Defining the Mechanism of the Tyrosine Kinase Inhibitor Pazopanib in Acute Myeloid Leukemia

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Defining the Mechanism of the Tyrosine Kinase Inhibitor Pazopanib in Acute Myeloid Leukemia
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Trujillo, Angelica
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Master's ( M.S.)
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University of Florida
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Medical Sciences, Molecular Genetics and Microbiology
Committee Chair:
Cogle, Christopher Ramin
Committee Members:
Rowe, Thomas C
Siemann, Dietmar W
Kraft, John

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aml -- inhibitor -- kinase -- leukemia -- tyrosine
Molecular Genetics and Microbiology -- Dissertations, Academic -- UF
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Acute myeloid leukemia (AML) arises from malignant transformation of hematopoietic stem cells (HSCs) and is difficult to treat with conventional chemotherapy due to high rate of refractory disease.Previously, we discovered a close developmental relationship between adult HSCs and blood vessels. In addition, mounting evidence indicates that HSCs can beextrinsically controlled by endothelial cells (ECs). Therefore, we hypothesizedthat (a) ECs provide sanctuary for AML cells from chemotherapy and that (b) targetinginteractions between ECs and AML cells leads to disease regression. We foundthat ECs protect AML cells from cytarabine chemotherapy by secretion of growth factors such as VEGF, PDGF, SCF, Angiopoietin 1 and 2, MCP-1 and IL-8. We alsofound that human AML cells express classically defined angiogenic receptors (e.g., VEGFRs, PDGFRs and c-Kit) and that activation of these receptors induces signaling through the Src pathway. Treatment of AML cells with the multipletyrosine kinase inhibitor pazopanib led to an enhanced additive cytotoxicity inleukemia cells and abrogated the protective effect of ECs. In a xenograft model of human AML, pazopanib mobilized AML cells into systemic circulation, decreased leukemia-associated angiogenesis in the bone marrow and significantly regressed AML engraftment. Given these results and the typical clinical presentation of initially high AML myeloblast burden, a time-sequential regimen of cytarabine-pazopanib-cytarabine was tested and showed significant leukemia regression. Together, these results support clinical studies of pazopanib in the treatment of patients with AML.
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by Angelica Trujillo.
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Thesis (M.S.)--University of Florida, 2012.
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Adviser: Cogle, Christopher Ramin.
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1 DEFINING THE MECHANI SM OF THE TYROSINE K INASE INHIBITOR PAZO PANIB IN ACUTE MYELOID LEU KEMIA By ANGELICA TRUJILLO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 A ngelica T rujillo

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3 To my family whose encouragement and positive energy have always been my rock

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4 ACKNOWLEDGMENTS I would like to thank Dr. Christopher Cogle, an exceptional and inspiring mentor who helped me develop to my ful l est potential and always foster ed and environment of scientifi c and personal growth. With gratitude, I would like to acknowledge my committee members, Dr. Thomas Rowe and Dr. Dietmar Sie man n for all their time and effort to help me complete my research goals. I would also like to thank m y wonderful lab mat es Amy, Liz, Leylah and Raphael. It has been a great experience to work with all of you. My most sincere thanks to Christie McGee for all her hard work and contributions to the project. I would like to thank my parents for teaching me to always work hard t o accomplish my goals, their constant love and support drives me. I would also like to recognize my sister for encouraging me to follow my dreams. To all my family whose love and positive energy reach me from the distance. To my great friends who have en couraged me along the way

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 Acute Myeloid Leukemia ................................ ................................ ......................... 11 Endothelial Cells in Support of Leukemia ................................ ............................... 12 Pazopanib: Targeting Tyrosine Kinases in AML ................................ ..................... 14 2 MATERIALS AND METHODS ................................ ................................ ................ 20 Human AML Cell Lines ................................ ................................ ........................... 20 Therapeutic Agents ................................ ................................ ................................ 20 Co Culture Studies ................................ ................................ ................................ 21 Cell Viability ................................ ................................ ................................ ............ 21 Apoptosis ................................ ................................ ................................ ................ 22 Human Angiogenes is Antibody Array ................................ ................................ ..... 22 cDNA and PCR ................................ ................................ ................................ ....... 23 Western Blot ................................ ................................ ................................ ........... 23 Mouse Xenograf t Model ................................ ................................ .......................... 24 Tissue Analysis ................................ ................................ ................................ ....... 25 Statistical Analysis ................................ ................................ ................................ .. 25 3 RESU LTS ................................ ................................ ................................ ............... 26 Endothelial Cells Protect Leukemia Cells from Chemotherapy ............................... 26 Angiogenic Cytokines are Up Regulated after Chemotherapy ................................ 27 Pazopanib Enhances Ara C Activity In Vivo and In Vitro ................................ ........ 27 Combination Therapy Overcomes the Endothelial Protective Effect ....................... 29 Pazopanib Activity is Dependent on Receptor Expression in Leukemia Cell Lines ................................ ................................ ................................ .................... 30 Pazopanib Downregulates c Kit Phosphorylation in KG1 Cells .............................. 31 Pazopanib Downregulates the Src/STAT Pathway, but has no Effect on ERK or AKT ................................ ................................ ................................ ..................... 31 4 DISCUSSION ................................ ................................ ................................ ......... 44

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6 LIST OF REFERE NCES ................................ ................................ ............................... 49 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 52

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7 LIST OF FIGURES Figure page 1 1 Endothelial Cells Protect Acute Myeloid Leukemia Cells from Chemotherapy. .. 17 1 2 Multiple Mechanisms of Angiogenic Pathways Regulate Acute Myeloid Leukemia Survival and Proliferation. ................................ ................................ .. 18 1 3 Pazopanib Chemical Structure ................................ ................................ ........... 19 1 4 Mechanism of Action for Tyrosine Kinase Inhibitors ................................ ........... 19 3 1 Endothelial Cells Protect Leukemia Cells from Chemotherapy Induced Apoptosis W hen Seeded in Direct Co Culture ................................ .................... 33 3 2 Endothelial Cells Protect Leukemia Cells from Chemotherapy In duced Apoptosis When Seeded in I ndirect Co Culture. ................................ ................ 3 4 3 3 Angiogenic Cytokines are Up Regulated After Chemotherapy ........................... 35 3 4 In Vitro Flow Cytometry Apoptosis Analysis Shows an Additive Effect of Treatment with Pazopanib and Cytarabine. ................................ ........................ 36 3 5 In Vivo Flow Cytometry Apoptosis Analysis Shows an Additive Effect of Treatment with Pazopanib and Cytarabine. ................................ ........................ 37 3 6 Pazopanib Treatment Mobilizes L eukemia Cells In Vivo Over Time .................. 38 3 7 Pazopanib Treatment Disrupts Microvessel Formation in Vivo. ......................... 39 3 8 Combination Treatment Overcomes th e Endothelial Protective Effect ............... 40 3 9 KG1 and HL60 Have Different Tyrosine Kinase Receptor Expression Profiles. 41 3 10 KG 1 Cell Proliferation is Inhibited at a Lower Dose of Pazopanib than HL 60 cells. ................................ ................................ ................................ ................... 41 3 11 Pazopanib Inhibits Tyrosine Autophosphorylation of c Kit in KG1 Cells ............ 42 3 12 Pazopanib Treatment Downregulates Activation of the Src/STAT Pathway. ...... 42 3 13 Pazopanib Treatment Has no Effect on ERK and AKT. ................................ ...... 43 3 14 Pazopanib Induces Cleavage of PARP and a Dose Dependent Downregul ation of Antiapoptotic Proteins ................................ ........................... 43 4 1 Bone M arrow from 12 C onsecutive AML P atients S howed that 33% of P atients had M alignant M yeloblasts that E xpressed VEGFR 1 .......................... 48

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8 LIST OF ABBREVIATION S AKT P ROTEIN K INASE B AML A CUTE M YELOID L EUKEMIA A RA C C YTARABINE A RABINOSYLCYTOSINE C KIT S TEM C ELL F ACTOR R ECEPTOR ERK E XTRACELLULAR S IGNAL R EGULATED K INASES GFP HL60 G REEN F LUORESCENT P ROTEIN TAGGED H UMAN P ROMYELOCYTIC L EUKEMIA C ELLS HL 60 H UMAN P ROMYELOCYTIC L EUKEMIA C ELLS HUVEC H UMAN U MBILICAL V EIN E NDOTHELIAL C ELL KG1 A CUTE M YELOGENOUS L EUKEMIA C ELL L INE D ERIVED BY K OEFFER AND G OLDE MCL 1 I NDUCED M YELOID L EUKEMIA C ELL D IFFERENTIATION P ROTEIN MECA 32 M OUSE E NDOTHELIAL C ELL A NTIGEN PARP P OLY (ADP RIBOSE ) P OLYMERASE PDGF P LATELET D ERIVED G ROWTH F ACTOR PDGFR P LATELET D ERIVED G ROWTH F ACTOR R ECEPTOR SCF S TEM C ELL F ACTOR SRC P ROTO O NCOGENE SHORT FOR S ARCOMA STAT 3 S IGNAL T RANSDUCERS AND A CTIVATORS OF T RANSCRIPTION 3 STAT 5 S IGNAL T RANSDUCERS AND A CTIVATORS OF T RANSCRIPTION 5 VEGF V ASCULAR E NDOTHELIAL G ROWTH F ACTOR VEGFR V ASCULAR E NDOTHELIAL G ROWTH F ACTOR R ECEPTOR XIAP X LINKED I NHIBITOR OF A POPTOSIS P ROTEIN

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9 A bstract of T hesis P resented to the G raduate S chool of the U niversity of F lorida in P artial F ulfillment of the R equirements for the D egree of M aster of S cience DEFINING THE MECHANI SM OF THE TYROSINE K INASE INHIBITOR PAZOPANIB IN ACUTE MYELOID LEU KEMIA By A ngelica T rujillo August 2012 Chair: Christopher R. Cogle Major: M edical Sciences Translational Biotechnology Acute myeloid leukemia (AML) arises from malignant transformation of hematopoietic stem cells (HSCs) and is difficult to treat with conventional chemotherapy due to high rate of refractory disease. Previously, we discovered a close developmental relationsh ip between adult HSCs and blood vessels. In addition, mounting evidence indicates that HSCs can be extrinsically controlled by endothelial cells (ECs). Therefore, we hypothesized that (a) ECs provide sanctuary for AML cells from chemotherapy and that (b) t argeting interactions between ECs and AML cells leads to disease regression. We found that ECs protect AML cells from cytarabine chemotherapy by secretion of growth factors such as VEGF, PDGF, SCF, Angiopoietin 1 and 2, MCP 1 and IL 8. We also found that h uman AML cells express classically defined angiogenic receptors (e.g., VEGFRs, PDGFRs and c Kit) and that activation of these receptors induces signaling through the Src pathway. Treatment of AML cells with the multiple tyrosine kinase inhibitor pazopanib led to an enhanced additive cytotoxicity in leukemia cells and abrogated the protective effect of ECs. In a xenograft model of human AML, pazopanib mobilized AML cells into systemic circulation, decreased leukemia associated

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10 angiogenesis in the bone marrow and significantly regressed AML engraftment. Given these results and the typical clinical presentation of initially high AML myeloblast burden, a time sequential regimen of cytarabine pazopanib cytarabine was tested and showed significant leukemia regress ion. Together, these results support clinical studies of pazopanib in the treatment of patients with AML.

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11 CHAPTER 1 INTRODUCTION While standard chemotherapy induces initial disease remission in many patients with acute myeloid leukemia (AML), most patients die of relapsed or refractory disease. In patients over 60 years old, conventional chemotherapy (such as cytarabine) bring about five year survival prognosis of only 20% 40% Refractory disease is a major challenge in AML and a better understandin g of resistance mechanisms and sanctuary sites are needed. Little information is available regarding the leukemia microenvironment and how it support s leukemogenesis and refractory disease. Prior reports indicate that up regulated microvessel density in pa tients with AML portend s for a worse prognosis Moreover, preliminary reports show that leukemia cells express recep tors for angiogenic cytokines [ 1 ] Finally, angiogenic factors within the leukemia microenvironment are strong indicators of disease relapse and early mortality [ 2 ] Together, these preliminary data indicate that extrinsic control of the leukemia microenvironment may prov ide a reservoir of resistant/refractory leukemia cells. Furthermore, given the new availability of angio inhibitory tyrosine kinase inhibitors (TKIs) initially developed fo r the treatment of solid tumors, these agents may also serve as therapeutic agents i n the treatment of patients with AML. Acute Myeloid Leukemia Acute myeloid leukemia (AML) is a cancer of the bone marrow. It is a result of a mutation in the hematopoietic stem or progenitor cell (HSPC), which leads to high proliferation and accumulation o f immature myeloid cells. The presence of these abnormal cells in the bone marrow results in abnormal peripheral blood counts (anemia, thrombocytopenia, leukocytosis due to the high number of my eloblasts and/or

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12 neutropenia) [ 3 ] Approximately 70% of young AML patients (< 65 years of age) and 30% of older AML patients (> 65 years of age) will achieve initial remission after treatment with standard induction chemotherapy such as seven days of cytarabine and three days of an anthracycline (e.g., idaraubicin, dau norubicin) Unfortunately, refractory and relapse disease remain a major challenge, especially in older patients. The 5 year survival prognosis for AML patients is only 20% 40% almost exclusively due to disease relapse [ 4 5 ] Despite a plethora of investigation in leukemia cell genetics, the mechanisms of relapse are elusive and still undefined. Disease relapse is thought to be driven by leukemi c myeloblasts insensitive to conventional chemotherapy. These drug insensitive properties may be related to the genetic mutations within the leukemia cell. However, clinical studies of sensitizing agents such as cyclosporine, have been disappointing [ 6 ] An alternative explanation is that sanctuary sites protect leukemia cells and promote their return. Given the importance of vascular networks in conferring resist ant solid tumors, the vascular niche within the bone mar row and leukemia cell response to angiogenic growth factors may also be important mechanisms in explaining the high rate of refractory AML By defining the mechanisms; we also highlight potential ther apeutic targets. Endothelial Cells in Support of Leukemia The study of leukemia mainly focuses on the leukemia cell itself. However, there is increasing evidence showing the importance of the bone marrow microenvironment in regulating hematopoiesis, making it extremely important to broaden the scope of investigation beyond the leukemia cell. The importance of the cancer microenvironment has been widely studied in solid tumors. Cancer cells interact with the stromal microenvironment in complex ways to

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13 promot e their own survival and proliferation [ 7 8 ] The bone marrow m icroenvironment is a very complex and not completely understood niche, but recent studies have been able to show the importance of the vascular niche in promoting leukemia cell proliferation and survival. Fiedler et al. [ 9 ] found an increased expression of vascular endothelial growth factor (VEGF), as well as it receptors VEGFR1 and VEGFR2, in AML patients. Furthermore, VEGF induced human umbilical endothelial cells (HUVECs) to secrete GM CSF, a known mitogen f or AML cells. These results suggest that AML cells are able to exploit angiogenic signaling for autocrine stimulation and are able to provoke endothelial cells to secrete preleukemic factors for survival and proliferation. Moreover, recent clinical evidence indicates that leukemia cells depend on angiogenesis in the bone marrow Studies by Hussong et al. [ 10 ] found an increased microvessel density in the bone marrow of untreated AML patients compared to a treated control group (p < 0.001). More importantly other studies have shown a strong correlation between increased bone marrow microvessel density and overa ll survival in AML patients [ 11 12 ] Furthermore blood vessel AML interactions may be important contributing factors in the development of refractory disease. Endothelial cells support adhesion and transmigration of subset s of normal CD34+ HSPCs. In vitro studies have shown that AML cells maintained in co culture with HUVECs, exhibit higher proliferation rates and are less susceptible to traditional chemotherape utic agents such as cytarabine [ 13 ] Our own studies support these findings; human acute promyeloc ytic cells (HL60) se eded in co culture with HUVECs appear to be less sensitive to cytarabine compared to HL60 alone (Figure 1 1) Cytarabine treatmen t of AML cells resulted in a significantly lower cell viability as

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14 compared to AML cells grown in culture with HUVECs ( 78.58 % vs. 127.99 %, P < 0.005 ) AML cells in co culture with HUVECs showed no decrease in viability ( 100 % vs. 126.83 %, P < 0.01 ). Taken together, these data provide compelling evidence to support the notion that endothelial cells protect leukemia and might provide a site of leukemia re initiation after chemotherapy. In a complementary manner some studies suggest that leukemia cells promo te endothelial cell survival. Hatfield et al [ 14 ] used transwell and direct contact assays using primary AML cells a nd dermal microvascular endothelial cells (DMEVCs) to show that e ndothelial cells cultured with AML blasts exhibited a higher proliferation rate o ver endothelial cells alone This co dependence is likely driven in part by secreted cytokines between leukemi a and endothelial cells. Some of these cytokines include, for example, GM CSF, CXCL8, and IL 6. Kruizinga et al. [ 15 ] evaluated the expression levels of several VEGF isoforms in AML patient samples. Various VEGF isoforms were present in the samples. Particularly, the VEGF 165 and VEGF 189 isoforms stimulated endothelial proliferation and angiogenesis. However, there was no clinical correlation between levels of VEGF expression and overall survival or relapse free survival. A cyclical positive feedback loop mechanism exists in which the endothelial cells promote the leukemia cell population and vice versa This interdepend ent system strongly favors the potential for endothelial cells to extrinsically control refractor y and relapsed AML (Figure 1 2 ). Pazopanib: Targeting Tyrosine Kinases in AML Angiogenesis is the formation of new blood vessels from pre existing ones. This p rocess is activated through a cascade of events initiated through the phosphorylation of the tyrosine kinase receptors such as vascular endothelial growth factor receptor

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15 (VEGFR), platelet derived growth factor receptor (PDGFR) and stem cell factor (SCF) receptor cKIT amongst others. Receptor tyrosine kinases (RTKs) are a family of transmembrane proteins which have an extracellular ligand binding domain as well as an intracellular kinase domain These kinases become phosphorylated when bound to their sp ecific ligands Orally administered, m ultitargeted tyrosine kinase inhibitors may be a n effective and convenient treatment option, that could help overcome redundancies in signaling pathways and effectively inhibit cancer cell growth [ 16 ] Pazopanib is an indazolylpyrimidine (Fig ure 1 3 ) that potently inhibits VE GFR1, 2 and 3, as well as PDGFR alpha and beta and cKIT [ 17 ] by competing with adenosine triphosphate (ATP) binding (Fig ure 1 4) In 2009 the FDA approved, p azopanib use for patients with renal cell carcinoma (RCC), after the drug showed promising results in a randomized, mu lticenter, phase III clinical trial In brief, adult patients with locally advanced and/or metastatic RCC were randomly assigned, in a 2:1 ratio, to receive pazopanib or placebo, with progression free survival being the primary end point. A total of 435 pa tients were enrolled in the study, 290 of them received pazopanib. Progression free survival was significantly enhanced with pazopanib treatment compared to placebo, 9.2 vs. 4.2 months [ 18 ] Pazopanib is used as an 800mg once daily tablet. Based on the potency of pazopanib in inhibiting intratumoral angiogenesis and in conjunction with leukemia associated reports of angiogenesis being an important microenvironment factor as well as angiogenic growth factor receptors expressed directly on leukemia cells, we hypothesized that a multi targeted tyrosine kinase inhibitor such as pazopan ib would regress leukemia. If true, pazopanib could be used

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16 for treatment of patients with AML and would represent a novel class of agents in this patient population.

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17 Figure 1 1. Endothelial Cells P rotect Acute Myeloid Leukemia Cells from C h emotherapy. Human acute promyelocytic leukemia cells (HL60) were culture d in two conditions: over plastic and in the presence of human umbilical vein endothelial cells (HUVECs). The leukemia cells were then exposed to cytarabine chemotherapy, which is comm only administered to patients with AML. Cell proliferation was subsequently measured by XTT assay. HL60 cells in co culture with HUVECs showed no decrease in cell proliferation after chemotherapy exposure (NS) as compared to HL60 cells cultured over plasti c (P<0.05).

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18 Figure 1 2 Multiple Mechanisms of Angiogenic Pathways Regulate Acute Myeloid Leukemia Survival and Proliferation. Acute myeloid leukemia cells exploit angiogenic mechanisms by (1) inducing angiogenesis directly, (2) expressing receptors for specific angiogenic growth factors (paracrine regulation), and (3) secreting their own angiogenic growth factor receptors (autocrine stimulation). Thus, angiogenesis has both c ell extrinsic and cell intrinsic significance in leukemia. Stem cell factor (SCF), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) are a few of many yet to be defined angiogenic factors that regulate leukemia cell survi val and proliferation.

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19 Figure 1 3 Pazopanib Chemical Structure Pazopanib is an indazolylpyrimidine, multi tyrosine kinase inhibitor It is bioavailable as an 800mg tablet, currently used for the treatment of renal cell carcinoma. Figure 1 4 Mechanism of Action for Tyrosine Kinase Inhibitors. Pazopanib acts by competitively binding the ATP active si te on several tyrosine kinases such as VEGFR1 VEGFR2 cKIT, PDGFR and PDGFR

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20 CHAPTER 2 MATERIALS AND METHOD S Human AML Cell Lines Releva nt AML cell lines were used for in vitro studies. Specifically, HL 60 and KG 1 cell lines were purchased from the American Tissue Culture Collection (Manassas, VA) and incubated with 5% CO 2 in air at 37 C. GFP tagged HL60 cells were used as well. HL 60 and KG 1 cell lines were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Invitrogen) supplemented with 20% heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals) and 1% pe nicillin/streptomycin (Cellgro) H uman Umbilical Vein Endothe lial Cells (HUVEC) were obtained from ATCC as well. The cell line was cultured in Endothelial Growth Medium 2 (EGM 2) (Lonza) supplemented with EGM 2 Bulletkit (Lonza) growth factors. Therapeutic Agents Pazopanib (Votrient) w as provided by GlaxoSmithKline Inc For in vitro experiments, pazopanib was dissolved in a 10 mM stock solution of dimethyl sulfoxide (DMSO) (Sigma Aldrich). Further dilution to indicated concentrations was done with DMSO. Cytarabine (Ara C) (Sigma Aldrich) was dissolved in a 10 mM sto ck solution to indicated concentrations with DMSO. For in vivo experiments, pazopanib was suspended in 0.5% hydroxypropylmethyl cellulose (HPMC) (Colorcon) and 0.1% Tween 80 in water as a vehicle (pH 1.3 1.5) and given daily by oral gavage. Cytarabine was given intraperitonea l Buffered Solution as a vehicle.

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21 Co Culture S tudies For direct contact co culture studies in a 96 well format, HUVEC cells were seeded at 2 x 10^ 4 cells per well in a 100uL of culture media and allowed t o att ach overnight. HUVEC cells were used in initial experiments as proof of principle, as well as their reliability and uniformity in cell culture propagation. Only early passage HUVECs were used for co culture experiments. Subsequently KG1 cells were see ded on direct contact at 1 x 10^ 5 cells per well in a 100uL of culture media. Like wise, for a 24 well format, HUVEC cells were seeded at 5 x 10^ 4 cells per well in a 1m L of culture media (EGM 2) and allowed to attach overnight. The next day, EGM 2 was aspirated and the KG1 cells were seeded on direct contact at 5 x 10^ 4 cells per well in 1mL of culture media. After treatment with cytarabine (AraC) pazopani b or a combination of both at indicated time and dose, assays were perfor med to determine cell viability or apoptosis. For transwell co culture studies HUVEC cells were seeded at 5 x 10^ 4 cells per well in a 1m L of culture media (EGM 2) and allowed to attach overnight. KG1 cells were seeded in transwell inserts the following d ay at 5 x 10^ 4 cells per well in 100uL of culture media. Cell viability was assed using flow cytometry, staining for PI and Annexin V (Biorad). Cell Viability Cell proliferation was measured using an XTT colorimetric assay (ATCC). Cells were seeded on a 96 well plate at 1 x 10^5 cells per well in 100 uL culture media. After a 24 hour incubation with treatment at indicated doses, 50 uL of XTT reagent was added to each well and plates were incubated at 37C for 3 hours. Plates were analyzed at a wavelength of 475 nm using an Infinite M200 Pro reader (Tecan). Pri s m

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22 (GraphPad) was used to calculate IC 50 values. This same procedure was followed for cells seeded in co culture as previously described. The XTT assay was used to quantify cell growth in the drug sensi tivity experiments [ 19 ] In brief, an absorbance microplate reader is used to measure the change in color that results when a tetrazodium dye is reduced to a soluble, highly pigmen ted, derivative by a mix of cellular effectors. Particularly, this assay uses PMS (N methyl dibenzopyrazine methyl sulfate) as an intermediate electron carrier which helps drive the formation of the XTT derivative. Apoptosis To measure apoptosis, HL 60 or KG1 cells were seeded at 2x10 ^ 5 cells/ml in culture medium and incubated with pazopanib and cytarabine for 48 hours at the concentrations indicated. Briefly, cells were collected and stained using propidium iodide (PI) and annexin V APC (BD Pharminogen ) according to the manufacturer's protocol. Analysis was performed using a Becton Dickinson FACSCanto II flow cytometer. The appearance of phosphatidylserine (PS) residues on the cell surface is considere d an early sign of apoptosis [ 20 ] These residues can be detected and bind the fluorescently labeled anticoagulant molecule Annexin V. Furthermore, as the apoptotic process progresses cell membrane integrity is loss. Propidium Iodide (PI) is a DNA specific viability dye. Apoptosis can t hen be determined using a cytometer to quantify the number of cells bound to either of the markers. This same procedure was followed for cells seeded in co culture as previously described. Human A ngiogenesis Antibody A rray Media supernatants were assayed f or the presence of angiogenic cytokines after stimulating the cells with chemotherapy The assay was performed using the Proteome

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23 Profiler Array from R&D Systems. This array has been previously validated [ 21 ] HUVEC and KG1 cells were seeded in co culture using a transwell system as previously described. Media was collected after 48 hours of treatment with increasing concentrations of cytarabine Briefly, the membrane containing immobilized angiogenesi s related antibodies was blocked with bovine serum albumin for 1 h on a rocking platform at room temperature. Membrane was then incubated with media supernatants along with Detection Antibody Cocktail overnight at 2C to 8C on a rocking platform. The memb rane was incubated with streptavidin horseradish peroxidase conjugate followed by chemiluminescent detection reagent. The membrane was scanned and pixel density was presented by quan tifying the mean spot densities. Density quantification was done using Ima ge J software. cDNA and PCR RNA isolation from KG 1 and HL 60 cell lines was performed following the RNeasy Plus Mini Kit protocol (Qiagen Sciences) and analyzed with a SmartSpec Plus Spectrophotometer (BioRad). Reverse transcription for cDNA was done following the High Capacity cDNa Reverse Transcription Kit protocol (Applied Biosciences). PCR was done using human primers to VEGFR 1, VEGFR 2, SCFR, PDGFR and PDGFR attained from Invitrogen Oligos and New England BioLabs reagents. Analysis was done following agarose gel electrophoresis procedure. Western Blot Cells were treated with various concentrations of pazopanib with or without the presence of recombinant human SCF or VEGF (R&D Systems). Cell extracts were isolated using RIPA buffer. F or phosphorylation studies, samples were first

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24 immunoprecipitated using protein A/G Agarose (Santa Cruz) in the presence of primary antibody against human SCFR and then separated on a 4 20% gel. Samples were then transferred onto a nitrocellulose membrane. The immunoprecipitated samples were probed with human anti pTYR antibody (Santa Cruz) and human anti SCF receptor antibody (Santa Cruz). For apoptosis studies whole cell lysates were separated on a 4 20% gel and subsequently blotted onto a nitrocellulose membrane. The membranes were incubated with primary antibodies against human PARP (Santa Cruz) MCL 1(Santa Cruz), XIAP ( Santa Cruz) and B actin (Santa Cruz). Horseradish peroxidase (HRP) marked immunoglobulins from Santa Cruz were used as secondary antib odies. Antibody binding was detected using chemiluminescence reagent (ECL Western Blotting Analysis System, Amersham). Furthermore in trying to elucidate a mechanism of action for the drug protein lysates were immunoblotted for total and phosphorylated Src (S anta Cruz) phosphorylated STAT3 (Cell Signaling) and phosphorylated STAT5 (Cell Signaling). Membranes were also blotted for total and phosphorylated ERK (Santa Cruz) and total and phosphorylated AKT (Cell Signaling). Mouse Xenograft Model All animal s tudies were performed according to approved protocols from the University of Florida IACUC. To test the efficacy of pazopanib and cytarabine in systemic AML mouse models, human leukemia chimeras were established using NOD/scid/ IL2R / (NSG) mice (Jackson Laboratories, Bar Harbor, Maine). 6 week old NSG mice were inoculated with 1x10 6 GFP HL 60 cells via tail vein injection. We allowed for four weeks of engraftment. The mice were divided into five groups of 10 mice each : p azopanib, Ara C, p azopanib + Ara C, v ehicle, and u ntreated. Treatments

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25 were 500mg/kg of body weight Ara C (IP) on day 1 and day 2, 100mg/kg of body weight p azopanib (oral gavage) daily for 8 days, and 100 uL of HPMC (vehicle control). Tissue Analysis After the last treatment, the liver, ki dney, spleen, lungs and femurs were harvested from all mice for the purpose of investigation. Engraftment was determined using flow cytometry staining for human CD45 and HLA (BD), as well as via probe to determine cell viability. CD 45, also known as the le ukocyte common antigen, is a highly specific marker for h ematopoietic cells lines. This marker, together with human specific anti HLA, was used in order to label the human leukemia cells present in the mouse bone marrow. Femurs were section ed and stained for MECA 32 (BD) for the assessment of blood vessel formation. Blood was collected from mice cheek pouch before the treatment, one week after treatment and at the end of treatment in order to assess the presence of leukemic blasts in circulat ion. The sample was analyz ed using flow cytometry by quantifying the percent of GFP tagged cells present in the sample Statistical Analysis test. P values of < 0.05 were considered stat istically significant.

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26 CHAPTER 3 RESULTS Endothelia l Cells Protect Leukemia Cells from C hemotherapy In previous studies I demonstrated that endothelial cells protect leukemia cells through direct contact In brief, HL60 cells were seeded in a 96 well plate alone or in direct contact with a HUVEC monolayer. The cells were in co culture for 24 hours and subsequently treated with cytarabine (1uM) overnight. Cell viability was assessed using an XTT viability assay (ATCC) as described above. Absorbance changes as cellular effectors break down the XTT and generate a color change in the media. Leukemia cells in co culture with endothelial cells appear to be less susceptible to cytarabine treatment as depicted in Figure 1 1 where there is no statistically significant difference in the level absorption from the samples treated with cytarabine and the untreated control ( 127.99 % vs. 126.83 %, P >0.5 ) We wanted to further study this idea to assess if direct contact was necessary for the protective effect to remain. Briefly, we co cultured leukemia and endothelial cells in direct co culture or a transwell system and assayed the leukemia cell population for apoptosis markers using flow cytometry analysis. There was a sign ificant difference in cell apoptotic status as low as 2uM Ara C concentration in both experimental settings L eukemia cells seeded in direct co culture were 49.2% viable over 17.1% for leukemia cells alone (P <0.0001 ) (Fig ure 3 1 ) .In the transwell system l eukemia cells in co culture were 77.40% viable over 44.13% for leukemia cells alone (P <0.0001 ) (Fig ure 3 2 ). This result leads us to believe that there is a paracrine effect generated from endothelial cells an exerted on leukemia cells

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27 Angiogenic Cytokines are Up R egulated after Chemotherapy With transwell experiments showing paracrine regulation, next w e used a human angiogenesis array kit to study the effect of chemotherapy treatment on endothelial cells and define secreted factors A sandwich E LISA technique was used ( R&D Systems Human Angiogenesis Array Kit ) and quantification of growth factors was accomplished by comparing mean pixel density in relationship to the negative and positive controls. There was a slight increase in the levels of VEG F and PDGF when the leukemia cells were in co culture with the HUVECs Furthermore w e found significant up regulation in the expression levels of relevant angiogenic cytokines such as VEGF, PDGF, IL 8 and MCP 1 in the co culture samples that had been trea ted with chemotherapy (Fig ure 3 3 ) Since cytarabine chemotherapy is the cornerstone standard treatment for all leukemia patients, our finding of increased angiogenic growth factors uncovers a mechanism of how ECs support AML cells during and after chemotherapy. Moreover, these specific angiogenic growth factors represent potential targets for AML therapy. Chemotherapy is still an important first line therapy to debulk the typically large leukemia burdens on patient presentation to the clinic; however, tyrosine kinase inhibitors such as pazopanib could play an important role in the treatment of leukemia and enhance the effects of conventional chemotherapy by blocking t he reactive angiogenesis signaling generated by activated ECs in the leukemia microenvironment. Pazopanib E nhances Ara C Activity In Vivo and In V itro Next, we wanted to study the effects of combining pazopanib with standard treatment to see what if any a dditive effects could be seen. In vitro there was a significant additive effect when cells were treated with both drugs. The percentage of

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28 apoptotic cells in the sample increased from 3 4 % in AraC alone treatment versus 66 % in the combined treatment (P <0.00 05 ) (Fig ure 3 4 ). For in vivo assays we transplanted immunocompromised NSG mice with HL60 cells U nfortunately the animals developed very rapid disease and many died prior to completion of our planned two weeks of pazopanib treatment. Evaluable animals sho wed leukemia regression in the pazopanib alone ( 1 4 % vs. 3 4 %, P <0.05 ) and combination treatment cohorts compared to untreated controls ( 11.4 % vs. 34 %, P <0.05 ) (Fig ure 3 5 ). Next, we questioned whether targeting angiogenic growth factors in leukemia would cause leukemia cells to seek refuge outside of the vascular niche and mobilize into the systemic circulation. Through serial blood monitoring for human leukemia cells in our xenograft model of human AML, w e found a trend t oward an increase in mobilization in the animals treated with pazopanib compared to the other treatment groups ( 0.5 % vs. 0.0 25 %, P =.0 618 ) We found no significant trend in mobilization in animals that were treated with cytarabine alone (0.06% vs. 0.025%, P =.3622) (Fig ure 3 6 ). These results could have a high impact since we hypothesize that leukemia cells leaving the protective vascular niche within the bone marrow could force leukemia cells into a less protective environment (peripheral circulation) that r enders the malignant cells more sensitive to systemic cytotoxic chemotherapy treatment. Pazopanib was initially developed as an angio inhibitory agent. Therefore, we were also interested in changes in microvessel density within the leukemia microenvironme nt the bone marrow. Femurs from mice transplanted with GFP HL60 cells and subsequently treated with pazopanib, were fixed and sectioned for staining

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29 with Meca 32 in order to assess blood vessel formation in the d ifferent treatment groups (Fig ure 3 7 ). Fe murs corresponding to animals treated with pazopanib showed a statistically significant decrease in bone marrow MVD in samples collected from animals who received any other treatment indicating that pazopanib had an effect on angiogenesis ( 18 .0 mean vessel density vs. 36.8 mean vessel density P<0.05 ) Even though there was a decrease in the number of bone marrow MVD in the combination therapy (pazopanib + cytarabine), the dr ugs were given in conju n ction. While pazopanib inh i bits angiogenesis, e xposing the cells to cytarabine up regulates angiogenic cytokines creating a feedback mechanism. Together, our findings support the regimen of applying the in vivo treatment in a sequential form (cytarabine first followed by pazopanib) rather than both dr ugs simultaneously. Furthermore, when considering the mobilization effect of pazopanib, our results also support following pazopanib with cytarabine thereby backing up a sequential sandwich treatment protocol of cytarabine pazopanib cytarabine. Combinat ion Therapy Overcomes the Endothelial Protective Effect Next we wanted to study the effects of combination therapy in the co culture system. Leukemia cells were seeded in co culture with the endothelial cells, as previously described and treated with pazop anib, chemotherapy or a combination of both The combination treatment was given in sequence, cytarabine on day one and pazopanib on day two. An XTT assay was used to assess cell proliferation. After 24 hours of treatment with either drug alone the leukemi a cells seeded in co culture with the endothelial cells had a higher proliferation rate than the cells seeded alone. In contrast, when the treatment was given in combination leukemia cells alone and in co culture where sensitive to the treatmen t and their proliferation rate was highly repressed

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30 ( 1.8258 % vs. 108.29 %, P <0.0001) (F ig ure 3 8 ). These results support our theory that using a tyrosine kinase inhibitor following chemotherapy could have important effects in vivo. Pazopanib Activity is Dependent on R ecep tor Expression in Leukemia Cell L ines As all leukemia clones are not alike, we were also interested in phenotyp i ng human AML cells for expression of growth factor receptors responsive to classically defined angiogenic cytokines. PCR analysis was used t o study the levels of receptor expression for VEGFR1, 2, c Kit and PDGFR alpha, beta in KG1 and HL60 cell lines. HL60 cells were positive for the expression of c Kit but none of the other receptors. On the other hand, KG1 cells were positive for the express ion of VEG FR1, 2, c Kit and ure 3 9 ). Based on these findings we assessed the difference in potency and selectivity associated with the different recept ors, as described by the drug manufacturer. It has the highest affinity for VEGFR 1 and VEGFR 2, exhibiting an IC 50 of 10 and 30nM respectively. Furthermore, it has an IC 50 of 71nM for PDGFR and 74nM for c KIT. It has the least affinity for PDGFR with an IC 50 of 84nM. Therefore, w e expected pazopanib to be the most potent for KG1 cells since they express most of the receptors targeted by the drug In contrast, we expected HL60 to be the least sensitive to pazopanib since they only express c Kit, and the drug exhibits a low affinity for this receptor. Briefly the cells were plated in 96 well plates and treated with varying concentrations of pazopanib and XTT assay was used to determine cell viability after 24 hours of treatment. As expected KG1 cells were the most sensitive yielding an IC50 of 0.2525uM versu s 27.71uM for HL60 cells (Fig ure 3 10 ). The HL60 cells might be somewhat resistant to low doses of pazopanib due to the differences in receptor

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31 selectivity mentioned above. This might explain the KG1 cel l line IC50 curve showing a consistent dose response as the drug concentration increases, while the HL60 curve does not show a response until the drug reaches higher concentrations. Pazopanib D ownregula tes c Kit Phosphorylation in KG1 C ells Recent studies on angiogenesis mechanisms in leukemia or solid tumors have focused on VEGFR 1 and 2 as necessary factors for anti angiogenic therapies to work. Since pazopanib had a regressive effect on HL60 cells even though they only express the c Kit receptor, we decided to study this mechanism further. Protein lysates from KG1 cells were collected and immunoprecipitated for SCF receptor, c Kit The IP lysates were run in a western blot and analyzed for receptor phosphorylation as well as total receptor. Receptor p hosphorylation was inhibited with increasing concentrations of pazopanib while t otal receptor expre ssion remained unchanged (Fig ure 3 11 ). Pazopanib D ownregulates the Src/STAT Pathway, but has no E ffect on ERK or AKT To study the downstream effects of SCFR (cKit) phosphorylation inhibition we decided to analyze the expression of Src after treatment with multiple doses of pazopanib. Src phosphorylation was inhibited in a dose dependent matter, while the total expressi on of Src did not change (Fig ure 3 12 ). The involvement of Src can have important implications. Studies suggest that Src plays a role in modulating the sensitivity of cancer cells to chemotherapy, even though the exact mechanism remains unknown. Due to the involvement of Src in the regulat ion of cell proliferation and survival, some have proposed growth arr est might be induced through Src signaling and could be a possible explanation for its role in chemotherapy resistance [ 22 ] By si gnaling growth factor receptors on the surface of AML cells and subsequently triggering Src signaling,

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32 endothelial cells could drive the leukemia cells to a G0 state and make them insensitive to chemotherapy. It would be necessary to conduct a cell cycle a nalysis to determine the validity of this idea. To study downstream effects of Src inhibition, we evaluated members of the STAT (Signal Transducers and Activators of T rans cription) family of proteins [ 23 ] Our interest in STAT proteins was also supported by prior reports indicating that Src kinases might promote AML survival through act ivation of the STAT proteins [ 24 ] Also, STAT 3 activity in AML has been associated with a poor prognosis which could be the result of increased resistance to chemotherapy [ 25 ] Using W estern blot to determine the state of STAT 3 and STAT 5. p hosphorylation we found significant inhibition of STAT 3 and STAT 5 after pazopanib exposure in a dose de pendent manner (Fig ure 3 12 ). We also studied the effects of pazopanib on ERK and AKT as these kinases are acti vated d ownstream of Src but there was no significant dif ference in phosphorylation levels (Fig ure 3 13 ) Redell et al [ 26 ] found similar results when looking at the effects of blocking STAT 3 phosphorylation in AML cell lines. We also analyzed these samples for an induced cleavage of PARP and downregulation of XIAP and MCL1 antiapoptotic proteins (Fig ure 3 1 4 ). Together, our results show that p azopanib l eads to the inhibition of the Src /STAT pathway which leads to decreased leukemia cell proliferation and apoptosis. Downregulation of Src/STAT also explains the enhanced susceptibility of AML cells when exposed to a combination of chemotherapy and pazopanib.

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33 Figure 3 1 Endothelial Cells Protect Leukemia Cells from Chemotherapy Induced Apoptosis When Seeded in Direct Co Culture. KG1 cells were seeded in direct co culture with endothelial cells and treated with varying concentrations of cytarabine. After 48 h ours of treatment the cells were collected and stained with Annexin V and PI antibodies. Apoptosis levels were determined using flow cytometry analysis. The leukemia cell line is more resistant to cytarabine treatment when it is in co culture with endothelial cells. (p<0.001)

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34 Figure 3 2 Endothelial Cells Protect Leukemia Cells from Chemotherapy Induced Apoptosis When Seeded in I ndirect Co Culture. KG1 cells were seeded in indirect co culture with endothelial cells and treated with varying concentrations of cytarabine. After 48 hours of treatment the cells were collected and stained with Annexin V and PI antibodies. Apoptosis levels w ere determined u sing flow cytometry analysis. The leukemia cell line is more resistant to cytarabine treatment when it is in co culture with endothelial cells, even though they are not in direct contact. (p<0.001)

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35 Figure 3 3 Angiogenic Cytokines are Up Regulated Afte r C hemotherapy. Leukemia cells where seeded in indirect co culture with e ndothelial cells. Media superna tants were collected and the presence of angiogenic cytokines quantified. After chemotherapy treatment the presence of cytokines such as PDGF AA, IL 8, VEGF and MCP 1 was increased.

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36 Figure 3 4 In V itro Flow Cytometry Apoptosis A na lysis Shows an Additive Effect of T reatment wi th Pazopanib and Cytarabine. Untreated HL 60 cells show less than 10% apoptosis. 80 uM pazopanib shows approximately 20% apoptosis of HL 60 cells. Cytarabine alone at 20 uM shows approximately 3 4 % cell apoptosis. In combinati on, pazopanib and c ytarabine yield approximately 66 % HL 60 cell apoptosis. p values <0.05 for all treatment samples, compared to untreated control.

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37 Figure 3 5 In Vivo Flow Cytometry Apoptosis A na lysis Shows an Additive Effect of T reatment wi th Pazopanib and Cytarabine. Pazopanib and cytarabine decreased the level of HL 60 engraftment. Decrease in engraftment for all treatment groups, determined by flow cytometry analysis. HL 60 engraftment for untreated mice was approximately 34%. HL 60 engraftments for treated mice were all between 12 18%. p values <0.05 for all treatment samples, compared to untreated control.

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38 Figure 3 6 Pazopanib Treatment Mobilizes Leukemia Cells In Vivo Over Time. Cell mobilization was assayed by quantif y ing GFP HL60 cells present in the peripheral blood collected at various time poi nts over the course of the treatment There is increased mobilization in the animals treated with pazopanib compared to the other treatment groups.

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39 A) B) Figure 3 7 Pazopanib Treatment Disrupts Microvessel Formation in Vivo. A) Micrograph of MECA 32 stain of mice femur section after treatment B) Pazopanib treatment leads to a reduction in the number of blood microvessels within leukemic cores compared to other treatment groups. Quantification of microvessels based on MECA 32 + Values represen t mean SEM. P < .05.

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40 Figure 3 8 Combination T reatment Overcomes the Endothelial Protective E ffect. Leukemia cells (KG1) were seeded in direct co culture with HUVECs and allowed to attach for 24 hours. The next day treatment was started Pazopanib alone (40uM), Cytarabine (20uM) or a combination of the two. XTT assay was used to assess cell proliferation after treatment. Cells seeded in the co culture system did not respond to treatment with either drug alone, but proliferation was inhibit ed when the cells were treated with a combination of the drugs. (P <0.0001)

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41 Figure 3 9 KG1 and HL60 Have Different Tyrosine Kinase Receptor Expression Profiles. Cell receptor expression was studied using PCR analysis. Neither HL 60 nor KG 1 cell lines express PDGFR c Kit (stem cell factor receptor). B KG 1 cells express VEGFR 1, VEGFR 2 whereas HL 60 cells express neither of the two receptors. These PCR studies show the pre sence of target receptors on AML cells for the purposes of interpreting susceptibility results with pazopanib. Figure 3 10. KG 1 Cell Proliferation is Inhibited at a Lower Dose of P azopanib than HL 60 cells Using escalating doses of pazopanib, XTT cel lular proliferation study analyses revealed an IC50 of 27.71 uM pazopanib for HL 60 cells and an IC50 of 0.2525 uM pazopanib for KG 1 cells. Considering that KG 1 cells express a greater number of target receptors (VEGFR1, VEGFR2, c Kit ) than HL 60 cells (c Kit), this suggests that expression of more target receptors on AML cells may result in increased sensitivity to multi targeted receptor tyrosine kinase inhibitors such as pazopanib.

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42 Figure 3 11 Pazopanib Inhibits Tyrosine A utophosphorylation of c Kit in KG1 C ells Cells were treated with different concentrations of pazopanib for 2 hours followed by stimulation with 100ng/mL human recombinant SCF. Whole cell lysates were immunoprecipitated for total c Kit and probed for pTYR or c Kit respectiv ely. Figure 3 12. Pazopanib Treatment Do wnregulates Activation of the Src /STAT Pathway. KG1 cells were treated with varying concentrations of pazopanib for 4 hours and subsequently stimulated with VEGF (10ng/mL) to activate downstream signaling cascade. Kinase phosphorylation state was assessed using western blot analysis. Membrane was incubated with primary antibodies to pSrc, Src pSTAT 3, pSTAT 5 and actin. Treatment with pazopanib inhibited phosphorylation of Src an d STAT kinases in a dose depende n t manner. actin is shown as a loading control.

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43 Figure 3 13. Pazopanib Treatment Has no Effect on ERK and AKT. KG1 cells were treated with varying concentrations of pazopanib overnight and subsequently stimulated with VEGF (10ng/mL) to activate downstream signaling cascade. Kinase phosphorylation state was assessed using western blot analysis. Membrane was incubated with antibodies to pERK, ERK, pAKT, and AKT. actin is shown as a loading control F igure 3 14 Pazopanib Induces C leavage of PARP and a Dose Dependent Downregulation of Antiapoptotic P roteins. Whole cell lysates were analyzed after 2 hour incubation of KG 1 cells with various concentrations of pazopanib as indicated actin is shown as a loading control.

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44 CHAPT ER 4 DISCUSSION One of the greatest challenges in treating AML is refractory and relapsing disease. Even though conventional chemotherapy is able to reduce the disease burden initially the majority of patients will die from a residual subpopulation of leu kemia cells which return Given the importance of vascular networks in extrinsically controlling relapsed cancer and the close developmental relationship between ECs and HSPCs as we have demonstrated previously, we hypothesized that e ndothelial cells serve as a sanctuary site for leukemia cells by protecting these cells from chemotherapy. Our studies showed that ECs secrete a panoply of growth factors VEGF PDGF SCF IL 8 and MCP 1, which although classically defined as angiogenic cytokines also serve to p romote leukemia cell viability and proliferation. Moreover, we found that certain human AML clones express receptors for these growth factors and that these growth factors protect AML cells from cytarabine chemotherapy. In a survey of 12 consecutive AML pa tients, Fig ure 4 1 [ 27 ] ). The refore, as we approach patient s with AML, careful attention should be made EC phenotype. In addition, the activation of ECs by cytarabine, providing a reactive growth response that protects AML cells, is a new finding and is an indictment on our current clinical practice. Our studies showed that endothelial cells protect leukemia cells from chemotherapy, in the transwell system supporting paracrine regulation as a mechanism of prov iding sanctuary Interestingly, this effect was further enhanced when we treated the leukemia cells with chemotherapy, which supports the notion that when the

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45 leukemia cells are in distress they are able to survive fostered by their relationship with the endothelial cells. Next we investigated the possibility of using a multi targe ted tyrosine kinase inhibitor as a therapy for AML. Pazopanib is an orally bioavailable, adenosine triphosphate competitive tyrosine kinase inhibitor, approved for use in renal c ell carcinoma. Pazopanib was able to directly impair AML cell viability in vitro and regress established disease in vivo In order to understand the mechanism of action for pazopanib, we investigated SRC si nce this kinase is activated th r o ugh pazopanib ta rgets ( c K it, VEGFRs, PDGFRs and c Kit) and once phosphorylated initiates a cascade of events leading to cell survival, proliferation, ang iogenesis and migration. By inhibiting c Kit pazopanib downregulates SRC phosphorylation. Downstream from Src STAT 3 and STAT 5 are also inactivated and explains the reduced cell survival and increased apoptosis seen in treated leukemia cells We studied the effects of pazopanib in two different AML cell lines. Since they only express one of the target receptors c KIT, HL60 were least susceptible to the drug compared to the KG1 cell line which express VEGFR1 and 2, c KIT and PDGFR Understanding the receptor profile among human AML patients is tantamount to developing vascular targeting agents such as pazaponib. T his lack of attention to AML phenotype may explain the modest improvement seen in recently reported TKI clinical trials to dat e [ 2 ] For a targeted therapy to be success ful the cancer cells should at least express the specific drug targets. This is made even more complicated when we acknowledge that patients with cancer harbor multiple malignant clones throughout the

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46 natural history of their disease [ 28 30 ] Future treatments might require a combination of targeted therapies to have more impactful results. We analyzed the effects of pazopanib treatment in combination with standard chemotherapy as this would be the most likely translation into the clinic. We found that pazopanib has an additive effe ct when given with cytarabine M ost importantly combination treatment was able to overcome the protective effect that existed with the endothelial ce lls. Interestingly pazopanib was able to mobilize the l eukemia cells probably as a result of the drugs effect on the feedback mechanism existing between the leukemia cells and endothelial cells in the microenvironment. In addition, pazopanib disrupted micr ovessel formation in the bone marrow, which points to a second effect on the angiogenic process within the leukemia microenvironment The results of this study serve as the basis for a phase I/ II clinical trial of pazopa nib in patients with AML. We propose to use cytarabine as initial treatment to reduce the disease burden, followed immediately by pazopanib to directly impair potentially resistant AML EC hybrid clones and mobilize the cells harbored in to the peripheral circulation and finally followed by a redosing of cytarabine to eradicate susceptible leukemia cells in circulation. Conclusions and Future Directions Endothelial cells in the bone marrow microenvironment have a protective effect on leukemia cells. They secrete angiogenic cytokines that ac tivate growth factor receptors on leukemia cells which stimulate t he Src /STAT pathway leading to increased cell survival. Treatment with chemotherapy activates ECs, inducing them to secrete large quantities of angiogenic cytokines. Taken together, these f indings implicate ECs as a sanctuary site of AML cells and show that leukemia clones can adopt a hybrid AML EC phenotype responsive to angiogenic cues.

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47 This mechanism could explain the high rates of relapse disease seen in AML patients. Novel therapies are needed to inhibit the effects of this protecti ve mechanism. Pazopanib is a mu l t i targeted tyrosine kinase inhibitor that has a two fold effect in AML: (a) directly regresses AML in vitro and in vivo and (b) decreases leukemia associated angiogenesis Towards clinical translation, pazopanib has an additive therapeutic effect in hu man AML and c o uld be used in the clinic with standard chemotherapy. Ongoing studies include an investigation of KG 1 cells, which show high expression of many receptors targete d by pazopanib. We expect to find significantly enhanced regression because of these additional receptor targets. If this is true, then this result would further underscore our claim that accurate phenotyping of AML clones is needed to identify patients at high risk for relapse and eligible for phase II clinical trial of pazopanib treatment. Additionally, o ur data suggest that combined therapies could have an important impact in the clinic Future studies will focus on combining multiple targeted therapies. For example the VEGF antibody b evacizumab (Avastin) has shown some clinical activity in various solid tumors [ 31 32 ] but its effect could be enhanced if used in conjunction with other therapies Future studies will investigate the effects of combining pazopanib to block receptor phosphorylation and bevaci zumab to block the ligand (above and below receptor starvation) Finally, our results will be strengthened by testing primary human AML cells.

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48 Fig ure 4 1. Bone M arrow from 12 C onsecutive AML P atients S howed that 33% of P atients had M alignant M yeloblasts that E xpressed VEGFR 1

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49 LIST OF REFERENCES 1. G.J. Madlambayan, e.a., Leukemia regression by vascular disruption and antiangiogenic therapy. Blood, 2010. 116 (9): p. 8. 2. A. Trujillo, C.M., CR. Cogle, Angiogenesis in acute myeloid leukemia and opportunities for novel therapies. Journal of Oncology, 2012. 3. E. J. Jabbour, E.E., and H. M. Kantarjian, Adult acute myeloid leukemia. Mayo Clinic Proceedings, 2006. 81 (2): p. 13. 4. B. Lowenberg, J.R.D., and A. Burnett, Acute Myeloid Leukemia. The New England Journal of Medicine, 1999. 341 (14): p. 11. 5. F. R. Appelbaum, H.G., D. R. Head et al., Age and acute myeloid leukemia. Blood, 2006. 107 (9): p. 4. 6. A.F. List, K.J.K., C.L. William, et al., Benefit of cyclosporine modulation of drug resistance in patients with poor risk acute myeloid leukemia: a Southwest Oncology Group study. Blood, 2001. 98 (12): p. 8. 7. S. M. Garrido, F.R.A., C. L. Willman, and D. E. Banker, Acute myeloid leukemia cells are protect ed from spontaneous and drug induced apoptosis by direct contact with a human bone marrow stromal cell line (HS 5). Experimental Haematology, 2001. 29 : p. 9. 8. M. B. Meads, L.A.H., and W. S. Dalton, The bone marrow microenvironment as a tumor sanctuary a nd contributor to drug resistance. Clinical Cancer Research, 2008. 14 (9). 9. W. Fiedler, e.a., Vascular endothelial growth factor, a possible paracrine growth factor in human acute myeloid leukemia. Blood, 1997. 89 (6): p. 5. 10. J.W. Hussong, G.M.R., P.J Shami, Evidence of increased angiogenesis in patients with acute myeloid leukemia. Blood, 2000. 95 : p. 4. 11. T. Ting Fang. Shih, e.a., Bone marrow angiogenesis magnetic resonance imaging in patients with acute myeloid leukemia: peak enhancement ratio i s an independent predictor for overall survival. Blood, 2009. 14 : p. 6. 12. T. Padro, R.M.M., et al, Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia. Blood, 2000. 95 : p. 7. 13. J.L. Liesveld, e.a., Acute myelogenous leukemia microenvironment interactions: role of endothelial cells and proteasome inhibition. Hematology, 2005. 10 (6): p. 11.

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50 14. K. Hatfield, e.a., Primary human acute myeloid leukemia cells increase the proliferation of microvascular endothelial cells thr ough the release of soluble mediators. British Journal of Haematology, 2009. 144 (1): p. 15. 15. R.C. Kruizinga, H.J.d.J., K.R. Kampen, A.M. Walenkamp, E.S. de Bont, Vascular Endothelial Growth Factor A Isoform mRNA expression in pediatric acute myeloid le ukemia. Pediatric Blood Cancer, 2011. 56 (2): p. 4. 16. Hutson, G.S.a.T.E., Pazopanib: A novel multitargeted tyrosine kinase inhibitor. Current Oncology Reports, 2007. 9 : p. 4. 17. K. Rakesh, V.B.K., et al., Pharmacokinetic pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity. Molecular Cancer Therapeutics, 2007. 6 : p. 9. 18. C. N. Sternberg, I.D.D., J. Mardiak, et al., Pazopanib in locally advanced or met astatic renal cell carcinoma: Results of a randomized phase III trial. Journal of Clinical Oncology, 2010. 28 (6): p. 7. 19. D.A. Scudiero, e.a., Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research, 1988. 48 : p. 4827. 20. M. Engeland, e.a., Annexin V affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry, 1998. 31 : p. 1. 21. T. Kanda, A.R., et al., MBP 1 inhibits breast cancer growth and metastasis in immunocompetent mice. Cancer Research, 2009. 69 (24): p. 5. 22. Gallick, D.P.L.a.G.E., Src family kinases as regulators of angiogenesis: Therapeutic implications. Current Cancer Therapy Reviews, 2005. 1 : p. 5. 23. Y. Okutani, e.a., Src directly tyrosine phosphorylates STAT5 on its activation site an is involved in erythropoietin induced signaling pathway. Oncogene, 2001. 20 24. Y. Ozawa, e.a., Src family kinases promote AML survival through activation of signal transducers and activators of transcription (STAT). Leukemia Research, 2008. 32 25. M. Benekli, Z.X., et al., Constitutive activity of signal transducer and activator of transcription 3 protein in acute myeloid leukemia blasts is associated with s hort disease free survival. Blood, 2002. 99 (1): p. 5.

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51 26. M. S. Redell, M.J.R., et al., Stat3 signaling in acute myeloid leukemia: ligand dependent and independent activation and induction of apoptosis by a novel small molecule Stat3 inhibitor. Blood, 20 11. 117 : p. 8. 27. S. Bais, E.W., E. M. Harris, et al., AML depends on VEGFR 1 and not VEGFR 2 for proliferation and protection from chemotherapy. In review. 28. L. Ding, T.J.L., D.E. Larson, et al., Clonal evolution in relapsed acute myeloid leukemia re vealed by whole genome sequencing. Nature, 2012. 481 (7382): p. 4. 29. J. Zhang, L.D., L. Holmfeldt, et al., The genetic basis of early T cell precursor acute lymphoblastic leukemia. Nature, 2012. 481 (7380): p. 6. 30. M.J. Walter, D.S., L. Ding, et al., C lonal architecture of secondary acute myeloid leukemia. New England Journal of Medicine, 2012. 366 (12): p. 8. 31. J. Karp, e.a., Targeting vascular endothelial growth factor for relapsed and refractory adult acute myelogenous leukemias: therapy with sequential 1 D arabinofuranosylcytosine, mitoxantrone, and bevacizumab. Clinical Cancer Research, 2004. 10 (11): p. 8. 32. R.M. Mesters, e.a., Bevacizumab reduces VEGF expression in patients with relapsed and refractory acute myeloid leukemia without cli nical antileukemic activity. Leukemia, 2007. 21 (6): p. 2.

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52 BIOGRAPHICAL SKETCH Angelica Trujillo was born in Barranquilla, Colombia. In 200 2 she moved to the United States after finishing high school in Colombia and was admitted into the Biotechnology Program at Santa Fe Community College. She graduated with honors and completed an Associate of Science in b iotechnology. Subsequently she was accepted into the University of Florida where she co mpleted her Bachelor of Science in m icrobiology and c ell s cience. As an undergraduate student Angelica worked as a researcher studying the effects of Focal Adhesion Kinase inhibition in Neuroblastoma. This research led to the publication of several a rticles. After finishing her bachelor s Angelica was hired as an assistant scientist at RTI Biologics, in Alachua, Florida. RTI provides sterile biological implants for various surgeries, by preparing donated human tissue and bovine tissue for transplantat ion. At RTI Angelica worked in the research and development department, completing the initial investigations for a stem cell based therapy product. In 2010 Angelica was accepted into the m aster concentrating in t ranslational b iotechnology That same year she joined the lab of Dr. Christopher Cogle and has focused her research efforts in defining the mechanism of action for a tyrosine kinase inhibitor in a cute myeloid leukemia.