Structure-Based Anti-Cancer Drug Development by Targeting Bcl-XL

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Title:
Structure-Based Anti-Cancer Drug Development by Targeting Bcl-XL
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1 online resource (96 p.)
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english
Creator:
Park, Dong-Kyoo
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences, Molecular Cell Biology (IDP)
Committee Chair:
Deng, Xingming
Committee Members:
Ishov, Alexander M
Liao, Daiqing
Narayan, Satya
Bloom, Linda B

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Subjects / Keywords:
anti-cancer -- apoptosis -- bcl-xl -- drug-development -- lung-cancer
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
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Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Bcl-XL is a major anti-apoptotic protein in the Bcl2 family whose overexpression is more widely observed in human lung cancer cells than that of Bcl2, suggesting that Bcl-XL is more biologically relevant and therefore a better therapeutic target for lung cancer.  Here, we screened small molecules that selectively target the BH3 domain (aa 90-LREAGDEFE-98) in binding pocket of Bcl-XL using the UCSF DOCK 6.1 suite of programs and the NCI chemical library database.  We identified two new Bcl-XL inhibitors (BXI-61 and BXI-72) that exhibit selective toxicity against lung cancer cells compared with immortalized normal human bronchial epithelial cells (BEAS-2B and HBEC-3KT).  Fluorescence polarization assay reveals that BXI-61 and BXI-72 preferentially bind to Bcl-XL protein but not Bcl2 in vitro with high binding affinities.  Treatment of lung cancer cells with BXI-72 results in disruption of Bcl-XL/Bak interaction, oligomerization of Bak and cytochrome c release from mitochondrial membrane.  These two BXI compounds potently repress lung cancer xenografts in vivo via apoptosis without significant normal tissue toxicity within effective does (i.e. 20~30mg/kg/d).  Importantly, BXI-72 can also overcome acquired radioresistance of lung cancer.  Based on our findings, the development of BXI(s) as a new class of anti-cancer agents is warranted and represents a novel strategy for improving lung cancer outcome.
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In the series University of Florida Digital Collections.
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Includes vita.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Dong-Kyoo Park.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Deng, Xingming.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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lcc - LD1780 2012
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UFE0043643:00001


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1 STRUCTURE BASED ANTI CANCER DRUG DEVELOPMENT BY TARGETING BCL XL By DONGKYOO PARK 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 201 2

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2 201 2 Dongkyoo Park

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3 To my parents for your love and encouragement

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4 ACKNOWLEDGMENTS This P h D research could not have been completed without the assistance of many individuals to whom I am very thankful I would like to present my great thanks to all of the following persons Most of all, I would like to express my special appreciation to my advisor Dr. Xingming Deng for his outstanding support and guidance. Dr. Deng has trained me to pro duce novel scientific idea, design the experiments, organize the data, make figures and graphics and write scientific articles and dissertation I am so lucky that he has guided m y P h D project, which will greatly benefit my future career and life I am also indebted to many researchers and my friends who provided me reagents, technical expertise and guidance. I would like to acknowledge Dr. Steven Weintraub, Dr. Chul Han, Dr. Kyung Suk Choi, Dr. Kiwon Ban and Dr. Inho Choi for their helpful discussion and encouragement In addition, I would like to thank everyone in the Deng lab for their friendship for years. Lastly, I would like to express my gratitude to Ms. Kimberly Hodges for her kind assistan ce and concern on handling all administrative works fo r my P h D studies I would like to truly thank my dissertation committee members, Dr. Satya Narayan, Dr. Daiqing Liao, Dr. Alexander Ishov and Dr. Linda Bloom for their constructive advice assistance patience and encouragement. I would also like to tha nk to our collaborators, Dr. Andrew Magis for his computer based drug screening and Dr. Gabriel Sica for his histopathological consultation. I would like to give my great thanks to my loving parents from the deep bottom of my heart. I am sure that I would never journey my ques t for scien tific knowledge

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5 without their support and sacrifice I thank my loving parents for their unconditional and endless love, encouragement and support for my whole life

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIA TIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Lung Cancer ................................ ................................ ................................ ........... 15 Apoptosis Pathways ................................ ................................ ............................... 17 Bcl2 Family Regulation of Apoptosis ................................ ................................ ...... 19 Regulation of Bcl XL/ Bak Heterodimerization and Bak Activation ......................... 22 Small Molecules Targeting Bcl2 Proteins ................................ ................................ 24 Aim and Significance of Study ................................ ................................ ................ 25 2 DISCOVERY AND CHARACTERIZATION OF NOVEL BCL XL INHIBITORS (BXI) FOR TREATMENT OF LUNG CANCER ................................ ....................... 32 Background ................................ ................................ ................................ ............. 32 Results ................................ ................................ ................................ .................... 33 Screening of small molecule Bcl XL inhibitors (BXIs) that target the BH3 domain of Bcl XL (aa 90 98) and suppress lung cancer cell death. .............. 33 Bcl XL is a required target for BXI suppression of human lung cancer. ........... 35 Treatment of human lung cancer cells with BXI results in disrupti on of Bcl XL/Bak association, Bak oligomerization and Cyt c release. ......................... 36 BXIs potently repress lung cancer growth in animal models. ........................... 37 Discussion ................................ ................................ ................................ .............. 38 3 EFFECT OF BXI ON ACQUIRED RADIORESISTANT LUNG CANCER ................ 63 Background ................................ ................................ ................................ ............. 63 Results ................................ ................................ ................................ .................... 64 BXI reverses radioresistance and restore radiation sensitivity of lung cancer cells. ................................ ................................ ................................ .............. 64 BXI overcomes a cquired radioresistance of lung cancer in animal models. ..... 64 Discussion ................................ ................................ ................................ .............. 65 4 DISCUSSION ................................ ................................ ................................ ......... 71

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7 5 MATERIALS AND METHODS ................................ ................................ ................ 75 Materials ................................ ................................ ................................ ................. 75 Screening of Bcl XL inhibitors ................................ ................................ ................. 75 Cell lines and cell culture ................................ ................................ ........................ 76 Sulforhodamine B colorimetric assays (SRB) ................................ ......................... 76 Colony formation assay ................................ ................................ .......................... 77 Analysis of apoptotic cell death ................................ ................................ ............... 77 Fluorescence polarization assays ................................ ................................ ........... 78 Wes tern blot analysis ................................ ................................ .............................. 78 Immunoprecipitaton assays ................................ ................................ .................... 79 RNA interference, plasmids and transfection ................................ .......................... 79 Bak oligomerization and Cytochrome c release ................................ ...................... 80 Lung cancer xenografts and treatments ................................ ................................ .. 81 Terminal deoxyn ucleotidyl transferase mediated dUTP nick end labeling assays (TUNEL) ................................ ................................ ................................ .............. 81 Immunohistochemistry (IHC) analysis ................................ ................................ ..... 82 Blood analysis for m ice ................................ ................................ ........................... 82 Establishment of ionizing radiation resistant (IRR) cell line ................................ ..... 82 IR resistant lung cancer xenografts and treatments ................................ ................ 83 Statistical analysis ................................ ................................ ................................ .. 84 LIST OF REFERENCES ................................ ................................ ............................... 85 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 96

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8 LIST OF TABLES Table page 1 1 Classification of Bcl2 family proteins ................................ ................................ .. 27 2 1 Molecular docking sco res of small molecules targeting the BH3 domain of Bcl XL ................................ ................................ ................................ ................. 42

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9 LIST OF FIGURES Figure page 1 1 Leading sites of new cancer related deaths for 2012 ................................ ......... 28 1 2 Intrinsic pathways in apoptosis ................................ ................................ ........... 29 1 3 Expression level of endogenous Bcl XL is up regulated in various lung cancer cell li nes ................................ ................................ ................................ .. 30 1 4 Proposed Model ................................ ................................ ................................ 31 2 1 Structural modeling of BXI 61 and BXI 72 in the BH3 domain binding pocket of Bcl XL protein and chemical structures of BXI 61 and BXI 72 ........................ 43 2 2 BXIs repress human lung cancer cell growth ................................ ..................... 44 2 3 BXIs show selective cytotoxic ity against lung cancer cells compared to immortalized normal human bronchial epithelial cells ................................ ......... 45 2 4 BXI 61 and BXI 72 preferentially bind to Bcl XL ................................ ................. 46 2 5 BXIs more effectively induce apoptosis than ABT 737 and cisplantin in H1299 cells ................................ ................................ ................................ ......... 47 2 6 BXIs more effectively inhibit growth of A549 and H1299 cells than ABT 737, ABT 263, cisplatin, and erlotinib ................................ ................................ ......... 48 2 7 BXI 72 shows much higher cytotoxic effects on A549 cells compared to H460 cells ................................ ................................ ................................ .................... 49 2 8 Up regulation of Bcl XL in H460 cells and down regulation of Bcl XL in H1299 cells ................................ ................................ ................................ ......... 51 2 9 Overexpression of Bcl XL in H460 cells sensitizes cells to the cell growth inhibition in response to BXI 61 or BXI 72 treatment ................................ .......... 52 2 10 Depletion of Bcl XL by RNAi reduces sensitivity of lung cancer cells to BXIs .... 53 2 11 Treatmen t of human lung cancer cells with BXI 72 results in Bcl XL/Bak dissociation, oilgomerization of Bak and Cyt c release ................................ ....... 54 2 12 BXI 72 activates caspases and induces PARP cleavage ................................ ... 56 2 13 Maximum tolerated dose of BXI 72 ................................ ................................ .... 57 2 14 BXI 72 potently represses lung cancer growth in vivo ................................ ........ 58

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10 2 15 Analysis of BXI 72 toxicity in vivo ................................ ................................ ....... 60 2 16 BXI 61 represses lung cancer growth in vivo ................................ ...................... 62 3 1 B XI 72 overcomes radioresistance of lung cancer in vivo ................................ .. 67 3 2 Analysis of toxicity for combination of BXi 72 and IR in vivo .............................. 69

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11 LIST OF ABBREVIATION S A1 Bcl2 r elated protein A1 AIF Apoptosis inducing factor APAF1 apoptotic protease activating factor 1 Bad Bcl2 associated death promoter Bak Bcl2 antagonist/killer 1 Bax Bcl2 associated x protein Bcl2 B cell CLL/Lymphoma 2 Bcl XL B cell lymphoma extra large BH doma ins Bcl2 homology domains B H3 domain Bcl2 homology 3 domain Bid BH3 interacting domain death agonist Bik Bcl2 interacting killer Bim Bcl2 interacting mediator of cell death Bok Bcl2 related ovarian killer protein BXI Bcl XL inhibitor Caspases Cysteine depe ndent aspartate specific proteases DISC Death inducing signaling complex FADD Fas associated death domain protein FasL Fas ligand IR Ionizing radiation NSCLC Non small cell lung cancer Mcl 1 Myeloid cell leukemia sequence 1 MOMP Mitochondrial outer membran e permeabilization Puma p53 upregulated modulator of apoptosis

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12 SCLC Small cell lung cancer Smac Small mitochondria derived activator of caspases tBID truncated BID TRAIL Tumor necrosis factor related apoptosis inducing ligand

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13 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 STRUCTURE BASED ANTI CANCER DRUG DEVELOPMENT BY TARGETING BCL XL By Dongkyoo Park August 201 2 Cha ir: Xingming Deng Major: Medical Sciences Molecular Cell Biology Bcl XL is a major anti apoptotic protein in the Bcl2 family whose overexpression is more wide ly o bserved in human lung cancer cells than that of Bcl2, suggesting that Bcl XL is more biologic ally relevant and therefore a better therapeutic target for lung cancer. Here, we screened small molecules that selectively target the BH3 domain (aa 90 LREAGDE F E 98) in binding pocket of Bcl XL using the UCSF DOCK 6.1 suite of program s and the NCI chemic al library database We identified two new B cl X L i n hibitor s (BXI 61 and B XI 72 ) that exhibit selective toxicity against lung cancer cells compared with immortalized normal human bronchial epithelial cells (BEAS 2B and HBEC 3KT ) F luorescence polarizatio n assay reveals that BXI 61 and BXI 72 preferentially bind to Bcl XL protein but not Bcl 2 in vitro with high binding affinities T reatment of lung cancer cells with BXI 72 results in disruption of Bcl XL/Bak interaction oligomerization of Bak and cytochr ome c release fr om mitochondria l membrane These two BXI compounds potently repress lung cancer xenografts in vivo via apoptosis without significant normal tissue toxicity within effective does ( i.e 20~30mg/kg/d). Importantly, BXI 72 can also overcome a cquired radioresistance of lung cancer Based on our findings, the development of BXI(s) as a new class of anti

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14 cancer agents is warranted and represents a novel strategy for improving lung cancer out come.

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15 CHAPTER 1 INTRODUCTION Lung Cancer Lung cancer is the major leading cause of cancer related death in the United states accounting for 28% of the total estimated cancer deaths ( Siegel et al., 2012 ) Lung cancer is the most common cause of cancer related death in male and the second in female ( Siegel et al., 2012 ) The estimated cancer related mortality from lung cancer alone has surpassed the combined mortality from prostate ( 11 %), colon (9% ) and pancreatic cancer (6%) in male cancer patients ( Siegel et al., 2012 ) (Figure 1 1) The known m ain causes of lung cancer are including cigarette smoking, ionizing radiation, radon, air pollution, family history of lung cancer, age over 65, etc ( Catelinois et al., 2006 ; Coyle et al., 2006 ; Hackshaw et al., 1997 ; Nyberg and Persha gen, 1998 ) Especially, smoking is accounting for 90% of lung cancer incidence ( Biesalski et al., 1998 ) and carcinogens derived from smoking damage lung epithelial cells by oxidative stress, leading to DNA damage ( Alavanja, 2002 ; Huang et al., 2011 ) The damaged cells in the lung s by the cumulative exposure to these risk factors may become cancerous over a l o ng term period. The major types of lung cancer are non small cell lung cancer (NSCLC, approximately 85%) and small cell lung cancer (SCLC, approximately 15%). These two types of lung cancer are categorized under a microscope based on how the lung cancer cells look. NSCLC is subclassified into adenocarcinoma, squamous cell carcinoma and large cell carcinoma. SCLC can occur in combination with adenocarcinoma or squamous cell carcinoma. This type of SCLC is subclassified as a combined small cell lung cancer (c SCLC). Contrary to NSCLC, c SCLC is the only subt ype of SCLC.

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16 Th es e two major categories of lung cancer, NSCLC and SCLC both have very dismal overall survival rates of 16% and 6%, respectively. Despite recent therapeutic advances, virtually all patients with advanced NSCLC eventually develop resistance to currently available cytotoxic and targeted therapy ( Amundson et al., 2000 ; Haura et al., 2004 ; Kobayashi et al., 2005 ) Traditional treatments for lung cancer depend on the type of lung cancer and its stage. Therefore, accurate diagnosis of lung tumor histology is the essential when deciding a treatment. Treatme nts include surgery, chemotherapy, radiotherapy, targeted therapy or a combination of treatments. For example, lung cancer patients with the limited stage of small cell lung cancer in which cancer is found in one side of lung s and its near tissue primaril y treated with radiotherapy and chemotherapy. Most patients with the extensive stage of small cell lung cancer in which cancer is found in the chest outside of lungs or in distant organs have chemotherapy only. Lung cancer patients with non small cell lu ng cancer may have surgery, chemotherapy, radiotherapy or a combination of treatments, depending on each lung cancer stage. Tumor tissues could be surgically removed for a small lung tumor. Before lung surgery, the stage must be reassessed to determine wh ether lung tumor is localized and can be remove d by surgery Chemotherapy refers to use anti cancer drugs for the treatment of lung cancer. There are a number of strategies in the drug administration scheme. Patients could be treated with chemotherapeut ic drugs in cycles and have a rest period after each treatment period. The length of the rest period and the number of cycles depend on the anti cancer drugs used f or lung cancer treatment. Radiotherapy is often given with chemotherapy. External beam ra diation is the most common type of

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17 radiotherapy for lung cancer. The amount of radiation varies depending on stage of lung caner. Lung cancer patients are usually treated for 5 day s a week for several weeks. A poptosis Pathways Apoptosis ( i.e. programmed cell death) is a well regulated critical process that occurs both in development and in response to stress to tightly maintain homeostasis. Apoptosis, coined by Currie and colleagues in 1972, was inspired from the release of apoptotic bodies ( Kerr et al., 1972 ) The first morphological description of apoptotic cell death was provided by Walter Flemming in 1885. Apoptosis results in cell volume reduction membrane blebbing chromatin condensat ion and DNA fragmentation, leading cells to shrunken corpses that are rapidly devoured by neighboring phagocytes. The concept of natural cell death was originally from developmental studies of Carl Vogt in 1842 ( Cotter, 2009 ) Programmed cell death is a fu ndamental part of normal development ( Jacobson et al., 1997 ) and normal physical processes Two major pathways of apoptosis, including intrinsic pathway ( i.e. mitochondrial pathway) mediated by permeabilization of the outer mitochondrial membrane and extrinsic pathwa y ( i.e. death receptor pathway) mediated by binding an extracelluar ligand to a transmembrane receptor, are responsible for processing the stress signals and executing cellular demolition. Because of the importance and lethal nature of apoptosis, it is hi ghly regulated by B cell CLL/Lymphoma 2 (Bcl2) family proteins (Table 1 1). Intrinsic apoptotic pathway is initiated by the release of cytochrome c (cyt c), which is normally sequestered between the inner and outer membranes of the mitochondria

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18 and a compo nent of the mitochondria electron transporting chain reaction producing ATP ( Green and Reed, 1998 ; Liu et al., 1996 ) Multiple apoptotic signals induce cyt c release from the mitochondria to activate apoptotic protease activating (Apaf 1) protein, leading to caspase activation ( Br une and Schneiderhan, 2003 ; Mattson and Chan, 2003 ; Popov et al., 2002 ) In response to a variety of pro apoptotic stimuli such as ionizing radiation cytotoxic agent, hypoxia, DNA damage and growth factor withdrawal, cyt c is released into the cytosol from the mitochondria l intermembrane space Th is critical step in apopto sis is the mitochondrial outer membrane permeabilizaltion (MOMP), which is induce d by oligomerization of pro apoptotic Bcl2 proteins such as Bax and Bak ( Yip and Reed, 2008 ) for the release of cyt c from mitochondria. Mitochondria sequester non Bcl2 pro apoptotic proteins such as cyt c, apoptosis inducing factor (AIF), small mitochondria derived activator of caspases (Smac), etc. These proteins initiate caspase activation by the formation of apoptosome, a large quaternary protein. Once cyt c is released into the cytosol, cyt c binds to Apaf 1 and then forms apoptosome. After apoptosome formed, apoptosome can activate pro caspase 9 and then thi s initiator caspase can trigger a cascade of events, leading to apoptosis ( Hengartner, 2000 ; Ow et al., 2008 ; Taylor et al., 2008 ) In contra st to intrinsic pathway, extrinsic apoptotic pathway is triggered by the binding of extracellular ligands such as Fas ligand (FasL) tumor necrosis factor related apoptosis in ducing ligand (TRAIL) or TNF and transmembrane death receptor such as Fas TRAIL R or TNF R 1 This ligation induces receptor clustering and the assembly of the death inducing signaling complex (DISC) and then the DISC can recruit multiple pro caspase 8 via Fas associated death doma in protein (FADD) ( Wajant, 2002 ) Active

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19 capase 8 can either directly activate downstream caspases such as pro capsase 3 or engage the intrinsic pathway via t run cated Bid (tBid) One of the well known hallmarks of cancer is the deregulation of programmed cell death ( i.e. apoptosis) resulting in evasion of apoptosis ( Hanahan and Weinberg, 2000 ; Hanahan and Weinberg, 2011 ) Impaired apoptosis is a c ritical step in tumor development and renders the tumor cells more resistance to conventional cytotoxic therapy. Despite the fre quent dysregulation of apoptosis in tumors, n early all tumors maintain the core apoptotic regulatory machinery : Bcl2 family proteins, cyt c caspases, etc Among these components of the apoptotic machinery, o verexpression of anti apoptotic Bcl2 family pro teins such as Bcl2, Bcl XL and Mcl 1 play s critical role s in mediating resistance t o apoptosis induced by chemotherapy or radiotherapy ( Datta et al., 1995 ; Niizuma et al., 2006 ; Taylor et al., 2006 ; Woo et al., 2005 ) Elevated expressions of anti apoptotic Bcl2 famil y proteins have been observed in lung cancers and are associated with chemo or radio resistance and poor prognosis ( Amundson et al., 2000 ; Haura et al., 2004 ) Bcl XL, an anti apoptotic member of the Bcl2 family, is widely expressed in both SCLC and NSCLC cells ( Amundson et al., 20 00 ; Karczmarek Borowska et al., 2006 ; Li et al., 2008 ) and is associated with resistance against chemotherapeutic agents ( Amundson et al., 2000 ) Therefore, attempt to overcome this inherent resistance against apoptosis by inactivating anti apoptotic molecules is a very attractive approach for anti cancer ther apeutics. Bcl2 Family Regulation of Apoptosis Bcl2 family members are the major regulators of the apoptotic pathway at the decision phase and Bcl2 family protein s share structural and functional characteristics. Bcl2 proteins share conserved Bcl2 homology ( BH ) domains ( i e. BH1, BH2 BH3 and

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20 BH4) ( Kelekar and Thompson, 1998 ) These four conserv ed domains are involved in the homodimerization and heterodimerization among the Bcl2 family proteins ( Sato et al., 1994 ; Zhang et al., 1995 ) Bcl2 proteins are further divided into pro apoptotic or anti apoptotic Bcl2 proteins. For example, Bcl2 B cell lymphoma extra large ( Bcl XL ) and Myeloid cell leukemia sequence 1 ( Mcl 1 ) have anti apoptotic activit ies and all four BH domains are present in anti apoptotic members ( Deng et al., 2006 ) The pro apoptotic Bcl2 family members are divided into subgroups including the multidoma in pro apoptotic members and BH3 only members. For example, Bax (Bcl2 associated x protein), Bak (Bcl2 antagonist/killer 1) Bok ( Bcl2 related ovarian killer protein) have BH1, BH 2 and BH 3 domains and pro apoptotic activit ies (Table 1 1 and Figure 1 2). BH3 only proteins have only BH3 domain a nd pro apoptotic activit ies ( Table 1 1 and Figure 1 2) Recent studies suggest that there are two different subgroups in the BH3 only proteins ( Chen et al., 2005 ; Kuwana et al., 2005 ; Pagliari et al., 2005 ) Among the BH3 only proteins, acti v ators such as Bid ( BH3 interactin g domain death agonist) and Bim ( Bcl2 interacting mediator of cell death) interact with Bax and Bak to induce MOMP whereas depressors neutralize the functions of anti apoptotic proteins such as Bcl2, Bcl XL and/or Mcl 1 ( Certo et al., 2006 ) in response to various apoptotic stimuli. There are 25 recognized members of Bcl2 family of proteins, each of which has either a pro or anti apoptotic function ( Reed an d Pellecchia, 2005 ) Of these four domains, BH3 domain is found in all Bcl2 proteins and Bcl2 proteins interact with each other members through their BH3 domain Bcl2 family members are not only constitutively localized on the outer membrane of mitochon dria but also found in the cytosol, the endoplasmic reticulum, nuclear

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21 envelope and microtubules ( Krajewski et al., 1993 ) The subcellular localizations of Bcl 2 proteins occur through heterodimerization, phosphorylation or proteolysis. For example, whereas Bax is localized in cytosol or loosely attached to the outer mitochondrial membrane in healthy cells, Bax in cytosol translocates to mitochondria and deeply inserts the membrane upon induction of apoptosis ( Goping et al., 1998 ; Hsu et al., 1997 ; Saikumar et al., 1998 ) Contrary to Bax, Bak permanently resides on the outer membrane of mitochondria in healthy cells. However, Bak undergoes conformational changes and oligomerization under apoptotic signals. Bcl2 is the firs t member of Bcl2 family, cloned from neoplastic B cells with the t(14;18) chromosome translocation ( Tsujimoto et al., 1984 ) It took years to understand how Bcl2 regulated the initiation of apoptosis. Bcl2 interacts with pro apoptotic prote ins such as Bax to form heterodimers on the mitochondrial membrane ( Oltvai et al., 1993 ) and then blocks the release of cyt c from mitochondria ( Yang et al., 1997 ) Bcl2 deficient mice studies demonstrated that Bcl2 is essential for survival of mature T and B lymphocytes ( Veis et al., 1993 ) Bcl2 is frequently overexpressed in cancers. Bcl XL was identified and characterized as a regulator of apoptotic cell death ( Boise et al ., 1993 ) and is essential for regulating survival of erythroid progenitor, neuronal cells and platelet ( Mason et al., 2007 ; Motoyama et al., 1995 ) Intriguingly, Bcl XL is more efficiently to associate with pro apoptotic proteins Bad and Bak as compared to Bcl2 Mutational studies of Bcl XL and Bak peptide complex suggested that BH3 domain of Bak binds to the h ydrophobic groove formed by BH1, BH2 and BH3 domains of Bcl XL ( Sattler et al., 1997 ) The interactions between hydrophobic residues of Bcl XL and Bak are critical in heterodimer formation. The hydrophobic groove in Bcl2

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22 is wider than in Bcl XL due to hydrophobic contacts in Bcl XL side chains ( Petros et al., 2004 ) In add ition, the differences in sequence position 104 (Ala in Bcl XL, Asp in Bcl2), 108 (Leu in Bcl xL, Met in Bcl2) and 122 (Ser in Bcl XL, Arg in Bcl2) change the electrostatic character of binding groove in Bcl XL and Bcl2 ( Petros et al., 2004 ) For example, Bak binds about 10 fold more strongly to Bcl XL than to Bcl2, suggesting each anti apoptotic proteins have a different specificity for binding to pro apoptotic pr oteins. Based on these different electrostatic characteristics, there are Bcl2 selective, Bcl XL selective, Mcl 1 selective, Bcl2/Bcl XL selective or Bcl2/Bcl XL/Mcl 1 selective small molecules ( Azmi and Mohammad, 2009 ; Chan et al., 2003 ; Kitada et al., 2008 ; Paoluzzi et al., 20 08 ) Mcl 1 is an anti apoptotic protein discovered as the second member of Bcl2 family from the human myeloid leukemia cell line, ML 1 ( Kozopas et al., 1993 ) Mcl 1 was predicted to play a critical role in the regulation of apoptosis because showing the sequence similarity to Bcl2 ( Kozopas et al., 1993 ) and further stud ies showed that Mcl 1 contributed to apoptotic cell death ( Reynolds et al., 1994 ) Mcl 1 is regulated at transcriptional and translational levels ( Akgul, 2009 ; Bingle et al., 2000 ) Mcl 1 has a short half life mediated by ubiq u itin depedent or ubiq u itin independent de gradation ( Nijhawan et al., 2003 ; Stewart et al., 2010 ) Mcl 1 is also overexpressed in a variety of human c ancer cell lines including lung cancer ( Plac zek et al., 2010 ) Regulation of Bcl XL/ Bak H eterodimerization and Bak Activation Bak, pro apoptotic protein, is sequestered by Bcl XL until displaced by BH3 only proteins ( Willis et al., 2005 ) In healthy cells, Bak is associated with Bcl XL or M cl 1 but not Bcl2, Bcl w or A1. This heterodimerization of Bcl XL and Bak requires the Bak BH3 domain which is also necessary for Bak activation in apoptotic cells. Mutational studies

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23 of Bcl XL and Bak peptide complex suggested that BH3 domain of Bak binds to the hydropho bic groove formed by BH1, BH2 and BH3 domains of Bcl XL ( Sattler et al., 1997 ) Thus, small molecule agents that can insert Bcl XL/Bak binding pocket may disrupt B cl XL/Bak binding leading to activation of Bak by its oligomerization. Bak is essential for apoptosis in response to diverse cell death signals. In healthy cells, Bak resides in the outer membrane of the mitochondria. After receiving various apoptotic s ignals, however, Bak undergoes conformational changes and oligomerization, leading to pore formation in the mitochondria and cyt c release into cytosol from mitochondria The Bak conformation al changes are initiated by the BH3 only members of the Bcl2 fam ily via three possible mechanisms ( i.e. direct activation model indirect activation model or priming capture displacement model ) First, Bak blocked by anti apoptotic proteins such as Bcl XL and Mcl 1 could be neutralized by BH3 only proteins such as B im and truncated Bid (tBid) leading to Bak activation ( i.e Bak oiligomerization ) (indirect activation model) ( Willis et al., 2007 ) Secondly Bim and tBid may directly bind non activated Bak to trigger Bak oligomerization (direct activation model) ( Dewson et al., 2009 ; Dewso n et al., 2008 ) In the priming capture displacement model, lastly, incorporate features of both direct and indirect activation models were proposed. Primed Bak is captured by anti apoptotic proteins such as Bcl XL until the primed Bak is displaced by further activated BH3 only proteins such as Bad or Bim, leading to Bak dim er ization and oligomerization. A cr itical unresolved issue in apoptosis is how Bak convert s from the non activated form into the oligomers inducing MOMP Dewson et al. (2008) recent ly showed that BH3 domain of Bak is transiently exposed and then inserted into the hydrophobic

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24 g r oove of another activated Bak monomer to form a dimer in addition to exposure of N terminal epitopes after apoptotic signaling. This interaction of two activa ted Bak monomers results in a symmetr ic dimer (face to face) To form Bak oligomers, dimers associate via crosslinking of cysteine residues placed in their distinct 6 helices (dimer multimerization model) The multiple 6 : 6 association s between symmetric dimers to form Bak oligomers containing at least 18 Bak proteins ( Dewson et al., 2009 ) In alternative model, active Bak binds to the proposed rear site of another activated Bak to form an asymmetric dimer (face to Back) producing a chain of monomers ( Shamas Din et al., 2011 ) S mall Molecules Targeting Bcl2 Proteins Several small molecule BH 3 mimetic compounds such as ABT 737 ABT 263 ( the oral form of ABT 737 ) AT 101, GX15 070, TW 37 have been developed and evaluated in clinical trials with limited success ( Azmi and Mohammad, 2009 ; Chan et al., 2003 ; Kitada et al., 2008 ; Li et al., 2008 ; Nguyen et al., 2007 ; Oakes et al., 2011 ; Oltersdorf et al., 2005 ; Paoluzzi et al., 2008 ; Tahir et al., 2007 ; Tse et al., 2008 ; Vogler et al., 2009a ; Vogler et al., 2008 ; Vogler et al., 2009b ) A lthough ABT 737 is considered a potent anti cancer drug, cancer cells expressing low level s of endogenous Bcl2 showed dramaticall y less sensitivity to ABT 737 ( Hann et al., 2008 ; Oltersdorf et al., 2005 ; Vogler et al., 2009a ) To overcome the limitation of Bcl2 inhibitors for lung cancer treatment, we have developed two additional effective anti cancer agents (BXI 61 and BXI 72) that specifically target Bcl XL for the treatment of lung cancer s and, potentially, other cancers with high expression levels of endogenous Bcl XL. Our findings demonstrate the efficacy of BXIs in potently repressing lung cancer and also overcoming acquired radioresistance of lung cancer cells both in vitro and in viv o

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25 In the present study, we selected NSC354961 (molecular weight, 446) and NSC334072 (molecular weight, 562) using cytotoxicity assays out of two hundred candidates, targeting the BH3 domain of Bcl XL, identified by DOCK suite of programs (version 6.1, U niversity of California, San Francisco CA). We termed NSC354961 B cl X L i nhibitor (BXI) 61 and NSC334072 BXI 72, respectively for the sake of brevity. We also evaluated the cytotoxicity of BXIs in various human lung cancer cell lines and anti tumor effic acy of BXIs in lung cancer xenograft models. Our studies suggest that BXIs are novel Bcl XL specific inhibitors inducing cancer cell death via apoptosis both in vitro cytotoxicity assays and in vivo tumor xenograft models. Aim and Significance of Study Lu ng cancer lead s the most cause of cancer related death s in men and the second most in women worldwide. Even with the best therapies, the overall survival for NSCLC and for SCLC is 16% and 6%, respectively. Induction of apoptosis in cancer cells is a crit ical mechanism for cancer treatment. Bcl XL, the major member of the Bcl2 family, suppresses apoptosis by binding to pro apoptotic proteins such as Bax and Bak M echanisms involved in this survival function of Bcl XL have been discerned for novel therape utic targets Since Bcl XL is over expressed in both SCLC and NSCLC cell s (Figure 1 3), this anti apoptotic protein should be an ideal target for treatment of human lung cancer. A major challenge affecting outcomes of patients with lung cancer is the devel opment of resistance to chemotherapy or radiotherapy. Therefore, we will develop more effective Bcl XL inhibitors that can overcome radio and chemoresistance leading to improved outcome of lung cancer. We hypothesized that small molecule drugs targetin g the BH3 domain of Bcl XL suppress tumor growth through apoptosis by disrupting Bcl XL/Bak association, activating Bak oligomeriazation and inducing

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26 cytochrome c release (Figure 1 4) To reach this goal, we will screen novel Bcl XL inhibitors (BXIs) usin g DOCK program suite determine the mechanism(s) by which BXIs induce apoptosis in human lung cancer cells determine anti lung cancer efficacy and toxicity of BXI compounds in animal models and determine whether BXIs overcome radioresistance of human lung cancer.

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27 Table 1 1. Classification of Bcl2 family proteins Apoptotic property Subclass Protein nomenclature Anti apoptotic Multidomain Bcl2 Bcl XL, Mcl 1, A1, Bcl w, Bcl B Pro apoptotic Multidomain Bax, Bak, Bok BH3 only (activator) Bid, Bim BH3 on ly (depressor) Bad, Bik, Puma, Noxa, others

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28 Figure 1 1 Leading sites of new cancer related deaths for 2012 In case of males, estimated mortality rate of lung cancer has been surpassed the sum of prostate, colon and pancreatic cancer related death ( Siegel et al., 2012 )

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29 Figure 1 2 Intrinsic pathways in apoptosis Intrinsic pathway is initiated by cytochrome c release into the cytosol from mitochondria l intermembrane space in response to a variety of pro apoptotic stimuli such as DNA damage, hypoxia, growth factor withdrawal, cytotoxic agents and ionizing radiatio n The critical step in apoptosis is the mitochondrial outer membrane permeabilization (MOMP) induced by oligomerization of Bax and Bak. Among the BH3 only proteins, activators such as Bim interact with Bax and Bak induce MOMP whereas depressors such as Ba d and Noxa neutralize anti apoptotic proteins such as Bcl2, Bcl XL and Mcl 1. The apoptotic pathways are not shown in full for simplicity. ABT 737 functions as a BH3 mimetic inhibiting Bcl2, resulting in Bcl2/Bax dissociation.

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30 Figure 1 3 Expression l evel of endogenous Bcl XL is up regulated in various lung cancer cell lines. Expression levels of Bcl XL, Bcl2 Bak and Bax in normal lung cells or various lung cancer cell lines were analyzed by Western blot using a Bcl XL, Bcl2, Bak, or Bax antibody, res pectively. Equal protein loading was confirmed by actin expression level. BEAS 2B, immortalized normal lung cell line; SCLC, small cell lung cancer; NSCLC non small lung cancer

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31 Figure 1 4. Proposed Model We hypothesized that small molecule drugs tar geting the BH3 domain of Bcl XL suppress tumor growth through apoptosis by disrupting Bcl XL/Bak association, activating Bak oligomeriazation and inducing cyt c release.

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32 CHAPTER 2 DISCOVERY AND CHARACTERIZATION OF NOVEL BCL XL INHIBITORS (BXI) FOR TREATM ENT OF LUNG CANCER Background O verexpression of anti apoptotic Bcl2 family proteins such as Bcl2, Bcl XL and Mcl 1 play s critical role s in resistance against apoptosis induced by chemotherapy or radiotherapy ( Datta et al., 1995 ) E levated expression l evels of anti apoptotic Bcl2 family proteins have been observed in lung cancers and are associated with radio and chemo resistance ( Amundson et al., 2000 ; Haura et al., 2004 ) Specially, Bcl XL is widely expressed in both SCLC and NSCLC cells ( Amundson et al., 2000 ; Karczmarek Borowska et al., 2006 ; Li et al., 2008 ) whereas Bcl2 is expressed mainly SCLC Therefore, Bcl2 inhibitors such as ABT 737 and ABT 263 under clinical t rials have shown limited success. In addition the expression level of Bcl XL strongly is associated with resistance against chemotherapeutic agents and contributes to tumor progression ( Amundson et al., 2000 ) Therefore, Bcl XL may be an attractive potential target for developing anti cancer drugs for lung cancer treatment. To overcome the limitations of currently available Bcl2 inhibitors for lung cancer treatment we have developed two effective anti cancer agents ( i.e. BXI 61 and BXI 72) that specifically targeting Bcl XL for the treatment of lung cancers and other cancers with high expression levels of endogenous Bcl XL. Our findings demonstrate the efficacy of Bcl XL inhibitors in repressing lung cancer in vitro and in vivo

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33 Results Screening of small molecule Bcl XL inhibitors (BXIs) that target the BH3 domain of Bcl XL (aa 90 98) and suppress lung cancer cell death. Our findings and those of others have shown that Bcl XL is more widely expressed than Bcl2 in SCLC and NSCLC cell lines (Figure 1 3) ( Amundson et al., 2000 ; Karczmarek Borowska et al., 2006 ; Li et al., 2008 ) suggesting that Bcl XL may be a more biologically relevant therapeutic target for lung cancer. To screen for small molecules that specifically targ et Bcl XL, a library containing a pproximately 300,000 small molecules from the NCI was used to dock the structural pocket of the BH3 domain (aa90 LREAGDEGE 9 8; accession number: 1LXL and 1MAZ) using the UCSF DOCK 6.1 program suite as described ( Ostrov et al., 2009 ) The small molecules were ranked according to their energy scores. The top 200 small molecules were selected for screening of cytotoxicity in human lun g cancer cells ( i.e H1299 or A549 cells) by SRB assay. Among these small molecules, two compounds ( i.e NSC354961 and NSC334072) had the most potent activities against human lung cancer cells. We named these two lead compounds B cl X L i nhibitor BXI 61 (C 20 H 19 ClN 7 O, MW: 408.86) and BXI 72 (C 27 H 29 ClN 6 O, MW: 489.01). In this report, we focus on characterizing BXI 61 and BXI 72. The molecular modeling of these two leads in complex with Bcl XL is shown in Figure 2 1. To compare sensitivities of BXIs with ABT 7 37 in human lung cancer cells, A549, H15 7 and H1299 cells were treated with increasing concentrations (0, 0.5, 1, 2, 5, 10, 20 and 30M) of BXI 61, BXI 72 or ABT 737 for 72h. The surviving cell fraction was determined using SRB assay as described ( Vichai and Kirtikara, 2006 ) SRB assay estimates cell density based on the amount of cellular protein content and is an established and opt imized assay for the toxicity screening of compounds on

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34 adherent cells in a 96 well format. Results indicate that BXI 61 and BXI 72 are superior to ABT 737 in suppressi ng lung cancer cell growth (Fig ure 2 2 ). BXI 72 showed the greatest potency based on i ts low IC 50 concentrations against the tested cell lines ( i.e H1299: 0.94 0.12 M; A549: 0.68 0.08 M ; Fig ure 2 2B ). Importantly, both BXI 61 and BXI 72 displayed selective cytotoxicity against lung cancer cells compared to transformed immortalized n ormal human bronchial epithelial cells ( BEAS 2B ) and non transformed immortalized normal human bronchial epithelial cells (HBEC 3KT) (Fig ure 2 3 ), indicating that these compounds are relatively selective for tumor cells. To directly measure BXI/Bcl XL bind ing we used an in vitro fluorescence polarization assay with a f luorescent Bak BH3 domain peptide (5 FAM GQVGRQLAIIGDDINR) and purified Bcl XL protein ( Bruncko et al., 2007 ; Enyedy et al., 2001 ; Wang et al., 2000 ; Zhang et al., 2002 ) W e found that BXI s directly bin d to Bcl XL with high binding affinity (BXI 61: Ki =14.8 1.54 nM; BXI 72: Ki = 0.9 0.15 nM ) ( Figure 2 4 ). Importantly, both BXI 61 and BXI 72 have very low binding affinity for Bcl2 protein ( Ki : BXI 61 1435 164.24 nM; BXI 72 283 37.33 nM), indicat ing the specificity of their binding to Bcl XL ( Figure 2 4 ). To determine if BXI 61 and BXI 72 induce apoptotic cell death in lung cancer cells, Annexin V assay was performed ( Martin et al., 1995 ) As expected, BXI 61 and BXI 72 treatments significantly increased Annexin V positive cells i n dose dependent manner (Figure 2 5). In addition, BXI 61 and BXI 72 more effectivel y inhibit growth of A549 and H1299 cells that ABT 737, ABT 263, ciplatin and erlotinib (Figure 2 6)

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35 Interestingly, BXI s showed much higher cytotoxic effects on A549 cells compared to H460 cells and these effects were Bcl XL expression level dependent mann er (Figure 1 3 and Figure 2 7). The complete inhibition of colony formation was observed at 0.5 M of BXI 72 in A549 cells whereas 6.67% inhibition observed in H460 cells ( Figure 2 7 ). There was over 4 0 fold difference in IC 50 of BXI 72 for H460 ( > 30 M) a nd A549 (0. 68 M) ( Figure 2 7A ) determined by SRB assays and 100 times more concentration (10M) of BXI 72 needed to induce 100% cell growth in hibition of H460 than that of A549 (0 .5 M) in colony formation assays (Figure 2 7B) BXI 61 also showed significa ntly different cytotoxic effect on H460 (IC 50 > 30 M) and H1299 (IC 50 =2.3 1 M) and required 1 5 times higher concentration (>15M) to completely inhibit colony formation of H460 than that of H1299 Bcl XL is a required target for BXI suppression of human lung cancer. To further test whether Bcl XL is an essential target for BXI suppression of human lung cancer, Bcl XL was overexpressed in H460 cells ( Figure 2 8A and Figure 2 9) or knocked down by RNAi using Bcl XL shRNA in H1299 cells ( Figure 2 8B and Fig ure 2 10) Up regulation of Bcl XL in H460 cells sensitized cells to the cell growth inhibition in response to BXI 61 (IC 50 = 25.45 M) (Fig ure 2 9B ) or BXI 72 (IC 50 = 8.55 M) (Fig ure 2 9B ) whereas control H460 cells were highly insensitive to BXI 61 and BXI 72 (IC 5 0 > 50 M, BXI 61 and BXI 72; Fig ure 2 2E and 2 9 B) determined by SRB assays In addition, BXI 61 and BXI 72 inhibited c olony formation of Bcl XL overexpressed H460 cells whereas control H460 cells were insensitive to BXI 61 and BXI 71 at indicated concentra tions ( Figure 2 9 C). T he effect of Bcl XL shRNA on Bcl XL expression was highly specific, as shown by the control shRNA having no effect. SRB and colony formation assays revealed that

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36 depletion of endogenous Bcl XL significantly reduced the sensitivity of H1299 cells to BXI 61 and BXI 72 (Figure 2 10 B, C), suggesting that Bcl XL is necessary for the anti cancer activity of BXIs. Treatment of human lung cancer cells with BXI results in disruption of B cl XL/Bak association, Bak oligomerization and Cyt c rele ase. Bcl XL forms a heterodimer with Bak through the BH3 domain and suppresses apoptosis ( Sattler et al., 1997 ; Wil lis et al., 2005 ) Since BXIs are predicted to target the BH3 binding pocket of Bcl XL, BXIs may disrupt the Bcl XL/Bak heterodimerization leading to dissociation of Bak from the Bcl XL/Bak complex and subsequent Bak homo oligomerization and activation. To test this hypothesis, H1299 cells expressing high levels of endogenous Bcl XL and Bak were treated with increasing concentrations of BXI 72 ( i.e. 0.1 5M) for 24h. Co immunoprecipitation experiments were performed using a Bcl XL antibody. Results in dicated that treatment of cells with BXI 72 resulted in a dose dependent Bcl XL/Bak dissociation (Figure 2 11A). To assess whether BXI dissociated Bak molecules form homo oligomers in the mitochondrial membrane, a cross linking study using BMH was carried out following treatment of cells with BXI 72 Intriguingly, treatment of cells with BXI 72 facilitated the formation of Bak dimers, trimers and multimers (Figure 2 11B). The molecular sizes of these oligomers obtained were esti mated to be multiples of ~ 28kDa, suggesting the formation of Bak homo oligomers. It is known that formation of Bak oligomers can result in c yt c release to induce apoptosis ( Dai et al., 2011 ; Wei et al., 2000 ) Our results show that BXI 72 initiated Bak oligomerization can also facilitate cyt c release from mitochondria (Figure 2 11C). BXI 72 also induced caspase activa tion and PARP cleavage in lung cancer cells (Figure 2 11D and Figure 2 12).

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37 BXIs potently repress lung cancer growth in animal models. In order to define the appropriate doses for in vivo experimentation, we first determined the maximum tolerated dose (MTD ) as previously described ( Kitada et al., 2008 ) Mice were treated in groups of six per dose level with increasing doses of BXI 72 (10 50mg/kg/d) intraperitoneally (i.p.) for up to 25 days. The 50mg/kg/d wa s uniformly lethal in the six mice within 10 days while 65% of mice treated at the 40mg/kg/d dose died within 25 days. The dose range between 10 and 30mg/kg/d was tolerable with no death recorded after 25 days of daily administration (Figure 2 13 ). We th erefore determined the MTD of BXI 72 (i.p.) with 25 day treatment to be approximately 30~40mg/kg/d. To test whether BXI is active in vivo we tested the anti lung cancer efficacy of BXI 72 in nude mice with subcutaneous (s.c.) lung tumor xenografts as desc ribed ( Oltersdorf et al., 2005 ; Puri et al., 2007 ) Briefly, 5 10 6 H1299 cells in a balanced salt solution were injected into s.c. tissue at the flank region of nude mice. Tumors were allowed to grow to an average volume of 225 230mm 3 prior to initiation of therapy. Three doses of BXI 72 (10mg/kg, 20mg/kg/d or 30mg/kg/d) were administered i.p. to mice continuously for two weeks (8 mice per dose). A control group received 0.5% DMSO injection with the same schedule. Tumor volume was estimated by caliper measurements (V=(LxW 2 )/2). Treatment with BXI 72 resulted in a dose dependent regression of established lung canc er xenografts (Fig ure 2 1 4 A ). To assess whether BXI 72 induced tumor growth regression via apoptosis in vivo representative samples from harvested tumor tissues were analyzed by IHC for active caspase 3 and by TUNEL assays as described ( Oltersdorf et al., 2005 ) A dose

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38 dependent apoptosis was observed in tumor tissues after BXI 72 treatment (Fig ure 2 14B and Figure 2 14C ). Importantly, doses of 20 30mg/kg not only potently suppressed tumor growth but were also well tolerated without weight loss ( Figure 2 14 and Figure 2 1 5A). There were no significant increases in ALT, AST and BUN or decreases in WBC, RBC, Hb and PLT (Fig ure 2 1 5B). Histopathology of harveste d normal tissues (heart, liver, lung, brain, spleen, kidney, intestine, etc.) revealed no evidence of normal tissue toxicities after treatment with doses of 10 30mg/kg/d (Fig ure 2 1 5C). However, treatment of mice with doses of 40 50mg/kg/d for two weeks r esulted in increases in ALT, AST and BUN, indicating renal and hepatic toxicities at these higher doses (Fig ure 2 1 5B). Elevated liver function test was associated with hepatocellular necrosis in mice treated with 50mg of BXI 72 (Fig ure 2 1 5C). These fin dings suggest that doses between 20mg/kg and 30mg/kg provide the optimal therapeutic index for BXI 72 for in vivo experimentation involving lung cancer xenografts To test the anti lung cancer efficacy of BXI 61, Nu/Nu nude mice with H1299 lung cancer xeno grafts were treated with 40mg/kg/d of BXI 61 for 14 days. 16 mice were randomly divided into two groups ( i.e control and BXI 61 treated groups). Tumor volume and body weight were measured once every 2 days during the treatment with BXI 61. BXI 61 repre ssed the growth of lung cancer without weight loss (Figure 2 16). Discussion The major findings of this study have shown that BXIs targeting the BH3 domain of Bcl XL could (i) specifically bind to Bcl XL proteins, (ii) more effectively induce cell death th an ABT 737 in various lung cancer cell lines, (iii) inhibit the growth of lung caner cells with Bcl XL expression dependent manner, (iv) induce cell death through clearly defined

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39 mitochondria mediated apoptotic mechanism of actions and (v) effectively repr ess tumor growth in xenograft models. Overexpression of anti apoptotic Bcl2 family members such as Bcl2, Bcl XL and Mcl 1 is associated with the development of cancers and the development of resistance to chemotherapies and radiotherapies ( Amundson et al., 2000 ; Campos et al., 1993 ; Haura et al., 2004 ; Kornblau et al., 1999 ; Reed et al., 1996 ) The development of BH3 mimetics targeting Bcl2 members has provided a novel therapeutic approach for the cancer treatment ( Hann et al., 2008 ; Kitada et al., 2008 ; Konopleva et al., 2006 ; Li et al., 2008 ; Nguyen et al., 2007 ; Oltersdorf et al., 2005 ; Paoluz zi et al., 2008 ; Tse et al., 2008 ) Especially, ABT 737 under evaluation in clinical trials has been extensively studied in vitro and in vivo and showed the higher cytotoxicity on cancer cells than other BH 3 mimetics ( Vogler et al., 2009a ; Vogler et al., 2009b ) However, it is likely that relatively high dose of ABT 737 is required to effectively inhibit the growth of lung cancer cells expressing low endogenous Bcl2 and high endogenous Bcl XL ( Hann et al., 2008 ; Oltersdorf et al., 2005 ; Tahir et al., 2007 ; Vogler et al., 2009a ) Non small cell lung cancer (NSCLC) cells more exten sively express Bcl XL than Bcl2 whereas small cell lung cancer (SCLC) cells express both Bcl XL and Bcl2 ( Hann et al., 2008 ; Li et al., 2008 ) In this study, therefore, Bcl XL was chosen as a therapeutically potential target for developing anti cancer drugs treating human lung cancer. Distinguishing feature of these Bcl2 family proteins is that they interact with one another to form heterodimers or homodimers through the Bcl2 homology 3 ( BH3 ) domain s ( Cosulich et al., 1997 ; Ottilie et al., 1997 ; Sattler et al., 1997 ) Anti apoptotic Bcl XL also binds to pro apoptotic proteins such as Bax and Bak through the BH3

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40 domain and inhibits their pro apoptotic functions ( Sattler et al., 1997 ) Therefore, we chose the BH3 domain in the hydrophobic cleft of Bcl XL as a docking site for screening of small molecules that may disrupt the anti apoptotic functi on of Bcl XL using computerized UCSF 6.1 DOCK suite of program s. Approximately, 30 0,000 small molecules from NCI database were docked into the BH3 domain of Bcl XL by the DOCK program After computer based virtual screening, two hundred small compounds w ere identified to potentially bind to the BH3 domain of Bcl XL with priority scores. The compounds with more negative score were considered to be better potential inhibitor s of Bcl XL ( Huo et al., 2002 ) Here, we report that BXIs specifically bind t o Bcl XL and display more potent effect on various lung cancer cells than ABT 737, one of the most potent Bcl2 family protein inhibitors under clinical trials, with the except of H460 cells expressing low Bcl XL. This observation is consistent with previo us reports indicating that the expression level of Bcl XL is low in H460 cells ( Amundson et al., 2000 ; Li et al., 2008 ) Interestingly, however, overexpression of Bcl XL in H460 cells sensitized cells to BXIs, suggesting that the expression level of Bcl XL could be a predictive marker of BXIs cytotoxicity. It is also possibly noted that BXIs induce cancer cell death via a poptosis i n Bcl XL expression dependent manner. Mechanically, apoptosis induction by BXIs was preceded by dissociation of Bak from Bcl XL and oligomerization of Bak as previously observed in apoptotic cells in response to various apoptotic stimuli such as cytokines, DNA damage, growth factor withdrawal, hypoxia, etc ( Dewson et al., 2009 ) Cyt c release, caspase activation and

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41 PARP cleavage were also observed in lung cancer cells treated with BXIs, indicating that BXIs induced cell death via mitochondria mediated apoptosis. In addition to t umor repression TUNEL and active casepase 3 positive c ells were detected in tumor tissue sections after 14 day BXI 72 treatment. In vivo results suggest that BXI 72 repressed tumor growth via apoptosis. Additionally, indicated concentrations of BXI 72 treatment did not cause weight loss or any other side ef fects. Collectively, our studies suggest that BXIs are novel Bcl XL specific inhibitors inducing lung cancer cell death both in vitro cytotoxicity assays and in vivo animal models via apoptosis.

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42 Table 2 1. Molecular docking scores of small molecules tar geting the BH3 domain of Bcl XL No. NSC No. Score No. NSC No. Score 1 36791 59.13923 26 39936 48.62978 2 36400 58.25262 27 30049 48.55549 3 177979 58.12625 28 334072 48.34986 4 371729 57.38224 29 35847 47.97976 5 10408 56.90021 30 149852 47. 91537 6 35450 56.72305 31 21235 47.73167 7 53040 55.64085 32 72381 47.12778 8 356363 55.59406 33 71204 46.94272 9 80640 55.57445 34 35843 46.85723 10 118977 55.36196 35 17721 46.77199 11 407323 54.77881 36 137877 46.59332 12 172971 53.9 2231 37 168615 46.54950 13 33450 53.91065 38 212556 46.36312 14 281708 53.16341 39 130813 46.31387 15 114057 53.00452 40 88647 46.02478 16 34740 52.57435 41 304712 45.77601 17 32895 51.51495 42 66184 45.69736 18 172855 51.14339 43 624906 45.49515 19 177977 50.65311 44 354961 45.33624 20 155877 50.49064 45 50469 45.12648 21 32237 50.08401 46 65372 44.94118 22 50077 49.93425 47 35024 44.82663 23 667746 49.52435 48 131869 44. 53159 24 348978 49.18729 49 80362 44. 52175 25 34 564 48.89887 50 38279 44. 10612

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43 Figure 2 1. Structural modeling of BXI 61 and BXI 72 in the BH3 domain binding pocket of Bcl XL protein and chemical structures of BXI 61 and BXI 72.

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44 Figure 2 2. BXIs repress human lung cancer cell growth. A) an d B) A549, H157 and H1299 cells were treated with increasing concentrations (0, 0.5, 1, 2, 5, 10, 20 or 30 M) of BXI 61 (circle), BXI 72 (rectangle) or ABT 737 (triangle) for 72h. Cell growth was analyzed by SRB assay. Error bars represent S.D from three different independent experiments.

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45 Figure 2 3 BXIs show selective cytotoxicity against lung cancer cells (A549 and H1299) compared to immortalized normal human bronchial epithelial cells (BEAS 2B and HBEC 3KT ). HBEC 3KT, BEAS 2B, A549 and H1299 cells were treated with 1 M of BXI 61 or BXI 72 for 72h. Cell growth was analyzed by SRB assay. Error bars represent S.D.

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46 Figure 2 4 BXI 61 and BXI 72 preferentially bind to Bcl XL. Fluorescent Bak B H 3 domain peptide (3nM) was incubated with purified hu man Bcl XL protein (6nM) or Bcl2 protein (6nM) in the absence or presence of increasing concentrations ( i.e 0.1 500nM) of BXI 61 or BXI 72 in the binding affinity assay buffer. Binding affinities of BXI/Bcl XL or BXI/Bcl2 were analyzed using a competition Chapter 5 Materials and Methods Reported values are the mean S.D. for three separate experiments run in duplicate. Dose dependent displacement of Flu Bak peptide from Bcl XL is indicated by a decrease i n polarization (mP).

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47 Figure 2 5 BXIs more effectively induce apoptosis than ABT 737 and cisplantin in H1299 cells. H1299 cells were treated with the indicated concentrations of BXI 61, BXI 72, ABT 737, or cisplatin (0, 0.1, 0.5, 1, 2, 5 or 10 M) for 24 h Apoptotic positive cells are determined by Annexin V assays. Data are mean SD from three independent experiments.

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48 Figure 2 6 BXIs more effectively inhibit growth of A549 and H1299 cells than ABT 737, ABT 263, cisplatin, and erlotinib. After tr eating A549 or H1299 cells with the indicated concentrations of BXI 61, BXI 72, ABT 737, ABT 263, cisplatin or erlotinib (0.1 or 0.5 M) for 10 days, colonies were stained with crystal violet (0.1% in 20% methanol) and counted

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49 Figure 2 7 BXI 72 show s much higher cytotoxic effects on A549 cells compared to H460 cells A) A549 and H460 cells were treated with BXI 72 ( closed rectangle, H460; open rectangle, A549) (0, 0.5, 1, 2, 5, 10 or 30 M) for 72 hours. The growth inhibitions of cells were determined using SRB assays. B) The complete inhibition of colony formation was observed at 0.5 M of BXI 72 in A549 cells whereas 6.67% inhibition observed in H460 cells. Data are mean SD from three independent experiments.

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50 Figure 2 7 Continued.

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51 Figure 2 8 Up regulation of Bcl XL in H460 cells and down regulation of Bcl XL in H1299 cells. A) Overexpression of Bcl XL proteins in H460 cells expressing relatively low level of endogenous Bcl XL B) Kn o ck down of Bcl XL proteins in H1299 cells expressing high r elatively high level of Bcl XL

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52 Figure 2 9 Overexpression of Bcl XL in H460 cells sensitize s cells to the cell growth inhibition in response to BXI 61 or BXI 72 treatment A) Expression level changes of Bcl2 family proteins after overexpression of Bc l XL in H460 cells. B) H460 control (closed circle or rectangle) and H460 Bcl XL overexpression cells (open circle or rectangle) were treated with in dicated concentration s of BXI 61 or BXI 72 (0, 0.5, 1, 2, 5, 10, or 30 M) for 72 hours. Cytotoxicity was de termined using B) SRB assays or C) colony formation assays. Data are mean SD from three independent experiments.

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53 Figure 2 10 Depletion of Bcl XL by RNAi reduces sensitivity of lung cancer cells to BXIs. A) Bcl XL shRNA or control shRNA was transfec ted into H1299 cells. Expression levels of Bcl XL, Bcl2 and Mcl 1 were analyzed by Western blot. B) and C) H1299 cells expressing Bcl XL shRNA or control shRNA were treated with indicated concentrations of BXI 61 or BXI 72 for 72h. Cell growth was determin ed by B) SRB assays or C) colony formation assays. Data are mean SD from three independent experiments.

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54 Figure 2 1 1 Treatment of human lung cancer cells with BXI 72 results in Bcl XL/Bak dissociation, oilgomerization of Bak and Cyt c release. A) H1 299 cells were treated with increasing concentrations of BXI 72 for 24h. C o immunoprecipitation (IP) was performed using a Bcl XL antibody. Bcl XL associated Bak and Bcl XL were analyzed by Western blot. Normal IgG was used as control for IP. B) and C) H12 99 cells were treated as in A). Bak oligomerization and c yt c Prohibitin was used as a mitochondrial marker The purity of cytosol fractions was confirmed by non detectable protein level of prohibitin. D) PA RP cleavage and caspase activation induced by BXI 72. PARP cleavage and caspase activation were evaluated using western analysis. Actin was probed as a loading control.

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55 Figure 2 1 1 Continued.

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56 Figure 2 1 2 BXI 72 activates caspases and induces PA RP cleavage Treatment of human lung cancer cells with BXI 72 results in activation of caspases in H358 cells and cleavage of PARP in H 6 9 cells Cells were treated with indicated concentration of BXI 72 for 24h. A) Activation of caspases in H358 cells B) Cleavage of PARP in H69 cells.

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57 Fig u re 2 1 3 Maximum tolerated dose of BXI 72 Nu/Nu nude mice were tested with increasing doses of BXI 72 for 25 days (6 mice each dose). BXI 72 was administered by i.p. (0, 10 20, 30, 40 and 50 mg/kg/d) for 25 days to determine the toxicity and administration dose of BXI 72 for lung cancer for xenograft models Percentage of survival of mice was calculated.

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58 Fig u re 2 1 4 BXI 72 potently repress es lung cancer growth in vivo A) Nu/Nu mice with H1299 lung cancer x enog raft s were treated with increasing doses (0 10, 20, and 30mg/kg/d) by i.p. for 14 days. Each group includes 8 mice. Tumor volume was measured once every 2 days. After 14 days, the mice were sacrificed and the tumors were removed and analyzed. Size bar rep resent s 10mm. B) Active caspase 3 was analyzed in tumor tissues at the end of C) TUNEL positive cells were counted at 400X magnification from three different fields of three independent tumor samples.

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59 Figure 2 14. continued.

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60 Figure 2 1 5 Analysis of BXI 72 toxicity in vivo A) Body weight of mice was measured once every other day during treatment with various doses of BXI 72 B) Blood analysis of mice after treatment with various doses of BXI 72. C) H&E histology of various organs from mice after treatment with various doses of BXI 72.

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61 Figure 2 1 5 continued.

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62 Figure 2 1 6 BXI 61 repress es lung cancer growth in vivo A) Nu/Nu nude mice with H1299 lung cancer xenografts were treated with BXI 61 (40mg/kg/d) by i.p. for 14 days. Each group includes 8 mice. Tumor volume w as measured once every 2 days. After 14 days, the mice were sacrificed. Tumors were then removed with picture taken. B) Body weight of mice was measured once ev ery other day during the treatment with BXI 6 1 (40mg/kg/d Size bar represents 10mm )

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63 CHAPTER 3 EFFECT OF BXI ON ACQUIRED RADIORESISTANT LUNG CANCER Background Radiation therapy is frequently used in the lung cancer treatment, either alone or in combinati on with surgery and/or chemotherapy ( Lyng et al., 2005 ) Ionizing radiation (IR) induces cell cycle arrest and ap optotic cell death ( Maity et al., 1994 ) Apoptotic cell death is caused mainly by IR induced DNA double strand breaks. In response to DNA damage induced by IR, autophosphorylated ataxia telangiectasia m utated (ATM) phosphorylates H2AX ( i.e H2AX), leading to recruitment of DNA repair complexes at double strand break (DSB) site ( Canman and Lim, 1998 ) During the cell cycle arrest, cell proliferation is resumed after the DNA damage is re paired at lower radiation does At higher radiation doses, apoptotic cell death occurs. However, r eiterative radiation therapy used alone or used in combination with other therapies might cause IR resistant tumors ( Nieder et al., 2000 ) Up regulation of Bcl XL and Bcl2 in IR resistant cells was associated with IR resistance ( Chao et al., 1995 ; Datta et al., 1995 ; Nelyudova et al., 2007 ) and the expression level of Bcl XL strongly correlates with resistance against chemotherapeutic agents ( Amundson et al., 2000 ) These elevated expressions of anti a poptotic Bcl2 family proteins have been observed in lung cancers and associated with radioresistance and chemoresistance ( Amundson et al., 2000 ; Haura et al., 2004 ) leading to poor prognosis Therefore, an attractive approach for anti cancer therapeutics is to overcome this inherent resistance against apoptosis by directly activating the core apoptotic cell death machi nery and Bcl XL may be a therapeutically potential target protein for developing anti cancer drugs treating human lung cancer.

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64 Results BXI reverses radio resistance and restore radiation sensitivity of lung cancer cells We established IR resistant lung can cer cell lines to determine if BXIs overcome IR resistance established by long term exposure of lung cancer cells to IR. To determine whether Bcl2 or Bcl XL contributes to acquired resistance to radiation, we established an A549 cell line with acquired re sistance to ionizing radiation ( i.e. A549 Chapter 5; Materials and ( Lee et al., 2010 ) Increased levels of Bcl XL were observed in A549 IRR cells as compared to A549 P cells (Fig ure 3 1A ). Similar findings were also observed in other lung cancer cells ( i.e H157 P/H157 IRR and H358 P/H358 IRR, Fig ure 3 1A ). A fter multiple IR exposures to A549 P H157 P and H358 P cells at 2Gy, Bcl XL p rotein was significantly up regulated in A549 IR R (139.24%), H157 IR R (130.43%) and H358 IR R cells (218.33%), respectively In addition to Bcl XL, Bcl2 protein was also increased about 2 fold in H157 IR R and H358 IR R cells (Fig ure 3 1 A). Importantly, par ental A549 (A549 P) cells remained sensitive but A549 IRR became insensitive to IR (Fig ure 3 1B and C ). These results provide strong evidence that IR enhanced Bcl XL contributes to acquired radioresistance. Intriguingly, both A549 P and A549 IRR cells re mained sensitive to BXI 61 and BXI 72 (Fig ure 3 1B and C ), suggesting that BXIs are able to overcome acquired radioresistance through their suppression of Bcl XL. BXI overcomes acquired radioresistance of lung cancer in animal models To further test wheth er BXI overcomes radioresistance in vivo NSCLC xenografts derived from A549 P and A549 IRR cell lines were treated with IR (2Gy 5) BXI 72 (20mg/kg/d 2 1) alone or in combination as indicated. Mice were treated with vehicle

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65 ( 0.5% DMSO, 100 L /d, i.p.) I R (2Gy/every two days at a rate of 0.8Gy/min, 5times), BXI 72 (2 0mg/kg/d, i.p ) or combination (IR and BXI 72). We observed that lung cancer xenografts derived from A549 IRR cells were resistant to IR treatment whereas xenografts derived from A549 P were s ensitive to IR treatment ( Figure 3 1D ). Consistent with the in vitro observation, BXI 72 repressed tumor s derived from both A549 P and A549 IRR cells for 3 week treatment indicating that BXI 72 can overcome acquired radioresistance in vivo Other than a slight decrease in WBC count in IR treated mice (Figure 3 2B) there were no significant normal tissue toxicities ( Figure 3 2 ). Discussion Although a combination of chemotherapy and radiotherapy is the standard treatment for lung cancer, the resistance to radiotherapy remains concerned due to the diversity of molecular abnormalities in lung cancer cells ( Sacco et al., 2011 ) Here, we established ionizing radiation (IR) resistant cell lines in order to test whether BXIs can overcom e the resistance to radiation therapy. Interestingly, we observed that Bcl XL and Bcl2 were increased in IR resistant cell lines. Therefore, we expected BXIs could effectively inhibit the growth of IR resistant cells because BXIs showed the potency in Bc l XL dependent manner. As we expected, BXIs potently overcome IR resistance whereas cytotoxic activity of cisplatin against IR resistant lung cancer cells decreased about 3 fold. Although the mechanisms of radioresistance are not regulated by a single pr otein ( Biard et al., 1994 ; Brachman et al., 1993 ) up regulation of anti apoptotic proteins such as Bcl XL and Bcl2 proteins in response to IR as shown in this study may contribute to resistance to IR.

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66 Tumor repression was observed after 2 1 day BXI 72 treatment. In vivo results suggest that BXI 72 overcame the acquired radioresistance resulting in tumor repression. Additionally, indicated concentrations of BXI 72 treatment did not cause weight loss or any other side effects It is known that WBCs contain nucleus but the nature red blood cells do not have nucleus. IR causes a reversible decrease in white blood cell s (WBCs). We previously discovered that Bcl2 and Bcl XL negatively regulate cell cycle by inhibiting G1/S transition ( Deng et al., 2003 ) Inhibition of Bcl XL by BXI may promote normal WBC proliferation. This may help explain why BXI 72 partially reverses IR reduced WBCs (Figure 3 2B). Collectively, our studies suggest that BXIs are novel Bcl XL specific inhibitors inducing lung cancer cell death both in vitro cytotox icity assays and in vivo animal models via apoptosis.

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67 Figure 3 1. BXI 72 overcomes radioresistance of lung cancer in vivo A) Expression levels of Bcl XL, Bcl2, Mcl 1, Bak and Bax in lung cancer parental cells (A549 P, H157 P and H358 P) and irradiat ion resistant cells (A549 IRR, H157 IRR and H358 IRR) were analyzed by Western blot. B) and C) A549 P and A549 IRR cells were treated with IR, BXI 61 or BXI 72 as indicated. Cell growth was analyzed by B) colony formation assay after 10 days or C) SRB assa y after 72h. D) Mice bearing A549 P or A549 IRR lung cancer xenografts were treated with IR, BXI 72 or th eir combination as indicated for 21 days. Each group includes 8 mice. Tumor volume was measured once every 2 days. After 21 days, the mice were sacrifi ced and the tumors were removed and ana ly zed

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68 Figure 3 1. Continued.

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69 Figure 3 2 Analysis of toxicity for combination of BXi 72 and IR in vivo A) Body weight of mice with A 5 49 P or A549 IRR xenografts was measured once ever y other day during treatment with 0.5% DMSO (control), IR (2Gy 5), BXI 72 (20mg/kg/d) or their combination for 21 days B) Blood analysis of mice after treatment with 0.5% DMSO (control), IR (2Gy 5), BXI 72 (20mg/kg/d) or their combination. C) H&E hist ology of various organs after treatment with 0.5% DMSO (control), IR (2Gy 5), BXI 72 (20mg/kg/d) or their combination. H&E h istological images are representative for 8 mice per group.

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70 Figure 3 2. Continued.

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71 CHAPTER 4 DISCUSSION The targeting BH3 domain in Bcl2 family proteins to treat cancers is a strategy that is presently being explored in the development of Bcl2 inhibitors as anticancer drugs ( Chongha ile and Letai, 2008 ; Kang and Reynolds, 2009 ) By binding to the hydrophobic cleft of Bcl2/Bcl XL, the BH3 mimetics function as competitive inhibitors ( Chonghaile and Letai, 2008 ; Oltersdorf et al., 2005 ) Three small molecule Bcl2 inhibitors, including gossypol (AT 101), obatoclax (GX15 070) and ABT 263, are in clinical trials (Phase I~II) ( Chonghaile and Letai, 2008 ; Kang and Reynolds, 2009 ) Gossypol and obatoclax are BH3 mimetics that act as pan Bcl2 inhibitors ( Kang and Reynolds, 2009 ; Nguyen et al., 2007 ; Reed, 2003 ) However, gossypol and obatoclax are not entirely dependent on Bax and Bak for apoptosis induction and show toxicity to normal cells ( Chonghaile and Letai, 2008 ; Konopleva et al., 2008 ) In contrast, the Bad BH3 mimetic ABT 737 was ineffective in inducing apoptosis in cells doubly deficient in Bax and Bak, indicating th at its mechanism of action may be predominantly through the Bcl2 family ( Chonghaile and Letai, 2008 ; van De lft et al., 2006 ) ABT 737 selectively binds to Bcl2, Bcl XL and Bcl W but not Mcl 1 and A1 ( Oltersdorf et al., 2005 ) However, ABT 737 resistance can be cau sed by expression of Mcl 1 and Bcl XL ( Konopleva et al., 2006 ; Lin et al., 2007 ; van Delft et al., 2006 ; Vogler et al., 2009a ) loss of Bax or Bak or reduction of BH3 only proteins ( Deng et al., 2007 ) Here we chose the BH3 domain of Bcl XL (aa 90 LREAGDEFE 98) as a docking site for screening of small molecules that may inactivate Bcl XL using the UCSF 6.1 DOCK program suit e and a database of small molecules from the NCI as d escribed ( Jaiswal et al., 2009 ) We found two new Bcl XL inhibitors (BXI 61 and BXI 72) that preferentially bind to Bcl XL with inhibitory constant ( K i) values

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72 a t nanomolar levels (Fig ure 2 4 ). These two compounds exhibited significantly lower binding affinity for Bcl 2 (Fig ure 2 4 ), indicating a more selective binding to Bcl XL. This is especially important because Bcl XL is more widely expressed in NSCLC and SC LC cells than Bcl 2 (Fig ure 1 3 ) ( Hann et al., 2008 ; Li et al., 2008 ) A relatively high dose of ABT 737 ( i.e. Bcl2 inhibi tor) is required to effectively inhibit the growth of lung cancer cells expressing low levels of endogenous Bcl2 and high levels of endogenous Bcl XL ( Hann et al., 2008 ; Oltersdorf et al., 2005 ; Tahir et al., 2007 ; Vogler et al., 2009a ) By contrast, our new Bcl XL in hibitors ( i.e BXI 61 and BXI 72) showed superior efficacy to ABT 737 against lung cancer cells that express high levels of endogenous Bcl XL (Fig ure 2 2 ) Consistent with our discovery approach, BXI repression of lung cancer growth occurs in a Bcl XL dep endent manner since depletion of Bcl XL significantly reduces sensitivity of lung cancer cells to BXI (Fig ure 2 10 ). Importantly, BXI 72, which showed a stronger Bcl XL binding affinity ( Ki 0.9 0.15 nM), also displayed greater cytotoxicity against huma n lung cancer cells as compared to BXI 61 ( Ki 14.8 1.54 nM) (Fig ure 2 2 ). This thus suggests that the anticancer potency of this new class of agents may be dependent on their Bcl XL binding affinity. A distinctive feature of Bcl2 family proteins is that they interact with one another to form heterodimers or homodimers through the Bcl2 homology (BH) domain s ( Cosulich et al., 1997 ; Ottilie et al., 1997 ; Sattler et al., 1997 ) Anti apoptotic Bcl XL preferentially interacts with Bak and forms a heterodimer that inhibits the pro apoptotic function of Bak ( Sattler et al., 1997 ) Bak is thought to drive apoptosis by forming homo oligomers that permeabilize mitochondria ( Dewson et al., 2008 ) This homo oligomerization of Bak is essential for activation of its pro apoptotic function. Oligomerization involves

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73 insertion of the BH3 domain of one Bak molecule into the groove of another and may p roduce symmetrical Bak dimers. Our results reveal that treatment of lung cancer cells with BXI 72 resulted not only in dissociation of Bak/Bcl XL complexes but also in Bak oligomerization and c yt c release (Fig ure 2 11 ), suggesting that the binding of BXI with Bcl XL could disrupt Bcl XL/Bak heterodimers and subsequently facilitate Bak activation via its homo oligomerization. This may be a key mechanism by which BXI activates apoptosis leading to cell death and tumor regression in vitro and in vivo respe ctively. This can also help explain why BXI suppresses lung cancer growth in a Bcl XL dependent manner (Fig ure 2 10 ). The anti tumor activity of BXI in vivo was evaluated in lung cancer xenografts. Both BXI 61 and BXI 72 potently repressed lung cancer in animal models (Fig ure 2 14 and Figure 2 16 ). We determined the MTD of BXI 72 with a 25 day treatment to be between 30~40mg/kg/d (Fig ure 2 13 ). Dose response experiments indicated that doses of BXI 72 between 20 and 30mg/kg/d potently repress lung cancer in vivo without normal tissue toxicity (Fig ure 2 14 and Figure 2 15 ), indicating that these doses should be effective and safe for further characterization of this compound in murine lung cancer models. Since a dose dependent increase of apoptosis in tumo r tissues was observed in the BXI treatment group, this suggests that repression of lung cancer by BXI occurs through induction of apoptosis in tumors (Fig ure 2 14B ). Radiotherapy is a major therapeutic intervention for patients with lung cancer and is adm inistered to up to 75% of lung cancer patients during the course of their disease ( Coate et al., 2011 ) A major challenge affecting outcomes of patients with lung cancer is the development of acquired radioresistance. To test whether BXI could overcome

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74 radioresistance of lung cancer, we established acquired radiation resistant lung cancer cell model systems (Fig ure 3 1 ). Elevated levels of the anti apoptotic prote ins Bcl XL and Bcl2 but not Mcl 1 were observed in ionizing radiation resistant cells ( i.e A549 IRR vs. A549 P, H157 IRR vs. H157 P, H358 IRR vs. H358 P) (Fig ure 3 1A ), suggesting that this upregulation could, at least in part, contribute to radioresistan ce Intriguingly, the BXI lead compound not only reversed radiation resistance in vitro but also overcame radiation resistance in vivo at a relatively low dose (i.e 20mg/kg/d), leading to the effective suppression of lung cancer xenografts that were resis tant to radiation (Fig ure 3 1 ). Mice tolerated the combination treatment with BXI and IR well without significant normal tissue toxicities except for a reversible decrease in white blood cells that resulted from radiotherapy (Fig ure 3 2 ). In summary, we h ave discovered new Bcl XL inhibitors (BXI 61 and BXI 72) that specifically bind the BH3 domain pocket of Bcl XL, disrupt Bcl XL/Bak heterodimerization, and facilitate Bak homo oligomerization leading to Bak activation and apoptosis in lung cancer cells. T hese lead compounds have potent activities against lung cancer in vitro and in vivo and potentially offer superior efficacy over the BH3 mimetic ABT 737 in lung cancer therapy. The increased levels of Bcl XL in lung cancer cells with acquired radioresist ance make Bcl XL an ideal target for overcoming radioresistance. Since our findings demonstrate that BXI can overcome acquired radioresistance of lung cancer in vitro and in vivo a combination of BXI with IR may represent an effective new strategy for th e treatment of lung cancer, including those patients who are resistant to radiotherapy, leading to long term tumor free survival.

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75 CHAPTER 5 MATERIALS AND METHOD S Materials Small molecules, including NSC354961 (BXI 61) and NSC334072 (BXI 72), were obtain ed from the Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (NCI Bethesda, MD ) ( http://dtp.nci.nih.gov/Reque stCompounds ). ABT 737 control shRNA, Bcl XL shRNA and antibodies ( i.e. PARP, Bax, Mcl 1, cytochrome c and Actin) were purchased from Santa Cruz (Santa Cruz, CA). Bcl2 antibody was purchased from Calbiochem (Darmstadt, Germany). Bcl XL and Bak antibodi es were purchased from Epitomics (Burlingame, CA). NanoJuice TM .Trasfection Kit was obtained from Novagene (Madison, WI). Active caspase 3 specific antibody was purchased from Cell Signaling Technology. Fluorescent Bak BH3 domain peptide (FAM GQVGRQLAIIG DDINR) and purified Bcl XL protein were purchased from NeoBioSci TM (Cambrige, MA). Purified Bcl2 protein was obtained from ProteinX Lab (San Diego, CA). Bis (maleimido) hexane (BMH) was purchased from Thermo Scientific (Rockford, IL). All other reagents used were obtained from commercial sources unless otherwise stated. Screening of Bcl XL inhibitors To identify the small molecules specifically targeting Bcl XL, a pproximately 300,000 small molecules from the NCI database were docked into a structural poc ket within the BH3 domain (aa 90 9 8 ) of Bcl XL and two hundred candidates were identified using the DOCK program s (version 6.1, University of California, San Francisco, CA) Subsequently, the se small molecules were screened on A549 and H1299 lung cancer

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76 c ells whether these candidate molecules induce lung cancer cell death using trypan blue exclusion method and Sulforhodamine B colorimetric assays ( SRB ) assays in vitro. Cell lines and cell culture Normal immortalized lung epithelial cell line s (BEAS 2B an d HBEC 3KT) and lung cancer cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA) and grown under standard cell culture conditions. BEAS 2B, HBEC 3KT, DMS53, DMS114, DMS153, H69, H126, H146, H209, A549, H157, H292, H358, H460 H1299, H1792, and H1944 cell lines were maintained at 37C containing 5% CO 2 DMS53, DMS114, and DMS153 were cultured in Waymouth s medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum ( FBS, Atlanta Biologicals Lawrenceville, GA) an d 5% bovine serum (BS, Gibco, Grand Island, NY). HBEC 3KT cells were cultured in Keratinocyte serum free medium (SFM, Gibco, Grand Island, NY) supplemented with 5ng/mL human recombinant epidermal growth factor 1 53 (EGF 1 53) and 50 g/mL bovine pituitary extract. BEAS 2B and A549 were cultured in DMEM/F 12 50/50 medium ( Mediatech, Inc., Manassas, VA ) supplemented with 10% FBS in 100mm plates until they approach confluence. H69, H292, H358, H460, H1299, H1792, and H1944 were cultured in RPMI 1640 medium ( Meidatech, Inc., Manassas, VA ) supplemented with 5% FBS and 5% BS in 100mm plates until they approach confluence. Sulforhodamine B colorimetric assays (SRB) To evaluate the cytotoxicity of BXIs SRB assay measuring cellular protein content was performed ( Vichai and Kirtikara, 2006 ) Briefly, cells (4000cells/well) were seeded in a volume of 100l/well in 96 well plates. After overni ght incubation, cells were treated with BXI 61, BXI 72, ABT 737, or cisplatin at various concentrations for 72 hours. Cells

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77 were fixed by adding 100l cold 10% TCA for 60 min at 4C after discarding the medium. Plates were washed 5 times with deionized w ater and air dried. 50l of 0.4% SRB solution in 1% acetic acid was added to each well and incubated for 10min at room temperature. Plates were washed 5 times with 1% acetic acid and air dried. 100l of 10mM unbuffered Tris Base (pH10.5) was added into each well to solubilize the bound dye and mixed for 5min on microtiter plate shaker. OD was read at a single wavelength 510 nm. Colony formation assay Cells (A549, H460, or H1299 single cell suspension) were plated in 6 well plates at a density of 500 per well as described ( Wang et al., 2008 ) On next day, cells were treated with BXI 61, BXI 72, ABT 737, ABT 263, cisplatin or erlotinib at various concentrations. T he medium was replaced with fresh medium containing the corresponding concentration of the potent compounds e very three days After 10 day treatment, the medium was removed and cell colonies were stained with crystal violet (0.1% in 20% methanol) and counted Analysis of apoptotic cell death A poptotic cells were assessed using an ApoAlert Annexin V kit (Clontech, Palo Alto, CA) detecting the exposed re distribution of phosphatidylserine (PS) to external surface of the plasma membra n e in apoptotic cells during early apoptosis according to ( Martin et al., 1995 ) The percentage of annexin V low cells (percentage of viable cells) or annexin V h igh cells (percentage of apoptotic cells) was determined by fluorescence activated cell sorter analysis (FACS).

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78 Fluorescence polarization assays Fluorescent Bak BH3 domain peptide and Bcl XL protein were purchased from NeoBioSci TM (Cambridge, MA). To meas ure the binding affinity of BXI to Bcl XL protein, a competition fluorescence polarization assay was employed as previously described ( Bruncko et al., 2007 ; Wang et al., 2000 ; Zhang et al., 2002 ) Fluorescent Bak BH3 domain peptide (3nM) was incubated with purified, human Bcl XL protein (6nM) in the absence or presen ce of increasing concentrations ( i.e 0.1~500nM) of BXI (s) in the binding affinity assay buffer (50mM Tris (pH8 .0 ), 150mM NaCl, 0.1% BSA, and 5mM DTT) in a 96 well assay plate. The plate was mixed on a shaker for 1 min and incubated at room temperature f or an additional 15 min. Polarization, defined as millipolarization units (mP), was measured at room temperature with a fluorescence microplate reader at 48 5 /5 3 0nm (Gemini XPS TM Molecular Devices, CA). A negative free peptide control (DMSO, 3nM peptide and assay buffer) and a positive bound peptide control (DMSO, 3nM peptide, 6nM Bcl XL and assay buffer) were used to determine the range of the assay. The percentage of inhibition was determined by the equation: 1 (mP value of well negative control)/ra nge) 100%. Inhibitory constant ( K i) value was determined by the formula: Ki = [ I ] 50 /([ L ] 50 / Kd + [ P ] 0 / Kd + 1) as described ( Bruncko et al., 2007 ; Nikolovska Coleska et al., 2004 ) Reported values are the mean S.D. for three separate experiments run in duplicate. Western blot analysis Cell lysates were prepared using EBC buffer (50mM Tris (pH 7.6), 120mM NaCl, 1mM EDTA, 1mM Na 3 VO 4 mercaptoethanol, 0.5% NP 40, and 100x protease inhibitors (10l/ml, added right before use)). Proteins were separated on

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79 12% or 15% SDS PAGE gels and transferred to nitrocellulose membranes (0.34m, GE ) Membranes were incubated overnig ht at 4C with primary antibodies. Protein expression analysis was performed using appropriate secondary horseradish peroxidase (HRP, Santa Cruz) conjugated antibodies and enhanced chem o luminescence (ECL, GE Healthcare, Little Chalfont, UK ) M embranes we re exposed to blue sensitive medical x ray film (HPI International Inc., Brooklyn, New York). Immunoprecipitaton assays Bcl XL/Bak co immunoprecipitations were performed in H1299 cells. After treating cells with drugs for 24h, cell lysates were prepared u sing Tris based lysis buffer (50mM Tis (pH7.5), 150mM NaCl, and 0.25% NP 40) and protease inhibitors. To immunoprecipitate endogenous Bcl XL and Bak in H1299 cells, immunoprecipitation was performed with a monoclonal Bcl XL antibody and protein A (Invitro gen Carlsbad, CA ) (overnight at 4C). Immunoprecipitates were subjected to 12% SDS PAGE gels and Bak was detected by immunoblot analysis as describe above. RNA interference, plasmids and transfection shRNAs against human Bcl XL were purchased from Santa Cruz (sc 43630 SH). Corresponding siRNA sequences: Sense: 5 GAC AAG GAG AUG CAG GUA Utt 3 ; Antisense: 5 AUA CCU GCA UCU CCU UGU Ctt 3 H1299 cells were stably transfected with control shRNA plasmids or human Bcl XL shRNA plasmids using NanoJuice TM Tra nsfection Kit (Novagen, Madison, WI) according The expression level of Bcl XL was analyzed using western blotting to confirm Bcl XL down regulation. To overexpress Bcl XL proteins in lung cancer cells, pSFFV neo or Bcl XL/ pSFFV plasmids were stably transfected into

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80 H460 cells using NanoJuice TM and the expression level of Bcl XL was analyzed using western blotting to confirm Bcl XL up regulation Human Bcl XL cDNA was ki ndly provide d from Dr. Steven J. Weintraub (Washington University, Saint Louis, MO). Bak oligomerization and Cytochrome c release Subcellular fraction was performed as previously described ( Jin et al., 2004 ) For cytochrome c release, H1299 cells were treated with BXI 72 at various concentrations and washed with cold 1X PBS, resuspended in isotonic mitochondrial buffer (210mM mannitol, 70mM sucrose, 1mM EGTA, and 10mM Hepes (pH7.5)) containing protease inhibitors, and then homogenized with a Polytron homogenizer, operating for six bursts of 10sec each at a setting of 5. The total cell lysate was centrifuged at 200Xg to pellet nuclei. The 1 st supernatant was centrifuge d at 150,000Xg to pellet mitochondrial fraction. The 2 nd supernatant containing cytosolic fraction and mitochondrial fraction were subjected to SDS PAGE gels and analyzed by western blotting using a cytochrome c antibody to detect cytochome c release into cytosol from mitochondria. 2 .8 mM B is(maleimido)hexane (BMH, Thermo Scientific, Rockford, IL) was added into mitochondrial fraction dissolved in crosslinking buffer ( 20mM HEPES (pH7. 5 ) 100nM Sucrose, 2.5mM MgCl 2 and 50mM KCl ) for crosslinking between sul fhydryl groups of Bak proteins. Reaction mixture was incubated for 1h at room temperature Reaction was stopped by adding quench solution (1M DTT) for 15min at room temperature Reaction was subjected to SDS PAGE gels and analyzed by western blotting us ing a Bak antibody to detect Bak oligomerization.

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81 Lung cancer xenografts and treatments Six week old female Nu/Nu Nude mice were purchased from Harlan Sprague Dawley, Inc. and hosted in the pathogen free conditions in microisolator cages. A ll a nimal treat ments were undertaken in accord ance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University. 3x10 6 H1299 cells in Hanks' Balanced Salt Solution (HBSS, Gibco) were injected intradermally into leg region of nud e mice to produce subcutaneous (s.c.) tumors. The tumors were allowed to grow to an average volume of ~250mm 3 prior to initiation of therapy as described ( Olters dorf et al., 2005 ) Mice were treated with BXI 72 or BXI 61 i.p. as indicated. BXI 72 was administered intraperitoneally (i.p.) once a day with 10 30mg/kg/day for 14 days. Mice were randomized into four groups (n=8 per group) as follows: (1) control (0 .5% DMSO, 100 L /d, i.p.); (2) BXI 72 10mg/kg/d, i.p.; (3) BXI 72 20mg/kg/d, i.p.; (4) BXI 72 30mg/kg/d. i.p. Mice were weighed and tumor s were measured by the digital caliper (Sigma) every two days. Tumor volume (V) was calculated with the following form ula: V=LxW 2 /2 (L: length; W: width) as described ( Vlahovic et al., 2007 ) After t he mice were euthanized at the indicated times tumors and organs were fixed in 4% formalin, embedded in paraffin, and stained with hematoxylin and eosin ( H&E). Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assays (TUNEL) For TUNEL assays, tumors were harvested and embedded in paraffin. Sections of paraffin e mbedded tumor tissues were labeled using TumorTACS TM In Situ Apoptosis Detection Kit (Trevigen, Inc., Gaithersburg MD) according to protocol to detect TUNEL positive cells. TUNEL positive cells were counted at 400X

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82 magnification. The average number of TUNEL positive cells was determined from three separated fields of three independent tumor samples. Immunohistochemistry (IHC) analysis Mice with established H1299 tumors were treated with BXI 72 (10 30mg/kg/d) for two weeks. Tumors were harvested, fixed in formalin and embedded in paraffin. Representative sections from paraffin embedded tumor tissues were analyzed by IHC staining using an active caspase 3 specific antibody. Active caspase positive cells in tumor tissues were scored at 4 00 X magnification. The average number of positive cells per 0.0625mm 2 area was determined from three separate fields in each of three independent tumor samples as described ( Oltersdorf et al., 2005 ) Blood analysis for mice Whole blood (250M) was collected in EDTA coated tubes via cardiac puncture of anesthetized mice for hematology studies. Specimens were analyzed for white blood cell (WBC), red blood cell (R BC), and Platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in Clinical Pathology Laboratory, University of Georgia ( Athens, GA ). For serum chemistries, whole blood (250M) was centrifuged at 1 5,000g for 2 min to separated serum from cells and fibrin after collecting whole blood in EDTA coated tubes. E stablishment of ionizing radiation resistant (IRR) cell line We chose A549, H157, and H358 To induce IR resistant cell lines, A549, H157 and H358 cell line s to establish ionizing radiation resistant lung cancer cell lines (A549 IRR, H157 IRR, and H358 IRR) as described ( Lee et al., 2010 ) Briefly, A549, H157, a nd H358 cells (1x10 6 ) were serially irradiated with 2Gy of X rays to a final dose of 100 Gy using X RAD 320 (Precision X ray Inc., North Branford, CT) at a rate of

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83 0.5Gy/min Culture medium was renewed immediately after each dose of radiation. After cells were allowed to grow to approximately 90% confluence, cells were trypsinized and passaged into new culture dishes. Re treatment of the newly passaged cells with 2Gy of X rays occurred at about 60% confluence and this was repeated 50 times over a period o f 5 months, for a total dose of 100Gy. The parental cells (A549 P, H157 P, and H358 P) were trypsinized, counted, and passaged under the same conditions without ionizing irradiation as described ( Lee et al., 2010 ) IR resistant l ung cancer xenografts and treatments Six week old male Nu/Nu Nude mice were purchased from Harlan Sprague Dawley, Inc. and hosted in the pathogen free animal facility at Winship Cancer Institut e, Emory University. Animal treatments were done according to institution approved protocols. For determining if BXI 72 overcomes IR resistance in vivo s.c. tumors were established by injecting 3x10 6 A549 P or A540 IR R cells in HBSS into the dorsal flan k area of nude mice. Treatments were initiated when tumors reached between 2 00 and 3 00mm 3 Mice were randomized into eight groups (n=6 per group) and treated with vehicle (0.5% DMSO, 100 L/d, i.p.), IR (2Gy/every two days at a rate of 0.8Gy.min, 5 times) BXI 72 (20mg/kg/d, i.p.), or combination (IR and BXI 72). For radio therapy, mice with A549 P or A549 IRR xenografts were irradiated with 2Gy every other day for 5 treatments using X RAD 320 irradiator (Precision X ray Inc., North Branford, CT ) to deliv er whole body irradiation to the mice at a rate of 0.8Gy per minutes as described ( Konstantinidou et al., 2009 ) During the treatment, tumor volume (V) was measured by caliper measurements once every two days and calculated with the formula: V=LxW 2 /2 (L: length; W: width) as described ( Vlahovic et al., 2007 ) Mice were sacrificed by inhale

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84 CO 2 at the end of treatment. Harvested tumors and organs were fixed in 4% formalin, embedded in paraffin, and stained with hematoxylin and eosin ( H&E). Statistical analysis The statistical significance of differences between g roups was analyzed with two sided unpaired student s t test Results were considered statistically significant at P<0.05. The IC 50 values were calculated using SPSS Statistics software 18 (IBM). All data are presented as mean sta ndard deviation (S.D.).

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96 BIOGRAPHICAL SKETCH After graduating from high school in 1992, Dongkyoo Park entered the Dongguk University in Seoul where he majored in Applied Biology. During his undergraduate study, Dongkyoo Park joined the laboratory of m icrobiology under Dr. Min Woong Lee s mentoring for four years. And he earned his Bachelor of Science degree in A pplied B iology in 1996. After he graduated, he worked in Research Institute for Natural Sciences (RINS), Dongguk University, Seoul, Korea and Clinical Research Institute (CRI), Seoul National University Hospital, Seoul, Korea. Dongkyoo Park was admitted by the Interdisciplinary Program (IDP) in Biomedical Scien ces in College of Medicine at the University of Florida, Gainesville, FL in 2005. He Medicine, Shands Cancer Center, University of Florida from November 2007 to August 2 009. In 2009, Dr. Deng accepted new position in Winship Cancer Institute, Emory University, Atlanta, GA. To continue his P h D dissertation project, he also joined Emory University with Dr. Deng star t ed from August 2009. instruction s, he h as d iscovered two novel Bcl XL inhibitors (BXIs), targeting BH3 domain of Bcl XL, which can induce apoptosis in various human cancer cells and repress lung cancer growth in xenograft animal models. In addition, he has identified that BXIs can overc ome acquired radioresistance of lung cancer both in vitro and in vivo Based on his research findings, these two compounds have great potential to be developed as a new class of anti cancer drugs. He will complete his Ph.D. degree program in Medical Scie nces Molecular Cell Biology in August of 201 2