had a partial response and 71% had stable disease. Sim ilar results for the 1 hour bolus have also been reported in malignant melanoma and multiple myleoma (79, 80). The only study of FP in pediatric patients also used this schedule and wa s performed by Whitlock, et al. in patients with solid tumors (81). No responses were obs erved despite achieving mean peak plasma concentrations of 3.71 and 9.11 M after doses of 37.5 mg/m2 and 80 mg/m2 respectively. Shorter infusion schedules for FP as outlined above were pursued by clinicians with the intention of increasing the peak pl asma concentration of the agent. Early trials of FP given as a 72 hour infusion were based on dr ug activity data generated from in vitro studies of FP that were performed in media supplemented with fetal bovi ne serum (FBS). Later studies showed that FP is highly bound to human plasma proteins ( 68, 82). Approximately 92-95% of FP is human plasma protein bound compared to 0-37% bound in FBS (82). This difference in protein binding results in a decrease in the in vitro cytotoxicity of FP. Studies of primary CLL cells have shown 1 hour and 24 hour LC50 values in FBS of 670 nM and 120 nM respectively, compared to 3,510 nM and 470 nM in human plasma or human serum (HS) (82). Based on these data and clinical pharmacology data, a pivotal study of the use of FP as a short infusion was performed (83). FP was given as a 30 minute bolus infusion followe d by a 4 hour continuous infusion in patients with CLL with the goal of achie ving a peak plasma concentr ation of 1.5 M. Patients were divided into cohorts, with the first receiving a 30 mg/m2 bolus dose followed by a 30 mg/m2 infusion. The second cohort received a 40 mg/m2 bolus followed by a 40 mg/m2 infusion. The maximum plasma FP concentrations achieved at these dose levels were 2,080 nM after 30 minutes and 960 nM after 4.5 hrs (84). A third cohort was given a 30 mg/m2 bolus followed by a 50 mg/m2 infusion. These dosages achieved peak pl asma levels of 1,950 nM after 30 minutes

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26 and 1,540 nM after 4.5 hours This study had to be te mporarily discontinued, as this schedule had high clinical activity that resulted in tumor lysis so severe that one patient died (85). The group implemented procedures for monitoring patient s for tumor lysis syndrome and continued the study which resulted in a 45% overall response rate in CLL patients. Biological Correlates of Clinical Activity Several clinical trials have in cluded studies to determine if the same mechanisms of action for FP observed in vitro could be achieved in vivo Previous in vitro studies have shown that FP inhibits CDK activity (14, 49-52, 86, 87), induc es apoptosis (54, 55, 87-96), reduces the transcription and/or expression of anti-apoptoti c proteins Bcl-2 (B-cell leukemia/lymphoma 2) and Mcl-1 (myeloid cell leukemia seque nce 1) (49, 87, 96-100), and binds to DNA (101). In a phase I study by Thomas, et al. FP was tested as a single agent given as a 72 hour infusion every two weeks in patients with a variety of tumor types (67). Peripheral blood lymphocytes were collected during treatment and analyzed via flow cytometry for evidence of apoptosis or changes in cell cycle kinetics. No evidence of changes in these measurements was found; however, the authors noted that there were ear ly signs of clinical activity. During a phase I trial of FP combined with docetaxel in patients with metastatic breast cancer, Tan and colleagues examined Ki67, p53, and phosphorylated pRb in paired patient tumor and buccal mucosa samples (102). Ki67 was used as an indication of cell proliferation and phosphorylated pRb was used as an indirect m easurement of CDK activity. The buccal mucosa biopsies of ten of the eleven patients enrolled in the study showed increased nuclear expression of p53 and decreased expression of phosphorylated pRb after treatmen t with FP as a single agent. The authors postulated that the increase in p53 e xpression could have been due to the ability of FP to bind to DNA (101) or the ability of FP to reduce transcription or down regulate MDM-2, based on the activity of other CDK inhibitors (50, 103). Six paired tumor samples showed no

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27 changes in p53, Ki67, or phosphorylated pRb. The aut hors concluded that the biological effect of FP was achieved in the buccal mucosa; however, the treatments tested were not feasible due to dose limiting toxicities. A similar phase I study used FP in combination with cisplatin or carboplatin in patients with advanced tumors (104). Peripheral blood mononuc lear cells were analyzed before and after FP treatment and found to have increased p53 ex pression and increased phosphorylated STAT3 (signal transducer and activator of transcription 3) levels. Trea tment had no effect on cyclin D1, phosphorylated RNA polymerase II (indicator of C DK activity), or Mcl-1. The authors felt that there was a possibility that th e increased p53 and pSTAT3 levels were due binding of FP to DNA and that the lack of an effect by FP on cy clin D1 expression, the phosphorylation of RNA polymerase II, or Mcl-1 expression might have been due to the inab ility of FP to inhibit P-TEFb (CDK 9) in vivo There was a lack of clinical activity observed during the tria l and it was further postulated that the lack of an effect on Mcl-1 expression by FP could have been an explanation for this low clinical response. Alternatively, the authors could not definitively say that the same effects that were observed in noncycling peripheral blood cells c ould be observed in tumor cells, as these were not tested. Finally, a phase II trial of re lapsed or refractory melano ma patients had disappointing clinical results that the authors partially attributed to a lack of biological activity in vivo (80). Western blot analyses very similar to those perf ormed in the studies cited above found that only one patient out of eight tested had the expected results of decreased Mcl-1 with increased p53 expression and increased expressi on of phosphorylated STAT3 as a result of FP treatment. Two additional patients had decreased Mcl-1 in combination with lower levels of p53 and

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28 phosphorylated STAT3. It was noted that the form er patient progressed after one cycle of FP treatment. All of the trials cited above used FP as eith er a 1, 24, or 72 hour infusion. This is contrary to the most recent use of FP as a 30 minute bolus followed by a 4 hour infusion found to be highly effective in CLL (85). Most of the in vitro studies to which the above authors were attempting to correlate biological activity in vivo were performed in FBS. As previously cited, the newer infusion schedule takes the high percenta ge of protein binding of FP that occurs in HS into account by achieving a higher plasma FP con centration in a shorter time than that achieved in previous trials. Studies have been conducted in vitro in CLL cells grown in the presence of HS that show that FP is biological ly active under these conditions when used at concentrations higher than those previously utilized in FBS (100). Clinical Trials of Paclitaxel (PAC) and Combining Flavopiridol with Paclitaxel With the exception of recent studies in CLL, FP has had limited efficacy in clinical trials when used as a single agent. However, as out lined previously, there have been promising preclinical results showing synergy between PAC and FP. Early clinical trials have also tested FP in combination with PAC in cancer types other than ALL. PAC has been found to be effective as a single agent in the treatment of several types of cancer including breast, ovari an, and lung cancer, and mela noma (105, 106). PAC also has considerable in vitro activity against ALL (107) and has been tested in both adults (15, 108) and children (16) with leukemia. Studies in adults used 3 doses of 100 minutes each repeated every three weeks (15) and a 24 hour infusion repeated every 3-4 weeks (108). A trial in pediatric leukemia patients used a 24 hour PAC infusion; achieving peak plasma concentrations of approximately 1,000 nM. Unfortunately, these studi es did not report any substantial clinical responses. Minimal responses have been reported using PAC as a single agent in children with

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29 solid tumors (109). This trial used varying dos es of PAC as a 24 hour infusion repeated every three weeks. Peak plasma concentrations were dose-dependent and ranged from approximately 1,000 nM to 7,000 nM. Two out of 31 total patients tr eated reported significant toxicity. PAC is 89-98% bound to plasma proteins in vivo (110). Perhaps similar to FP, a shorter infusion schedule with the goal of obtaining a high p eak PAC plasma concentration might prove beneficial in the treatment of ALL. Promising results have been achieved when FP was combined with PAC in patients with a variety of solid tumor types (111). Clinical resp onses were observed in patients with esophagus, lung and prostate cancer, some of whom had progressed on PAC single agent treatment. It should be noted that the agents were given in the specific sequence of PAC followed by FP treatment. Project Rationale Preclinical in vitro studies or clinical studies usin g FP have never been conducted in relation to childhood ALL. Relapsed ALL patient s become increasingly refractory to agents typically used in the treatment of ALL, thus creating a need to investigate new drugs. I examined FP because of the high frequency of p15/p16 abno rmalities and altered expression of other cell cycle regulatory proteins in rela psed ALL. Results from preclini cal studies suggest that FP can act similarly to these molecules in that it inhibits CDK activity and induces cell cycle arrest. FP can also induce apoptosis in human cancer cells. Based on these findings, I performed in vitro studies of FP at different times of exposur e to mimic prolonged infusion and newer bolus schedules. During these studies I examined the ce ll death and alterations in cell cycle progression induced by FP. Clinical responses measured dur ing trials of FP as a single ag ent have shown that it has limited efficacy when used in a 72-, 24or 1 hour dosing schedule. Recent data from studies in

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30 CLL have suggested that prolonged FP infusions have a low overall response rate due to failure to achieve an effective free FP concentration as a result of secondary protein binding in human plasma. A shorter infusion of a higher dose of FP was found to be very promising. In order to model this new infusion strategy, this project includes experime nts using cultures that were grown in medium supplemented with human serum in place of fetal bovine serum. A higher concentration of FP is also administered over a shorter period of time when compared to previous experiments. This project has not only served as a means to determine the potential efficacy of FP when used as a single agent in ALL cell lines, but has al so served as a study to determine the effects of combining FP with PAC. In vitro studies and clinical trials in patients with types of cancer other than ALL have shown that FP can enhance the ac tivity of PAC. In some cases using FP in combination with PAC can ha ve a synergistic effect on in vitro treatment. This enhancement is dependent on the sequence in which the drugs ar e administered. In the case of FP combination treatment with PAC, this sequen ce dependence has been reported to be due to the ability of PAC to activate CDK 1 activity coupled to the inhib itory action of FP against this same CDK (56). Because PAC is not typically used in the treatment of ALL, I first established that ALL cell lines were sensitive to PAC treatment. I also tested whether FP enhances the efficacy of PAC in vitro and if this enhancement was dependent on the sequence in which the two drugs were administered. This drug combination could offer a treatment regimen to children with relapsed ALL that would utilize two agents to which the pa tients will not have been previously exposed. In the future, data from this project could be used to develop a clinical trial which would utilize either FP as a single agent or PAC in combination with FP, both in the schedule that I have found to be most efficacious. This project al so provides data to indicate the mechanism of

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31 action behind the efficacy of FP in ALL in order to provide a biological basis for the clinical study.

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32 Induction: 1 month; Results in comple te remission in 99% of patients Treatments can include the following: --L-asparaginase --vincristine --steroid --anthracycline for high-risk patients (daunorubicin) Intrathecal therapy-2 doses in the first month sin ce diagnosis and 4-6 doses during the next 1 or 2 months. Agents utilized include --methotrexate --hydrocortisone and cytosine arabinoside (ara-C) added for high-risk patients Patients with high white blood cell (WBC) count (hi gh risk) or WBC in the cerebral spinal fluid receive radiation to the brain and possibly the spinal cord. Ma y also administer high dose intrathecal methotrexate with leucovorin to treat side effects. Consolidation: 4-8 months; Reduces the remaining number of leukemic blasts Standard risk patients receive --methotrexate --6-mercaptopurine or 6-thioguanine --optional: vincristine and prednisone High risk patients receive: --L-asparaginase, doxorubicin, etoposide, cy clophosphamide, ara-C and dexamethasone substituted for prednisone (possibly two rounds) Maintenance: 1.5 to 2.5 yrs --methotrexate, 6-mercaptopurine --vincristine; prednisone or de xamethasone (every 4-8 weeks) Figure 1-1. Treatment of Childhood Acute Lymphobl astic Leukemia (ALL): 2-3 yrs. total (3)

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33 Figure 1-2. Cyclin dependent kinase (CDK) inhibitors func tion in the transition from G1 (Gap 1) to S (DNA synthesis) phase of the cell cy cle. p15 and p16 inhibit CDK 4 and CDK 6, as do p21 and p27. The restriction point of the cell cycle is located at the transition from G0 to G1 and marks the point at which the ce ll is no longer sensitive to external agents such as growth factors or a drug. Pursuing permission from American Association for Cancer Research: [Clinical Cancer Research] Shah MA, Schwartz GK. Cell cycle mediated drug resistance: an emerging concept in cancer therapy. Clinical Cancer Research 2001; 7:2168-2181., copyright 2001, origin ally published at Figure 1, p. 2169.

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34 Figure 1-3. p16 works in concer t with pRb to regulate the G1-S transition. p16 inhibits the activity of CDK 4 and CDK 6, thus prev enting the phosphorylation of pRb. In the hypophosphorylated state, pRb can prevent bindi ng of E2F/DP transcription factors to genes involved in pr ogression through the G1-S transition. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Cancer] Classon M, Harlow E. The retinoblastoma tumour suppressor in de velopment and cancer. Nature Reviews Cancer 2002; 2:910-917, copyright 2002, or iginally published as Figure 1, p.911.

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35 Figure 1-4. Flavopiridol is a pa n-CDK inhibitor. FP inhibits CDK 4, CDK 6 and CDK 2, thus inducing a G1-S arrest. FP can also inhibi t CDK 1 (cdc2) and induce G2-M arrest Reprinted by permission from Meniscus Ltd: [Horizons in Cancer Therapeutics: From Bench to Bedside] Shah MA, Schwartz GK. Cell cycle modulation: an emerging target for cancer therapy. Horizons in Ca ncer Therapeutics: From Bench to Bedside 2004; 4(3):3-21., copyright 2004, origin ally published as Figure 5, p.7.

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36 CHAPTER 2 FLAVOPIRIDOL DISPLAYS PRECLINICAL ACTIVITY IN ACUTE LYMPHOBLASTIC LEUKEMIA Introduction One of the most commonly used methods of evaluating the potential efficacy of chemotherapeutic agents prior to their use in patie nts is to determine the ability of the agent to prevent growth of cancer cells in vitro Results from methyl-thiazol -tetrazolium (MTT) assays have shown a correlation between in vitro sensitivity of leukemia cells taken from peripheral blood and bone marrow of patient s and clinical outcome (112-119) Hongo et al. (112) found that using agents determined to be efficacious in MT T assays resulted in better outcome for patients with ALL or acute nonlymphoblas tic leukemia when compared with patients whose treatment regimens were determined via conventional met hods of the time. Approxi mately 82% (n=11) of patients treated with agents determined to be e fficacious in MTT assays had complete or partial remissions as compared with 40% (n=15) of pa tients treated by conventional means. I have chosen to use a modified MTT assay as my initia l means of determining the sensitivity of ALL cell lines to FP. I have expanded my studies by testing the ability of FP to induce apoptosis and cell cycle arrest in ALL cell lines as cell proliferati on assays merely measure an increase or decrease in viable cell number. The mechanism of action of a chemotherapeutic agent is an integral part of determining how the agent will be used as well as what side effects might occur as a result of its use. Knowing the mechanism of action can also help to target cancer cell s without affecting noncancerous tissue. The ultimate fate of a cell, ie. cell death or senescence as a result of cell cycle arrest can have an effect on the progression of the cancer. Cells which senesce might have the ability to secrete signaling molecules which prom ote the growth of other cancer cells in the surrounding area (120). For some researchers, th is possibility makes apoptosis a preferred

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37 mechanism of action for anti-neoplastic agents. I have determined that FP has the ability to reduce ALL cell proliferation via a modified MTT assay and have fu rther investigated the ability of FP to induce cell cycle arrest and apoptosis. Through Western blot anal ysis I have observed a correlation between the concentration dependent effects of FP on cell proliferation and the endogenous phosphorylation of pRb Correlating these mechanisms with drug concentration and the effect that FP has on cell cycle regulatory el ements will serve as important information when deciding the use of FP as a single agent or in comb ination with other agents in the treatment of childhood ALL. Methods In Vitro Drug Sensitivity Testing In vitro drug sensitivity assays were modeled af ter those described originally by Pieters and colleagues (113). Cell lines were grown in RPMI 1640 (Mediatech, Inc. Herndon, VA) with 10% fetal bovine serum (FBS, Mediatech) or 10% human AB serum (H S, Mediatech) and 1% penicillin/streptomycin (Mediatech) at 37 C with 5% CO2. Nalm-6 (B-precursor ALL) was originated by Minowada, et al (121). Molt-4 and Jurkat (both T-cell ALL) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). RCH-ACV is a B-precursor ALL cell line provided by Dr. Seshadri (122) K562 (ATCC) is a chronic myelogenous leukemia (CML) cell line commonly used for in vitro testing in the NCI 60 cell line test set and was included in this study as a control. Exponentially growing cell cultures were plated in flat bottomed 96-well dishes in 100 L of cell culture medium with a diluti on of drug in vehicle appropriate for each agent. Vehicles included ethanol for dexamethasone (Sigma, St. Louis, MO), water for doxorubicin (Sigma), and dimet hyl sulfoxide (DMSO) for FP (Sanofi-Aventis, Bridgewater, NJ). All were further diluted in RPMI 1640. Samples for each drug concentration were tested in quadruplicate for each experiment Also tested in parallel were appropriate

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38 dilutions of vehicle without drug, which functione d as an untreated control for calculation of IC50. For the purpose of my study IC50 was defined as the concentr ation of drug at which cell proliferation was inhibited by 50% as compared to an untreated control. Ce ll lines were plated at a concentration of 1X105 cells/mL in 100 L for RCH-ACV, Molt-4, and Jurkat. Nalm-6 and K562 were plated at a concentration of 5X104 cells/mL in 100 L. Different cell concentrations were used in order to maintain the cultures in log phase growth th roughout the period of the experiments. Cell lines were incubated with dr ug for 96 hours, at which time WST-1 (4-[3-(4Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene disulfonate ) reagent (Roche, Indianapolis, IN) was directly added to each well according to manufacturers instructions. WST-1 is a modified version of the MTT (methy l-thiazol-tetrazolium) reagent. Absorbance was measured on a Molecular Devices (Sunnyvale, CA) Vmax kinetic microplate reader at 450nm, subtracting a reference wavelength of 650nm. IC50 was calculated by plotting leukemic cell survival (LCS) against drug concentration. The drug concentration at which LCS equaled 50% was defined as the IC50. LCS was calculated as follows: blank control blank treatedabs abs abs abs X 100%. (2-1) Results are the mean of at leas t two independent experiments. Western Blot Analyses ALL cell lines were tested for p16 protein expr ession with HeLa cells used as a positive control. HeLa extract was diluted into ex tract from Nalm-6 (previously found to be p16 deleted via Southern blot (40 )) in order to simulate a low leve l or variable amount of p16 protein expression. Protein extracts were prepared using Radio-Immunoprecipitation Assay (RIPA) Buffer (Sigma) with sodium orthovanadate (San ta Cruz, Santa Cruz, CA), phenylmethylsulfonyl fluoride (PMSF, Santa Cruz), and a protease in hibitor cocktail (Sigma). Fifty micrograms of

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39 protein was loaded onto a 4-20% gradient po lyacrylimide gel (Biorad, Hercules, CA) and subjected to sodium dodecyl su lfate-polyacrylimide gel electr ophoresis (SDS-PAGE). Proteins were transferred to a 0.2 M pore nitrocellulo se membrane (Biorad). After transfer, the membrane was blocked with 5% dry non-fat milk in TBS with 0.1% Tween-20 (TBS-T) for one hour with gentle agitation. Fo llowing blocking, the membrane was incubated at room temperature (RT) with mouse monoclonal IgG1 antibody to full length p16 protein (50.1, catalog number sc-9968, Santa Cruz) at a dilution of 1:375 in 5% non-fat milk for one hour. The membrane was then washed 3 X 15 minutes in TBS-T and incubated for one hour in goat antimouse IgG secondary antibody conjugated with horseradish peroxidase in 5% milk. Retinoblastoma protein phosphor ylated on serine 795 (pp-Rbser795) and retinoblastoma protein phosphorylated on serines 807 and 811 (pp-Rbser807/811) were resolved via SDS-PAGE after loading 25 g protein lysate. The proteins were transferred to a nitrocellulose membrane which was blocked as described and incubated with rabbit polyclonal antibodies to pp-Rbser795 and ppRbser807/811 (product numbers 9301 and 9308, Cell Signaling Technology, Danvers, MA) 1:1000 in 5% bovine serum albumin (BSA) overnight and treated as previously described. Detection of total p-Rb expression was performed using mouse monoclonal IgG1 antibody (IF8, catalog number sc-102, Santa Cruz) 1:200 in 1% BSA after blocking for 1 hour at RT with 1% BSA. Detection of actin (isoform non-specific) (C2, catalog number sc-8432, Santa Cruz) was used as a loading control on all membrane s. After washing 3 X 15 minutes, proteins were visualized on radiographic film via ECL or ECL Plus reagen t (Amersham, Piscataway, NJ). Results from Western analyses were obtained from at least two separate experiments. Measurement of Cell Death Two methods were utilized to detect cell deat h and/or apoptosis in drug treated samples. Samples were stained with Annexin V (Pharmin gen, San Diego, CA) and Propidium Iodide (PI)

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40 (Roche) as recommended by Pharmingen. Direct TUNEL (terminal deoxynucleotidyltransferase dUTP nick end labeling) staining was also perf ormed according to manufacturers instructions (Apo-Direct Kit, Pharmingen). Samples were analyzed via flow cytometry using a Becton Dickinson (San Jose, CA) FACSort flow cytomete r. Percentages of cell death/apoptosis were measured by obtaining the sum of the upper right and lower right quadrants of the scatterplot generated by analysis of samples stained with A nnexinV/PI. Percentages of apoptotic cells were measured via TUNEL by obtaining the percenta ge of the cell population staining positive for FITC-dUTP. Results were obtained from at least three independent experiments. Cell Cycle Analysis To determine cell cycle kinetics as a result of FP treatment, cell lin es were analyzed for DNA content using PI staining and flow cytometric analysis essentially as described by Ormerod (123). Data were generated using ModFit LT fo r Mac version 3.1 software (Verity Software House, Topsham, ME) Results are representative of at l east three independent experiments. Results ALL Cell Lines Used for in Vitro Testing Lack p16 Protein Expression I determined p16 protein expression in the cell lines used for in vitro drug sensitivity testing via Western blot. Nalm-6, REH, Molt-4, and Jurkat have been reported previously to have homozygous p16 deletions (33, 40, 124, 125), while the Hunger laboratory has found RCH-ACV to have intact p16 via Southern blot (126). HeLa cells we re used as a positive control. HeLa lysate was diluted into Nalm-6 (p16 deleted) lysate in order to simulate 10% and 1% p16 expression. I found that none of the ALL cell line s tested expressed a detectable amount of p16 protein, including RCH-ACV (Figure 2-1a).

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41 In Vitro Drug Sensitivity in ALL Cell Lines I determined the sensitivity (IC50) of Nalm-6, Molt-4, Jurkat RCH-ACV and K562 to a continuous 96 hour exposure to dexamethasone (Dex) and doxorubicin (Dox), two agents commonly used in the treatment of childhood ALL, and FP. Each of the four ALL cell lines and K562 were highly resistant to dexamethasone a nd variably sensitive to doxorubicin (Figure 21b). Each of the cell lines tested showed sensitivity to FP, with IC50s ranging from 99.5 nM in Molt-4 to 312.5.1 nM in K562. These values are similar to concentrations achieved in vivo in phase I/II trials of FP administered bo th as a 1 hour and a 72 hour infusion (67-69, 72, 77, 127) FP Induces Apoptosis in ALL Cell Lines WST-1 assays measure the numbers of viable cells present following exposure to drug. Decreased numbers of viable ce lls could be due to apoptosis, decreased cell proliferation, or both. I performed Annexin V/PI staining and subse quent flow cytometric analysis on cell lines which were exposed to drug for 72 hours to dete rmine whether FP induced apoptosis. First, I compared the cell death induced by FP and Dox in Nalm-6 and RCH-ACV at concentrations approximating the IC50 of each drug (FP:150nM; Dox:10ng/mL). At these concentrations, FP induced a substantially lower percentage of cel l death than Dox in both Nalm-6 and RCH-ACV (Figure 2-2a). I then examined apoptosis i nduced by 300 nM FP and observed much higher rates of apoptotic cell death, 93% and 83% in Nalm-6 and RCH-ACV respectively. I expanded these studies and confirmed that FP induces apoptosis by performing parallel Annexin V/PI and TUNEL analysis in Nalm-6, RCH-ACV, Molt-4 and Jurkat following 72 hours exposure to FP at various concentrations (Figure 2-2b and 2-2c). Fo r each cell line tested, modest levels (<25%) of apoptosis were induced by 72 hours exposure to 150 nM FP and high levels (>80%) were observed following exposure to 300 nM FP. Sim ilar results were seen via Annexin V/PI and

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42 TUNEL assays, confirming the apop totic nature of the observed ce ll death. Taken together, these data establish that FP treatment induces modest apoptotic cell death in B-precursor and T-cell ALL lines at lower concentrations and is a poten t inducer of apoptosis at higher concentrations. FP Induces Cell Cycle Arrest in ALL Cell Line s which Correlates with Effects on pp-Rb Protein Expression My results demonstrated that the inhibition of cell proliferatio n observed with WST-1 assays can only partially be attributed to apopt osis when cell lines are treated with 150 nM FP; however, 300 nM FP fully induces apoptosis. I hypot hesized that the rema ining inhibition at 150 nM FP could be due to cell cycle arrest. In orde r to test this hypothesi s I performed cell cycle analysis of samples treated with 0, 50, 150 a nd 300 nM FP for 24 and 48 hours. Treatment with 50 nM FP did not induce arrest when compared to an untreated control in RCH-ACV (Figure 23a) and Nalm-6 (Figure 2-4). I observed a transient G1-S arrest after 150 nM treatment that was present at 24 hours, but resolved by 48 hours. Sustained G1-S and G2-M arrest were induced after treatment with 300 nM FP; which was apparent at 24 hours and more pronounced at 48 hours. In order to address the possibility that the transient nature of the arrest induced by 150 nM treatment was due loss of drug potency over time, I performed experiments in which treated cells were exposed to FP for 24 hours, at which tim e the growth medium and drug were replaced (Figure 2-3b). Cultures were allowed to incubate in parallel with those established 24 hours prior for an additional 24 hours. Following incubati on all cultures were ev aluated for cell cycle kinetics. Data showed similar cell cycle phase dist ributions between cultures treated with FP for 48 hours and those which had medium and drug replaced after 24 hours. To investigate the mechanism of the observed ce ll cycle arrest, I dete rmined expression of total pRb and specific phospho-pRb forms (pp-Rbser795 and pp-Rbser807/811) in parallel to cell cycle analysis (Figure 2-3c). Phos phorylation of pRb on ser 795 has been largely linked to CDK 4

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43 activity and regulation of the G1-S transition by pRb (128). Phos phorylation of ser 807/811 has also been linked to CDK 4 activ ity (129). Treatment of ALL cell lines with 300 nM FP resulted in a sustained decrease in pp-Rbser795 and pp-Rbser807/811 protein expression, which correlates with the G1-S arrest observed at this drug concentration. Total pRb protein le vels indicate stable levels of total protein and a decrease in the expressi on of the hyperphosphorylated form of pRb (upper band) that is dependent on drug concentration an d time of exposure to FP. Treatment with a low level of FP (50 nM) did not re sult in a decrease in the phos phorylation of pRb; however, treatment with 150 nM and 300 nM FP did resu lt in a decrease in the expression of the phosphorylated form of pR b after 48 hours treatment. Apoptotic Effects of FP in Human Serum Others have shown that FP is 92-95% protein bound in human plasma and that there is a decrease in the activity of FP in CLL cells when grown in human plasma or serum vs. FBS (82). In order to determine if supplementation with hu man serum (HS) would have a similar effect on FP efficacy in ALL cell lines, I tested the abil ity of FP to induce apoptosis in Nalm-6, RCHACV, Jurkat, and Molt-4 grown in medium suppl emented with FBS and compared this to cell death of cell lines grown in HS. In order to mimic the peak drug levels that occur with FP infusion schedules with high activity against CLL cells (30 minute bolus followed by a 4 hour infusion), I measured cell death at 4.5 hrs (Figur e 2-5a). For measurement of cell death at a subpeak level, I analyzed after 24 hours drug exposur e (Figure 2-5b). Varying concentrations of FP were used in keeping with those found to be achieved in CLL patients treated with the above schedule at approximately these time points (85) After 4.5 hours, a modest percentage of cell death is induced in Nalm-6 and RCH-ACV ( 15-20% and 10%, respectively). In contrast, approximately 55-60% cell death is induced in Ju rkat and Molt-4 at 4.5 hours. When comparing the cell death induced in cultures supplemented with FBS to that of HS, I show that the

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44 differences in the percentage of apoptosis induced in media containing FBS vs. HS are not substantial for Nalm-6, RCH-ACV and Molt-4; however, more ce ll death was induced by FP in Jurkat cells grown in media containing FBS vs. HS. The percentage of apoptosis observed after 24 hours FP treatment was higher than that at 4.5 hours in all four cell lines tested. When comparing the cell death achieved in cultures supp lemented with FBS to that of HS at the 24 hour timepoint, I observed substantial differenc es between media containing FBS and HS for RCH-ACV at all FP concentrations tested and at the lowest FP concentration (300 nM) in Nalm6 and Jurkat. My results show no differ ences between FBS and HS for Molt-4. Discussion The poor outcome of children with ALL w ho experience a bone marrow relapse despite intensive chemotherapy and/or stem cell transplant, makes it imperative to identify agents with novel mechanisms of action. Based on the frequent acquisition of p16 deletions at relapse (27) and alterations in expression of genes that encode for cell cycle regulatory proteins at relapse (18), I performed preclinical studi es of FP in ALL cell lines. My results support the use of these cell lines as a model of relapsed ALL in that no ne of the lines expresse d p16 protein and all were resistant to dexamethasone and variably sensi tive to doxorubicin, two agents commonly used in the treatment of ALL and to which relapsed patien ts frequently become re sistant (130). I report that childhood ALL cell lines are sensitive to FP providing a biological rationale for clinical trials of FP in relapsed ALL. I found that FP was active in a concentration dependent manner against ALL cell lines. At a concentration approximating the IC50 determined in WST-1 assays (150 nM), FP induced transient cell cycle arrest with a limited percentage of apoptosis. At approximately twice this concentration, FP was a potent inducer of cell death. This information provides a dual mechanistic explanation for the decrease in viable cell number that I observed in WST-1 assays.

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45 CDKs phosphorylate pRb and ther efore regulate its ability to sequester transcription factors involved in cell proliferation during the G1-S phase transition. Phosphorylation of pRb on approximately 16 different serine and/or thre onine residues can be attrib uted to the activity of specific CDKs (131). My Western blot an alysis shows that expression of pp-Rbser795 and ppRbser807/811 is reduced after 24 and 48 hours treatment with 300nM FP. These data correlate with the sustained G1-S arrest observed after 300 nM FP treatme nt and show that FP treatment results in a reduction of endogenous CDK activity. Evaluation of total pR b protein expression showed consistent expression across all treatment levels, indicating that the decrease in phospho-specific pRb was not due to loss of total pRb. Treatment with 150 and 300 nM FP resulted in a decrease in the hyperphosphorylated form of total protei n after 48 hours drug expo sure. This decrease further illustrates the reduction in CDK activity as a result of FP treatment in ALL cell lines. This also suggests a mechanism for the transient G1-S arrest observed after 150 nM FP treatment and the G2-M arrest I observed after 300 nM treatm ent. The apoptosis and cell cycle arrest induced by FP treatment in ALL cel l lines provide two potential modes of treatment in ALL. FP could be used as a single agent to induce a cytoto xic effect or be utili zed in combination with another chemotherapeutic agent that would complement the abil ity of FP to induce cell cycle arrest. My observation that FP inhibits CDK activ ity and induces cell cycl e arrest in ALL cells suggests that there may be sche dule dependent differences in ac tivity if FP is combined with other agents, particularly those with cell cycle specific activity. Initial phase I/II trials of FP in many human cancer s were disappointing. While in vitro studies showed that FP was efficaceous against a di verse variety of tumors at concentrations of 100-300 nM, no significant clinical activity was s een with prolonged infusion regimens, despite achievement of similar FP concentrations in vivo (69-73). Shinn and colleagues hypothesized

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46 that the disparity between in vitro and in vivo activity might be due to differences in binding of FP to plasma proteins present in FBS used in vitro vs. those present in human plasma (82). They confirmed that for CLL cells the FP IC50 was significantly (approximately 10-fold) higher in vitro when experiments were performed in human plasma rather than bovine serum. This suggested that infusion schedules that produced high peak FP concentrations might be more effective than prolonged lower dose infusion schedules Early phase clinical trials confirmed this hypothesis in CLL using a 30 minute bolus dose followed by a 4 hour infusion (85). Expanded studies are ongoing. Based on these observations, I also examined the relative efficacy of FP in vitro in experiments using human serum compared to bovine serum. In my experiments I observed fewer differences between ALL cell line se nsitivity to FP in HS vs. FBS than observed by Shinn and colleagues. Importantly, despite some differences among the cell lines tested, substantial amounts of apoptosis were induced by FP in all cell lines under conditions that are very similar to what might be observed clin ically, particularly at the 1000 nM and 2000 nM levels at 4.5 hours and 300 nM le vel at 24 hours. These data sugge st that the newer FP infusion schedules found to be very promising in CLL should be utilized to test FP against relapsed ALL.

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47 Figure 2-1. Fifty percent i nhibitory concentration (IC50) determinations via WST-1 in cell lines that lack p16 protein expre ssion. A) Western blot for p16 pr otein expression; Lane 1) 100% HeLa (positive control), 2) empty la ne, 3)10% HeLa, 4) 1% HeLa, 5) Nalm-6, 6) RCH-ACV, 7) REH, 8) Molt-4, 9) Jurk at B) IC50 valuesSD for Dex, Dox and FP after 96 hours drug exposure measured via WST-1 cell proliferation assays.

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48 Figure 2-2. Flavopiridol induces apoptosis in ALL cell lines in a concentration dependent manner. A) Scatter plots from flow cytome tric analysis of Annexin V/PI stained samples of Nalm-6 and RCH-ACV co mparing cell death induced by 72 hours exposure to 150 nM and 300 nM FP to the cell death induced by 72 hours exposure to 10 ng/mL Dox, an agent known to induce apoptos is. Percentage to right of each plot represents the sum of the lo wer right quadrant (cells in the early stage of apoptosis) and upper right quadrant (late stage apoptosis) of each plot B) Sca tterplots generated using two different staining methods afte r 72 hours continuous exposure to 300 nM FP show similar results; AnnexinV/PI (left) and TUNEL st aining (right) C) Comparison of results from AnnexinV/PI to TUNEL in cell lines treated with 0, 150, and 300 nM FP respectively.

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49 Figure 2-3. Flavopiridol induces G1-S and G2-M (Gap 2-mitotic) arre st in RCH-ACV with reduced phosphorylation of pRb A) Cell cycle data after 24 and 48 hours exposure to 0, 50, 150 and 300 nM FP. Treatment with 50 nM FP has no effect on cell cycle kinetics when compared to untreated control. Data show a transient G1-S arrest after 24 hours exposure to 150 nM FP and a sustained G1-S arrest after 24 and 48 hours treatment with 300 nM FP. Also shown is a sustained G2-M arrest following 300 nM FP exposure. B) Release from G1-S arrest post-24 hours expos ure is not due to loss of drug potency. RCH-ACV cells were treate d with FP for 24 hours, at which time medium and drug were freshly repla ced. Samples collected 24 hours after replacement (48 hours total time of drug expos ure) show cell cycle kinetics that are comparable to samples without drug replacement. C) Western blot showing a sustained decrease in expression of pp-Rbser 795 and pp-Rbser807/811 after exposure to 300 nM FP. Total p-Rb expression remain s constant with a decrease in the hyperphosphorylated form (upper band) after 150 and 300 nM treatment.

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50 Figure 2-4. Flavopiridol induces transient G1-S arrest in Nalm-6. Ce ll cycle data after 24 and 48 hours exposure to 0, 50, and 150 nM FP. Treatment with 50 nM FP has no effect on cell cycle kinetics when compared to untreated control. Data show a G1-S arrest after 24 hours treatment with 150 nM. Cell cycle kine tics return to baseline after 48 hours treatment.

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51 Figure 2-5. Efficacy of FP in human serum. Ce ll lines were exposed to clinically achievable concentrations of FP for 4.5 hours and 24 hours in medium supplemented with FBS or HS. A) More cell death was induced in Jurkat cells treated for 4.5 hours and supplemented with FBS than those in HS ; however, the cell death induced in the remaining cell lines was approximately equal between the two types of sera. B) After 24 hours treatment of RCH-ACV, more cell death was induced by FP treatment at all concentrations tested in cells supplemente d with FBS than those supplemented with HS. Differences in cell death between the tw o types of sera were also observed in Nalm-6 and Jurkat after treatment with 300 nM FP. No differences between sera were observed in Molt-4.

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52 CHAPTER 3 PRECLINICAL STUDIES OF FLAVOPIRIDO L COMBINED WITH PACLITAXEL IN ACUTE LYMPHOBLASTIC LEUKEMIA Introduction Clinical trials of FP involving cancer types ot her than ALL have shown that FP has limited efficacy when used as a single agent. As outlined in Chapter 1, several in vitro studies have shown that the efficacy of traditional chemothe rapy agents can be increased when used in combination with FP. One such traditional agen t is paclitaxel (PAC). Few clinical trials involving FP combination therapy have been pe rformed; however, promising results have been obtained in a variety of solid tumor patients us ing FP in combination with PAC (111). PAC has shown in vitro toxicity in leukemia cell lines; however, has had limited efficacy in clinical trials in children and adults with leukemia (15, 16) PAC and FP represent two drugs to which ALL patients will not have been previous ly exposed. This fact as well as in vitro data showing synergy between FP and PAC in other cell types lead me to question if PAC/FP combination therapy could hold promise in the treatment of ALL. It has been previously found that the interaction between PAC and FP is dependent on the sequence in which the two drugs are administered in vitro. Combination therapy is most efficacious when PAC precedes FP in the treatment sequence (PAC FP), as opposed to the reverse or concurrent therapy (56, 60). I have confirmed these findings in ALL cell lines as well as determined optimal treatment duration for each agent prior to testing combination therapy. In an effort to maintain the clinical relevance of my findings I have also taken the currently accepted in vivo infusion schedule for each drug into account when designing my experiments. The recommended schedule for PAC administration is either 3 hour or 24 hour infusion (110). I have tested 6 and 24 hours exposure to PAC. Earl y trials involving FP used a 72 hour continuous infusion schedule. My experiments reflect this, as in experiments in which PAC was combined

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53 with FP, cell lines were exposed to FP for 72 hours. Later studies by others have shown that FP is highly protein bound and that supplementing ce ll culture medium with human serum (HS) in place of fetal bovine serum (FBS) d ecreases the sensitivity of CLL cells to FP (82). In order to address this I have conducte d experiments in media supplem ented with both FBS and HS. Methods Materials ALL cell lines were obtained and cultured as described previously (132). PAC (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxi de (DMSO) and freshl y diluted in RPMI 1640 prior to each experiment. FP (Sanofi-Aventis, Bridgewater, NJ) was dissolved in DMSO and further diluted in RPMI 1640 no more than 30 days prior to each experiment. Single Agent In Vitro Sensitivity Assays Nalm-6 and RCH-ACV were exposed to 0-300 nM FP in RPMI 1640 with 10% fetal bovine serum (FBS; Mediatech, Inc. Herndon, VA) continuously for 72 hours. Cell death was measured every 24 hours via flow cytometric an alysis of Annexin V (Pharmingen, San Diego, CA)/Propidium Iodide (PI, Roche, Indianapolis IN) as described previously (132). Treatment duration and sensitivity to PAC were determ ined by exposing Nalm-6 and RCH-ACV to 0-100 nM PAC for 6 hours and 24 hours in parallel followed by cell death measurements every 24 hours for a total of 72 hrs (Figure 3-1). In a separate experiment, Nalm-6 was exposed to 01,000 nM PAC in RPMI 1640 supplemented with 10% human serum (HS; Mediatech) for a period of 6 hours in parallel to samples treated in media supplemented w ith 10% FBS with cell death measured every 24 hours. In order to determine the effect of HS on untreated cell proliferation and viability, growth curves were generated using trypan blue staining (Sigma) of samples grown with HS compared to FBS.

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54 Drug Combination Studies FP was combined with PAC using a drug concen tration ratio of 1:10 (PAC:FP) in Nalm-6 and RCH-ACV. Due to greater sensitivity to PAC, Molt-4 and Jurkat were treated at a ratio of 1:20 to allow for a lower concentration of PAC to be utilized. Cell lines were exposed to PAC in cell culture medium supplemented with 10% FBS for approximately six hours, washed, and then treated with FP for an additional 72 hours. Tr eatment duration was selected based on PAC and FP single agent experiments. Sing le drug controls for PAC consis ted of six hours incubation with PAC followed by incubation in drug free medium fo r approximately 72 hours. Cell lines used for single drug treatment with FP were incubated in drug free RPMI 1640 for approximately six hours; after which the cell lines were incubated with FP for an additional 72 hours. At the completion of the 72 hour incuba tion, cell death was evaluated fo r all samples. In order to confirm that results similar to that found in medium supplemented with FBS could be achieved in cultures supplemented with HS, Nalm-6 wa s exposed to PAC for 6 hours followed by FP for 72 hours at a concentration ratio of 1:3. Cont rol samples were treated and cell death was evaluated in the same manner as descri bed for combination studies in FBS. Treatment Sequence Nalm-6 and RCH-ACV were used to determin e the optimal treatment sequence. One 1:10 combination was chosen from the drug combination studies in FBS in order to confirm that PAC followed by FP (20nM PAC 200nM FP) was indeed the most efficacious treatment sequence. Briefly, for the samples in which FP treatment followed PAC treatment (PAC FP), cell lines were cultured in RPMI 1640 with and without 20 nM PAC for 6 hours, then washed and transferred to RPMI 1640 with a nd without 200 nM FP. In sample sets in which FP treatment preceded PAC treatment (FP PAC), cell lines were treated similarly, with the sequence of drug exposure reversed. Cell death measurements we re taken immediately after FP treatment for

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55 PAC FP and its controls. For FP PAC and its controls, cell death was determined approximately 18 hours following completion of PAC treatment. Concurrent exposure experiments were performed separately; during whic h cell lines were treated with both agents for a total of 72 hours. Cell death was measured at 24 hour intervals via flow cytometry as previously described. Statistical Analysis All experiments, excluding concurrent expos ure to PAC and FP, were performed with three independent replicates. A mixed model was used to analyze each experiment. The replicates for each experiment were consider ed a random factor. If there was a significant interaction (p<0.05) between the variables in each experiment, mainly cell line, treatment, time of exposure, and drug concentration depending on the type of experiment, then Least Squares Means of the treatment combinations were co mpared using a Students t-test or F test. Results Single Agent FP Treatment In order to determine the time of exposure to FP that resulted in the maximum cell death response in ALL cell lines, I incubated Nalm-6 and RCH-ACV with 0-300 nM FP with cell death measured at 24, 48, and 72 hours. There was a significant con centration dependent response in both cell lines (p<0.000 1; Figure 3-2 a). I compared the cell death induced after 24 hours treatment to the cell death induced after 72 hours. There were no significant differences in cell death based on time of exposure at the 50 nM concentration in either Nalm-6 or RCH-ACV. In Nalm-6, there was a significant difference in cell death between 24 and 72 hours exposure to 300 nM FP (p<0.0001), while in RCH-ACV these di fferences were observed between 24 and 72 hours exposure to 150 nM (p=0.0034) as well as between 24 and 72 hours exposure to 300 nM FP (p<0.0001).

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56 Single Agent PAC Treatment I exposed Nalm-6 and RCH-ACV to PAC for 6 or 24 hours and measured apoptosis at 24, 48, and 72 hours following initial ex posure. I observed concentra tion dependent cell killing in Nalm-6 and RCH-ACV samples treated for both 6 and 24 hours with a greater percentage of cell death observed after 24 hours vs. 6 hours drug exposu re (Figure 3-2 b). Further, I show that cell lines treated for 6 hours then transferred to drug-free medium show a greater gradation in response between 10 nM PAC and 100 nM PAC than those treated for 24 hour s, particularly in Nalm-6. Cell death peaked 48 hours post-treatment and remained consistent with no substantial change at 72 hours post-treatment in both cell lines. Statistical analysis showed that time of exposure (6 or 24 hours) to PAC wa s a significant factor in the pe rcentage of cell death observed in both Nalm-6 (p=0.0001) and RCH-ACV (p=0.0138). The post-treatment time at which cell death measurements were taken was also a si gnificant factor in both Nalm-6 (p<0.0001) and RCH-ACV (p=0.0008). The interaction between the f actors of time of exposure and sample time was significant in Nalm-6 (p=0.0427); however, this interaction was not significant in RCHACV (p=0.6812). My results indica te that even though a 24 hour drug exposure time resulted in a greater percentage of cell death, a shorter exposure period of 6 hours still resulted in a substantial amount of cell d eath. Importantly, incubation fo r 48-72 hours post-treatment was needed in order to achieve a maximum response. Combination Treatment with FP and PAC Based on my single agent studies I treated f our cell lines with PAC for 6 hours; then transferred the cultures to media containing FP for 72 hours and measured cell death at the end of this period. Nalm-6 and RCH-ACV were tr eated with a drug concentration ratio of 1:10 (PAC:FP) and Molt-4 and Jurkat at a ratio of 1:20. I observed a concentration dependent cell death response for both the single agent treatments as well as each combination (Figure 3-3). My

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57 results demonstrate that when PAC is combined w ith FP the cell death that results is significantly higher than when either of the two agents is ut ilized by itself. I found a statistically significant difference (p<0.05 or p<0.0001) between single agen t and combination treatment in the range of 10-30 nM PAC and 100-300 nM FP in Nalm-6. In RCH-ACV these differences were present for treatments in the range of 15-25 nM PAC and 100-250 nM FP. Significant differences (p<0.0001) were present at 510 nM PAC and 100-150 nM FP in Molt-4 and Jurkat. Determination of Optimal Schedule for PAC+FP I tested PAC FP, the reverse sequence, and each of th e single agent cont rols to confirm that the former was the most effective treatm ent sequence. The most promising dose level (20 nM PAC 200 nM FP) was selected for more detailed analysis. Combining PAC with FP in the sequence PAC FP results in significantly greater cell death in Nalm-6 than FP PAC or single agent treatment (Figure 3-4 a). Th e cell death resulting from the PAC FP was significantly higher than the other treatments with p valu es ranging from p<0.0001 to p=0.001. I observed a similar response in RCH-ACV with p values ranging from p<0.0001 to p<0.01 (Figure 3-5). I also examined concurrent exposure to 20 nM PAC and 200 nM FP and observed no enhancement (Figure 3-4 b) in activity. Indeed, the cell death induced af ter treatment with both agents is less than that observed wi th FP alone across three days of testing. Activity of PAC in Human Serum Studies have found that 89-98% of PAC is protein bound in human serum (110). This prompted us to investigate how the efficacy of PAC in ALL cell lines would be affected if culture medium was supplemented with human AB serum (HS) in place of fetal bovine serum (FBS). I show that Nalm-6 cells exposed to 10, 100, and 1,000 nM PAC for 6 hours in media supplemented with HS underwent significantly le ss apoptosis than observed when experiments were performed using media supplemented with FBS (Figure 3-6). These differences in PAC

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58 sensitivity were not due to intrinsic differences in cell growth in media supplemented with FBS vs. HS (Figure 3-6 d). Combination Studies in Human Serum Shinn and colleagues have reported that 63-100% of FP is free (non-protein bound) in FBS, as compared to only 4.7-7.9% free in human plasma in vitro (82). Based on this information and the high level of protein bindi ng by PAC to plasma prot eins, I treated Nalm-6 with PAC combined with FP at a concentration ra tio (PAC:FP) of 1:3 in medium supplemented with HS in order to confirm that the enhancem ent of PAC activity by FP that I observed in FBS could also be achieved under these culture cond itions (Figure 3-7). Highe r concentrations of PAC were used in order to compensate for the lo wer sensitivity of ALL cell lines to PAC in the presence of HS. I show that FP significantly enha nces the efficacy of PAC at all concentrations tested (p<0.0001). Combination treatment was significantly different from FP single agent treatment at the lowest concentration tested (p<0.05); however, signifi cant differences did not exist at higher concentrations of FP most probably due to a high percentage of cell death induced from single agent FP treatment using this prolonged exposure schedule. Discussion There is a significant need to identify novel agents and new combination treatments for relapsed ALL. Others have shown that PAC and FP have limited efficacy when utilized as single agents in leukemia patients ( 16, 75, 133); however, together thes e drugs have mechanisms of action that complement each othe r and might effectively target aberrations in cell cycle regulation that are commonly presen t in ALL cells at relapse. My studies demonstrate that ALL cell lines are sensitive to both PAC and FP in vitro, and that FP enhances the efficacy of PAC in a sequence specific manner.

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59 My observation that the cell d eath induced after treatment with 1,000 nM PAC in HS is comparable to cell death achieved by treatment w ith 100 nM PAC in FBS is consistent with prior studies by others showing a high de gree of PAC binding by proteins pr esent in HS. In contrast to data of Shinn and colleagues in CLL (82), my prev ious studies showed rela tively little difference between the in vitro activity of FP when experiments are pe rformed in HS vs. FBS. My current observations confirm that despite a decrease in sensitivity to PAC in the presence of HS, FP enhances the efficacy of PAC under these treatment conditions. The time of actual drug exposure utilized for PAC treatment (6 hours) in my experiments was in keeping with the recommended clinical administration of 3 or 24 hours infusion (110). My findings show that 48-72 hours of incubati on are needed to achieve a maximum cell death response to PAC treatment. I also found that the amount of cell death induced by FP in the concentration ranges studied was dependent upo n the duration of exposure, with 72 hours inducing a peak response. Based on these two f actors, I studied a 72 hour FP exposure in the combination studies. This schedule of FP exposure is different than newer FP dosing strategies that administer a 30 minute FP bolus followed by a 4 hour infusion to produce much high peak FP concentrations than those achieved with other infusion schedules, which have yielded promising early results in patients with refracto ry chronic lymphocytic le ukemia (CLL) (83). In other studies, I have found that shorter in vitro exposure of the same AL L cell lines, cultured in media containing either FBS or HS to high FP concentrations similar to those attained in CLL clinical trials induced substant ial amounts of apoptosis. Based on the results attained in the current studies, I anticipate that administrati on of PAC prior to the FP bolus should enhance ALL cell death and suggest that this combination should be investigat ed in clinical trials for relapsed ALL.

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60 Figure 3-1. Experimental design for PAC single agent treatment.

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61 Figure 3-2. Cell death induced by treatment with FP or PAC in Nalm-6 and RCH-ACV. A) Percent cell death induced after 72 hours treatment with 0, 50, 150, 200, and 300 nM FP measured every 24 hours via flow cytometric analysis of Annexin V/PI stained samples of Nalm-6. Results are the mean of three independent experimentsSD. Significant differences in time of exposure at identical drug concentrations are indicated; *p<0.0001 and #p=0.0034. B) Cell death induced after 6 hours or 24 hours treatment with 0, 1.0, 10, 100 nM PAC measured every 24 hours thereafter for a total of 72 hours. Resu lts are the mean of three independent experimentsSD. Significant differences at identical concentrations between the 24 and 72 hour sample times are indicated; p values ranged from 0.01 to <0.0001 for all symbols.

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62 Figure 3-3. Flavopiridol enhances the efficacy of PAC in ALL cell lines. FP was combined with PAC in the sequence PAC FP at a concentration ratio of 1:10 in Nalm-6 and RCH-ACV and 1:20 in Molt-4 and Jurkat. Cell lines were treated with PAC for 6 hours immediately followed by FP for 72 hours. Cell death was meas ured at the conclusion of treatment via flow cytometric analysis of Annexin V/PI stained samples. Results represent averages from at least three independent experimentsSD.

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63 Figure 3-4. PAC FP is a more efficacious treatment sequence than FP PAC or concurrent exposure in Nalm-6. A) Nalm-6 was tr eated for 6 hours with 20 nM PAC followed by 200 nM FP for 72 hours or the revers e sequence. Both sequences included appropriate single agent contro ls and an untreated control. Cell death was measured via flow cytometric analysis of Annexin V/PI stained samples immediately following FP treatment for PAC FP and approximately 18 hours after PAC treatment for FP PAC. Results are the mean of three in dependent experimentsSD. Statistical significance was measured by comparing PAC20 FP200 to the remaining treatment sequences. *p 0.0001; #p=0.001. B) Concurrent e xposure to PAC and FP in Nalm6. Cell lines were simultaneously exposed to 20 nM PAC and 200 nM FP for a total of 72 hours with cell death measured every 24 hours.

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64 Figure 3-5. PAC FP is a more efficacious treatment sequence than FP PAC or concurrent exposure in RCH-ACV. A) RCH-ACV was treated for 6 hours with 20 nM PAC followed by 200 nM FP for 72 hours or the reverse sequence. Both sequences included appropriate single ag ent controls and an untreated control. Cell death was measured via flow cytometric analysis of Annexin V/PI staine d samples immediately following FP treatment for PAC FP and approximately 18 hours after PAC treatment for FP PAC. Results are the mean of th ree independent experimentsSD. Statistical significance was measured comparing PAC20 FP200 to the remaining treatment sequences. *p 0.01. B) Concurrent exposure to PAC and FP in RCH-ACV. Cell lines were simultaneously exposed to 20 nM PAC and 200 nM FP for a total of 72 hours with cell death measured every 24 hours

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65 Figure 3-6. Efficacy of PAC in Nalm-6 in the pr esence of human serum. Cell death resulting from 6 hours exposure to PAC in the concen trations shown in medium supplemented with HS compared to that in FBS measured every 24 hours for a total of 72 hours. a) 10 nM PAC b) 100 nM PAC c) 1000 nM PA C d) growth curve from untreated cell cultures comparing HS and FBS supplements All results are th e mean of three independent experimentsSD; *p<0.05, #p 0.0002.

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66 Figure 3-7. Flavopiridol enhances the efficacy of PAC in human serum. PAC was combined with FP at a concentration ratio of 1:3 in Nalm-6 cultured in the presence of HS. Nalm-6 was treated and sampled as in previous assays in FBS. Results are the mean of three independent experimentsSD.

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67 CHAPTER 4 CONCLUSIONS AND DISCUSSION FP Single Agent Studies Establishing an in Vitro Treatment Model of ALL In this project I have focused on determining the potential efficacy of FP both as a single agent and in combination with PAC in ALL ce ll lines. I tested these agents based on their mechanisms of action. FP could ta rget defects in cell cycle regul ation produced by mutations in human cancer, such as p16; which is frequently altered at the gene a nd/or protein expression level in relapsed ALL (124). The action of PA C as an inducer of CDK1 and microtubule depolymerization inhibitor complements this acti vity. Following the first chapter of background, the second chapter of this dissert ation details the resu lts that I obtained when testing FP as a single agent. As part of my initial studies, I es tablished that the cell lines to be used for sensitivity testing were an accu rate model of relapsed ALL by determining their level of p16 expression and their sensitivity to dexamethasone and doxorubicin, agents typically used to treat ALL (3). I found that despite the fact that one of th e cell lines had an intact p16 gene, none of the cell lines expressed p16 (Figure 2-1) I also showed that the cell lines were highly resistant to dexamethasone and variably sensitive to doxor ubicin; findings not unl ike what would be obtained in a patient (2). Importantly, the cell line s were sensitive to FP at concentrations similar to those found to be effective in vitro by others. Drug Sensitivity Testing via Cell Proliferation Assays Results for drug sensitivity were obtained us ing WST-1; a modified version of the MTT cell proliferation assay. As intr oduced in the se cond chapter, in vitro sensitivity to chemotherapeutic agents measured via this t ype of assay has been found to correlate to in vivo efficacy (112-119). In order to use these assays, I fi rst needed to establish that the cell lines could

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68 be maintained untreated in log phase growth ove r the standard time course of four days. I found that using a cell concentration of 1X104 cells/well in 96 well plates gave the best exponential growth over time (Figure 4-1 a, b). Nalm-6 was la ter established at a lower concentration due its lower doubling time (Chapters 2 and 3). In addition, as part of my preliminary studies I showed that measuring cell proliferation over time via a cell proliferation assay result ed in the same type of exponential growth curve as when the numbe r of viable cells over time was measured by trypan blue exclusion (Figure 41 c); thus validating the assay. Cell proliferation assays were used to generate growth curves to establish the IC50 concentrations reported in Chapter 2 (Figure 2-1). Examples of these growth curves may be found in Figure 4-2. The Mechanism of Cell Death Indu ced by FP in ALL Cell Lines Also in Chapter 2, I established that FP i nduces apoptosis in A LL cell lines. FP induced less apoptosis at its IC50 than a similar concentration of doxorubicin, a known cytotoxic agent (Figure 2-2). I also showed that FP treatment re sulted in apoptosis consis tently across four ALL cell lines (Figure 2-2). Results were obtained after 72 hours treatment to reflect treatment strategies current at the time. These measurements were taken using flow cytometric analysis of both Annexin V/PI and TUNEL stained samples in order to confirm that the level of cell death measured using Annexin V/PI assays was consistent with another method. Based on the ability of FP to induce cell cycle arrest in cancer cell types other than ALL (14, 53) and the apparent disp arity in cell death at the IC50s for doxorubicin and FP, I conducted a study of cell cycle kinetics afte r treatment with both low and hi gh concentrations of FP. I found that at the IC50, FP induced a transient G1-S block that appeared af ter 24 hours treatment; with cell cycle kinetics re turning to baseline by 48 hours treatment (Figure 2-3). This arrest gave an explanation for the decrease in cell proliferation observed at this FP concentration despite a lack of apoptosis. Treatment with a concentration of FP twice the IC50 resulted in a sustained G1-S

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69 and G2-M arrest over the course of 48 hours. Drug re placement studies showed that the transient nature of the G1-S arrest at the IC50 was not due to loss of drug potency over time. Further, I established that FP prevents th e phosphorylation of specific serine residues on pRb, an indication of a decrease in endogenous CDK activity. FP Activity in Human Serum Recent findings have shown that FP is highly protein bound in human plasma and that this low drug availability can decrease in vitro efficacy (82). Researchers hypothesized that this could explain the disappointing result s obtained in clinical trials using FP as a 72, 24 or 1 hour infusion, despite obtaining plas ma concentrations similar to that found to be effective in vitro (20-400 nM) (69-81, 124). As explained in Chapte rs 2 and 3, Byrd and colleagues from Ohio State University (OSU) designed a clinical trial fo r patients with refractory CLL with the goal of obtaining a plasma FP concentration of 1.5 M after a 30 minute bolus dose followed by a 4 hour infusion (83). The trial was quite successful despite issues with tumor lysis syndrome, with an overall response rate of 45%. In order to determine if protein binding would have an effect on the cell death induced by FP treatment in ALL cell lines, I tested in vitro sensitivity in the presence of HS and compared it to FBS. Cell death measurements were taken after 4.5 hours and 24 hours continuous exposure to mimic peak a nd trough concentrations in the OSU infusion schedule (Figure 2-5). I found that despite the high level of protein binding in human serum reported by others, it is possible to achieve a high percentage of cell death in ALL cell lines in vitro with concentrations that mimic those expect ed to be produced by the treatment schedule utilized by Byrd and colleagues.

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70 PAC+FP Combination Studies Note about Statistical Analysis Chapter 3 details my studies of FP in comb ination with PAC. To make meaningful comparisons between treatments, statistical analys is was applied to the data. The analyses of each data set followed the same basic pattern; be ginning with a mixed model analysis of variance (ANOVA) followed by post-tests to determine if there were significant differences between treatments, times of exposure, or treatment sequences depending on the variables of the individual experiment. ANOVA was chosen as the method of an alysis based on the fact that each type of experiment involved multiple comparisons. If a Students t-test had been applied to each individual measurement within each data set, the probability of obtaining significant p values would have been artificially high (134). By definition ANOVA compares the actual resu lts to the data that would have been obtained if the null hypothesis were correct. ANOVA was used to test the significance of the interaction between the variable s for each experiment. If the nu ll hypothesis were correct, there would be no interaction between any of the variab les in the experiment. Each p value given from the mixed model analysis assigns a percentage to the probability that the interaction present was due to chance. If significant interactions betw een variables were presen t, results based on one variable could not be analyzed for significance without taking the other variables into account. The mixed model designation to the ANOVA simp ly states that there were fixed and random factors in each of the experiment s. The variables tested in each experiment were defined as being fixed effects and the number of replicates wa s taken as a random effect. The mixed model analyses are given in tables which are di scussed throughout the te xt that follows.

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71 If the interaction between the factors in each experiment was significant, then the Least Squares Means of the replicates from each experime nt were compared using a Students t-test or an F-test. These analyses are also in cluded in the discussion that follows. Enhancement of PAC Activity by FP PAC and FP have complementary modes of ac tion in that PAC enhances CDK 1 activity while FP is a pan-CDK inhibitor. This is the hypothesis behind why the enhancement of PAC by FP is dependent on the order in which the drugs are administered (56) As summarized in Chapter 3, others have shown that FP can enhance the activity of PAC in vitro in cancer types other than ALL (56, 60). Promising results have al so been obtained during a clinical trial using patients with various types of can cer (111). I chose to test PAC in combination with FP due to the aberrations in cell cycle regul atory proteins frequently fou nd in relapsed ALL patients and because this treatment regimen would offer a ne w possibility to children with relapsed ALL using two agents to which they would not have been previously exposed. In order to conduct these experiments I first needed to establish whether ALL cell lines were sensitive to PAC and determine a treatment schedule for each of the drugs as single agents. After testing FP in two cell lines over 72 hours, I was able to determine that the time of exposure had a significant effect on the percentage of cell d eath induced by FP. Statistical analysis was conducted by first determining if there was a si gnificant interaction between the factors of FP concentration and time of exposure (Tables 41 and 4-2). When it was determined that a significant interaction existed, the data were fu rther analyzed by determ ining if there were significant differences in cell deat h at different times of exposure for a given FP concentration (Tables 4-3 and 4-4). With the resu lts given in Chapter 3 (Figure 3-2) I was able to conclude that 72 hours FP treatment gave a peak cell death response.

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72 Through single agent PAC studies I established that ALL cell lines were sensitive to this agent. I chose to measure cell death as a resu lt of 6 hours and 24 hours treatment based on earlier pharmacokinetic studies by others (110, 135, 136) Following drug exposure, apoptosis was measured every 24 hours for a total of 72 hours. Statis tical analysis of my da ta showed that there was a significant interaction betw een PAC concentration, time of exposure, and sample time in Nalm-6 but not RCH-ACV (Tables 4-5 and 4-6). I chose to use a 6 hour e xposure to PAC for my combination studies, as this resulted in a better range of choices for drug concentrations to test given the gradation in response between 10 nM and 100 nM when compared to 1.0 and 10 nM after 24 hours exposure. Also, as detailed in Chapter 3, my data showed that 48-72 hours incubation were required to achieve maximum cell death after treatment (Figure 3-2). Statistical analysis of PAC single agent treatment ma y be reviewed in Tables 4-7 and 4-8. Methods of Determining Synergy I combined FP with PAC to determine if the combination would increase the efficacy of the single agents. Synergy can be defined as when the effect of a combination of agents is greater than the sum of the effects of each of the single agents (137) There are various methods to determine if the effect of treatment with multiple agents is synergistic, including isobologram analysis, fractional effect, and median-effect an alysis (138). Isobologram analysis begins by measuring the dose of each drug required to pro duce the same effect, e.g. 50% cell death. These doses are plotted against each other and a line is drawn connecting the two doses (Figure 4-3) (139). The line is said to repres ent the doses of the two drugs which are equipotent. If a dose combination produces the designated effect and is plotted far below the line, this combination is considered synergistic, e.g. point Q in Figure 4-3. If the drug combination is plotted far above the line, it is considered to be antagonistic (point R) Points very close to the line represent additivity (point P). The isolobolgram met hod requires a large number of meas urements, applies only if the

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73 drugs have similar modes of action (mutually excl usive) and can only be used for combinations of two drugs (138). The fractional product method is very intu itive in that one simply multiplies the percentages represented by the una ffected fraction (e.g. percent vi able cells post-treatment) for each single agent (138). If the co mbination of drugs results in a pe rcentage that is equal to the product of the two single agents, then the two ag ents are additive. The requirements of this method are that the drugs must have different mo des of action (mutually non-exclusive) and that the dose-effect curves for the agents are hyperbolic. The most common method in current literatur e used to evaluate for synergy between agents is based on the median-effect principle authored by Chou and Talalay (61). One uses median-effect analysis to determine a Combinat ion Index (CI) value for each drug combination. According to median effect analysis, if CI=1.0 th is indicates an additive relationship between the two drugs. If CI<1.0, synergy is present and if CI >1.0 antagonism is indicated. This method has the advantage of allowing the researcher to ev aluate a minimal number of drug concentrations and determine the relationship betw een greater than two drugs if de sired. In addition, one is not limited to evaluating agents with only the same or different modes of action. Both mutually exclusive and mutually non-exclusive agents can be analyzed. To understand median-effect analysis, let us firs t examine the median effect principle. This principle is based on the IC50 for each individual agent. Consider statement 4-1: 2 1 2 1fu fa= 2 2 1 1fu fa fu fa = 2 2 1 1Dm D Dm D (4-1) The term (fa)X is the fraction affected by drug (percen t cell death after tr eatment with drug), fu is the fraction unaffected by drug (percent viable cells), D is the dose of a single drug in the

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74 combination and Dm is the IC50 for that drug if it were used as a single agent. If drugs 1 and 2 were combined at their IC50s, 2 2 1 1fu fa fu fa =0.5+0.5=1.0. (4-2) Now consider two examples from PAC combined with FP in Nalm-6 at a ratio of 1:10. In the first, 10 nM PAC was combined with 100 nM FP. The IC50 for PAC was 28.95 nM based on the single agent controls in the experiment and the IC50 for FP was 279.1 nM. Thus, according to statement 4-1: 2 2 1 1Dm D Dm D 1 279 100 95 28 10 =0.703 (4-3) In the second example, 15 nM PAC was co mbined with 150 nM FP with the same IC50s as in the first example for a sum equaling 1.055. Compara tively, the sum in the first example is 30% below 1.0, whereas the sum in the second exam ple is 6% above 1.0. Through the use of the median-effect principle, one can conclude that th e combination in the first example is synergistic and the second combination is additive, providing that both agents have the same mode of action. From this information one understands how CI values based on 1.0 were derived. From statement 1, median effect analysis defines CI as: CI= 2 2 1 1Dx D Dx D (4-4) D is the dose of each drug used in the combination and Dx is the dose of each single agent that would be required to induce the same percentage of cell death caused by the drug combination. In the case of two or more agents having differe nt modes of action, Equation 4-4 is modified to: CI= 2 1 2 1 2 2 1 1Dx Dx D D Dx D Dx D (4-5)

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75 FP Combined with PAC Combination studies were initiated by first establishing a concentra tion ratio for PAC and FP. Under median-effect analysis, it is suggest ed that agents are combined using a set drug concentration ratio (137), in this case PAC:FP. Se veral ratios were tested in Nalm-6 including 1:5, 1:10, 1:12, and 1:15 (Figure 4-4). I f ound that FP enhanced the activity of PAC most dramatically when the two agents were used in the ratios of 1:10 and 1:12. I performed medianeffect analysis to generate Combination Index (C I) values for the four ratios tested. A CI<1.0 indicated synergy, while CI>1.0 indicated an tagonism and CI=1.0 indicated an additive relationship between PAC and FP (61). Table 4-9 shows CI values at the 50% effective dose for the drug combination (ED50), ED75, and ED90 for each ratio. When evaluating combination data using median-effect analysis, two sets of CI valu es are generated based on the modes of action of the two drugs tested: mutually exclusive and mutua lly non-exclusive CI valu es. I chose to utilize the mutually non-exclusive CI values, as PAC and FP have different modes of action. Though the CI results were <1.0 when PAC was combined with FP at a ratio of 1:12, the actual cell death measurements were more compelling when the two agents were combined at a ratio of 1:10 (see Figure 4-4). This ratio also allowed for the us e of lower concentrations of both agents. Nalm-6 and RCH-ACV were test ed using a ratio of 1:10. Wh en testing Molt-4 and Jurkat, I used a ratio of 1:20 to account for the fact that these cell lines were exquisitely sensitive to PAC. This allowed for the use of a lower PAC concentration. Results from cell death measurements shown in Chapter 3 indicated a si gnificant difference in the cell death induced by single agent controls when compared to each comb ination in all four cell lines tested (Figure 33). A mixed model analysis similar to what was employed for FP and PAC single agent studies was utilized to evaluate the overall significance of the results (Tables 4-10 and 4-11). Nalm-6 and RCH-ACV were evaluated under the same analys is, as these cell lines were treated with the

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76 same concentration ratio (1:10). Mo lt-4 and Jurkat were subjected to a separate analysis, as both were treated with a drug concentr ation ratio of 1:20. It was f ound that there was a significant interaction between the factors cell line, treatment (PAC, FP, or PACFP) and drug concentration in all four cell lines tested. Based on the overall significance of the results, the cell death induced by each single agent control was co mpared to its respective combination. The results for which there were significant diffe rences are reported in Tables 4-12 and 4-13. Representative CI values for the ED50, ED75, and ED90 for each cell line are reported in Table 414. The data ranged from being slightly synergistic (Nalm-6 ED90 CI=0.939) to antagonistic (Jurkat ED50 CI=1.59), with half of the drug combinations showing near additivity to slight antagonism. The CI values reported in this chapter represent those obtained based on PAC and FP having different modes of action and clearly show that the degree of synergy between PAC and FP is very slight where it is present. In Chapte r 3 I show that FP enhan ces PAC activity and vise versa by measuring the simple effects of the dr ugs. In order for this enhancement to be considered synergistic via median-effect analysis the differences between single agent treatment and combination therapy would need to be several orders of magnitude highe r than the data that I obtained (61). This can be explained by the median-eff ect plot which graphs log (D) vs. log fu fa and ultimately connects to the median-effect equa tion from which all of the above equations are derived. Stated more simply, analysis of comb ined drug effects through median-effect analysis requires log order differences be tween single agent and combinati on treatment. For example, if instead of obtaining the data repor ted in Chapter 3 at a drug concentration ratio of 1:10, I had found similar results using a ratio of 1:100 or 1: 1000, my CI values would have been much lower and therefore more synergistic.

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77 Sequence Dependent Enhancement Results reported previously by others show that enhancement of PAC activity by FP is dependent on the sequence in which the agents are administered (56). In order to confirm this in ALL cell lines, I chose the most promising treatme nt from my combination studies to determine if PACFP, FPPAC or concurrent exposure would resu lt in the highest pe rcentage of cell death. As reported in Chapter 3, the percentage of cell death re sulting from standard treatment (PACFP) was compared to the reverse sequence and single agent controls. An ANOVA was utilized to analyze the statistical significan ce of treatment sequence prior to comparing the individual effects of treatment. A one-way anal ysis of variance was used for Nalm-6 and a weighted one-way analysis was used for RCH -ACV (Tables 4-15 and 4-16) to make this determination. The weighted analysis was used due to an inconsistent sample size for some of the treatment conditions for this cell line. The term one-way connotes that the experiments were categorized in one way: by treatment sequence instead of by treatment sequence and cell line. Treatment sequence was a significant factor in the percentage of cel l death resulting from the various treatments tested. Statistics were not applied to the data fr om concurrent exposure experiments, as these data were generated from one experiment. I confirmed that PACFP was the most efficacious treatment sequence in Nalm -6 (Figure 3-4 a). The statistical analysis comparing 20 nM PAC200 nM FP to the reverse sequence and single agent controls can be found in Table 4-17. I also confirmed the proper treatment sequence in RCH-ACV (Figure 3-5 a, Table 4-18). I showed in Figure 3-4 b and Figure 3-5 b that concu rrent exposure is not a feasible option for this drug combination, as it resulted in less cell death than the sum of the two single agents.

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78 Drug Sensitivity in Human Serum Due to the reported difference in binding by FP to human plasma proteins vs. proteins in FBS and the resulting decrease in sensitivity in C LL cells, I compared the sensitivity of ALL cell lines to FP in FBS and HS and found that ther e was not a substantial difference in sensitivity between the two sera (Figure 2-5). PAC is al so highly plasma protein bound and I report in Chapter 3 that there is a 10-fold decrease in th e sensitivity of ALL cell lines to PAC in the presence of HS when compared to FBS (Figure 3-6) Statistical analysis s howed that the type of serum used had a significant impact on the results (p=0.0370; Tabl e 4-19). Statistical comparisons between the two types of sera at specific PAC concentrations were performed to supplement the data shown in Chapter 3 (Table 4-20) I reported that the di fference in sensitivity between FBS and HS was not due to a significant difference in cell proliferation in the two sera. The statistical analysis on which this conc lusion was based is repor ted in Table 4-21. Based on these results I wanted to confirm that the enhancement in PAC activity by FP that I had observed in FBS could also be achieved in the presence of HS. I chose to use a concentration ratio of 1:3 based on preliminary experiments using a variety of ratios (Figure 45). This concentration ratio allowed for the use of higher concentrations of PAC to compensate for lower sensitivity in HS. However, these concen trations are still substantially lower than the plasma concentrations reported during clinical trials of PAC in children with leukemia or solid tumors (16, 109). I found that FP enhances the efficacy of PAC in a manner similar to the enhancement found in FBS using a ratio of 1:10 or 1:20. Statistical analysis showed that despite a lack of significance in the in teraction between treatment and dr ug concentration, there was an enhancement of PAC activity by FP (Tables 4-22 and 4-23).

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79 Placing Perspective on this Project Potential Side Effects of Single Agent and Combination Therapy As previously discussed in Chapters 2 and 3, severe tumor lysis syndrome (TLS) resulted during the initial clinical studies of the curr ently used administration schedule for FP (83). Though this was a clear sign of the efficacy of FP in CLL, the toxicity that resulted caused the death of one patient enrolled in the study. Steps have since be en taken to prevent TLS through the use of prophylactic therapy prior to the admini stration of FP and monitoring of patients while on therapy. An algorithm for monitoring for hyperkal emia has also been instituted as part of the study (140). According to official monitoring criteria from the NCI, the level of hyperkalemia that has resulted has been low in both inpati ents and outpatients; however, pre-treatment, potassium chelation therapy, and dialysis have still been required in some cases. Concern might be raised about the potential side effects of combining PAC with FP. Any dose limiting toxicities reported during trials of PAC in patients with leukemia have occurred at concentrations in the micromolar range; much higher than the concentrations used in my experiments. It should also be noted that these concentrations were achieved after a 24 hour infusion. I am proposing a shorter infusion time for PAC in my combination studies. Where Does FP Fit into the Treatment Scheme of ALL? When a novel agent becomes available as a possi ble addition to the regimen used to treat ALL, researchers and clinicians must determine fo r what stage of therapy the new agent is best suited. Because childhood ALL has such a high cure rate, it is difficult to measure a significant improvement as a result of the addition of a new ag ent to the initial stages of therapy. Some feel that novel agents should replace current therapies with the goal of decrea sing toxic side effects rather than increasing the cure rate; particularly when the drug is a targeted agent that would be used in a subgroup that alrea dy has a positive prognosis (141). While it might be somewhat

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80 beyond the scope of this dissertation, consid ering whether FP would have a place in the treatment of ALL is relevant to proposing a clin ical trial. Some might question whether a panCDK inhibitor has a place in an age of cancer drug discovery characterized by targeted therapies. FP has been used with success in trials of CLL patients. Others have s hown that the biological mechanism behind the capability of FP to kill CLL ce lls is its ability to decrease the transcription and protein expression levels of short-lived anti-apoptotic molecules such as Bcl-2 and Mcl-1 (100). These molecules were targeted based on th e need of CLL cells, wh ich are non-cycling, to express them continuously in order to remain in a state of senescence. While ALL is characterized by many types of chromosomal translocations and other genetic aberrations, there is not one specific molecule that can be targeted across subgroups of patients, such as the BCR-ABL tyrosine kinase pr oduced by the 9;22 translocation that has made imatinib mesylate (Gleevec) so successful in patients with chronic myelogenous leukemia. As previously discussed in Chapters 2 and 3, mo re modern studies using microarray technology have found significant differences in the expressi on of genes that regulat e cell cycle, DNA repair and apoptosis between the times of diagnosis and relapse in ALL; however, more work is necessary to discern a clear patte rn in gene expression that woul d reveal which aberrations lead to relapse (18). Several studies remain that show that p16 is altered at the gene and/or protein expression level in up to 50% of ALL cases. Because FP can function in the p16 pathway and there is a lack of available targ ets that affect a comparable per centage of ALL patients, the fact that FP is not a precisely targeted therapy should not be a hindrance to its possible use in ALL. Future Directions This work represents an initial study of the efficacy of FP as a single agent and combined with PAC in ALL cell lines. It might be benefici al to expand these studies into patient samples, in order to determine if the treatments woul d be efficacious in samples which are far less

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81 removed from a patient than immortalized cell li nes. I would also propos e testing the biological basis of the apoptosis induced by FP as a single agent by determining the activation of caspases and downregulation of antiapoptoti c molecules such as Bcl-2 and Mcl-1 as result of treatment. Importantly, and perhaps in contrast to the work performed by others, I would only propose the further biological studies after FP had been su ccessfully tested in a patient population. The studies of FP contained herein, perhaps with the addition of single ag ent studies in patient samples, provide a biological just ification for a clinical trial of FP in children with ALL. I have shown that FP can induce apoptosis and cell cy cle arrest, both mechanisms that inhibit proliferation of cancer cells. If FP was found to be successful in tr eating children with ALL, then further studies into its mechanism of action in vivo would be warranted. These studies could hold the possibility of assisting researchers in discov ery of new targets for more potent agents on the horizon. Also, this information would provide a basis for using FP in combination with other agents such as PAC.

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82 Figure 4-1. Growth curves used to establish cell concentration for pro liferation assays. Trypan blue exclusion and WST-1 were utilized to measure the number of viable cells per well in a 96-well plate over a 4 day time period. a)growth curves generated from RCH-ACV; b)REH; c)growth curves gene rated using WST-1 for comparison to trypan blue exclusion.

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83 Figure 4-2. Representative dose-response curv es generated from cell proliferation assays.

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84 Figure 4-3. Illustration of isobologram analysis of combined drug effects. Given the combination of drugs A and B at equipoten t concentrations, possible responses from the drug mixture are shown. Point Q represents a synergistic effect, point P an additive effect and point R represents antagonism Reprinted by permission from American Society for Pharmacology and Experimental Therapeutics: [Journal of Pharmacology and Experimental Therapeutic s] Tallarida RJ. Drug synergism: its detection and applications. Journal of Pharmacology and Experimental Therapeutics 2001; 298(3):865-872, copyright 2001, origin ally published as Figure 1, p.866.

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85 Figure 4-4. Preliminary combination data at a variety of ratios in Nalm-6. Cell death measurements after treatment with PAC for 6 hours followed by FP for 72 hours at concentration ratios (PAC:FP) of 1:5, 1:10, 1:12, and 1:15.

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86 Figure 4-5. Preliminary combination data at a va riety of ratios in the presence of human serum. Cell death measurements after treatment with PAC for 6 hours followed by FP for 72 hours at concentration ratios (P AC:FP) of 1:2, 1:3, 1:4, and 1:5. The 1:3 ratio was chosen for further study.

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87 Table 4-1. Mixed model analysis for FP treatment duration in Nalm-6 Numerator DF Denominator DF F value P value FP Concentration 4 10 20.04 <0.0001 Day 2 20 25.93 <0.0001 Day*FP concentration 8 20 26.25 <0.0001 Table 4-2. Mixed model analysis for FP treatment duration in RCH-ACV Numerator DF Denominator DF F value P value FP concentration 4 10 7.90 0.0038 Day 2 20 67.28 <0.0001 Day*FP concentration 8 20 19.42 <0.0001 Table 4-3. Differences between treatment dur ation at a given FP concentration in Nalm-6 Sample timeFP concentration (nM) Sample timeFP concentration (nM) Difference in mean cell death T value P value Day 1 FP 150 Day 2 FP 150 0.3033 0.26 0.7978 Day 1 FP 150 Day 3 FP 150 0.6033 0.52 0.6113 Day 1 FP 200 Day 2 FP 200 0.2533 0.22 0.8305 Day 1 FP 200 Day 3 FP 200 1.6367 1.40 0.1766 Day 1 FP 300 Day 2 FP 300 -11.8500 -10.14 <0.0001 Day 1 FP 300 Day 3 FP 300 -18.5167 -15.85 <0.0001 Day 1 FP 50 Day 2 FP 50 0.4200 0.36 0.7230 Day 1 FP 50 Day 3 FP 50 0.8667 0.74 0.4669 Day 2 FP 150 Day 3 FP 150 0.3000 0.26 0.8000 Day 2 FP 200 Day 3 FP 200 1.3833 1.18 0.2503 Day 2 FP 300 Day 3 FP 300 6.6667 5.71 <0.0001 Day 2 FP 50 Day 3 FP 50 0.4467 0.38 0.7063 Table 4-4. Differences between treatment dur ation at a given FP concentration in RCH-ACV Sample time-FP concentration (nM) Sample timeFP concentration (nM) Difference in mean cell death T value P value Day 1 FP 150 Day 2 FP 150 6.5633 1.64 0.1162 Day 1 FP 150 Day 3 FP 150 -13.2733 3.32 0.0034 Day 1 FP 200 Day 2 FP 200 -22.0367 5.51 <0.0001 Day 1 FP 300 Day 2 FP 300 -38.4000 9.61 <0.0001 Day 1 FP 300 Day 3 FP 300 -56.9200 -14.24 <0.0001 Day 1 FP 50 Day 2 FP 50 0.8600 0.22 0.8318 Day 1 FP 50 Day 3 FP 50 1.7233 0.43 0.6709 Day 2 FP 150 Day 3 FP 150 6.7100 1.68 0.1087 Day 2 FP 300 Day 3 FP 300 -18.5200 4.63 0.0002 Day 2 FP 50 Day 3 FP 50 2.5833 0.65 0.5254

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88 Table 4-5. Mixed model analysis of PAC single agent treatment in Nalm-6 Numerator DF Denominator DF F value P value PAC concentration 3 14 174.68 <0.0001 Time of exposure (6 hours or 24 hours) 1 14 27.77 0.0001 PAC*Time 3 14 10.52 0.0007 Sample time (Day) 2 32 15.42 <0.0001 Day*PAC 6 32 31.63 <0.0001 Day*Time 2 32 3.48 0.0427 Day*PAC*Time 6 32 3.16 0.0150 Table 4-6. Mixed model analysis of PAC single agent treatment in RCH-ACV Numerator DF Denominator DF F value P value PAC concentration 3 14 108.48 <0.0001 Time of Exposure (6 hours or 24 hours) 1 14 7.91 0.0138 PAC*Time 3 14 5.90 0.0081 Sample Time (Day) 2 32 9.08 0.0008 Day*PAC 6 32 6.12 0.0002 Day*Time 2 32 0.39 0.6812 Day*PAC*Time 6 32 0.71 0.6454

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89 Table 4-7. Differences in cel l death based on incubation time af ter 6 or 24 hours PAC treatment in Nalm-6 Day PAC (nM) Time (hours) Day P value Difference in mean cell death Day 1 0 6 Day 2 0.1876 0.4799 Day 1 0 24 Day 2 0.0318 0.8074 Day 1 1 6 Day 2 0.1835 0.7225 Day 1 1 24 Day 2 0.7622 0.2860 Day 1 10 6 Day 2 0.9098 0.1913 Day 1 10 24 Day 2 <0.0001 -36.8805 Day 1 100 6 Day 2 <0.0001 -52.0495 Day 1 100 24 Day 2 <0.0001 -54.6743 Day 1 0 6 Day 3 0.0133 0.8677 Day 2 0 6 Day 3 0.2114 0.3879 Day 1 0 24 Day 3 0.0884 0.6504 Day 2 0 24 Day 3 0.6296 0.1571 Day 1 1 6 Day 3 0.0015 1.6311 Day 2 1 6 Day 3 0.0433 0.9085 Day 1 1 24 Day 3 0.0059 2.3689 Day 2 1 24 Day 3 0.0125 2.0829 Day 1 10 6 Day 3 0.1085 2.4893 Day 2 10 6 Day 3 0.0871 2.6806 Day 1 10 24 Day 3 <0.0001 -34.7488 Day 2 10 24 Day 3 0.8238 2.1317 Day 1 100 6 Day 3 <0.0001 -57.5053 Day 2 100 6 Day 3 0.6047 5.4558 Day 1 100 24 Day 3 <0.0001 -59.4446 Day 2 100 24 Day 3 0.6758 4.7703

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90 Table 4-8. Differences in cel l death based on incubation time af ter 6 or 24 hours PAC treatment in RCH-ACV Day PAC (nM) Time (hours) Day P value Difference in mean cell death Day 1 0 6 Day 2 0.4185 0.9311 Day 1 0 24 Day 2 0.8931 0.1687 Day 1 1 6 Day 2 0.3051 1.3073 Day 1 1 24 Day 2 0.0582 1.7955 Day 1 10 6 Day 2 0.6557 0.9633 Day 1 10 24 Day 2 0.3989 3.9142 Day 1 100 6 Day 2 0.0065 -14.7008 Day 1 100 24 Day 2 0.0196 -16.2206 Day 1 0 6 Day 3 0.795 0.3189 Day 2 0 6 Day 3 0.5811 0.6121 Day 1 0 24 Day 3 0.9794 0.0317 Day 2 0 24 Day 3 0.8727 0.2003 Day 1 1 6 Day 3 0.4016 1.0914 Day 2 1 6 Day 3 0.8488 0.2160 Day 1 1 24 Day 3 0.436 1.0129 Day 2 1 24 Day 3 0.0096 2.8084 Day 1 10 6 Day 3 0.5508 1.3161 Day 2 10 6 Day 3 0.8795 0.3527 Day 1 10 24 Day 3 0.0812 9.3543 Day 2 10 24 Day 3 0.3514 5.4401 Day 1 100 6 Day 3 <0.0001 -37.3090 Day 2 100 6 Day 3 0.0265 -22.6082 Day 1 100 24 Day 3 0.0001 -37.2492 Day 2 100 24 Day 3 0.0724 -21.0286 Table 4-9. Combination Index (CI) values for drug combination studies us ing a variety of ratios Nalm-6 PAC:FP ED50 ED75 ED90 1:5 1.53 1.65 1.80 1:10 1.25 1.08 0.939 1:12 1.14 0.885 0.715 1:15 1.39 1.26 1.16 Table 4-10. Mixed model analysis of Nalm-6 and RCH-ACV combination data Numerator DF Denominator DF F value P value Cell Line 1 2 20.83 0.0448 Treatment 2 61 150.97 <0.0001 Cell Line*Treatment 2 61 14.43 <0.0001 Drug concentration 4 61 114.93 <0.0001 Treatment*Concentration 8 61 5.30 <0.0001 Cell Line*Treatment*Concentration 9 61 3.61 0.0012

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91 Table 4-11. Mixed model analysis of Molt-4 and Jurkat combination data Numerator DF Denominator DF F value P value Cell line 1 2 0.03 0.8727 Treatment 2 56 188.13 <0.0001 Cell line*Treatment 2 56 9.70 0.0002 Concentration 4 56 1002.86 <0.0001 Treatment*Concentration 8 56 36.00 <0.0001 Cell line*Treatment*Concentration 12 56 2.32 0.0174 Table 4-12. Significant differen ces in treatment for a given cell line and drug concentration Cell line Combo treatment PAC (nM):FP (nM) Single agent control SE T value P value Nalm-6 10:100 100 nM FP 5.8083 2.93 0.0048 Nalm-6 10:100 10 nM PAC 5.8083 2.24 0.0286 Nalm-6 15:150 150 nM FP 5.8083 7.27 <0.0001 Nalm-6 15:150 15 nM PAC 5.8083 6.08 <0.0001 Nalm-6 20:200 200 nM FP 5.8083 8.49 <0.0001 Nalm-6 20:200 20 nM PAC 5.8083 8.41 <0.0001 Nalm-6 25:250 250 nM FP 5.8083 7.52 <0.0001 Nalm-6 25:250 25 nM PAC 5.8083 8.24 <0.0001 Nalm-6 30:300 300 nM FP 5.8083 3.97 0.0002 Nalm-6 30:300 30 nM PAC 5.8083 6.86 <0.0001 RCH-ACV 10:100 100 nM FP 5.0301 3.43 0.0011 RCH-ACV 15:150 150 nM FP 5.0301 4.86 <0.0001 RCH-ACV 15:150 15 nM PAC 5.0301 3.67 0.0005 RCH-ACV 20:200 200 nM FP 5.0301 4.57 <0.0001 RCH-ACV 20:200 20 nM PAC 5.0301 3.36 0.0014 RCH-ACV 25:250 250 nM FP 5.0301 3.63 0.0006 RCH-ACV 25:250 25 nM PAC 5.0301 5.45 <0.0001 Table 4-13. Significant differen ces in treatment for a given cell line and drug concentration Cell line Combo treatment PAC (nM):FP (nM) Single agent control SE T value P value Jurkat 5:100 100 nM FP 3.6222 12.58 <0.0001 Jurkat 5:100 5 nM PAC 3.6222 8.03 <0.0001 Jurkat 7.5:150 150 nM FP 3.6222 13.55 <0.0001 Jurkat 7.5:150 7.5 nM PAC 3.6222 5.99 <0.0001 Jurkat 10:200 200 nM FP 3.6222 4.87 <0.0001 Jurkat 10:200 10 nM PAC 3.6222 5.32 <0.0001 Molt-4 5:100 100 nM FP 3.6222 9.99 <0.0001 Molt-4 5:100 5 nM PAC 3.6222 7.08 <0.0001 Molt-4 7.5:150 150 nM FP 3.6222 11.21 <0.0001 Molt-4 7.5:150 7.5 nM PAC 3.6222 10.71 <0.0001 Molt-4 10:200 10 nM PAC 3.6222 6.67 <0.0001

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92 Table 4-14. Combination Index (CI) values for drug combination studies ED50 ED75 ED90 Nalm-6 1.25 1.08 0.939 RCH-ACV 1.18 1.15 1.15 Molt-4 1.58 1.34 1.14 Jurkat 1.59 1.33 1.11 Table 4-15. One-Way Analysis of Vari ance of treatment sequence in Nalm-6 Numerator DF Denominator DF F value P value Treatment Sequence 6 14 46.29 <0.0001 Table 4-16. Weighted One-Way Analysis of Variance of treatment sequence in RCH-ACV Numerator DF Denominator DF F value P value Treatment Sequence 6 14 16.86 <0.0001 Table 4-17. Significant differen ces between standard treatment sequence, reverse treatment sequence, and single agent controls in Nalm-6 Treatment 1 Treatment 2 Treatment 1 meanTreatment 2 mean SE T value P value 0FP 200 PAC 20FP 200 -29.3867 5.6021 -5.25 0.0001 0PAC 20 PAC 20FP 200 -44.0467 5.6021 -7.86 <0.0001 FP 200PAC 20 PAC 20FP 200 -22.9500 5.6021 -4.10 0.0011 FP 2000 PAC 20FP 200 -23.4267 5.6021 -4.18 0.0009 PAC 20FP 200 PAC 200 70.6333 5.6021 12.61 <0.0001 PAC 20FP 200 untreated 74.0600 5.6021 13.22 <0.0001

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93 Table 4-18. Significant differen ces between standard treatment sequence, reverse treatment sequence, and single agen t controls in RCH-ACV Treatment 1 Treatment 2 Treatment 1 meanTreatment 2 mean SE T value P value 0FP 200 PAC 20FP 200 -20.7433 6.8754 -3.02 0.0092 0PAC 20 PAC 20FP 200 -40.4933 6.8754 -5.89 <0.0001 FP 2000 PAC 20FP 200 -31.6603 7.3598 -4.30 0.0007 FP 200PAC 20 PAC 20FP 200 -30.9227 7.4244 -4.16 0.0010 PAC200 PAC20FP 200 -54.2467 6.8754 -7.89 <0.0001 PAC 20FP 200 untreated 58.4167 6.8754 8.50 <0.0001 Table 4-19. Mixed model analysis for comparis on of cell death induced by PAC in FBS vs. HS Numerator DF Denominator DF F value P value Type of serum 1 2 25.57 0.0370 PAC (nM) 4 14 62.75 <0.0001 Type*PAC 4 14 5.08 0.0097 Day 3 54 69.74 <0.0001 Day*Type 3 54 8.75 <0.0001 Day*PAC 12 54 18.85 <0.0001 Day*Type*PAC 12 54 1.69 0.0941

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94 Table 4-20. Comparison of cell deat h induced by PAC in FBS vs. HS Day Type of serum PAC concentration (nM) Day Type of serum PAC concentration (nM) P value Difference in mean cell death 0 FBS 0 0 HS 0 1 -1.49E-14 0 FBS 1 0 HS 1 1 6.59E-15 0 FBS 10 0 HS 10 1 -2.94E-14 0 FBS 100 0 HS 100 1 -2.20E-14 0 FBS 1000 0 HS 1000 1 1.69E-17 1 FBS 0 1 HS 0 0.5418 0.6095 1 FBS 1 1 HS 1 0.3828 1.076 1 FBS 10 1 HS 10 0.0363 4.455 1 FBS 100 1 HS 100 0.0002 12.94 1 FBS 1000 1 HS 1000 0.0235 11.24 2 FBS 0 2 HS 0 0.2391 1.029 2 FBS 1 2 HS 1 0.2649 1.540 2 FBS 10 2 HS 10 0.0578 3.416 2 FBS 100 2 HS 100 <0.0001 42.72 2 FBS 1000 2 HS 1000 0.0223 35.55 3 FBS 0 3 HS 0 0.9267 -0.1275 3 FBS 1 3 HS 1 0.9722 -0.0537 3 FBS 10 3 HS 10 0.2053 2.605 3 FBS 100 3 HS 100 <0.0001 42.38 3 FBS 1000 3 HS 1000 0.1712 27.57 Table 4-21. Mixed model analysis of cell viability FBS vs. HS Numerator DF Denominator DF F value P value Day 3 12 16.07 0.0002 Type of serum 1 2 1.09 0.4055 Day*Type 3 12 1.27 0.3294 Table 4-22. Mixed model analysis of combination studies in human serum Numerator DF Denominator DF F value P value Treatment 2 16 151.97 <0.0001 Drug concentration 3 6 12.12 0.0059 Treatment*Concentration 6 16 1.25 0.3349

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95 Table 4-23. Significant differe nces in treatment for combination studies in human serum Treatment 1 Treatment 2 P value Difference in mean cell death 300 nM FP 100 nM PAC, 300 nM FP 0.0112 -24.4877 100 nM PAC, 300 nM FP 100 nM PAC <0.0001 37.8793 375 nM FP 125 nM PAC, 375 nM FP 0.061 -32.0078 125 nM PAC, 375 nM FP 125 nM PAC <0.0001 63.9835 450 nM 150 nM PAC, 450 nM FP 0.4802 -16.7949 150 nM PAC, 450 nM FP PAC 150 <0.0001 78.2333 600 nM FP 200 nM PAC, 600 nM FP 0.8746 4.2426 200 nM PAC 200 nM PAC, 600 nM FP <0.0001 23.6096

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106 118. Hongo T, Yajima S, Sakurai M, Horikoshi Y, Hanada R. In vitro drug sensitivity testing can predict induction failure and early relapse of childhood lymphoblastic leukemia. Blood 1997;89(8):2959-2965. 119. Kaspers GJL, Veerman AJP, Van Zantw ijk CH, Smets LA, van Wering ER, Van Der Does-Van Den Berg A. In vitro cellular dr ug resistance and prognosis in newly diagnosed childhood acute lymphoblastic le ukemia. Blood 1997;90(7):2723-2729. 120. Roninson IB, Broude EV, Chang B. If not apoptosis, then what? Treatment induced senescence and mitotic catastrophe in tumor cells. Drug Resistance Updates 2001;4:303-313. 121. Minowada J, Tsubota T, Nakazawa S, Srivas tava BIS, Huang CC, Oshimura M, et al. Establishment and characterization of leukemic Tcell lines, B-cell lines and null-cell lines: A progress report on surface antigen study of fresh lymphatic leukemias in man. In: Thierfelder S, Rodt H, Thiel E, editors. Immunological dia gnosis of leukemias and lymphomas. Berlin, Heidelberg, New York: Springer-Verlag; 1977. p. 241. 122. Jack I, Seshadri R, Garson M, Michael P, Callen D, Zola H, et al. RCH-ACV: A lymphoblastic leukemia cell line with chromosome translocation 1;19 and trisomy 8. Cancer Genet Cytogenet 1986;19:261-269. 123. Ormerod MG. Analysis of DNA-genera l methods. In: Ormerod MG, editor. Flow Cytometry-Practical Approach. Third ed. Oxford : Oxford University Press; 2000. p. 83-97. 124. Drexler H. Review of alterations of the cy clin-dependent kinase inhibitor INK4 family genes p15, p16, p18, and p19 in human leuke mia-lymphoma cells. Leukemia 1998;12:845-859. 125. Quesnel B, Preudhomme C, Lepelley P, Hetuin D, Vanrumbeke M, Bauters F, et al. Transfer of p16inka/CDKN2 gene in leukaemic cell lines inhib its cell proliferation. British Journal of Haematology 1996;95:291-298. 126. Maloney KW, McGavran L, Odom LF, H unger SP. Different patterns of homozygous p16INK4A and p15INK4B deletions in childhood acute lymphoblas tic leukemias containing distinct E2A translocations. Leukemia 1998;12(9):1417-1421. 127. Zhai S, Sausville EA, Senderowicz AM, Ando Y, Headlee D, Messmann R, et al. Clinical pharmacology and pharmcogenetics of flavopiridol 1h i.v. infusion in pa tients with refractory neoplasms. Anti-Cancer Drugs 2003;14(2):125-135. 128. Connell-Crowley L, Harper JW, Goodrich DW. Cyclin D1/Cdk4 regul ates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. Molecular Biology of the Cell 1997;8:287-301. 129. Zarkowska T, Mittnacht S. Differential phos phorylation of the reti noblastoma protein by G1-S cyclin-dependent kinases. Journal of Biological Chemistry 1997;272(19):12738-46.

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107 130. Berg SL, Poplack DG. Pharmacology of antineoplastic agents and multidrug resistance. In: Nathan DG, Orkin SH, Ginsberg D, Look AT editors. Nathan and Oski's Hematology of Infancy and Childhood. Philadelphia: W. B. Saunders Company; 2003. p. 1274-1306. 131. Adams PD. Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks. Biochimica et Biophysica Acta 2001;1471:M123-M133. 132. Jackman KM, Hunger SP. Flavopiridol di splays preclinical activity in acute lymphoblastic leukemia. SUBMITTED 2007. 133. Colburn DE, Thomas DA, Giles FJ. Phase II study of single agent paclitaxel in adult patients with relapsed acute lymphocytic leuke mia. Investigational New Drugs 2003;21(1):109111. 134. Motulsky H. Intuitive Biostatistics. New York: Oxford University Press; 1995. 135. Gianni L, Kearns CM, Giani A, Capri G, Vigano L, Locatelli A, et al. Nonlinear pharmacokinetics and metabolism of paclitax el and its pharmacoki netic/pharmacodynamic relationships in humans. Journal of Clinical Oncology 1995;13(1):180-190. 136. Sonnichsen DS, Hurwitz CA, Pratt CB, Shuster JJ, Relling MV. Saturable pharmacokinetics and paclitaxel pharmacodynamics in children with solid tumors. Journal of Clinical Oncology 1994;12(3):532-538. 137. Chou T-C, Hayball MP. CalcuSyn. In. 2.0 ed. Ferguson, MO: Biosoft; 1996. 138. Chou T-C, Talalay P. Analysis of combin ed drug effects: a new look at a very old problem. TIPS 1983;4:450-454. 139. Tallarida RJ. Drug synergism: its detecti on and applications. Journal of Pharmacology and Experimental Therapeutics 2001;298(3):865-872. 140. Moran M, Fischer B, Broering S, Blum KA, Lin TS, Byrd JC, et al. Successful management (Mgt) of hyperkalemia associated with tumor lysis syndrome (TLS) in refractory chronic lymphocytic leukemia (CLL) patients (pts) receiving fla vopiridol on an active pharmacologically derived schedul e. Blood 2005;106(11):Abstract #2124. 141. Hunger SP. NOTCH1: prognostic factor or molecu lar target? Blood 2006;108(4):11171118.

PAGE 108

108 BIOGRAPHICAL SKETCH Kelly Marie Jackman was born in December 1976 in Jacksonville, Florida where she was raised and educated until movi ng to Gainesville to pursue her graduate study. She graduated from Mandarin High School in 1995 and earned a B.S. in biology from Jacksonville University in 1999. Kelly attended St. Vincents School of Medical Technology; beco ming a board certified and licensed Medical Technologist (M T) in late 2000. She then worked for a short time as an MT before enrolling in the Interdisciplinary Program in Biomedical Sciences in the College of Medicine at the University of Florida in 2001. Her graduate research comprised laboratory study of a novel chemotherapy agent for treatment of children with acute lymphoblastic leukemia. Kelly hopes to use her education to contribute to the positive transformation of therapies made available to cancer patients.


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A PRECLINICAL STUDY OF FLAVOPIRIDOL IN THE TREATMENT OF ACUTE
LYMPHOBLASTIC LEUKEMIA



















By

KELLY MARIE JACKMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































O 2007 Kelly Marie Jackman


































To those whose lives have been touched by cancer; especially women who have lost their
fathers. Even though our loved ones have moved on, a small part of them is still here in us.









ACKNOWLEDGMENTS

I would first like to thank my mentor, Dr. Hunger. I appreciate the time that he has given to

assist me in my writing and scientific development. Because of his patient guidance and honesty,

my ability to communicate in a more sophisticated and organized manner has matured

tremendously. My professional demeanor has also changed a great deal during my graduate

career; part of which I owe to the example set by Dr. Hunger. I will always be grateful for his

mentorship.

I would also like to thank the members of my committee, Drs. Rowe, Kilberg, and Fletcher

for their input into this work and their contribution to making sure that it progressed in a timely

fashion.

My appreciation also goes to the past and present members of the Hunger laboratory.

Through discussions of various topics, both professional and non-scientific, Dr. Victor Prima has

helped me to learn how to articulate and defend my ideas; skills which are integral to the

graduate experience. Dr. Mi Zhou was and still is a wonderful friend and an important source of

personal support. Both of these individuals have taught me so much about the cultures of

Ukraine and China, respectively, which has made my time in the lab a truly unique experience. I

would also like to thank Carole Frye for her valuable advice and technical assistance with my

experiments. My thanks also go to Amanda Rice, who became a great friend in the short time

that she worked in the lab.

Without the tireless help of the individuals in the Flow Cytometry Core Lab, this work

would not have been possible. I would like to thank Neil Benson, Bhavna Bhardwaj, and Steve

McClellan for assisting me with my experiments. Bhavna and Steve were willing spirits as they

performed most of the raw data analyses contained in this dissertation, for which I was always










grateful. I would also like to thank Linda Young in the Department of Statistics for patiently

helping me through all of the statistics required to properly analyze my data.

Other members of the College of Medicine that I wish to thank are Judy Adams, my

graduate secretary, as well as Cathy Hymon, secretary for Pediatric Hematology/Oncology.

Without these ladies I would not have been able to navigate the huge system that is UF. I would

also like to remember my fellow students and members of GSO.

Finally, and most importantly, I would like to thank my mom. She has been there through

everything; so many events that it becomes difficult to list them all. She has held my hand

literally and figuratively when it was time for many of the special people in my life, and hers, to

leave us. She has been there through the frustrations and triumphs of my life and academic career

and has always supported me. My mom is truly my best friend and I know that without her I

would not have gotten as far as I have. I hope that my achievement brings to her a sense of

satisfaction that a small part of the plan that she and my father set into motion many years ago

continues on.











TABLE OF CONTENTS



page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ............ ...... ._._ ...............8....

LIST OF FIGURES .............. ...............10....


AB S TRAC T ........._._ ............ ..............._ 12...

CHAPTER


1 BACKGROUND .............. ...............14....


General Treatment of Acute Lymphoblastic Leukemia (ALL) ................. ......................14
Relapsed ALL: The Clinical Problem .............. ...............15....
Important Regulators of Cell Cycle ................. ...............16........... ...
Cell Cycle Regulators in Cancer and ALL ................ ...............17........... ..
Flavopiridol (FP) .............. ..... .. .. .. ..... ... ...................2
In Vitro Testing of Flavopiridol in Combination with Other Agents: Sequence of
Administration and Synergy ............... ...............22....
Efficacy of Flavopiridol in Clinical Trials............... ...............23.
Biological Correlates of Clinical Activity .............. .... ........... .... ...... .......2
Clinical Trials of Paclitaxel (PAC) and Combining Flavopiridol with Paclitaxel .................28
Proj ect Rationale. ................. ...............29..............

2 FLAVOPIRIDOL DISPLAYS PRECLINICAL ACTIVITY IN ACUTE
LYMPHOBLASTIC LEUKEMIA ............ ..... ._ ...............36....


Introducti on ............ ..... ._ ...............36....
M ethods .............. .. ... .... ..................3
In Vitro Drug Sensitivity Testing .............. ...............37....
Western Blot Analyses .............. ...............38....
Measurement of Cell Death ............ ..... ._ ...............39...
Cell Cycle Analysis .............. ...............40....
R e sults............ _... ... .. .._ ...... .. .._... .. ... .... ... ............4
ALL Cell Lines Used for in Vitro Testing Lack pl6 Protein Expression ................... ....40
In Vitro Drug Sensitivity in ALL Cell Lines................ ...............41.
FP Induces Apoptosis in ALL Cell Lines.............. .. .........................4
FP Induces Cell Cycle Arrest in ALL Cell Lines which Correlates with Effects on
pp-Rb Protein Expression .............. ...............42....
Apoptotic Effects of FP in Human Serum ................. ....___ .......... .........4
Discussion ................. ...............44........ ......












3 PRECLINICAL STUDIES OF FLAVOPIRIDOL COMBINED WITH PACLITAXEL
INT ACUTE LYMPHOBLASTIC LEUKEMIA .................... ...............5


Introducti on ................. ...............52.................
M ethods .............. ...............53....
M materials .................. .. .. .... ...... .... ....... .............5

Single Agent in Vitro Sensitivity Assays .............. ...............53....
Drug Combination Studies .............. ...............54....
Treatment Sequence .............. ...............54....
Statistical Analysis .............. ...............55....
R e sults................... ... .. ....... .. ........ .............5

Single Agent FP Treatment .............. ...............55....
Single Agent PAC Treatment ................... .......... ...............56......
Combination Treatment with FP and PAC ................. ...............56...............
Determination of Optimal Schedule for PAC+FP ................. .............................57
Activity of PAC in Human Serum. ................ ........... ......... ........ ..........57
Combination Studies in Human Serum .............. ...............58....
Discussion ........._._._..... ..... ...............58....


4 C ONCLU SIONS AND DI SCU SSION .............. ...............67....


FP Single Agent Studies .........._.... .. ......_._... ............_ ............6
Establishing an in Vitro Treatment Model of ALL ......____ ..... ... ................67
Drug Sensitivity Testing via Cell Proliferation Assays............... ...............67.
The Mechanism of Cell Death Induced by FP in ALL Cell Lines .............. ...............68
FP Activity in Human Serum .............. ...............69....
PAC+FP Combination Studies .............. ...............70....
Note about Statistical Analysis............... ...............70
Enhancement of PAC Activity by FP ......__....._.__._ ......._._. ...........7
Methods of Determining Synergy .................... ...............7
FP Combined with PAC .............. ...............75...

Sequence Dependent Enhancement. ...._ ......_____ .......___ ............7
Drug Sensitivity in Human Serum .............. ...............78....
Placing Perspective on this Proj ect..........._...._ ....... ...___ .... .....___ ...........7
Potential Side Effects of Single Agent and Combination Therapy ................ ...............79
Where Does FP Fit into the Treatment Scheme of ALL? ............__.. ...___...........79
Future Directions ................. ...............80.................


LI ST OF REFERENCE S ................. ...............96................


BIOGRAPHICAL SKETCH ................. ...............108......... ......










LIST OF TABLES


Table page

4-1. Mixed model analysis for FP treatment duration in Nalm-6 ................ .......___...........87

4-2. Mixed model analysis for FP treatment duration in RCH-ACV............__ ..........__ .....87

4-3. Differences between treatment duration at a given FP concentration in Nalm-6 .................87

4-4. Differences between treatment duration at a given FP concentration in RCH-ACV..........87

4-5. Mixed model analysis of PAC single agent treatment in Nalm-6 ................... ...............8

4-6. Mixed model analysis of PAC single agent treatment in RCH-ACV .............. ..................88

4-7. Differences in cell death based on incubation time after 6 or 24 hours PAC treatment in
N alm -6 .............. ...............89....

4-8. Differences in cell death based on incubation time after 6 or 24 hours PAC treatment in
RCH-ACV. ........... ..... .._ ...............90....

4-9. Combination Index (CI) values for drug combination studies using a variety of ratios .......90

4-10. Mixed model analysis of Nalm-6 and RCH-ACV combination data ........._...... ........._.....90

4-11. Mixed model analysis of Molt-4 and Jurkat combination data ................... ...............9

4-12. Significant differences in treatment for a given cell line and drug concentration ...............91

4-13. Significant differences in treatment for a given cell line and drug concentration ...............91

4-14. Combination Index (CI) values for drug combination studies .............. ....................9

4-15. One-Way Analysis of Variance of treatment sequence in Nalm-6 ........._.._. ........._.._.....92

4-16. Weighted One-Way Analysis of Variance of treatment sequence in RCH-ACV ..............92

4-17. Significant differences between standard treatment sequence, reverse treatment
sequence, and single agent controls in Nalm-6 ................. ...............92..............

4-18. Significant differences between standard treatment sequence, reverse treatment
sequence, and single agent controls in RCH-ACV ....__ ................. ................ ...93

4-19. Mixed model analysis for comparison of cell death induced by PAC in FBS vs. HS ........93

4-20. Comparison of cell death induced by PAC in FBS vs. HS............... ...................9

4-21. Mixed model analysis of cell viability FBS vs. HS............... ...............94...










4-22. Mixed model analysis s of combination studies in human serum ................. ............... ....94

4-23. Significant differences in treatment for combination studies in human serum..................95










LIST OF FIGURES


Figure page

1-1. Treatment of Childhood Acute Lymphoblastic Leukemia (ALL) ................. ................ ...32

1-2. Cyclin dependent kinase (CDK) inhibitors function in the transition from G1 (Gap 1) to
S (DNA synthesis) phase of the cell cycle............... ...............33.

1-3. pl6 works in concert with pRb to regulate the G1-S transition. .............. ....................3

1-4. Flavopiridol is a pan-CDK inhibitor. ................. ...............35........ ..

2-1. Fifty percent inhibitory concentration (ICso) determinations via WST-1 in cell lines that
lack pl6 protein expression. ............. ...............47.....

2-2. Flavopiridol induces apoptosis in ALL cell lines in a concentration dependent manner......48

2-3. Flavopiridol induces G1-S and G2-M (Gap 2-mitotic) arrest in RCH-ACV with reduced
phosphorylation of pRb ............. ...............49.....

2-4. Flavopiridol induces transient G1-S arrest in Nalm-6. ........... ...............50......

2-5. Efficacy of FP in human serum. ............. ...............51.....

3-1. Experimental design for PAC single agent treatment. ............. ...............60.....

3-2. Cell death induced by treatment with FP or PAC in Nalm-6 and RCH-ACV. ...................61

3 -3. Flavopiridol enhances the efficacy of PAC in ALL cell lines. ........... _... ...._._...........62

3-4. PAC+FP is a more efficacious treatment sequence than FP&PAC or concurrent
exposure in Nalm-6. ........... ..... .._ ...............63...

3-5. PAC+FP is a more efficacious treatment sequence than FP&PAC or concurrent
exposure in RCH-ACV. .............. ...............64....

3-6. Efficacy of PAC in Nalm-6 in the presence of human serum. .........___ ...... .._. ...........65

3-7. Flavopiridol enhances the efficacy of PAC in human serum. ..........__..... ._ ..............66

4-1. Growth curves used to establish cell concentration for proliferation assays.........................82

4-2. Representative dose-response curves generated from cell proliferation assays. ...................83

4-3. Illustration of isobologram analysis of combined drug effects.. ............... ............. .......84

4-4. Preliminary combination data at a variety of ratios in Nalm-6. ........._._ .... ...._.._........85










4-5. Preliminary combination data at a variety of ratios in the presence of human serum...........86









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

A PRECLINICAL STUDY OF FLAVOPIRIDOL IN THE TREATMENT OF ACUTE
LYMPHOBLASTIC LEUKEMIA

By

Kelly Marie Jackman

May 2007

Chair: Stephen P. Hunger
Major: Medical Sciences--Physiology and Pharmacology

Approximately 80% of children with acute lymphoblastic leukemia (ALL) will be cured;

however, it is essential to study novel agents and new combinations of existing therapies for their

potential use in relapsed patients. Loss of pl6 function might play a part in the progression of

ALL, which makes this pathway an interesting target for novel therapeutics. I have chosen to

study flavopiridol (FP), a semi-synthetic flavonoid that targets the pl6 pathway. FP acts as a

pan-cyclin dependent kinase inhibitor with the ability to induce apoptosis and cell cycle arrest in

human cancer cells. My studies have shown that at a concentration approximately equal to the

IC5o, FP induces a transient G1-S arrest and a low percentage of apoptosis in ALL cell lines. At

approximately twice the ICso, FP induces a sustained G1-S and G2-M arrest with a high

percentage of apoptosis. My work has also shown that FP treatment decreases the

phosphorylation of retinoblastoma protein on specific serine residues; an indication of a

reduction in endogenous CDK activity. Further, despite a high level of binding by FP to proteins

present human serum and subsequent reduction in its in vitro activity reported by others, I show

that there is not a substantial difference in FP activity in the presence of human serum when

compared to fetal bovine serum.









Based on disappointing results from early clinical studies of FP by others, I chose to test

FP in combination with paclitaxel. PAC has a mechanism of action which is complementary to

that of FP. PAC enhances the activity of CDK 1, inhibits microtubule depolymerization, and

induces G2-M arrest. Others have reported that FP enhances the efficacy of PAC in a sequence-

dependent manner in cell types other than ALL. My results show that FP enhances the efficacy

of PAC in ALL cell lines and that this enhancement is dependent on the sequence of

administration. In this study I established optimal times of exposure for each of the agents when

used in combination and confirmed that the enhancement of PAC activity by FP is present both

in fetal bovine serum and human serum.









CHAPTER 1
BACKGROUND

General Treatment of Acute Lymphoblastic Leukemia (ALL)

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer,

accounting for approximately 30% of pediatric malignancies (1). With current multi-agent

chemotherapy regimens, approximately 80% of patients are cured of their disease; however,

relapse remains a significant clinical problem (2). Treatment of children with ALL consists of

three phases: induction, consolidation and maintenance (Figure 1-1) (2, 3). The total length of

treatment lasts 2-3 yrs. The purpose of the first phase of treatment, which lasts approximately

one month, is to induce a complete remission or an absence of morphologically detectable

leukemic blast cells in the blood or bone marrow. This is successfully achieved in 99% of

patients with three or four drugs (2, 3). The consolidation phase of therapy lasts 4-8 months and

is designed to reduce the number of remaining leukemic blast cells using the agents listed in

Figure 1-1 (3). Maintenance therapy (1.5 to 2.5 years) consists of methotrexate and 6-

mercaptopurine; in addition to vincristine and either prednisone or dexamethasone.

Patients with recurrent ALL receive more intensive therapy involving any or all of the

agents previously outlined in other phases with other active agents such as ifosfamide, etoposide

or teniposide often added. Stem cell transplant is also frequently performed for patients whose

disease recurs during treatment or within 6 months of completing therapy. In the case of relapse

outside of the bone marrow, such as leukemic blasts found in the central nervous system or

testes, radiation can be administered at that site if it has not been previously administered.

There are agents which are currently used on an experimental basis in children with ALL.

These include newer cytotoxic agents such as clofarabine, a nucleoside analog, agents which

target tyrosine kinases and those that target histone deacetylases (2). Imatinib mesylate (Gleevec)









targets the tyrosine kinase formed by the BCR-ABL fusion protein resulting from a translocation

between chromosome number 9 and chromosome number 22 (Philadelphia chromosome) as well

as other tyrosine kinases. Use of this agent has induced remissions in BCR-ABL positive ALL

(4-6). Other therapies under investigation include the use of RNA interference technology, gene

therapy and immunotherapy (2).

Relapsed ALL: The Clinical Problem

Relapse can occur in a number of sites, including but not limited to the bone marrow,

central nervous system, and testes. Survival rates after bone marrow relapse range from 5% to

57% and are especially poor for those with a relapse within 36 months of initial diagnosis (7-11).

Increased dosage, the use of other existing chemotherapy agents not typically used in primary

treatment (etoposide, ifosfamide, and others) and widespread use of stem cell transplantation

have not significantly improved outcome for these patients. In addition, while complete

remission rates for children who relapse more than 3 years after diagnosis are similar to those

seen at initial diagnosis (>95%), patients who relapse less than 3 years after diagnosis often fail

to attain a second remission (12).

Thus, there is a need to develop novel agents and/or new combinations of existing agents

in order to improve the outcome of relapsed pediatric ALL patients. Many new agents have been

developed that have novel modes of action. Some of these include the cyclin-dependent kinase

inhibitors (CDKIs), examples being flavopiridol and UCN-01 (13, 14). Other drugs which are

typically used in other types of cancers, such as the microtubule depolymerization inhibitors

paclitaxel and docetaxel, have been used experimentally in ALL with mixed results (15-17). It

becomes essential to study the biology that makes relapsed ALL different from ALL at initial

diagnosis so that priority can be given to the study of the most promising agents.









Important Regulators of Cell Cycle

Gene expression profiling reveals that several key pathways are altered at the time of ALL

relapse vs. at initial diagnosis, including cell cycle regulation, DNA repair and apoptosis (18).

This proj ect has focused on preclinical testing of agents that target the aberrations in cell cycle

regulation present in relapsed ALL. Regulatory proteins can be broadly separated into those

which regulate the transition from G1 (Gap 1) to S (DNA synthesis) phase and from G2 (Gap 2)

to M (mitosis) phase. CDK 2, CDK 4, and CDK 6 regulate the transition from G1 to S and CDK

1 (cdc2) regulates the transition from G2 to M (Figure 1-2) (19). Most molecules of interest in

this proj ect function in the restriction point from G1 to S or the point at which the cell is

committed to divide with or without the presence of growth factors (20), as beyond this point the

cell is less likely to respond to external stimuli such as a drug. CDK 4 and CDK 6 become

functional after cyclin DI, cyclin D2, or cyclin D3 binding (19). These kinases phosphorylate

retinoblastoma protein (pRb) at specific serine and/or threonine residues. This phosphorylation is

normally prevented by pl6 (cyclin-dependent kinase 4 inhibitor A; INK4A), which binds to

CDK 4 and CDK 6 in the place of the cyclin (21). pl6 is part of the INK4 family of proteins,

including pl5 (INK4B), pl8(INK4C), and pl9(INK4D), which work to inhibit CDK 4 and CDK

6, along with members of the CIP/KIP family, including p27 (cyclin dependent kinase inhibitor

IB; KIPl), p57 (cyclin dependent kinase inhibitor IC; KIP2) and p21 (cyclin dependent kinase

inhibitor 1A; WAFl/CIPl) (Figure 1-2) (22). When pl6 is present, the hypophosphorylated form

of pRb acts as a tumor suppressor by binding to E2F transcription factor, making E2F unable to

bind to DP-1 and 2 (Figure 1-3). These molecules function as transcription factors that act in

DNA synthesis and nucleotide metabolism (22).

pl6 has been a maj or interest in this proj ect; however, other molecules such as pl5, p21,

and p27 have been studied by the Hunger lab and others for their possible roles in the









progression of ALL (see below). pl5 shares great homology with pl6, is found within 25kb of

the pl6 gene on chromosome 9 (23), and acts as a TGF-P (transforming growth factor-p) induced

inhibitor of CDK 4 and CDK 6 (24). p21 and p27 regulate not only CDK 4 and CDK 6, but also

CDK 2, which functions in concert with CDK 4 and CDK 6 to phosphorylate pRb (19). It is

CDK 2 that actually completes the hyperphosphorylation of pRb. This inhibits the tumor

suppressive nature of pRb and allows progression of the cell cycle through S-phase. p21 is

activated by the p53 transcription factor (22). P53 is regulated by MDM-2 (mouse double minute

2; HDM-2 in humans) which inactivates the transcriptional activity of p53, flags it for

ubiquitylation, and ensures its transport from the nucleus into the cytoplasm. An alternate

reading frame of the pl6 locus produces pl4 (alternate reading frame; ARF) which acts as a

tumor suppressor by preventing the p53 suppression activity of MDM-2.

Cell Cycle Regulators in Cancer and ALL

Deletion of pl6 is the most common form of genetic alteration in cancer among cell cycle

regulators (25). Studies have shown that greater than 30% of ALL cases have pl5 and pl6

deletions, with that percentage increasing to greater than 50% in T-cell ALL and remaining

greater than 20% in B-precursor ALL (26). It has been found by the Hunger lab and others that a

substantial number of patients develop pl5 and/or pl6 deletions in the bone marrow between the

time of initial diagnosis and relapse of ALL (27, 28). The pl5 promoter has also been studied

and has been found to undergo methylation between diagnosis and relapse, much more

commonly than the pl6 promoter (29-3 1). This pl5 methylation takes place in CpG islands at the

5' end of the gene, which results in loss of transcription in the promoter region (32). A study

from the Hunger laboratory used 18 matched specimen pairs from children with ALL at initial

diagnosis and first relapse to determine if pl5/pl6 deletions or hypermethylation of the pl5









promoter occurred between diagnosis and relapse (27). Results showed that out of 14 pairs that

were germline at diagnosis, three developed homozygous deletions of both pl5 and pl6 and two

developed homozygous pl6 deletions and retained germline pl5 status between the time of

initial diagnosis and relapse. pl5 promoter hypermethylation developed in two patients between

diagnosis and relapse. Out of the eighteen total cases, seven had homozygous pl5 deletions, nine

had homozygous pl6 deletions, and two of eight cases tested had pl5 promoter

hypermethylation at relapse. Similar findings have been reported by Carter, et al., showing that

out of a group of 25 pediatric ALL patients, at diagnosis 32% and 20% had homozygous and

hemizygous pl6 (exon 2) deletions respectively (28). The incidence of homozygous pl6 deletion

at relapse increased to 64%, illustrating the potential importance of the loss pl6 in the

progression of ALL.

The prevalence of pl5 and pl6 alterations is much higher than the level of p21 and p2 7

alterations found in ALL (33, 34). Both p21 and p27 function to inhibit CDK 2 and CDK 4 (35-

37). p21 is regulated by p53 in order to control cell growth (38). Hayette, et al. performed a study

of alterations of molecules which inhibit CDKs in leukemia, using bone marrow or peripheral

blood from 121 newly diagnosed ALL cases, 85 newly diagnosed acute myeloid leukemia cases,

and 42 newly diagnosed B-cell chronic lymphocytic leukemia cases (34). Via Southern blot this

group found that pl6 was inactivated in 25 of 38 T-cell ALL cases and 28 of 83 B-lineage ALLs.

After testing 40 ALL samples with a pl6 aberration, it was found that 22 cases (55%) had

biallelic pl5 deletions and 11 cases (28%) had monoallelic deletions. All cases with a pl5

deletion also had an anomaly in the pl6 gene. There were no alterations found in p21 and

monoallelic deletion of p2 7 was present in 4 of 85 acute myeloid leukemia cases tested. These

data show that p21 and p2 7 alterations are much less prevalent in leukemia than deletions of pl5









and pl6. Another study by Kawamura, et al. further illustrates this point by analyzing 71 primary

T-ALL samples and 18 T-ALL cell lines for alterations in pl5, pl6, p21, p53, and RAS via

polymerase chain reaction-single strand conformation polymorphism analysis (33). They found

that none had alterations in p21. In contrast, 18 of 47 (3 8%) newly diagnosed patients had pl6

alterations and 7 of 14 (50%) patients had pl6 alterations at relapse.

Gene deletion is not the only cause of loss of functional pl6. Many samples have been

found to have an intact pl6 gene, but no protein expression. An interesting study by Nakamura,

et al. notes that when pl6 expression was investigated in childhood ALL samples via Western

blot, 18 of 22 samples with an intact pl6 gene did not express pl6 protein; however, protein

expression was able to be induced after treatment with a demethylating agent, indicating that the

loss of pl6 protein expression was due to gene hypermethylation (39). Others have reported

similar results in T-cell ALL and/or AML (40-42). A separate study of pediatric T-cell ALL

patients reported that only 9 of the 45 samples with intact pl6 expressed pl6 protein (43). This

study found that pl6 was altered at the DNA, RNA or protein level in 1 15 of the 124 (93%)

samples tested and concluded that alteration in both pl6 and pl5 were essential to the

progression of T-cell ALL. Most recently a study of adults with untreated ALL found that not

one of the samples tested (n=91) expressed pl6 protein (44).

A study performed by Carter, et al. on 45 patient samples via quantitative PCR techniques

found that ALL patients with a hemizygous deletion of pl6 at diagnosis were 6.5 times more

likely (P=0.00687) to relapse and those with a homozygous deletion had an even higher risk ratio

of 1 1.5 (P=0.000539) (45). In contrast to the findings of Carter et al., Einsiedel et al. found that

there was no association between pl6 deletions and event free survival in ALL (46). pl5 and pl6

status could be correlated to two maj or prognostic indicators: T-cell immunophenotype and first









remission duration. This study did not assess for hemizygous deletions, as Carter did, because of

theoretic and methodologic considerations. Another group compared wildtype pl6 to

hemizygous deletions and found no difference in potential for event free survival (47).

Given this information it becomes apparent that despite the fact that its prognostic value is

still somewhat controversial, pl6 alterations occur commonly in relapsed ALL and are often

acquired during disease progression. Deletion of pl6 and hypermethylation of the pl5 promoter

region occur much more frequently in ALL than alterations in other cell cycle regulatory

molecules such as p21 or p27. Therefore the pl5/pl6 pathway is an attractive target for

therapeutic intervention in relapsed ALL. Several agents exist that modulate cell cycle

progression and are logical candidates to test in relapsed ALL. One such agent is flavopiridol, a

description of which follows.

Flavopiridol

Flavopiridol (FP) is a semisynthetic flavonoid derived from rohitukine, an alkaloid isolated

from a plant indigenous to India (48). Flavopiridol has a variety of mechanisms of action;

however, most relevant to my studies is the ability of FP to decrease the activity of CDKs and

induce cell cycle arrest (Figure 1-4). Cell cycle regulatory elements such as the CDK inhibitors

pl5 and pl6 are altered in ALL between diagnosis and relapse, indicating that this loss of

checkpoint control in the cell cycle could be a critical factor in the progression of the disease and

an attractive target for novel therapeutic agents. FP competitively binds to the ATP binding cleft

of the CDK (14) and is capable of reducing the activity of CDK 1, CDK 2, CDK 4, CDK 6, and

CDK 7 with IC5o values in the range of 20-400 nM (49). FP also reduces the activity of CDK 9

(50-52).

FP induces cell cycle arrest at the G1-S phase border as well as during G2-M. Inhibition of

CDK 2 and CDK 4 has been correlated to G1 arrest in MCF-7 breast carcinoma cells (14).









Another group had similar findings using MDA-468 breast carcinoma cells that were

synchronized either in G1 phase with aphidicolin or synchronized in M phase with nocodazole

(53). MDA-468 cells treated with 200 nM FP after release from aphidicolin G1 block arrested in

G2-M after 24 hours. Cultures released from nocodazole M phase block and treated with 200 nM

FP showed a G1-S arrest when compared to control cultures not treated with FP.

The ability of FP to induce cell death has been tested in vitro in a variety of cancer cell

types, including adult leukemia. An early study in a variety of solid tumor cell types and HL-60

leukemia cells found that FP was cytotoxic as measured by trypan blue exclusion and colony

formation assays (54). Previous studies had only shown that FP was cytostatic (53). The former

study also found that 90% cell death was induced 72 hours following a 24 hour exposure to 250-

300 nM FP compared to 50% cell death induced immediately following the 24 hour drug

exposure, thus showing that more time was needed to achieve a maximum cell death response.

This group also showed that both logarithmically growing and cytostatic cell lines were affected

by FP treatment. Similar results have been found by others testing non-small cell lung carcinoma

cell lines (55). It was found that seven different cell lines were sensitive to FP at concentrations

ranging from 100-500 nM; regardless of whether the cell lines were in logarithmic growth phase

or cytostatic. This group also showed that cell cycle arrest preceded cell death in most cases and

that maximal cell death occurred 72 hours post-treatment with concentrations of FP 500 nM or

below. These data illustrate the cytotoxic action of FP during a prolonged exposure. This activity

combined with the ability of FP to inhibit CDK activity and induce cell cycle arrest contribute to

this proj ect' s focus on testing the efficacy of FP as a potential treatment for ALL.









In Vitro Testing of Flavopiridol in Combination with Other Agents: Sequence of
Administration and Synergy

It has been found that administering FP with traditional antineoplastic agents can improve

the efficacy of those agents and that in some cases this interaction is synergistic. The

enhancement of a traditional agent by FP has been shown to be dependent on the sequence in

which the drugs are given, such as the enhancement of paclitaxel (PAC) activity by FP (56).

Paclitaxel (trade name Taxol) prevents microtubule depolymerization (57) and induces G2-M

phase arrest (58). An example of the enhancement of PAC activity by FP can be found in work

by Motwani, et al. in which MKN-74 human gastric carcinoma cells and MCF-7 human breast

carcinoma cells were exposed to PAC, FP, or both agents either sequentially or simultaneously

(56). When MKN-74 cells were exposed to PAC and FP for 24 hours, the level of apoptosis

increased from 3 +/- 1% with FP alone to 8 +/- 1%. A significant increase was then seen when

the drugs were used sequentially. MKN-74 cells were exposed to PAC for 18 hours followed by

FP for 24 hours and the level of apoptosis was 40 +/- 2%; however, when using FP followed by

PAC the level was 8 +/- 1%, which was not significantly different from the amount of apoptosis

found after exposing the cells to FP for 24 hours followed by no drug for 18 hours. Caspase-3,

the final activator of the apoptotic cascade was activated when MKN-74 and MCF-7 were treated

with PAC followed by FP. Without FP, PAC only minimally activated caspase-3. If the sequence

of administration was reversed, FP inhibited the function of PAC by preventing mitosis and

CDK 1 activity. Similar results in regard to cytotoxicity have been achieved when using FP in

conjunction with docetaxel in vitro and in xenograft tumor models (59).

When testing eight agents against a human non-small cell lung carcinoma cell line (A549),

Bible and Kaufmann found that seven of the eight agents had synergy with FP that was sequence

specific (60). These authors extensively studied the possibility that treatment with PAC and FP









could show sequence dependent synergy. Their finding that the effects of PAC were more

pronounced when administered before FP treatment as opposed to after or concomitantly was

particularly intriguing. A marked decrease in clonigenic cell survival over PAC alone and FP

alone was seen when PAC treatment was followed by FP. Synergy was assessed through the use

of combination index or CI. A CI of 1.0 indicates that the relationship between the drugs being

studied is nearly additive, while a CI of <1.0 indicates synergy and a CI of >1.0 indicates

antagonism (61). At the concentration at which cell proliferation was inhibited by 75% (IC75)

and 95% (IC95), COmbination indices of 0.49 +/- 0.21 and 0.20 +/- 0. 14 were found, respectively,

indicating synergy if PAC was given before FP in the treatment sequence (60). Antagonism was

found if PAC followed FP.

Others have tested many agents in conjunction with FP in myeloid leukemia cell lines.

These agents have included phorbol 12-myristate 13-acetate (PMA), imatinib mesylate

(Gleevec), bryostatin 1, bortezomib (Velcade) and suberoylanilide hydroxamic acid (SAHA)

(62-66). All have shown promising results for the ability of FP to enhance the activity of other

agents.

Efficacy of Flavopiridol in Clinical Trials

Based on its action as a CDK inhibitor and promising preclinical activity, FP was tested in

phase I human trials. Studies designed to obtain clinical pharmacology data after giving FP as a

72 hour infusion readily achieved plasma concentrations that were comparable to that found to

be effective in vitro (67, 68). However, most clinical trials involving cancer patients gave FP as a

72 hour infusion every 2-3 weeks and found that it had limited efficacy as a single agent (69-75).

One of these trials found that FP had antitumor activity in certain patients with renal, prostate,

and colon cancer, and non-Hodgkin's lymphoma (69). The two maximum tolerated doses

(MTDs) found in this phase I study gave peak plasma concentrations of 271 nM and 344 nM, the









second after antidiarrheal prophylaxis. The concentrations of FP needed to inhibit cyclin

dependent kinase function (200 to 400 nM) were safely achieved in this study. Despite both in

vitro and in vivo data showing that FP was cytostatic and cytotoxic in non-small cell lung

carcinoma cells, Shapiro and colleagues stopped a phase II study after only 20 of 45 patients

proj ected to be in the study were treated, as no responses were observed in these individuals (72).

This study also noted that a mean steady-state plasma concentration of 200+89.9nM was

achieved, which was well within the FP concentration range found to be effective in vitro.

Questions regarding dose and length of treatment have been the main focus of many

clinical trials involving FP. In addition to the traditional 72 hour infusion schedule, FP has also

been tested as a 24 hour continuous infusion given every two weeks and a 1 hour bolus

administered over a range of schedules. Flinn, et al. found that FP had no clinical activity in

patients with fludarabine refractory chronic lymphocytic leukemia (CLL) when given as a 24

hour infusion (76). A study from the same group compared FP activity in CLL when the agent

was administered as a 72 hour infusion to a 1 hour bolus and found that the 72 hour schedule did

not result in any patient responses; however, the bolus dose did result in slight clinical activity

(75). A separate phase I study using FP as a single agent in patients with advanced neoplasms

tested FP at varying 1 hour infusion doses over 5-days, 3-days and 1-day every 3 weeks (77).

During the trial, median peak total concentrations at the MTD of 1.7 CIM (range 1.3 to 4.2 CIM)

for 5-day administration, 3.2 CIM (range 1.7 to 4.8 CIM) for 3-day administration, and 3.9 CIM

(range 1.8 to 5.1 CIM) for 1-day administration were found. Twelve of the 55 patients studied had

stable disease for greater than or equal to three months with a median duration of six months

(range, three to eleven months). A similar study was conducted by the National Cancer Institute

of Canada using FP as a bolus infusion over 3 days in patients with untreated or relapsed mantle-









cell lymphoma (78). No complete responses were observed; however, 1 1% of patients had a

partial response and 71% had stable disease. Similar results for the 1 hour bolus have also been

reported in malignant melanoma and multiple myleoma (79, 80). The only study of FP in

pediatric patients also used this schedule and was performed by Whitlock, et al. in patients with

solid tumors (81). No responses were observed despite achieving mean peak plasma

concentrations of 3.71 and 9. 11 CIM after doses of 37.5 mg/m2 and 80 mg/m2 TOSpectively.

Shorter infusion schedules for FP as outlined above were pursued by clinicians with the

intention of increasing the peak plasma concentration of the agent. Early trials of FP given as a

72 hour infusion were based on drug activity data generated from in vitro studies of FP that were

performed in media supplemented with fetal bovine serum (FBS). Later studies showed that FP

is highly bound to human plasma proteins (68, 82). Approximately 92-95% of FP is human

plasma protein bound compared to 0-37% bound in FBS (82). This difference in protein binding

results in a decrease in the in vitro cytotoxicity of FP. Studies of primary CLL cells have shown

1 hour and 24 hour LCso values in FBS of 670 nM and 120 nM respectively, compared to 3,510

nM and 470 nM in human plasma or human serum (HS) (82). Based on these data and clinical

pharmacology data, a pivotal study of the use of FP as a short infusion was performed (83). FP

was given as a 30 minute bolus infusion followed by a 4 hour continuous infusion in patients

with CLL with the goal of achieving a peak plasma concentration of 1.5 CIM. Patients were

divided into cohorts, with the first receiving a 30 mg/m2 bolus dose followed by a 30 mg/m2

infusion. The second cohort received a 40 mg/m2 bolus followed by a 40 mg/m2 infuSion. The

maximum plasma FP concentrations achieved at these dose levels were 2,080 nM after 30

minutes and 960 nM after 4.5 hrs (84). A third cohort was given a 30 mg/m2 bolus followed by a

50 mg/m2 infuSion. These dosages achieved peak plasma levels of 1,950 nM after 30 minutes









and 1,540 nM after 4.5 hours This study had to be temporarily discontinued, as this schedule had

high clinical activity that resulted in tumor lysis so severe that one patient died (85). The group

implemented procedures for monitoring patients for tumor lysis syndrome and continued the

study which resulted in a 45% overall response rate in CLL patients.

Biological Correlates of Clinical Activity

Several clinical trials have included studies to determine if the same mechanisms of action

for FP observed in vitro could be achieved in vivo. Previous in vitro studies have shown that FP

inhibits CDK activity (14, 49-52, 86, 87), induces apoptosis (54, 55, 87-96), reduces the

transcription and/or expression of anti-apoptotic proteins Bcl-2 (B-cell leukemia/1ymphoma 2)

and Mcl-1 myeloidd cell leukemia sequence 1) (49, 87, 96-100), and binds to DNA (101). In a

phase I study by Thomas, et al. FP was tested as a single agent given as a 72 hour infusion every

two weeks in patients with a variety of tumor types (67). Peripheral blood lymphocytes were

collected during treatment and analyzed via flow cytometry for evidence of apoptosis or changes

in cell cycle kinetics. No evidence of changes in these measurements was found; however, the

authors noted that there were early signs of clinical activity.

During a phase I trial of FP combined with docetaxel in patients with metastatic breast

cancer, Tan and colleagues examined Ki67, p53, and phosphorylated pRb in paired patient tumor

and buccal mucosa samples (102). Ki67 was used as an indication of cell proliferation and

phosphorylated pRb was used as an indirect measurement of CDK activity. The buccal mucosa

biopsies of ten of the eleven patients enrolled in the study showed increased nuclear expression

of p53 and decreased expression of phosphorylated pRb after treatment with FP as a single agent.

The authors postulated that the increase in p53 expression could have been due to the ability of

FP to bind to DNA (101) or the ability of FP to reduce transcription or down regulate MDM-2,

based on the activity of other CDK inhibitors (50, 103). Six paired tumor samples showed no









changes in p53, Ki67, or phosphorylated pRb. The authors concluded that the biological effect of

FP was achieved in the buccal mucosa; however, the treatments tested were not feasible due to

dose limiting toxicities.

A similar phase I study used FP in combination with cisplatin or carboplatin in patients

with advanced tumors (104). Peripheral blood mononuclear cells were analyzed before and after

FP treatment and found to have increased p53 expression and increased phosphorylated STAT3

(signal transducer and activator of transcription 3) levels. Treatment had no effect on cyclin DI,

phosphorylated RNA polymerase II (indicator of CDK activity), or Mcl-1. The authors felt that

there was a possibility that the increased p53 and pSTAT3 levels were due binding of FP to

DNA and that the lack of an effect by FP on cyclin Dl expression, the phosphorylation of RNA

polymerase II, or Mcl-1 expression might have been due to the inability of FP to inhibit P-TEFb

(CDK 9) in vivo. There was a lack of clinical activity observed during the trial and it was further

postulated that the lack of an effect on Mcl-1 expression by FP could have been an explanation

for this low clinical response. Alternatively, the authors could not definitively say that the same

effects that were observed in non-cycling peripheral blood cells could be observed in tumor cells,

as these were not tested.

Finally, a phase II trial of relapsed or refractory melanoma patients had disappointing

clinical results that the authors partially attributed to a lack of biological activity in vivo (80).

Western blot analyses very similar to those performed in the studies cited above found that only

one patient out of eight tested had the expected results of decreased Mcl-1 with increased p53

expression and increased expression of phosphorylated STAT3 as a result of FP treatment. Two

additional patients had decreased Mcl-1 in combination with lower levels of p53 and










phosphorylated STAT3. It was noted that the former patient progressed after one cycle of FP

treatment.

All of the trials cited above used FP as either a 1, 24, or 72 hour infusion. This is contrary

to the most recent use of FP as a 30 minute bolus followed by a 4 hour infusion found to be

highly effective in CLL (85). Most of the in vitro studies to which the above authors were

attempting to correlate biological activity in vivo were performed in FBS. As previously cited,

the newer infusion schedule takes the high percentage of protein binding of FP that occurs in HS

into account by achieving a higher plasma FP concentration in a shorter time than that achieved

in previous trials. Studies have been conducted in vitro in CLL cells grown in the presence of HS

that show that FP is biologically active under these conditions when used at concentrations

higher than those previously utilized in FB S (100).

Clinical Trials of Paclitaxel (PAC) and Combining Flavopiridol with Paclitaxel

With the exception of recent studies in CLL, FP has had limited efficacy in clinical trials

when used as a single agent. However, as outlined previously, there have been promising

preclinical results showing synergy between PAC and FP. Early clinical trials have also tested

FP in combination with PAC in cancer types other than ALL.

PAC has been found to be effective as a single agent in the treatment of several types of

cancer including breast, ovarian, and lung cancer, and melanoma (105, 106). PAC also has

considerable in vitro activity against ALL (107) and has been tested in both adults (15, 108) and

children (16) with leukemia. Studies in adults used 3 doses of 100 minutes each repeated every

three weeks (15) and a 24 hour infusion repeated every 3-4 weeks (108). A trial in pediatric

leukemia patients used a 24 hour PAC infusion; achieving peak plasma concentrations of

approximately 1,000 nM. Unfortunately, these studies did not report any substantial clinical

responses. Minimal responses have been reported using PAC as a single agent in children with









solid tumors (109). This trial used varying doses of PAC as a 24 hour infusion repeated every

three weeks. Peak plasma concentrations were dose-dependent and ranged from approximately

1,000 nM to 7,000 nM. Two out of 31 total patients treated reported significant toxicity. PAC is

89-98% bound to plasma proteins in vivo (110). Perhaps similar to FP, a shorter infusion

schedule with the goal of obtaining a high peak PAC plasma concentration might prove

beneficial in the treatment of ALL.

Promising results have been achieved when FP was combined with PAC in patients with a

variety of solid tumor types (111). Clinical responses were observed in patients with esophagus,

lung and prostate cancer, some of whom had progressed on PAC single agent treatment. It

should be noted that the agents were given in the specific sequence of PAC followed by FP

treatment.

Project Rationale

Preclinical in vitro studies or clinical studies using FP have never been conducted in

relation to childhood ALL. Relapsed ALL patients become increasingly refractory to agents

typically used in the treatment of ALL, thus creating a need to investigate new drugs. I examined

FP because of the high frequency of pl5/pl6 abnormalities and altered expression of other cell

cycle regulatory proteins in relapsed ALL. Results from preclinical studies suggest that FP can

act similarly to these molecules in that it inhibits CDK activity and induces cell cycle arrest. FP

can also induce apoptosis in human cancer cells. Based on these findings, I performed in vitro

studies of FP at different times of exposure to mimic prolonged infusion and newer bolus

schedules. During these studies I examined the cell death and alterations in cell cycle progression

induced by FP.

Clinical responses measured during trials of FP as a single agent have shown that it has

limited efficacy when used in a 72-, 24- or 1 hour dosing schedule. Recent data from studies in









CLL have suggested that prolonged FP infusions have a low overall response rate due to failure

to achieve an effective free FP concentration as a result of secondary protein binding in human

plasma. A shorter infusion of a higher dose of FP was found to be very promising. In order to

model this new infusion strategy, this proj ect includes experiments using cultures that were

grown in medium supplemented with human serum in place of fetal bovine serum. A higher

concentration of FP is also administered over a shorter period of time when compared to

previous experiments.

This proj ect has not only served as a means to determine the potential efficacy of FP when

used as a single agent in ALL cell lines, but has also served as a study to determine the effects of

combining FP with PAC. In vitro studies and clinical trials in patients with types of cancer other

than ALL have shown that FP can enhance the activity of PAC. In some cases using FP in

combination with PAC can have a synergistic effect on in vitro treatment. This enhancement is

dependent on the sequence in which the drugs are administered. In the case of FP combination

treatment with PAC, this sequence dependence has been reported to be due to the ability of PAC

to activate CDK 1 activity coupled to the inhibitory action of FP against this same CDK (56).

Because PAC is not typically used in the treatment of ALL, I first established that ALL cell lines

were sensitive to PAC treatment. I also tested whether FP enhances the efficacy of PAC in vitro

and if this enhancement was dependent on the sequence in which the two drugs were

administered. This drug combination could offer a treatment regimen to children with relapsed

ALL that would utilize two agents to which the patients will not have been previously exposed.

In the future, data from this proj ect could be used to develop a clinical trial which would

utilize either FP as a single agent or PAC in combination with FP, both in the schedule that I

have found to be most efficacious. This proj ect also provides data to indicate the mechanism of









action behind the efficacy of FP in ALL in order to provide a biological basis for the clinical

study .












Induction: 1 month; Results in complete remission in 99% of patients


Treatments can include the following:
--L-asparaginase
--vincristine
--steroid
--anthracycline for high-risk patients (daunorubicin)

Intrathecal therapy-2 doses in the first month since diagnosis and 4-6 doses during the next 1 or 2
months. Agents utilized include
--methotrexate
--hydrocortisone and cytosine arabinoside (ara-C) added for high-risk patients

Patients with high white blood cell (WBC) count (high risk) or WBC in the cerebral spinal fluid
receive radiation to the brain and possibly the spinal cord. May also administer high dose
intrathecal methotrexate with leucovorin to treat side effects.

Consolidation: 4-8 months; Reduces the remaining number of leukemic blasts
Standard risk patients receive
--methotrexate
--6-mercaptopurine or 6-thioguanine
--optional: vincristine and prednisone

High risk patients receive:
--L-asparaginase, doxorubicin, etoposide, cyclophosphamide, ara-C and dexamethasone
substituted for prednisone (possibly two rounds)


Maintenance: 1.5 to 2.5 yrs
--methotrexate, 6-mercaptopurine
--vincristine; prednisone or dexamethasone (every 4-8 weeks)


Figure 1-1. Treatment of Childhood Acute Lymphoblastic Leukemia (ALL): 2-3 yrs. total (3)

















Synthesel
Degradalloq

Restriction 11
Fpint -3


Cycln
D1,2.3
+CDK(4
+ CDK6


rs~-Cc~`J


Dephosphorylation I


\ + Cdo2


1Cs


I
-C
+-- "


CDKls


Figure 1-2. Cyclin dependent kinase (CDK) inhibitors function in the transition from G1 (Gap 1)
to S (DNA synthesis) phase of the cell cycle. pl5 and pl6 inhibit CDK 4 and CDK 6,
as do p21 and p27. The restriction point of the cell cycle is located at the transition
from Go to G1 and marks the point at which the cell is no longer sensitive to external
agents such as growth factors or a drug. Pursuing permission from American
Association for Cancer Research: [Clinical Cancer Research] Shah MA, Schwartz
GK. Cell cycle mediated drug resistance: an emerging concept in cancer therapy.
Clinical Cancer Research 2001; 7:2168-2181., copyright 2001, originally published at
Figure 1, p. 2169.


















Fiue -.pl orsi cner ih ~ t eult teG-Staniin.p6 niit h







Figur genes1 inolved in prgesonetwt ~ th rogugh the G 1-S transition. Rpr6intedbyt p heriso

from Macmillan Publishers Ltd: [Nature Reviews Cancer] Classon M, Harlow E. The
retinoblastoma tumour suppressor in development and cancer. Nature Reviews
Cancer 2002; 2:910-917, copyright 2002, originally published as Figure 1, p.911.










Synthesis


Degradation)


Cyclin D1,23,3
+ CDK4
+ CDK6


(Cyclin B
+ Cdc2


Figure 1-4. Flavopiridol is a pan-CDK inhibitor. FP inhibits CDK 4, CDK 6 and CDK 2, thus
inducing a G1-S arrest. FP can also inhibit CDK 1 (cdc2) and induce G2-M arrest
Reprinted by permission from Meniscus Ltd: [Horizons in Cancer Therapeutics: From
Bench to Bedside] Shah MA, Schwartz GK. Cell cycle modulation: an emerging
target for cancer therapy. Horizons in Cancer Therapeutics: From Bench to Bedside
2004; 4(3):3-21., copyright 2004, originally published as Figure 5, p.7.









CHAPTER 2
FLAVOPIRIDOL DISPLAYS PRECLINICAL ACTIVITY IN ACUTE LYMPHOBLASTIC
LEUKEMIA

Introduction

One of the most commonly used methods of evaluating the potential efficacy of

chemotherapeutic agents prior to their use in patients is to determine the ability of the agent to

prevent growth of cancer cells in vitro. Results from methyl-thiazol-tetrazolium (MTT) assays

have shown a correlation between in vitro sensitivity of leukemia cells taken from peripheral

blood and bone marrow of patients and clinical outcome (1 12-1 19). Hongo et al. (1 12) found that

using agents determined to be efficacious in MTT assays resulted in better outcome for patients

with ALL or acute nonlymphoblastic leukemia when compared with patients whose treatment

regimens were determined via conventional methods of the time. Approximately 82% (n=1 1) of

patients treated with agents determined to be efficacious in MTT assays had complete or partial

remissions as compared with 40% (n=15) of patients treated by conventional means. I have

chosen to use a modified MTT assay as my initial means of determining the sensitivity of ALL

cell lines to FP. I have expanded my studies by testing the ability of FP to induce apoptosis and

cell cycle arrest in ALL cell lines, as cell proliferation assays merely measure an increase or

decrease in viable cell number.

The mechanism of action of a chemotherapeutic agent is an integral part of determining

how the agent will be used as well as what side effects might occur as a result of its use.

Knowing the mechanism of action can also help to target cancer cells without affecting non-

cancerous tissue. The ultimate fate of a cell, ie. cell death or senescence as a result of cell cycle

arrest can have an effect on the progression of the cancer. Cells which senesce might have the

ability to secrete signaling molecules which promote the growth of other cancer cells in the

surrounding area (120). For some researchers, this possibility makes apoptosis a preferred









mechanism of action for anti-neoplastic agents. I have determined that FP has the ability to

reduce ALL cell proliferation via a modified MTT assay and have further investigated the ability

of FP to induce cell cycle arrest and apoptosis. Through Western blot analysis I have observed a

correlation between the concentration dependent effects of FP on cell proliferation and the

endogenous phosphorylation of pRb. Correlating these mechanisms with drug concentration and

the effect that FP has on cell cycle regulatory elements will serve as important information when

deciding the use of FP as a single agent or in combination with other agents in the treatment of

childhood ALL.

Methods

In Vitro Drug Sensitivity Testing

In vitro drug sensitivity assays were modeled after those described originally by Pieters

and colleagues (113). Cell lines were grown in RPMI 1640 (Mediatech, Inc. Herndon, VA) with

10% fetal bovine serum (FBS, Mediatech) or 10% human AB serum (HS, Mediatech) and 1%

penicillin/streptomycin (Mediatech) at 370C with 5% CO2. Nalm-6 (B-precursor ALL) was

originated by Minowada, et al. (121). Molt-4 and Jurkat (both T-cell ALL) were obtained from

the American Type Culture Collection (ATCC, Rockville, MD). RCH-ACV is a B-precursor

ALL cell line provided by Dr. Seshadri (122). K562 (ATCC) is a chronic myelogenous leukemia

(CML) cell line commonly used for in vitro testing in the NCI 60 cell line test set and was

included in this study as a control. Exponentially growing cell cultures were plated in flat

bottomed 96-well dishes in 100 CIL of cell culture medium with a dilution of drug in vehicle

appropriate for each agent. Vehicles included ethanol for dexamethasone (Sigma, St. Louis,

MO), water for doxorubicin (Sigma), and dimethyl sulfoxide (DMSO) for FP (Sanofi-Aventis,

Bridgewater, NJ). All were further diluted in RPMI 1640. Samples for each drug concentration

were tested in quadruplicate for each experiment. Also tested in parallel were appropriate










dilutions of vehicle without drug, which functioned as an untreated control for calculation of

ICso. For the purpose of my study IC5o was defined as the concentration of drug at which cell

proliferation was inhibited by 50% as compared to an untreated control. Cell lines were plated at

a concentration of 1X105 cells/mL in 100 CIL for RCH-ACV, Molt-4, and Jurkat. Nalm-6 and

K562 were plated at a concentration of 5X104 CellS/mL in 100 CL. Different cell concentrations

were used in order to maintain the cultures in log phase growth throughout the period of the

experiments. Cell lines were incubated with drug for 96 hours, at which time WST-1 (4-[3-(4-

lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazoi]1,3-benzene disulfonate) reagent (Roche,

Indianapolis, IN) was directly added to each well according to manufacturer' s instructions.

WST-1 is a modified version of the MTT (methyl-thiazol-tetrazolium) reagent. Absorbance was

measured on a Molecular Devices (Sunnyvale, CA) Vmax kinetic microplate reader at 450nm,

subtracting a reference wavelength of 650nm. ICso was calculated by plotting leukemic cell

survival (LCS) against drug concentration. The drug concentration at which LCS equaled 50%

was defined as the ICso. LCS was calculated as follows:

abstreated absblank
X 100%. (2-1)
abscontrol absblank

Results are the mean of at least two independent experiments.

Western Blot Analyses

ALL cell lines were tested for pl6 protein expression with HeLa cells used as a positive

control. HeLa extract was diluted into extract from Nalm-6 (previously found to be pl6 deleted

via Southern blot (40)) in order to simulate a low level or variable amount of pl6 protein

express si on. Protein extracts were prepared using Radi o-Immunoprecipitati on As say (RIPA)

Buffer (Sigma) with sodium orthovanadate (Santa Cruz, Santa Cruz, CA), phenylmethylsulfonyl

fluoride (PMSF, Santa Cruz), and a protease inhibitor cocktail (Sigma). Fifty micrograms of









protein was loaded onto a 4-20% gradient polyacrylimide gel (Biorad, Hercules, CA) and

subjected to sodium dodecyl sulfate-polyacrylimide gel electrophoresis (SDS-PAGE). Proteins

were transferred to a 0.2 CIM pore nitrocellulose membrane (Biorad). After transfer, the

membrane was blocked with 5% dry non-fat milk in TBS with 0.1% Tween-20 (TBS-T) for one

hour with gentle agitation. Following blocking, the membrane was incubated at room

temperature (RT) with mouse monoclonal IgG1 antibody to full length pl6 protein (50.1, catalog

number sc-9968, Santa Cruz) at a dilution of 1:375 in 5% non-fat milk for one hour. The

membrane was then washed 3 X 15 minutes in TB S-T and incubated for one hour in goat anti-

mouse IgG secondary antibody conjugated with horseradish peroxidase in 5% milk.

Retinoblastoma protein phosphorylated on serine 795 (pp-Rbser795) and retinoblastoma protein

phosphorylated on series 807 and 811 (pp-Rb""ser/sovi) were resolved via SDS-PAGE after

loading 25 Clg protein lysate. The proteins were transferred to a nitrocellulose membrane which

was blocked as described and incubated with rabbit polyclonal antibodies to pp-Rbser795 and pp-

Rbserso7isll (product numbers 9301 and 9308, Cell Signaling Technology, Danvers, MA) 1:1000

in 5% bovine serum albumin (B SA) overnight and treated as previously described. Detection of

total p-Rb expression was performed using mouse monoclonal IgG1 antibody (IF8, catalog

number sc-102, Santa Cruz) 1:200 in 1% BSA after blocking for 1 hour at RT with 1% BSA.

Detection of actin (isoform non-specific) (C2, catalog number sc-8432, Santa Cruz) was used as

a loading control on all membranes. After washing 3 X 15 minutes, proteins were visualized on

radiographic film via ECL or ECL Plus reagent (Amersham, Piscataway, NJ). Results from

Western analyses were obtained from at least two separate experiments.

Measurement of Cell Death

Two methods were utilized to detect cell death and/or apoptosis in drug treated samples.

Samples were stained with Annexin V (Pharmingen, San Diego, CA) and Propidium Iodide (PI)










(Roche) as recommended by Pharmingen. Direct TUNEL (terminal deoxynucleoti dyltransferase

dUTP nick end labeling) staining was also performed according to manufacturer' s instructions

(Apo-Direct Kit, Pharmingen). Samples were analyzed via flow cytometry using a Becton

Dickinson (San Jose, CA) FACSort flow cytometer. Percentages of cell death/apoptosis were

measured by obtaining the sum of the upper right and lower right quadrants of the scatterplot

generated by analysis of samples stained with AnnexinV/PI. Percentages of apoptotic cells were

measured via TUNEL by obtaining the percentage of the cell population staining positive for

FITC-dUTP. Results were obtained from at least three independent experiments.

Cell Cycle Analysis

To determine cell cycle kinetics as a result of FP treatment, cell lines were analyzed for

DNA content using PI staining and flow cytometric analysis essentially as described by Ormerod

(123). Data were generated using ModFit LT for Mac version 3.1 software (Verity Software

House, Topsham, ME). Results are representative of at least three independent experiments.

Results

ALL Cell Lines Used for in Vitro Testing Lack pl6 Protein Expression

I determined pl6 protein expression in the cell lines used for in vitro drug sensitivity

testing via Western blot. Nalm-6, REH, Molt-4, and Jurkat have been reported previously to have

homozygous pl6 deletions (33, 40, 124, 125), while the Hunger laboratory has found RCH-ACV

to have intact pl6 via Southern blot (126). HeLa cells were used as a positive control. HeLa

lysate was diluted into Nalm-6 (pl6 deleted) lysate in order to simulate 10% and 1% pl6

expression. I found that none of the ALL cell lines tested expressed a detectable amount of pl6

protein, including RCH-ACV (Figure 2-la).









In Vitro Drug Sensitivity in ALL Cell Lines

I determined the sensitivity (IC5o) of Nalm-6, Molt-4, Jurkat, RCH-ACV and K562 to a

continuous 96 hour exposure to dexamethasone (Dex) and doxorubicin (Dox), two agents

commonly used in the treatment of childhood ALL, and FP. Each of the four ALL cell lines and

K562 were highly resistant to dexamethasone and variably sensitive to doxorubicin (Figure 2-

lb). Each of the cell lines tested showed sensitivity to FP, with ICSOs ranging from 99~111.5 nM

in Molt-4 to 312.51159.1 nM in K562. These values are similar to concentrations achieved in

vivo in phase I/II trials of FP administered both as a 1 hour and a 72 hour infusion (67-69, 72, 77,

127).

FP Induces Apoptosis in ALL Cell Lines

WST-1 assays measure the numbers of viable cells present following exposure to drug.

Decreased numbers of viable cells could be due to apoptosis, decreased cell proliferation, or

both. I performed Annexin V/PI staining and subsequent flow cytometric analysis on cell lines

which were exposed to drug for 72 hours to determine whether FP induced apoptosis. First, I

compared the cell death induced by FP and Dox in Nalm-6 and RCH-ACV at concentrations

approximating the ICso of each drug (FP: 150nM; Dox: 10ng/mL). At these concentrations, FP

induced a substantially lower percentage of cell death than Dox in both Nalm-6 and RCH-ACV

(Figure 2-2a). I then examined apoptosis induced by 300 nM FP and observed much higher rates

of apoptotic cell death, 93% and 83% in Nalm-6 and RCH-ACV respectively. I expanded these

studies and confirmed that FP induces apoptosis by performing parallel Annexin V/PI and

TUNEL analysis in Nalm-6, RCH-ACV, Molt-4 and Jurkat following 72 hours exposure to FP at

various concentrations (Figure 2-2b and 2-2c). For each cell line tested, modest levels (<25%) of

apoptosis were induced by 72 hours exposure to 150 nM FP and high levels (>80%) were

observed following exposure to 300 nM FP. Similar results were seen via Annexin V/PI and









TUNEL assays, confirming the apoptotic nature of the observed cell death. Taken together, these

data establish that FP treatment induces modest apoptotic cell death in B-precursor and T-cell

ALL lines at lower concentrations and is a potent inducer of apoptosis at higher concentrations.

FP Induces Cell Cycle Arrest in ALL Cell Lines which Correlates with Effects on pp-Rb
Protein Expression

My results demonstrated that the inhibition of cell proliferation observed with WST-1

assays can only partially be attributed to apoptosis when cell lines are treated with 150 nM FP;

however, 300 nM FP fully induces apoptosis. I hypothesized that the remaining inhibition at 150

nM FP could be due to cell cycle arrest. In order to test this hypothesis I performed cell cycle

analysis of samples treated with 0, 50, 150 and 300 nM FP for 24 and 48 hours. Treatment with

50 nM FP did not induce arrest when compared to an untreated control in RCH-ACV (Figure 2-

3a) and Nalm-6 (Figure 2-4). I observed a transient G1-S arrest after 150 nM treatment that was

present at 24 hours, but resolved by 48 hours. Sustained G1-S and G2-M arrest were induced after

treatment with 300 nM FP; which was apparent at 24 hours and more pronounced at 48 hours. In

order to address the possibility that the transient nature of the arrest induced by 150 nM

treatment was due loss of drug potency over time, I performed experiments in which treated cells

were exposed to FP for 24 hours, at which time the growth medium and drug were replaced

(Figure 2-3b). Cultures were allowed to incubate in parallel with those established 24 hours prior

for an additional 24 hours. Following incubation all cultures were evaluated for cell cycle

kinetics. Data showed similar cell cycle phase distributions between cultures treated with FP for

48 hours and those which had medium and drug replaced after 24 hours.

To investigate the mechanism of the observed cell cycle arrest, I determined expression of

total pRb and specific phospho-pRb forms (pp-Rbser795 and pp-Rbserso7/sll) in parallel to cell cycle

analysis (Figure 2-3c). Phosphorylation of pRb on ser 795 has been largely linked to CDK 4









activity and regulation of the G1-S transition by pRb (128). Phosphorylation of ser 807/811 has

also been linked to CDK 4 activity (129). Treatment of ALL cell lines with 300 nM FP resulted

in a sustained decrease in pp-Rbser795 and pp-Rbserso7/sll protein expression, which correlates with

the G1-S arrest observed at this drug concentration. Total pRb protein levels indicate stable levels

of total protein and a decrease in the expression of the hyperphosphorylated form of pRb (upper

band) that is dependent on drug concentration and time of exposure to FP. Treatment with a low

level of FP (50 nM) did not result in a decrease in the phosphorylation of pRb; however,

treatment with 150 nM and 300 nM FP did result in a decrease in the expression of the

phosphorylated form of pRb after 48 hours treatment.

Apoptotic Effects of FP in Human Serum

Others have shown that FP is 92-95% protein bound in human plasma and that there is a

decrease in the activity of FP in CLL cells when grown in human plasma or serum vs. FBS (82).

In order to determine if supplementation with human serum (HS) would have a similar effect on

FP efficacy in ALL cell lines, I tested the ability of FP to induce apoptosis in Nalm-6, RCH-

ACV, Jurkat, and Molt-4 grown in medium supplemented with FBS and compared this to cell

death of cell lines grown in HS. In order to mimic the peak drug levels that occur with FP

infusion schedules with high activity against CLL cells (30 minute bolus followed by a 4 hour

infusion), I measured cell death at 4.5 hrs (Figure 2-5a). For measurement of cell death at a sub-

peak level, I analyzed after 24 hours drug exposure (Figure 2-5b). Varying concentrations of FP

were used in keeping with those found to be achieved in CLL patients treated with the above

schedule at approximately these time points (85). After 4.5 hours, a modest percentage of cell

death is induced in Nalm-6 and RCH-ACV (15-20% and 10%, respectively). In contrast,

approximately 55-60% cell death is induced in Jurkat and Molt-4 at 4.5 hours. When comparing

the cell death induced in cultures supplemented with FB S to that of HS, I show that the









differences in the percentage of apoptosis induced in media containing FB S vs. HS are not

substantial for Nalm-6, RCH-ACV, and Molt-4; however, more cell death was induced by FP in

Jurkat cells grown in media containing FBS vs. HS. The percentage of apoptosis observed after

24 hours FP treatment was higher than that at 4.5 hours in all four cell lines tested. When

comparing the cell death achieved in cultures supplemented with FBS to that of HS at the 24

hour timepoint, I observed substantial differences between media containing FB S and HS for

RCH-ACV at all FP concentrations tested and at the lowest FP concentration (300 nM) in Nalm-

6 and Jurkat. My results show no differences between FBS and HS for Molt-4.

Discussion

The poor outcome of children with ALL who experience a bone marrow relapse despite

intensive chemotherapy and/or stem cell transplant, makes it imperative to identify agents with

novel mechanisms of action. Based on the frequent acquisition of pl6 deletions at relapse (27)

and alterations in expression of genes that encode for cell cycle regulatory proteins at relapse

(18), I performed preclinical studies of FP in ALL cell lines. My results support the use of these

cell lines as a model of relapsed ALL in that none of the lines expressed pl6 protein and all were

resistant to dexamethasone and variably sensitive to doxorubicin, two agents commonly used in

the treatment of ALL and to which relapsed patients frequently become resistant (130). I report

that childhood ALL cell lines are sensitive to FP, providing a biological rationale for clinical

trials of FP in relapsed ALL.

I found that FP was active in a concentration dependent manner against ALL cell lines. At

a concentration approximating the ICso determined in WST-1 assays (150 nM), FP induced

transient cell cycle arrest with a limited percentage of apoptosis. At approximately twice this

concentration, FP was a potent inducer of cell death. This information provides a dual

mechanistic explanation for the decrease in viable cell number that I observed in WST-1 assays.









CDKs phosphorylate pRb and therefore regulate its ability to sequester transcription

factors involved in cell proliferation during the G1-S phase transition. Phosphorylation of pRb on

approximately 16 different serine and/or threonine residues can be attributed to the activity of

specific CDKs (13 1). My Western blot analysis shows that expression of pp-Rbser795 and pp-

Rbserso7isll is reduced after 24 and 48 hours treatment with 300nM FP. These data correlate with

the sustained G1-S arrest observed after 300 nM FP treatment and show that FP treatment results

in a reduction of endogenous CDK activity. Evaluation of total pRb protein expression showed

consistent expression across all treatment levels, indicating that the decrease in phospho-specific

pRb was not due to loss of total pRb. Treatment with 150 and 300 nM FP resulted in a decrease

in the hyperphosphorylated form of total protein after 48 hours drug exposure. This decrease

further illustrates the reduction in CDK activity as a result of FP treatment in ALL cell lines.

This also suggests a mechanism for the transient G1-S arrest observed after 150 nM FP treatment

and the G2-M arrest I observed after 300 nM treatment. The apoptosis and cell cycle arrest

induced by FP treatment in ALL cell lines provide two potential modes of treatment in ALL. FP

could be used as a single agent to induce a cytotoxic effect or be utilized in combination with

another chemotherapeutic agent that would complement the ability of FP to induce cell cycle

arrest. My observation that FP inhibits CDK activity and induces cell cycle arrest in ALL cells

suggests that there may be schedule dependent differences in activity if FP is combined with

other agents, particularly those with cell cycle specific activity.

Initial phase I/II trials of FP in many human cancers were disappointing. While in vitro

studies showed that FP was efficaceous against a diverse variety of tumors at concentrations of

100-300 nM, no significant clinical activity was seen with prolonged infusion regimens, despite

achievement of similar FP concentrations in vivo (69-73). Shinn and colleagues hypothesized









that the disparity between in vitro and in vivo activity might be due to differences in binding of

FP to plasma proteins present in FBS used in vitro vs. those present in human plasma (82). They

confirmed that for CLL cells the FP ICso was significantly (approximately 10-fold) higher in

vitro when experiments were performed in human plasma rather than bovine serum. This

suggested that infusion schedules that produced high peak FP concentrations might be more

effective than prolonged lower dose infusion schedules. Early phase clinical trials confirmed this

hypothesis in CLL using a 30 minute bolus dose followed by a 4 hour infusion (85). Expanded

studies are ongoing. Based on these observations, I also examined the relative efficacy of FP in

vitro in experiments using human serum compared to bovine serum. In my experiments I

observed fewer differences between ALL cell line sensitivity to FP in HS vs. FBS than observed

by Shinn and colleagues. Importantly, despite some differences among the cell lines tested,

substantial amounts of apoptosis were induced by FP in all cell lines under conditions that are

very similar to what might be observed clinically, particularly at the 1000 nM and 2000 nM

levels at 4.5 hours and 300 nM level at 24 hours. These data suggest that the newer FP infusion

schedules found to be very promising in CLL should be utilized to test FP against relapsed ALL.


















Dmianerlhsluone Doxorubida Flnlopirido~l inlM
(ughL) (nfinL)

RCH-ACV >10 1318.6 131.3+33.8

Naint-6 >10 10.612.9 142.5246.0
Muolt-4l >10 9.7+3.8 89911.5
Jurkat >10 771.3142.1 300135.4
IEW2 >10 39.2t28.7 312.50 59.1


acti~ -C ~L


p15 ~ ~ I--


1234 5


6 78 9


Figure 2-1. Fifty percent inhibitory concentration (ICso) determinations via WST-1 in cell lines
that lack pl6 protein expression. A) Western blot for pl6 protein expression; Lane 1)
100% HeLa (positive control), 2) empty lane, 3)10% HeLa, 4) 1% HeLa, 5) Nalm-6,
6) RCH-ACV, 7) REH, 8) Molt-4, 9) Jurkat B) IC50 values+1SD for Dex, Dox and
FP after 96 hours drug exposure measured via WST-1 cell proliferation assays.

















































Celideath Apoptosis
measured via measured via
annowinv/DI T(JNEL(%8)

Nalm-6
Untreated 2.47 0.51
150 nM FP 19.72 22 22
300 nM FP 81.45 89.02
RCH-ACV
Untmeated I < 0.58
150 nM FP 23 46 22.33
300 nM FP 8 16 80,04
Molt-4
Untr~ence i.91 1 81

300 nMF 905116 51 0
Jurkat
Untrated 4.948 0.541
150 nM FP 11.12 3.07
300 nM FP 79.28 82.03


Figure 2-2. Flavopiridol induces apoptosis in ALL cell lines in a concentration dependent

manner. A) Scatter plots from flow cytometric analysis of Annexin V/PI stained

samples of Nalm-6 and RCH-ACV comparing cell death induced by 72 hours

exposure to 150 nM and 300 nM FP to the cell death induced by 72 hours exposure to

10 ng/mL Dox, an agent known to induce apoptosis. Percentage to right of each plot

represents the sum of the lower right quadrant (cells in the early stage of apoptosis)

and upper right quadrant (late stage apoptosis) of each plot B) Scatterplots generated

using two different staining methods after 72 hours continuous exposure to 300 nM

FP show similar results; AnnexinV/PI (left) and TUNEL staining (right) C)

Comparison of results from AnnexinV/PI to TUNEL in cell lines treated with 0, 150,

and 300 nM FP respectively.


A rr.lm-a RcH-ncv
vhi*rar~ slftraa wa~o~a~ 'P~mD~


larr r


renurr ~arruw lur~rr ~oc~rr
1


''it~J n~n r~l ,, ~E~ ,,



B.
Nth-6 3MI rM FP REH-nn, 3W *n FP
~

,,,
..,,, ifl ,,
,,,,

~olr-r 3W nl~ FP Illrtiat 300 nbl FP


,, ,,
,,
~
~












A. Untre.aed 50 nN FP 150 nMl FP 3001 aMF
14 has*~ j 850 4.6 a5.6% 41



Untreated 50 nN FP 150 nN FP 200 mM FP












48du to.Drg Rpl re

Unteatd 50slF






D M IM 3@ 0 2 150 3MrMF

24 rs, 40

Figure 2-3. Flavopridol induces Gi-SandG- Gp -ioi) reti RHAVwt
reucd hsporltin fp~ A el cce at ftr 4an 4 ous xosret

0,5,10 n 0 n P ratmen wih5 MF hsn fet nclyl
kinetics when compared to untrureatead cnrl aaso rnin Sars fe
24 our exosue t 10 n FPanda sstine GiS ares ater24 nd 8 hur
treamen wih 30 nMFP.Als shwn s a ustine G2M arestfolowig 30 n










Figre2-. lcomparable tosampes without adrug2 reaplacement.C) Wrestern blot showing
seustied decphrelasei xrsion of p p-Rbsel yer9 dandp-beroai after a 4 or exposure to
0,5,10ad300 nM FP. TotlpR xressin emiscntnt with 5 F as doeecrs in thel ye

hyperhous ephosryaed for (uppe band)assand 1Sars after 150 and 300 nMretmnt













5 11strpated S LnLifR 15 L~r hif2










Figre -4.Flvopridl idues raniet.19 Gi-S 1 ares in Nal-6.Cl ccedtaatr24ad4

hour exosue t 0,50, and 50 M F. Tramn ih5 M Phsn feto

cell ccle knetic when ompard to ntreatd conrol. ataso G- res fe
24 ous retmntwih 50nM Cllcylekietcsreur t bsein ate 4 hur
treatment













in0 100


0g 300 10 29 30 100 2






0 300 1000 2000I 0 300 1000 2000
FP(nM) FP(nMI)



orHS DA) More24n c rrelldeth a i ndce in Jurat cells o~ t ~reatdfr 45husandi I
suplm ene wihFSta hs n S oeeteclldahidcdih


remaining_ cel lie a proiaeyeua ewe h totpso sr.B fe
24! hor ramn fRHAV oecl eahwsidcdb Pteteta l
cocetrtin tete incll uplmnedwt F Sthntos uplmnedwt
HS Difeene in celdahbtentetotps fsr eeas bevdi
Nlm- n uktafe ramn wt 0 MF.Nodfeecsbewe eawr
obsre in Molt-4.I~


Cel Death Post-4.5 hrs. Treatment in HS









CHAPTER 3
PRECLINICAL STUDIES OF FLAVOPIRIDOL COMBINED WITH PACLITAXEL IN
ACUTE LYMPHOBLASTIC LEUKEMIA

Introduction

Clinical trials of FP involving cancer types other than ALL have shown that FP has limited

efficacy when used as a single agent. As outlined in Chapter 1, several in vitro studies have

shown that the efficacy of traditional chemotherapy agents can be increased when used in

combination with FP. One such traditional agent is paclitaxel (PAC). Few clinical trials

involving FP combination therapy have been performed; however, promising results have been

obtained in a variety of solid tumor patients using FP in combination with PAC (111). PAC has

shown in vitro toxicity in leukemia cell lines; however, has had limited efficacy in clinical trials

in children and adults with leukemia (15, 16). PAC and FP represent two drugs to which ALL

patients will not have been previously exposed. This fact as well as in vitro data showing

synergy between FP and PAC in other cell types lead me to question if PAC/FP combination

therapy could hold promise in the treatment of ALL.

It has been previously found that the interaction between PAC and FP is dependent on the

sequence in which the two drugs are administered in vitro. Combination therapy is most

efficacious when PAC precedes FP in the treatment sequence (PAC+FP), as opposed to the

reverse or concurrent therapy (56, 60). I have confirmed these Eindings in ALL cell lines as well

as determined optimal treatment duration for each agent prior to testing combination therapy. In

an effort to maintain the clinical relevance of my findings I have also taken the currently

accepted in vivo infusion schedule for each drug into account when designing my experiments.

The recommended schedule for PAC administration is either 3 hour or 24 hour infusion (110). I

have tested 6 and 24 hours exposure to PAC. Early trials involving FP used a 72 hour continuous

infusion schedule. My experiments reflect this, as in experiments in which PAC was combined









with FP, cell lines were exposed to FP for 72 hours. Later studies by others have shown that FP

is highly protein bound and that supplementing cell culture medium with human serum (HS) in

place of fetal bovine serum (FB S) decreases the sensitivity of CLL cells to FP (82). In order to

address this I have conducted experiments in media supplemented with both FBS and HS.

Methods

Materials

ALL cell lines were obtained and cultured as described previously (132). PAC (Sigma, St.

Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) and freshly diluted in RPMI 1640

prior to each experiment. FP (Sanofi-Aventis, Bridgewater, NJ) was dissolved in DMSO and

further diluted in RPMI 1640 no more than 30 days prior to each experiment.

Single Agent Inz Vitro Sensitivity Assays

Nalm-6 and RCH-ACV were exposed to 0-300 nM FP in RPMI 1640 with 10% fetal

bovine serum (FBS; Mediatech, Inc. Herndon, VA) continuously for 72 hours. Cell death was

measured every 24 hours via flow cytometric analysis of Annexin V (Pharmingen, San Diego,

CA)/Propidium Iodide (PI, Roche, Indianapolis, IN) as described previously (132). Treatment

duration and sensitivity to PAC were determined by exposing Nalm-6 and RCH-ACV to 0-100

nM PAC for 6 hours and 24 hours in parallel followed by cell death measurements every 24

hours for a total of 72 hrs (Figure 3-1). In a separate experiment, Nalm-6 was exposed to 0-

1,000 nM PAC in RPMI 1640 supplemented with 10% human serum (HS; Mediatech) for a

period of 6 hours in parallel to samples treated in media supplemented with 10% FBS with cell

death measured every 24 hours. In order to determine the effect of HS on untreated cell

proliferation and viability, growth curves were generated using trypan blue staining (Sigma) of

samples grown with HS compared to FBS.









Drug Combination Studies

FP was combined with PAC using a drug concentration ratio of 1:10 (PAC:FP) in Nalm-6

and RCH-ACV. Due to greater sensitivity to PAC, Molt-4 and Jurkat were treated at a ratio of

1:20 to allow for a lower concentration of PAC to be utilized. Cell lines were exposed to PAC in

cell culture medium supplemented with 10% FBS for approximately six hours, washed, and then

treated with FP for an additional 72 hours. Treatment duration was selected based on PAC and

FP single agent experiments. Single drug controls for PAC consisted of six hours incubation with

PAC followed by incubation in drug free medium for approximately 72 hours. Cell lines used for

single drug treatment with FP were incubated in drug free RPMI 1640 for approximately six

hours; after which the cell lines were incubated with FP for an additional 72 hours. At the

completion of the 72 hour incubation, cell death was evaluated for all samples. In order to

confirm that results similar to that found in medium supplemented with FBS could be achieved

in cultures supplemented with HS, Nalm-6 was exposed to PAC for 6 hours followed by FP for

72 hours at a concentration ratio of 1:3. Control samples were treated and cell death was

evaluated in the same manner as described for combination studies in FBS.

Treatment Sequence

Nalm-6 and RCH-ACV were used to determine the optimal treatment sequence. One 1:10

combination was chosen from the drug combination studies in FBS in order to confirm that PAC

followed by FP (20nM PAC+200nM FP) was indeed the most efficacious treatment sequence.

Briefly, for the samples in which FP treatment followed PAC treatment (PAC+FP), cell lines

were cultured in RPMI 1640 with and without 20 nM PAC for 6 hours, then washed and

transferred to RPMI 1640 with and without 200 nM FP. In sample sets in which FP treatment

preceded PAC treatment (FP&PAC), cell lines were treated similarly, with the sequence of drug

exposure reversed. Cell death measurements were taken immediately after FP treatment for









PAC+FP and its controls. For FP&PAC and its controls, cell death was determined

approximately 18 hours following completion of PAC treatment. Concurrent exposure

experiments were performed separately; during which cell lines were treated with both agents for

a total of 72 hours. Cell death was measured at 24 hour intervals via flow cytometry as

previously described.

Statistical Analysis

All experiments, excluding concurrent exposure to PAC and FP, were performed with

three independent replicates. A mixed model was used to analyze each experiment. The

replicates for each experiment were considered a random factor. If there was a significant

interaction (p<0.05) between the variables in each experiment, mainly cell line, treatment, time

of exposure, and drug concentration depending on the type of experiment, then Least Squares

Means of the treatment combinations were compared using a Student' s t-test or F test.

Results

Single Agent FP Treatment

In order to determine the time of exposure to FP that resulted in the maximum cell death

response in ALL cell lines, I incubated Nalm-6 and RCH-ACV with 0-300 nM FP with cell

death measured at 24, 48, and 72 hours. There was a significant concentration dependent

response in both cell lines (p<0.0001; Figure 3-2 a). I compared the cell death induced after 24

hours treatment to the cell death induced after 72 hours. There were no significant differences in

cell death based on time of exposure at the 50 nM concentration in either Nalm-6 or RCH-ACV.

In Nalm-6, there was a significant difference in cell death between 24 and 72 hours exposure to

300 nM FP (p<0.0001), while in RCH-ACV these differences were observed between 24 and 72

hours exposure to 150 nM (p=0.0034) as well as between 24 and 72 hours exposure to 300 nM

FP (p<0.0001).









Single Agent PAC Treatment

I exposed Nalm-6 and RCH-ACV to PAC for 6 or 24 hours and measured apoptosis at 24,

48, and 72 hours following initial exposure. I observed concentration dependent cell killing in

Nalm-6 and RCH-ACV samples treated for both 6 and 24 hours with a greater percentage of cell

death observed after 24 hours vs. 6 hours drug exposure (Figure 3-2 b). Further, I show that cell

lines treated for 6 hours then transferred to drug-free medium show a greater gradation in

response between 10 nM PAC and 100 nM PAC than those treated for 24 hours, particularly in

Nalm-6. Cell death peaked 48 hours post-treatment and remained consistent with no substantial

change at 72 hours post-treatment in both cell lines. Statistical analysis showed that time of

exposure (6 or 24 hours) to PAC was a significant factor in the percentage of cell death observed

in both Nalm-6 (p=0.0001) and RCH-ACV (p=0.0138). The post-treatment time at which cell

death measurements were taken was also a significant factor in both Nalm-6 (p<0.0001) and

RCH-ACV (p=0.0008). The interaction between the factors of time of exposure and sample time

was significant in Nalm-6 (p=0.0427); however, this interaction was not significant in RCH-

ACV (p=0.6812). My results indicate that even though a 24 hour drug exposure time resulted in

a greater percentage of cell death, a shorter exposure period of 6 hours still resulted in a

substantial amount of cell death. Importantly, incubation for 48-72 hours post-treatment was

needed in order to achieve a maximum response.

Combination Treatment with FP and PAC

Based on my single agent studies I treated four cell lines with PAC for 6 hours; then

transferred the cultures to media containing FP for 72 hours and measured cell death at the end

of this period. Nalm-6 and RCH-ACV were treated with a drug concentration ratio of 1:10

(PAC:FP) and Molt-4 and Jurkat at a ratio of 1:20. I observed a concentration dependent cell

death response for both the single agent treatments as well as each combination (Figure 3-3). My









results demonstrate that when PAC is combined with FP the cell death that results is significantly

higher than when either of the two agents is utilized by itself. I found a statistically significant

difference (p<0.05 or p<0.0001) between single agent and combination treatment in the range of

10-30 nM PAC and 100-300 nM FP in Nalm-6. In RCH-ACV these differences were present for

treatments in the range of 15-25 nM PAC and 100-250 nM FP. Significant differences

(p<0.0001) were present at 5-10 nM PAC and 100-150 nM FP in Molt-4 and Jurkat.

Determination of Optimal Schedule for PAC+FP

I tested PAC+FP, the reverse sequence, and each of the single agent controls to confirm

that the former was the most effective treatment sequence. The most promising dose level (20

nM PAC+200 nM FP) was selected for more detailed analysis. Combining PAC with FP in the

sequence PAC+FP results in significantly greater cell death in Nalm-6 than FP&PAC or single

agent treatment (Figure 3-4 a). The cell death resulting from the PAC+FP was significantly

higher than the other treatments with p values ranging from p<0.0001 to p=0.001. I observed a

similar response in RCH-ACV with p values ranging from p<0.0001 to p<0.01 (Figure 3-5).

I also examined concurrent exposure to 20 nM PAC and 200 nM FP and observed no

enhancement (Figure 3-4 b) in activity. Indeed, the cell death induced after treatment with both

agents is less than that observed with FP alone across three days of testing.

Activity of PAC in Human Serum

Studies have found that 89-98% of PAC is protein bound in human serum (110). This

prompted us to investigate how the efficacy of PAC in ALL cell lines would be affected if

culture medium was supplemented with human AB serum (HS) in place of fetal bovine serum

(FB S). I show that Nalm-6 cells exposed to 10, 100, and 1,000 nM PAC for 6 hours in media

supplemented with HS underwent significantly less apoptosis than observed when experiments

were performed using media supplemented with FBS (Figure 3-6). These differences in PAC









sensitivity were not due to intrinsic differences in cell growth in media supplemented with FBS

vs. HS (Figure 3-6 d).

Combination Studies in Human Serum

Shinn and colleagues have reported that 63-100% of FP is free (non-protein bound) in

FBS, as compared to only 4.7-7.9% free in human plasma in vitro (82). Based on this

information and the high level of protein binding by PAC to plasma proteins, I treated Nalm-6

with PAC combined with FP at a concentration ratio (PAC:FP) of 1:3 in medium supplemented

with HS in order to confirm that the enhancement of PAC activity by FP that I observed in FB S

could also be achieved under these culture conditions (Figure 3-7). Higher concentrations of

PAC were used in order to compensate for the lower sensitivity of ALL cell lines to PAC in the

presence of HS. I show that FP significantly enhances the efficacy of PAC at all concentrations

tested (p<0.0001). Combination treatment was significantly different from FP single agent

treatment at the lowest concentration tested (p<0.05); however, significant differences did not

exist at higher concentrations of FP most probably due to a high percentage of cell death induced

from single agent FP treatment using this prolonged exposure schedule.

Discussion

There is a significant need to identify novel agents and new combination treatments for

relapsed ALL. Others have shown that PAC and FP have limited efficacy when utilized as single

agents in leukemia patients (16, 75, 133); however, together these drugs have mechanisms of

action that complement each other and might effectively target aberrations in cell cycle

regulation that are commonly present in ALL cells at relapse. My studies demonstrate that ALL

cell lines are sensitive to both PAC and FP in vitro, and that FP enhances the efficacy of PAC in

a sequence specific manner.









My observation that the cell death induced after treatment with 1,000 nM PAC in HS is

comparable to cell death achieved by treatment with 100 nM PAC in FBS is consistent with prior

studies by others showing a high degree of PAC binding by proteins present in HS. In contrast to

data of Shinn and colleagues in CLL (82), my previous studies showed relatively little difference

between the in vitro activity of FP when experiments are performed in HS vs. FBS. My current

observations confirm that despite a decrease in sensitivity to PAC in the presence of HS, FP

enhances the efficacy of PAC under these treatment conditions.

The time of actual drug exposure utilized for PAC treatment (6 hours) in my experiments

was in keeping with the recommended clinical administration of 3 or 24 hours infusion (1 10).

My findings show that 48-72 hours of incubation are needed to achieve a maximum cell death

response to PAC treatment. I also found that the amount of cell death induced by FP in the

concentration ranges studied was dependent upon the duration of exposure, with 72 hours

inducing a peak response. Based on these two factors, I studied a 72 hour FP exposure in the

combination studies. This schedule of FP exposure is different than newer FP dosing strategies

that administer a 30 minute FP bolus followed by a 4 hour infusion to produce much high peak

FP concentrations than those achieved with other infusion schedules, which have yielded

promising early results in patients with refractory chronic lymphocytic leukemia (CLL) (83). In

other studies, I have found that shorter in vitro exposure of the same ALL cell lines, cultured in

media containing either FBS or HS, to high FP concentrations similar to those attained in CLL

clinical trials induced substantial amounts of apoptosis. Based on the results attained in the

current studies, I anticipate that administration of PAC prior to the FP bolus should enhance ALL

cell death and suggest that this combination should be investigated in clinical trials for relapsed

ALL.
















24 hours


Sample and stain
with Annexin VIPI
(Dayl1) Sample and stain
with Annexin V/PI
Sanpl~nd 8dn ~(DaIys 2 and 3)
with Annexln VIPI
(Day 1)


Spin 5 min. 18 hrs.
6 hours 1,000 RPM
r Replace medium


Set-up flasks with PAC

Sample and stain
wvith Annexin VIPI (Day 0)


Spin 5 min.
1,000 RPM
Replace medium





Figure 3-1. Experimental design for PAC single agent treatment.
















Cll Drth Inducld ~ Inenarins Cenerntraolonr ef
PP [n ~Ulln4
~I In
Il*L ~)~:
r.l
~II
m II ~I I ri
or
11'1
u ;;I

041 041 D43


Cll Drth Inducd by Inc nalnp Conf Nmrdonl at
FP In RCH~C\I
+ o:rl~bn
rr

r o;r ~
f
13
V 2:
1:

Dsyl Ogl D43


B. I NRCH CTreatedwnmhPadharr ala Hoer .ACVTnreal th Padkanel24 hts.

100 100It IP









A) Perce to- cel det indcedaferll 72hustetetwt 0 5,20 n 0
nMFPmesuedevry24hor via~n flow ctoercalyiofAnxnVP

stie smlsofN l-. eut arth menotreidpnet
expriensi SD. Sinfcn ifrncsi ieo xour tietcld



hours or 24 IVTI* horstramentLrll wih 0 .0 10,C~r 100 n PACllnl mesrd vr 2 or


thrafe oratt alof7 hou lrs.Rslts ar hema o heeidpedn

exeimnsi D Sinfcn diferece at idniacoetrinsbwenhe2
andl 72r horsml ie r nictd ausrne r m0.1t<.01foal
symbols.I~p ~ n




































fPAC


Sequential Treatment with Paciltaxel and Flavoplrldol In
RCHdNC
1-I






*p 10, 100 15, 150 20, 200 25, 250 *pio~uorl
PAC (nM) FP (nl@


Sequential Treatment with Padiltaxel and Flavoplrldol
In Jurket


Sequential Treatment with Paelitaxel and Flavopiridol
in Molt-4









2.5, SO 5, 100 7.5, 150) 10, 200
PAC (nl@,FP (n) ap~o.Dool


2.5. 50 5, 100 7.5, 150
PAC (nl@, FP (nM)


10, 200

Cp

Figure 3-3. Flavopiridol enhances the efficacy of PAC in ALL cell lines. FP was combined with
PAC in the sequence PAC+FP at a concentration ratio of 1:10 in Nalm-6 and RCH-ACV

and 1:20 in Molt-4 and Jurkat. Cell lines were treated with PAC for 6 hours immediately

followed by FP for 72 hours. Cell death was measured at the conclusion of treatment via
flow cytometric analysis of Annexin V/PI stained samples. Results represent averages
from at least three independent experiments+1SD.


100
00
70
~ so
~511
cl 40
130
20
10













Nalm-6 Sequence Experiment


100





PPACa2>FP200 PAC2&tO >FP200 FP2D~aPAC20 FP20M)O &PAC2 urstraded



Nalm-6 Concurrent Excposure

Treatment Day D Day 1 Day 2 Day 3
untreated 3.19 4.13 2.683 1.64
PAC 20+FP
200 3.19 31 66~.63 53.63
PAC 20 3.19 44.33 40.69 41.58
FP 200 3.19 65.46 66~.77 67.45





Figure 3-4. PAC+FP is a more efficacious treatment sequence than FP&PAC or concurrent
exposure in Nalm-6. A) Nalm-6 was treated for 6 hours with 20 nM PAC followed
by 200 nM FP for 72 hours or the reverse sequence. Both sequences included
appropriate single agent controls and an untreated control. Cell death was measured
via flow cytometric analysis of Annexin V/PI stained samples immediately following
FP treatment for PAC+FP and approximately 18 hours after PAC treatment for
FP&PAC. Results are the mean of three independent experiments+1SD. Statistical
significance was measured by comparing PAC20+FP200 to the remaining treatment
sequences. *p<0.0001; #p=0.001. B) Concurrent exposure to PAC and FP in Nalm-
6. Cell lines were simultaneously exposed to 20 nM PAC and 200 nM FP for a total
of 72 hours with cell death measured every 24 hours.












A.
RCH-ACV Sequence Experiment





PrAC2>P0 AE SPm F2>ACD P2> >A2 rra

B,
so RC-C ocretEpsr







PACAC 20crrn 4.51661 2.2 4.1



FP 200 4.5 15.23 18.7 19.52





Figure 3-5. PAC+FP is a more efficacious treatment sequence than FP&PAC or concurrent
exposure in RCH-ACV. A) RCH-ACV was treated for 6 hours with 20 nM PAC
followed by 200 nM FP for 72 hours or the reverse sequence. Both sequences
included appropriate single agent controls and an untreated control. Cell death was
measured via flow cytometric analysis of Annexin V/PI stained samples immediately
following FP treatment for PAC+FP and approximately 18 hours after PAC
treatment for FP&PAC. Results are the mean of three independent experiments SD.
Statistical significance was measured comparing PAC20+FP200 to the remaining
treatment sequences. *p<0.01. B) Concurrent exposure to PAC and FP in RCH-ACV.
Cell lines were simultaneously exposed to 20 nM PAC and 200 nM FP for a total of
72 hours with cell death measured every 24 hours














Treatment with 10 nM PAC MBS v. HS

100
90
Sso
E 70

20



S100~
9 0
80



Day0 Day1 DaY2 Day3


Treatment with 1000 nM PAC FBS v. HS

100
90


20

S10

OayD 041 DWZ D y3


Averagen Growt F00n A BS v. HS






C 0 00E+0
Day Da1 Dy


*












*












C,












D,


Figure 3-6. Efficacy of PAC in Nalm-6 in the presence of human serum. Cell death resulting
from 6 hours exposure to PAC in the concentrations shown in medium supplemented
with HS compared to that in FBS measured every 24 hours for a total of 72 hours.

a) 10 nM PAC b) 100 nM PAC c) 1000 nM PAC d) growth curve from untreated cell
cultures comparing HS and FB S supplements. All results are the mean of three
independent experiments+1SD; *p<0.05, #p<0.0002.














Sequential Treatment with Paditaxel and Flavopiridol
in Nalm-6


S80


B3 450- 6FP


100, 300 125. 375 150. 450 200. 600 "p<0.0
PAC (nM), FP (nM) p000


Figure 3-7. Flavopiridol enhances the efficacy of PAC in human serum. PAC was combined
with FP at a concentration ratio of 1:3 in Nalm-6 cultured in the presence of HS.
Nalm-6 was treated and sampled as in previous assays in FBS. Results are the mean
of three independent experiments SD.









CHAPTER 4
CONCLUSIONS AND DISCUSSION

FP Single Agent Studies

Establishing an in Vitro Treatment Model of ALL

In this proj ect I have focused on determining the potential efficacy of FP both as a single

agent and in combination with PAC in ALL cell lines. I tested these agents based on their

mechanisms of action. FP could target defects in cell cycle regulation produced by mutations in

human cancer, such as pl6; which is frequently altered at the gene and/or protein expression

level in relapsed ALL (124). The action of PAC as an inducer of CDK1 and microtubule

depolymerization inhibitor complements this activity. Following the first chapter of background,

the second chapter of this dissertation details the results that I obtained when testing FP as a

single agent. As part of my initial studies, I established that the cell lines to be used for

sensitivity testing were an accurate model of relapsed ALL by determining their level of pl 6

expression and their sensitivity to dexamethasone and doxorubicin, agents typically used to treat

ALL (3). I found that despite the fact that one of the cell lines had an intact pl6 gene, none of the

cell lines expressed pl6 (Figure 2-1). I also showed that the cell lines were highly resistant to

dexamethasone and variably sensitive to doxorubicin; findings not unlike what would be

obtained in a patient (2). Importantly, the cell lines were sensitive to FP at concentrations similar

to those found to be effective in vitro by others.

Drug Sensitivity Testing via Cell Proliferation Assays

Results for drug sensitivity were obtained using WST-1; a modified version of the MTT

cell proliferation assay. As introduced in the second chapter, in vitro sensitivity to

chemotherapeutic agents measured via this type of assay has been found to correlate to in vivo

efficacy (112-119). In order to use these assays, I first needed to establish that the cell lines could









be maintained untreated in log phase growth over the standard time course of four days. I found

that using a cell concentration of 1X104 CellS/well in 96 well plates gave the best exponential

growth over time (Figure 4-1 a, b). Nalm-6 was later established at a lower concentration due its

lower doubling time (Chapters 2 and 3). In addition, as part of my preliminary studies I showed

that measuring cell proliferation over time via a cell proliferation assay resulted in the same type

of exponential growth curve as when the number of viable cells over time was measured by

trypan blue exclusion (Figure 4-1 c); thus validating the assay. Cell proliferation assays were

used to generate growth curves to establish the ICso concentrations reported in Chapter 2 (Figure

2-1). Examples of these growth curves may be found in Figure 4-2.

The Mechanism of Cell Death Induced by FP in ALL Cell Lines

Also in Chapter 2, I established that FP induces apoptosis in ALL cell lines. FP induced

less apoptosis at its IC5o than a similar concentration of doxorubicin, a known cytotoxic agent

(Figure 2-2). I also showed that FP treatment resulted in apoptosis consistently across four ALL

cell lines (Figure 2-2). Results were obtained after 72 hours treatment to reflect treatment

strategies current at the time. These measurements were taken using flow cytometric analysis of

both Annexin V/PI and TUNEL stained samples in order to confirm that the level of cell death

measured using Annexin V/PI assays was consistent with another method.

Based on the ability of FP to induce cell cycle arrest in cancer cell types other than ALL

(14, 53) and the apparent disparity in cell death at the ICsos for doxorubicin and FP, I conducted

a study of cell cycle kinetics after treatment with both low and high concentrations of FP. I found

that at the ICso, FP induced a transient G1-S block that appeared after 24 hours treatment; with

cell cycle kinetics returning to baseline by 48 hours treatment (Figure 2-3). This arrest gave an

explanation for the decrease in cell proliferation observed at this FP concentration despite a lack

of apoptosis. Treatment with a concentration of FP twice the ICso resulted in a sustained G1-S









and G2-M arrest over the course of 48 hours. Drug replacement studies showed that the transient

nature of the G1-S arrest at the ICso was not due to loss of drug potency over time. Further, I

established that FP prevents the phosphorylation of specific serine residues on pRb, an indication

of a decrease in endogenous CDK activity.

FP Activity in Human Serum

Recent findings have shown that FP is highly protein bound in human plasma and that this

low drug availability can decrease in vitro efficacy (82). Researchers hypothesized that this could

explain the disappointing results obtained in clinical trials using FP as a 72, 24 or 1 hour

infusion, despite obtaining plasma concentrations similar to that found to be effective in vitro

(20-400 nM) (69-81, 124). As explained in Chapters 2 and 3, Byrd and colleagues from Ohio

State University (OSU) designed a clinical trial for patients with refractory CLL with the goal of

obtaining a plasma FP concentration of 1.5 CIM after a 30 minute bolus dose followed by a 4

hour infusion (83). The trial was quite successful despite issues with tumor lysis syndrome, with

an overall response rate of 45%. In order to determine if protein binding would have an effect on

the cell death induced by FP treatment in ALL cell lines, I tested in vitro sensitivity in the

presence of HS and compared it to FBS. Cell death measurements were taken after 4.5 hours and

24 hours continuous exposure to mimic peak and trough concentrations in the OSU infusion

schedule (Figure 2-5). I found that despite the high level of protein binding in human serum

reported by others, it is possible to achieve a high percentage of cell death in ALL cell lines in

vitro with concentrations that mimic those expected to be produced by the treatment schedule

utilized by Byrd and colleagues.









PAC+FP Combination Studies


Note about Statistical Analysis

Chapter 3 details my studies of FP in combination with PAC. To make meaningful

comparisons between treatments, statistical analysis was applied to the data. The analyses of

each data set followed the same basic pattern; beginning with a mixed model analysis of variance

(ANOVA) followed by post-tests to determine if there were significant differences between

treatments, times of exposure, or treatment sequences depending on the variables of the

individual experiment. ANOVA was chosen as the method of analysis based on the fact that each

type of experiment involved multiple comparisons. If a Student' s t-test had been applied to each

individual measurement within each data set, the probability of obtaining significant p values

would have been artificially high (134).

By definition ANOVA compares the actual results to the data that would have been

obtained if the null hypothesis were correct. ANOVA was used to test the significance of the

interaction between the variables for each experiment. If the null hypothesis were correct, there

would be no interaction between any of the variables in the experiment. Each p value given from

the mixed model analysis assigns a percentage to the probability that the interaction present was

due to chance. If significant interactions between variables were present, results based on one

variable could not be analyzed for significance without taking the other variables into account.

The "mixed model" designation to the ANOVA simply states that there were fixed and random

factors in each of the experiments. The variables tested in each experiment were defined as being

fixed effects and the number of replicates was taken as a random effect. The mixed model

analyses are given in tables which are discussed throughout the text that follows.









If the interaction between the factors in each experiment was significant, then the Least

Squares Means of the replicates from each experiment were compared using a Student' s t-test or

an F-test. These analyses are also included in the discussion that follows.

Enhancement of PAC Activity by FP

PAC and FP have complementary modes of action in that PAC enhances CDK 1 activity

while FP is a pan-CDK inhibitor. This is the hypothesis behind why the enhancement of PAC by

FP is dependent on the order in which the drugs are administered (56). As summarized in

Chapter 3, others have shown that FP can enhance the activity of PAC in vitro in cancer types

other than ALL (56, 60). Promising results have also been obtained during a clinical trial using

patients with various types of cancer (1 11). I chose to test PAC in combination with FP due to

the aberrations in cell cycle regulatory proteins frequently found in relapsed ALL patients and

because this treatment regimen would offer a new possibility to children with relapsed ALL

using two agents to which they would not have been previously exposed.

In order to conduct these experiments I first needed to establish whether ALL cell lines

were sensitive to PAC and determine a treatment schedule for each of the drugs as single agents.

After testing FP in two cell lines over 72 hours, I was able to determine that the time of exposure

had a significant effect on the percentage of cell death induced by FP. Statistical analysis was

conducted by first determining if there was a significant interaction between the factors of FP

concentration and time of exposure (Tables 4-1 and 4-2). When it was determined that a

significant interaction existed, the data were further analyzed by determining if there were

significant differences in cell death at different times of exposure for a given FP concentration

(Tables 4-3 and 4-4). With the results given in Chapter 3 (Figure 3-2), I was able to conclude that

72 hours FP treatment gave a peak cell death response.









Through single agent PAC studies I established that ALL cell lines were sensitive to this

agent. I chose to measure cell death as a result of 6 hours and 24 hours treatment based on earlier

pharmacokinetic studies by others (110, 135, 136). Following drug exposure, apoptosis was

measured every 24 hours for a total of 72 hours. Statistical analysis of my data showed that there

was a significant interaction between PAC concentration, time of exposure, and sample time in

Nalm-6 but not RCH-ACV (Tables 4-5 and 4-6). I chose to use a 6 hour exposure to PAC for my

combination studies, as this resulted in a better range of choices for drug concentrations to test

given the gradation in response between 10 nM and 100 nM when compared to 1.0 and 10 nM

after 24 hours exposure. Also, as detailed in Chapter 3, my data showed that 48-72 hours

incubation were required to achieve maximum cell death after treatment (Figure 3-2). Statistical

analysis of PAC single agent treatment may be reviewed in Tables 4-7 and 4-8.

Methods of Determining Synergy

I combined FP with PAC to determine if the combination would increase the efficacy of

the single agents. Synergy can be defined as when the effect of a combination of agents is greater

than the sum of the effects of each of the single agents (137). There are various methods to

determine if the effect of treatment with multiple agents is synergistic, including isobologram

analysis, fractional effect, and median-effect analysis (138). Isobologram analysis begins by

measuring the dose of each drug required to produce the same effect, e.g. 50% cell death. These

doses are plotted against each other and a line is drawn connecting the two doses (Figure 4-3)

(139). The line is said to represent the doses of the two drugs which are equipotent. If a dose

combination produces the designated effect and is plotted far below the line, this combination is

considered synergistic, e.g. point Q in Figure 4-3. If the drug combination is plotted far above the

line, it is considered to be antagonistic (point R). Points very close to the line represent additivity

(point P). The isolobolgram method requires a large number of measurements, applies only if the









drugs have similar modes of action (mutually exclusive) and can only be used for combinations

of two drugs (138).

The fractional product method is very intuitive in that one simply multiplies the

percentages represented by the unaffected fraction (e.g. percent viable cells post-treatment) for

each single agent (13 8). If the combination of drugs results in a percentage that is equal to the

product of the two single agents, then the two agents are additive. The requirements of this

method are that the drugs must have different modes of action (mutually non-exclusive) and that

the dose-effect curves for the agents are hyperbolic.

The most common method in current literature used to evaluate for synergy between

agents is based on the median-effect principle authored by Chou and Talalay (61). One uses

median-effect analysis to determine a Combination Index (CI) value for each drug combination.

According to median effect analysis, if CI=1.0 this indicates an additive relationship between the

two drugs. If CI<1.0, synergy is present and if CI>1.0 antagonism is indicated. This method has

the advantage of allowing the researcher to evaluate a minimal number of drug concentrations

and determine the relationship between greater than two drugs if desired. In addition, one is not

limited to evaluating agents with only the same or different modes of action. Both mutually

exclusive and mutually non-exclusive agents can be analyzed.

To understand median-effect analysis, let us first examine the median effect principle. This

principle is based on the ICso for each individual agent. Consider statement 4-1:


+ + (4-1)
().( i)l ( i)l (Dml), (Dmz)

The term (fa)x is the fraction affected by drug (percent cell death after treatment with drug), ft~ is

the fraction unaffected by drug (percent viable cells), D is the dose of a single drug in the









combination and Dm is the ICso for that drug if it were used as a single agent. If drugs 1 and 2

were combined at their ICsos,


+ -0.5+0.5=1.0. (4-2)


Now consider two examples from PAC combined with FP in Nalm-6 at a ratio of 1:10. In the

first, 10 nM PAC was combined with 100 nM FP. The ICso for PAC was 28.95 nM based on the

single agent controls in the experiment and the ICso for FP was 279.1 nM. Thus, according to

statement 4-1:

(D)l (D)Z 10 100
+ = + 0.703 (4-3)
(Dmz), (Dmz) 28.95 279.1

In the second example, 15 nM PAC was combined with 150 nM FP with the same ICsos as in the

first example for a sum equaling 1.055. Comparatively, the sum in the first example is 30%

below 1.0, whereas the sum in the second example is 6% above 1.0. Through the use of the

median-effect principle, one can conclude that the combination in the first example is synergistic

and the second combination is additive, providing that both agents have the same mode of action.

From this information one understands how CI values based on 1.0 were derived. From

statement 1, median effect analysis defines CI as:

(D), (D)L
CI- + (4-4)
(Dx), (Dx),

D is the dose of each drug used in the combination and Dx is the dose of each single agent that

would be required to induce the same percentage of cell death caused by the drug combination.

In the case of two or more agents having different modes of action, Equation 4-4 is modified to:

(D), (D)Z (D), (D)2
CI= + + (4-5)
(Dx), (Dx), (Dx), (Dx),









FP Combined with PAC

Combination studies were initiated by first establishing a concentration ratio for PAC and

FP. Under median-effect analysis, it is suggested that agents are combined using a set drug

concentration ratio (137), in this case PAC:FP. Several ratios were tested in Nalm-6 including

1:5, 1:10, 1:12, and 1:15 (Figure 4-4). I found that FP enhanced the activity of PAC most

dramatically when the two agents were used in the ratios of 1:10 and 1:12. I performed median-

effect analysis to generate Combination Index (CI) values for the four ratios tested. A CI<1.0

indicated synergy, while CI>1.0 indicated antagonism and CI=1.0 indicated an additive

relationship between PAC and FP (61). Table 4-9 shows CI values at the 50% effective dose for

the drug combination (ED5o), ED75, and ED90 for each ratio. When evaluating combination data

using median-effect analysis, two sets of CI values are generated based on the modes of action of

the two drugs tested: mutually exclusive and mutually non-exclusive CI values. I chose to utilize

the mutually non-exclusive CI values, as PAC and FP have different modes of action. Though

the CI results were <1.0 when PAC was combined with FP at a ratio of 1:12, the actual cell death

measurements were more compelling when the two agents were combined at a ratio of 1:10 (see

Figure 4-4). This ratio also allowed for the use of lower concentrations of both agents.

Nalm-6 and RCH-ACV were tested using a ratio of 1:10. When testing Molt-4 and Jurkat,

I used a ratio of 1:20 to account for the fact that these cell lines were exquisitely sensitive to

PAC. This allowed for the use of a lower PAC concentration. Results from cell death

measurements shown in Chapter 3 indicated a significant difference in the cell death induced by

single agent controls when compared to each combination in all four cell lines tested (Figure 3-

3). A mixed model analysis similar to what was employed for FP and PAC single agent studies

was utilized to evaluate the overall significance of the results (Tables 4-10 and 4-1 1). Nalm-6

and RCH-ACV were evaluated under the same analysis, as these cell lines were treated with the









same concentration ratio (1:10). Molt-4 and Jurkat were subj ected to a separate analysis, as both

were treated with a drug concentration ratio of 1:20. It was found that there was a significant

interaction between the factors cell line, treatment (PAC, FP, or PAC+FP) and drug

concentration in all four cell lines tested. Based on the overall significance of the results, the cell

death induced by each single agent control was compared to its respective combination. The

results for which there were significant differences are reported in Tables 4-12 and 4-13.

Representative CI values for the ED5o, ED75, and ED90 for each cell line are reported in Table 4-

14. The data ranged from being slightly synergistic (Nalm-6 ED90 CI=0.939) to antagonistic

(Jurkat ED5o CI=1.59), with half of the drug combinations showing near additivity to slight

antagomism.

The CI values reported in this chapter represent those obtained based on PAC and FP

having different modes of action and clearly show that the degree of synergy between PAC and

FP is very slight where it is present. In Chapter 3 I show that FP enhances PAC activity and vise

versa by measuring the simple effects of the drugs. In order for this enhancement to be

considered synergistic via median-effect analysis, the differences between single agent treatment

and combination therapy would need to be several orders of magnitude higher than the data that I


obtained (61). This can be explained by the median-effect plot which graphs log (D) vs. log


and ultimately connects to the median-effect equation from which all of the above equations are

derived. Stated more simply, analysis of combined drug effects through median-effect analysis

requires log order differences between single agent and combination treatment. For example, if

instead of obtaining the data reported in Chapter 3 at a drug concentration ratio of 1:10, I had

found similar results using a ratio of 1:100 or 1:1000, my CI values would have been much lower

and therefore more synergistic.










Sequence Dependent Enhancement

Results reported previously by others show that enhancement of PAC activity by FP is

dependent on the sequence in which the agents are administered (56). In order to confirm this in

ALL cell lines, I chose the most promising treatment from my combination studies to determine

if PAC+FP, FP&PAC or concurrent exposure would result in the highest percentage of cell

death. As reported in Chapter 3, the percentage of cell death resulting from standard treatment

(PAC+FP) was compared to the reverse sequence and single agent controls. An ANOVA was

utilized to analyze the statistical significance of treatment sequence prior to comparing the

individual effects of treatment. A one-way analysis of variance was used for Nalm-6 and a

weighted one-way analysis was used for RCH-ACV (Tables 4-15 and 4-16) to make this

determination. The weighted analysis was used due to an inconsistent sample size for some of

the treatment conditions for this cell line. The term "one-way" connotes that the experiments

were categorized in one way: by treatment sequence instead of by treatment sequence and cell

line. Treatment sequence was a significant factor in the percentage of cell death resulting from

the various treatments tested. Statistics were not applied to the data from concurrent exposure

experiments, as these data were generated from one experiment. I confirmed that PAC+FP was

the most efficacious treatment sequence in Nalm-6 (Figure 3-4 a). The statistical analysis

comparing 20 nM PAC+200 nM FP to the reverse sequence and single agent controls can be

found in Table 4-17. I also confirmed the proper treatment sequence in RCH-ACV (Figure 3-5 a,

Table 4-18). I showed in Figure 3-4 b and Figure 3-5 b that concurrent exposure is not a feasible

option for this drug combination, as it resulted in less cell death than the sum of the two single

agents.









Drug Sensitivity in Human Serum

Due to the reported difference in binding by FP to human plasma proteins vs. proteins in

FBS and the resulting decrease in sensitivity in CLL cells, I compared the sensitivity of ALL cell

lines to FP in FBS and HS and found that there was not a substantial difference in sensitivity

between the two sera (Figure 2-5). PAC is also highly plasma protein bound and I report in

Chapter 3 that there is a 10-fold decrease in the sensitivity of ALL cell lines to PAC in the

presence of HS when compared to FBS (Figure 3-6). Statistical analysis showed that the type of

serum used had a significant impact on the results (p=0.0370; Table 4-19). Statistical

comparisons between the two types of sera at specific PAC concentrations were performed to

supplement the data shown in Chapter 3 (Table 4-20). I reported that the difference in sensitivity

between FBS and HS was not due to a significant difference in cell proliferation in the two sera.

The statistical analysis on which this conclusion was based is reported in Table 4-21.

Based on these results I wanted to confirm that the enhancement in PAC activity by FP

that I had observed in FBS could also be achieved in the presence of HS. I chose to use a

concentration ratio of 1:3 based on preliminary experiments using a variety of ratios (Figure 4-

5). This concentration ratio allowed for the use of higher concentrations of PAC to compensate

for lower sensitivity in HS. However, these concentrations are still substantially lower than the

plasma concentrations reported during clinical trials of PAC in children with leukemia or solid

tumors (16, 109). I found that FP enhances the efficacy of PAC in a manner similar to the

enhancement found in FB S using a ratio of 1:10 or 1:20. Statistical analysis showed that despite

a lack of significance in the interaction between treatment and drug concentration, there was an

enhancement of PAC activity by FP (Tables 4-22 and 4-23).









Placing Perspective on this Project

Potential Side Effects of Single Agent and Combination Therapy

As previously discussed in Chapters 2 and 3, severe tumor lysis syndrome (TLS) resulted

during the initial clinical studies of the currently used administration schedule for FP (83).

Though this was a clear sign of the efficacy of FP in CLL, the toxicity that resulted caused the

death of one patient enrolled in the study. Steps have since been taken to prevent TLS through

the use of prophylactic therapy prior to the administration of FP and monitoring of patients while

on therapy. An algorithm for monitoring for hyperkalemia has also been instituted as part of the

study (140). According to official monitoring criteria from the NCI, the level of hyperkalemia

that has resulted has been low in both inpatients and outpatients; however, pre-treatment,

potassium chelation therapy, and dialysis have still been required in some cases.

Concern might be raised about the potential side effects of combining PAC with FP. Any

dose limiting toxicities reported during trials of PAC in patients with leukemia have occurred at

concentrations in the micromolar range; much higher than the concentrations used in my

experiments. It should also be noted that these concentrations were achieved after a 24 hour

infusion. I am proposing a shorter infusion time for PAC in my combination studies.

Where Does FP Fit into the Treatment Scheme of ALL?

When a novel agent becomes available as a possible addition to the regimen used to treat

ALL, researchers and clinicians must determine for what stage of therapy the new agent is best

suited. Because childhood ALL has such a high cure rate, it is difficult to measure a significant

improvement as a result of the addition of a new agent to the initial stages of therapy. Some feel

that novel agents should replace current therapies with the goal of decreasing toxic side effects

rather than increasing the cure rate; particularly when the drug is a targeted agent that would be

used in a subgroup that already has a positive prognosis (141). While it might be somewhat










beyond the scope of this dissertation, considering whether FP would have a place in the

treatment of ALL is relevant to proposing a clinical trial. Some might question whether a pan-

CDK inhibitor has a place in an age of cancer drug discovery characterized by targeted therapies.

FP has been used with success in trials of CLL patients. Others have shown that the biological

mechanism behind the capability of FP to kill CLL cells is its ability to decrease the transcription

and protein expression levels of short-lived anti-apoptotic molecules such as Bcl-2 and Mcl-1

(100). These molecules were targeted based on the need of CLL cells, which are non-cycling, to

express them continuously in order to remain in a state of senescence.

While ALL is characterized by many types of chromosomal translocations and other

genetic aberrations, there is not one specific molecule that can be targeted across subgroups of

patients, such as the BCR-ABL tyrosine kinase produced by the 9;22 translocation that has made

imatinib mesylate (Gleevec) so successful in patients with chronic myelogenous leukemia. As

previously discussed in Chapters 2 and 3, more modern studies using microarray technology

have found significant differences in the expression of genes that regulate cell cycle, DNA repair

and apoptosis between the times of diagnosis and relapse in ALL; however, more work is

necessary to discern a clear pattern in gene expression that would reveal which aberrations lead

to relapse (18). Several studies remain that show that pl6 is altered at the gene and/or protein

expression level in up to 50% of ALL cases. Because FP can function in the pl6 pathway and

there is a lack of available targets that affect a comparable percentage of ALL patients, the fact

that FP is not a precisely targeted therapy should not be a hindrance to its possible use in ALL.

Future Directions

This work represents an initial study of the efficacy of FP as a single agent and combined

with PAC in ALL cell lines. It might be beneficial to expand these studies into patient samples,

in order to determine if the treatments would be efficacious in samples which are far less









removed from a patient than immortalized cell lines. I would also propose testing the biological

basis of the apoptosis induced by FP as a single agent by determining the activation of caspases

and downregulation of antiapoptotic molecules such as Bcl-2 and Mcl-1 as result of treatment.

Importantly, and perhaps in contrast to the work performed by others, I would only propose the

further biological studies after FP had been successfully tested in a patient population. The

studies of FP contained herein, perhaps with the addition of single agent studies in patient

samples, provide a biological justification for a clinical trial of FP in children with ALL. I have

shown that FP can induce apoptosis and cell cycle arrest, both mechanisms that inhibit

proliferation of cancer cells. If FP was found to be successful in treating children with ALL, then

further studies into its mechanism of action in vivo would be warranted. These studies could hold

the possibility of assisting researchers in discovery of new targets for more potent agents on the

horizon. Also, this information would provide a basis for using FP in combination with other

agents such as PAC.














A RMl-AGVI Cal Gumih in SSWall Plate
2.00E+06




1.0ECE O
I 00E*00



0 00E*00


B. REN CdlGrowh in 8HhIB Plate


2.00E+06
1.80E*M *
1.60E+06

1.20E*06
1.00E+05


4.00E+05
200E+05 .

01234
Dyes


, 1X10n3



I 1X10^5
ce&ssual


* 1X1*

celsluel


(irxioN


c- Abrsorbance ovrer TlrnelX10^4 cerlls/ell

2500
iR, + ROACV






01234
Days






Figure 4-1. Growth curves used to establish cell concentration for proliferation assays. Trypan
blue exclusion and WST-1 were utilized to measure the number of viable cells per
well in a 96-well plate over a 4 day time period. a)growth curves generated from
RCH-ACV; b)REH; c)growth curves generated using WST-1 for comparison to

trypan blue exclusion.




















82











Nalm.G

S100




120

0 50 100 19) 200 250
FP (n M)


RCHALVIC

S100






0 50 100 190 200 250
F P (n M)


Figure 4-2. Representative dose-response curves generated from cell proliferation assays.














so4 -







Dos6e A

Figure 4-3. Illustration of isobologram analysis of combined drug effects. Given the
combination of drugs A and B at equipotent concentrations, possible responses from
the drug mixture are shown. Point "Q" represents a synergistic effect, point "P" an
additive effect and point "R" represents antagonism Reprinted by permission from
American Society for Pharmacology and Experimental Therapeutics: [Journal of
Pharmacology and Experimental Therapeutics] Tallarida RJ. Drug synergism: its
detection and applications. Journal of Pharmacology and Experimental Therapeutics
2001; 298(3):865-872, copyright 2001, originally published as Figure 1, p.866.













Sequential Treatment wkth PaalItXel and Flavopiridel Sequential T reament with Paditaxel and FlavopirIdo
in Nalm M in Nalm4 1:10






PA (n .F FP( A n P(M


Figure 4-4. Preliminary combination data at a variety of ratios in Nalm-6. Cell death
measurements after treatment with PAC for 6 hours followed by FP for 72 hours at
concentration ratios (PAC:FP) of 1:5, 1:10, 1:12, and 1:15.



























Figure 4-5. Preliminary combination data at a variety of ratios in the presence of human serum.
Cell death measurements after treatment with PAC for 6 hours followed by FP for 72
hours at concentration ratios (PAC:FP) of 1:2, 1:3, 1:4, and 1:5. The 1:3 ratio was
chosen for further study.










Table 4-1. Mixed model analysis for FP treatment duration in Nalm-6
Numerator DF Denominator DF F value P value
FP Concentration 4 10 20.04 <0.0001
Day 2 20 25.93 <0.0001
Day*FP 8 20 26.25 <0.0001
concentration

Table 4-2. Mixed model analysis for FP treatment duration in RCH-ACV
Numerator DF Denominator DF F value P value
FP concentration 4 10 7.90 0.0038
Day 2 20 67.28 <0.0001
Day*FP 8 20 19.42 <0.0001
concentration

Table 4-3. Differences between treatment duration at a given FP concentration in Nalm-6
Sample time- FP Sample time- FP Difference in T value P value
concentration (nM) concentration mean cell death
(nM)
Day 1 FP 150 Day 2 FP 150 0.3033 0.26 0.7978
Day 1 FP 150 Day 3 FP 150 0.6033 0.52 0.6113
Day 1 FP 200 Day 2 FP 200 0.2533 0.22 0.8305
Day 1 FP 200 Day 3 FP 200 1.6367 1.40 0. 1766
Day 1 FP 300 Day 2 FP 300 -11.8500 -10.14 <0.0001
Day 1 FP 300 Day 3 FP 300 -18.5167 -15.85 <0.0001
Day 1 FP 50 Day 2 FP 50 0.4200 0.36 0.7230
Day 1 FP 50 Day 3 FP 50 0.8667 0.74 0.4669
Day 2 FP 150 Day 3 FP 150 0.3000 0.26 0.8000
Day 2 FP 200 Day 3 FP 200 1.3833 1.18 0.2503
Day 2 FP 300 Day 3 FP 300 6.6667 5.71 <0.0001
Day 2 FP 50 Day 3 FP 50 0.4467 0.38 0.7063

Table 4-4. Differences between treatment duration at a given FP concentration in RCH-ACV
Sample time-FP concentration (nM) Sample time- Difference T value P value
FP in mean cell
concentration death


(nM)
Day 2 FP 150
Day 3 FP 150
Day 2 FP 200
Day 2 FP 300
Day 3 FP 300
Day 2 FP 50
Day 3 FP 50
Day 3 FP 150
Day 3 FP 300
Day 3 FP 50


Day 1 FP 150
Day 1 FP 150
Day 1 FP 200
Day 1 FP 300
Day 1 FP 300
Day 1 FP 50
Day 1 FP 50
Day 2 FP 150
Day 2 FP 300
Day 2 FP 50


- 6.5633
-13.2733
-22.0367
-38.4000
-56.9200
0.8600
-1.7233
- 6.7100
-18.5200
- 2.5833


-1.64
- 3.32
- 5.51
- 9.61
-14.24
0.22
- 0.43
-1.68
- 4.63
- 0.65


0.1162
0.0034
<0.0001
<0.0001
<0.0001
0.8318
0.6709
0.1087
0.0002
0.5254























Table 4-6. Mixed model analysis of PAC single agent treatment in RCH-ACV
Numerator DF Denominator F value P value
DF
PAC concentration 3 14 108.48 <0.0001
Time of Exposure (6 1 14 7.91 0.0138
hours or 24 hours)
PAC*Time 3 14 5.90 0.0081
Sample Time (Day) 2 32 9.08 0.0008
Day*PAC 6 32 6.12 0.0002
Day*Time 2 32 0.39 0.6812
Day*PAC*Time 6 32 0.71 0.6454


Table 4-5. Mixed model analysis of PAC single agent treatment in Nalm-6
Numerator DF Denominator DF F value P value
PAC concentration 3 14 174.68 <0.0001
Time of exposure (6 1 14 27.77 0.0001


hours or 24 hours)`
PAC*Time
Sample time (Day)
Day*PAC
Day*Time
Day*PAC*Time


10.52
15.42
31.63
3.48
3.16


0.0007
<0.0001
<0.0001
0.0427
0.0150














Day 2
Day 2
Day 2
Day 2
Day 2
Day 2
Day 2
Day 2
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3


0.1876
0.0318
0.1835
0.7622
0.9098
<0.0001
<0.0001
<0.0001
0.0133
0.2114
0.0884
0.6296
0.0015
0.0433
0.0059
0.0125
0.1085
0.0871
<0.0001
0.8238
<0.0001
0.6047
<0.0001
0.6758


rs PAC treatment


Table 4-7. Differences in cell death based on incubation time after 6 or 24 hou
in Nalm-6
Day PAC (nM) Time (hours) Day P value


Difference in
mean cell death
0.4799
0.8074
0.7225
0.2860
- 0.1913
-36.8805
-52.0495
-54.6743
0.8677
0.3879
0.6504
- 0.1571
1.6311
0.9085
2.3689
2.0829
2.4893
2.6806
-34.7488
2.1317
-57.5053
- 5.4558
-59.4446
- 4.7703


Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2










Table 4-8. Differences in cell death based on incubation time after 6 or 24 hours PAC treatment


in RCH-ACV
PAC (nM)


Day


Time
(hours)
6
24
6
24
6
24
6
24
6
6
24
24
6
6
24
24
6
6
24
24
6
6
24
24


Day

Day 2
Day 2
Day 2
Day 2
Day 2
Day 2
Day 2
Day 2
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3
Day 3


P value

0.4185
0.8931
0.3051
0.0582
0.6557
0.3989
0.0065
0.0196
0.795
0.5811
0.9794
0.8727
0.4016
0.8488
0.436
0.0096
0.5508
0.8795
0.0812
0.3514
<0.0001
0.0265
0.0001
0.0724


Difference in mean cell
death
0.9311
- 0.1687
1.3073
1.7955
- 0.9633
- 3.9142
-14.7008
-16.2206
0.3189
- 0.6121
0.0317
0.2003
1.0914
- 0.2160
-1.0129
- 2.8084
-1.3161
- 0.3527
- 9.3543
- 5.4401
-37.3090
-22.6082
-37.2492
-21.0286


Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2


0
0
1
1
10
10
100
100
0
0
0
0
1
1
1
1
10
10
10
10
100
100
100
100


Table 4-9. Combination Index (CI) values for drug
Nalm-6 PAC:FP EDso El


combination studies using a variety of ratios
Dys ED90
65 1.80
08 0.939
885 0.715


1:5
1:10
1:12
1:15


1.53
1.25
1.14
1.39


1.1
1.(


1.26


1.16


Table 4-10. Mixed model analysis of Nalm-6 and RCH-ACV combination data
Numerator Denominator F value
DF DF


P value


Cell Line 1
Treatment 2
Cell Line*Treatment 2
Drug concentration 4
Treatment* Concentrati on 8
Cell Line*"Treatment* Concentrati on 9


20.83 0.0448
150.97 <0.0001
14.43 <0.0001
114.93 <0.0001
5.30 <0.0001
3.61 0.0012










Table 4-11i. Mixed model analysis of Molt-4 and Jurkat combination data
Numerator DF Denominator F value P value
DF


Cell line 1
Treatment 2
Cell line*Treatment 2
Concentration 4
Treatment* Concentrati on 8
Cell line*Treatment* Concentration 12


0.03
188.13
9.70
1002.86
36.00
2.32


0.8727
<0.0001
0.0002
<0.0001
<0.0001
0.0174


Table 4-12. Significant differences in treatment for a given cell line and drug concentration


Cell line


Combo treatment PAC
(nM):FP (nM)
10:100
10:100
15:150
15:150
20:200
20:200
25:250
25:250
30:300
30:300
10:100
15:150
15:150
20:200
20:200
25:250
25:250


Single agent
control
100 nM FP
10 nM PAC
150 nM FP
15 nM PAC
200 nM FP
20 nM PAC
250 nM FP
25 nM PAC
300 nM FP
30 nM PAC
100 nM FP
150 nM FP
15 nM PAC
200 nM FP
20 nM PAC
250 nM FP
25 nM PAC


SE T value P value


Nalm-6
Nalm-6
Nalm-6
Nalm-6
Nalm-6
Nalm-6
Nalm-6
Nalm-6
Nalm-6
Nalm-6
RCH-ACV
RCH-ACV
RCH-ACV
RCH-ACV
RCH-ACV
RCH-ACV
RCH-ACV


5.8083
5.8083
5.8083
5.8083
5.8083
5.8083
5.8083
5.8083
5.8083
5.8083
5.0301
5.0301
5.0301
5.0301
5.0301
5.0301
5.0301


2.93
2.24
7.27
6.08
8.49
8.41
7.52
8.24
3.97
6.86
3.43
4.86
3.67
4.57
3.36
3.63
5.45


0.0048
0.0286
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0002
<0.0001
0.0011
<0.0001
0.0005
<0.0001
0.0014
0.0006
<0.0001


Table 4-13. Significant differences in treatment for a given cell line and drug concentration
Cell Combo treatment PAC (nM):FP Single agent SE T P value
line (nM) control value


Jurkat
Jurkat
Jurkat
Jurkat
Jurkat
Jurkat
Molt-4
Molt-4
Molt-4
Molt-4
Molt-4


5:100
5:100
7.5:150
7.5:150
10:200
10:200
5:100
5:100
7.5:150
7.5:150
10:200


100 nM FP
5 nM PAC
150 nM FP
7.5 nM PAC
200 nM FP
10 nM PAC
100 nM FP
5 nM PAC
150 nM FP
7.5 nM PAC
10 nM PAC


3.6222
3.6222
3.6222
3.6222
3.6222
3.6222
3.6222
3.6222
3.6222
3.6222
3.6222


12.58
8.03
13.55
5.99
4.87
5.32
9.99
7.08
11.21
10.71
6.67


<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001











ED5o ED75 ED90
Nalm-6 1.25 1.08 0.939
RCH-ACV 1.18 1.15 1.15
Molt-4 1.58 1.34 1.14
Jurkat 1.59 1.33 1.11

Table 4-15. One-Way Analysis of Variance of treatment sequence in Nalm-6
Numerator DF Denominator DF F value P value
Treatment 6 14 46.29 <0.0001
Sequence

Table 4-16. Weighted One-Way Analysis of Variance of treatment sequence in RCH-ACV
Numerator DF Denominator DF F value P value
Treatment 6 14 16.86 <0.0001
Sequence

Table 4-17. Significant differences between standard treatment sequence, reverse treatment
sequence, and single agent controls in Nalm-6


Table 4-14.


Combination Index (CI) values for drug combination studies


Treatment 1 SE
mean-
Treatment 2
mean
-29.3867 5.6021


Treatment 1


Treatment 2


T value


P value


0+FP 200

0+PAC 20

FP
200+PAC
20
FP 200~00

PAC 20+FP
200
PAC 20+FP
200


PAC 20+FP
200
PAC 20+FP
200
PAC 20+FP
200

PAC 20+FP
200
PAC 20~00

untreated


-5.25

-7.86

-4.10


-4.18

12.61

13.22


0.0001

<0.0001

0.0011


0.0009

<0.0001

<0.0001


-44.0467

-22.9500


-23.4267

70.6333

74.0600


5.6021

5.6021


5.6021

5.6021

5.6021










Table 4-18. Significant differences between standard treatment sequence, reverse treatment


sequence, and single
Treatment 1 Treatment 2




0+FP 200 PAC 20+FP
200
0+PAC 20 PAC 20+FP
200
FP 20000 PAC 20+FP
200
FP PAC 20+FP
200+PAC 20 200
PAC2000 PAC20+FP
200
PAC 20+FP untreated
200


agent controls in RCH-ACV
Treatment 1 SE


T value




-3.02

-5.89

-4.30

-4.16

-7.89

8.50


P value




0.0092

<0.0001

0.0007

0.0010

<0.0001

<0.0001


mean-
Treatment 2
mean
-20.7433

-40.4933

-31.6603

-30.9227

-54.2467

58.4167


6.8754

6.8754

7.3598

7.4244

6.8754

6.8754


Table 4-19. Mixed model analysis for comparison of cell death induced by PAC in FBS vs. HS
Numerator DF Denominator DF F value P value
Type of serum 1 2 25.57 0.0370
PAC (nM) 4 14 62.75 <0.0001
Type*PAC 4 14 5.08 0.0097
Day 3 54 69.74 <0.0001
Day*Type 3 54 8.75 <0.0001
Day*PAC 12 54 18.85 <0.0001
Day*Type*PAC 12 54 1.69 0.0941







































Table 4-21. Mixed model analysis of cell viability FBS vs. HS
Numerator DF Denominator DF F value P value
Day 3 12 16.07 0.0002
Type of serum 1 2 1.09 0.4055
Day*Type 3 12 1.27 0.3294

Table 4-22. Mixed model analysis of combination studies in human serum


Table 4-20. Comparison of cell
Day Type of PAC
serum concentration
(nM)
0 FBS 0
0 FBS 1
0 FBS 10


death induced by PAC in FBS vs. HS
Day Type of PAC
serum concentration
(nM)
0 HS 0
0 HS 1
0 HS 10
0 HS 100
0 HS 1000
1 HS 0
1 HS 1
1 HS 10
1 HS 100
1 HS 1000
2 HS 0
2 HS 1
2 HS 10
2 HS 100
2 HS 1000
3 HS 0
3 HS 1
3 HS 10
3 HS 100
3 HS 1000


P value Difference
in mean cell


death
-1.49E-14
6.59E-15
-2.94E-14
-2.20E-14
1.69E-17
0.6095
1.076
4.455
12.94
11.24
1.029
1.540
3.416
42.72
35.55
-0.1275
-0.0537
2.605
42.38
27.57


1
1
1
1
1
0.5418
0.3828
0.0363
0.0002
0.0235
0.2391
0.2649
0.0578
<0.0001
0.0223
0.9267
0.9722
0.2053
<0.0001
0.1712


FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS
FBS


100
1000
0
1
10
100
1000
0
1
10
100
1000
0
1
10
100
1000


Numerator DF Denominator F value


P value

<0.0001
0.0059
0.3349


Treatment
Drug concentration
Treatment* Concentrati on


151.97
12.12
1.25









Table 4-23. Significant differences in treatment for combination studies in human serum
Treatment 1 Treatment 2 P value Difference in mean
cell death
300 nM FP 100 nM PAC, 300 nM 0.0112 -24.4877
FP
100 nM PAC, 300 nM 100 nM PAC <0.0001 37.8793
FP
375 nM FP 125 nM PAC, 375 nM 0.061 -32.0078
FP
125 nM PAC, 375 nM 125 nM PAC <0.0001 63.9835
FP
450 nM 150 nM PAC, 450 nM 0.4802 -16.7949
FP
150 nM PAC, 450 nM PAC 150 <0.0001 78.2333
FP
600 nM FP 200 nM PAC, 0.8746 4.2426
600 nM FP
200 nM PAC 200 nM PAC, 600 nM <0.0001 23.6096
FP









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