STRUCTURE-BASED ANTI-CANCER DRUG DEVELOPMENT BY TARGETING BCL-XL

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 2012 1

2012 Dongkyoo Park 2

To my parents for your love and encouragement 3

ACKNOWLEDGMENTS This Ph.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 produce 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 my Ph.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 assistance and concern on handling all administrative works for my Ph.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 thank 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 quest for scientific knowledge 4

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

TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES... 8 LIST OF FIGURES... 9 LIST OF ABBREVIATIONS... 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 disruption 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 acquired radioresistance of lung cancer in animal models.... 64 Discussion... 65 4 DISCUSSION... 71 6

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 Western 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 deoxynucleotidyl transferase mediated dutp nick end labeling assays (TUNEL)... 81 Immunohistochemistry (IHC) analysis... 82 Blood analysis for mice... 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 7

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

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 lines... 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 cytotoxicity 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 Treatment 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 9

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

LIST OF ABBREVIATIONS A1 AIF Bcl2 related protein A1 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 BH domains BH3 domain Bid Bik Bim Bok BXI Caspases DISC FADD FasL IR NSCLC B-cell lymphoma-extra large Bcl2 homology domains Bcl2 homology 3 domain BH3 interacting domain death agonist Bcl2 interacting killer Bcl2 interacting mediator of cell death Bcl2 related ovarian killer protein Bcl-XL inhibitor Cysteine-dependent aspartate specific proteases Death inducing signaling complex Fas-associated death domain protein Fas ligand Ionizing radiation Non-small cell lung cancer Mcl-1 Myeloid cell leukemia sequence 1 MOMP Puma Mitochondrial outer membrane permeabilization p53 upregulated modulator of apoptosis 11

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

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STRUCTURE-BASED ANTI-CANCER DRUG DEVELOPMENT BY TARGETING BCL- XL By Dongkyoo Park August 2012 Chair: Xingming Deng Major: Medical Sciences-Molecular Cell Biology 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- 13

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

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 main 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 Pershagen, 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 lungs by the cumulative exposure to these risk factors may become cancerous over a long 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 subtype of SCLC. 15

These 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. Treatments 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 lungs and its near tissue primarily 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 lung 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 whether lung tumor is localized and can be removed 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 chemotherapeutic 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 for lung cancer treatment. Radiotherapy is often given with chemotherapy. External beam radiation is the most common type of 16

radiotherapy for lung cancer. The amount of radiation varies depending on stage of lung caner. Lung cancer patients are usually treated for 5 days a week for several weeks. Apoptosis 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 term apoptosis originated from Greek means to fall away from. The first morphological description of apoptotic cell death was provided by Walter Flemming in 1885. Apoptosis results in cell volume reduction, membrane blebbing, chromatin condensation 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 fundamental 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 pathway (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 highly 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 17

and a component 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 (Brune 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 mitochondrial intermembrane space. This critical step in apoptosis is the mitochondrial outer membrane permeabilizaltion (MOMP), which is induced 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 procaspase 9 and then this initiator caspase can trigger a cascade of events, leading to apoptosis (Hengartner, 2000; Ow et al., 2008; Taylor et al., 2008) In contrast to intrinsic pathway, extrinsic apoptotic pathway is triggered by the binding of extracellular ligands such as Fas ligand (FasL), tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or TNFα and transmembrane death receptor such as Fas, TRAIL-R or TNF-R1. This ligation induces receptor clustering and the assembly of the death inducing signaling complex (DISC) and then the DISC can recruit multiple procaspase 8 via Fas-associated death domain protein (FADD) (Wajant, 2002). Active 18

capase 8 can either directly activate downstream caspases such as pro-capsase 3 or engage the intrinsic pathway via truncated 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 critical step in tumor development and renders the tumor cells more resistance to conventional cytotoxic therapy. Despite the frequent dysregulation of apoptosis in tumors, nearly all tumors maintain the core apoptotic regulatory machinery: Bcl2 family proteins, cyt c, caspases, etc. Among these components of the apoptotic machinery, overexpression of antiapoptotic Bcl2 family proteins such as Bcl2, Bcl-XL and Mcl-1 plays critical roles in mediating resistance to 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 family 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., 2000; 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 therapeutics. Bcl2 Family Regulation of Apoptosis Bcl2 family members are the major regulators of the apoptotic pathway at the decision phase and Bcl2 family proteins share structural and functional characteristics. Bcl2 proteins share conserved Bcl2 homology (BH) domains (i.e. BH1, BH2, BH3 and 19

BH4) (Kelekar and Thompson, 1998). These four conserved 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 activities and all four BH domains are present in anti-apoptotic members (Deng et al., 2006). The proapoptotic Bcl2 family members are divided into subgroups including the multidomain 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, BH2 and BH3 domains and pro-apoptotic activities (Table 1-1 and Figure 1-2). BH3-only proteins have only BH3 domain and pro-apoptotic activities (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, activators such as Bid (BH3 interacting 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 proor anti-apoptotic function (Reed and 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 mitochondria but also found in the cytosol, the endoplasmic reticulum, nuclear 20

envelope and microtubules (Krajewski et al., 1993). The subcellular localizations of Bcl2 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 first 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 proteins 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 hydrophobic 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 21

is wider than in Bcl-XL due to hydrophobic contacts in Bcl-XL side chains (Petros et al., 2004). In addition, 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 proteins. Based on these different electrostatic characteristics, there are Bcl2-selective, Bcl-XLselective, 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., 2008). 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 studies 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 ubiquitin-depedent or ubiquitin-independent degradation (Nijhawan et al., 2003; Stewart et al., 2010). Mcl-1 is also overexpressed in a variety of human cancer cell lines including lung cancer (Placzek et al., 2010). Regulation of Bcl-XL/ Bak Heterodimerization 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 Mcl-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 22

of Bcl-XL and Bak peptide complex suggested that BH3 domain of Bak binds to the hydrophobic 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 Bcl-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 signals, however, Bak undergoes conformational changes and oligomerization, leading to pore formation in the mitochondria and cyt c release into cytosol from mitochondria. The Bak conformational changes are initiated by the BH3- only members of the Bcl2 family 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 Bim 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; Dewson et al., 2008). In the priming-capturedisplacement 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 dimerization and oligomerization. A critical unresolved issue in apoptosis is how Bak converts from the non-activated form into the oligomers inducing MOMP. Dewson et al. (2008) recently showed that BH3 domain of Bak is transiently exposed and then inserted into the hydrophobic 23

groove of another activated Bak monomer to form a dimer in addition to exposure of N- terminal epitopes after apoptotic signaling. This interaction of two activated Bak monomers results in a symmetric dimer (face to face). To form Bak oligomers, dimers associate via crosslinking of cysteine residues placed in their distinct α6 helices (dimer multimerization model). The multiple α6:α6 associations 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). Small Molecules Targeting Bcl2 Proteins Several small molecule BH3 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). Although ABT-737 is considered a potent anticancer drug, cancer cells expressing low levels of endogenous Bcl2 showed dramatically 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 cancers 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 vivo. 24

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, University of California, San Francisco, CA). We termed NSC354961 Bcl- XL inhibitor (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 antitumor efficacy 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 Lung cancer leads the most cause of cancer-related deaths 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 critical 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. Mechanisms involved in this survival function of Bcl-XL have been discerned for novel therapeutic targets. Since Bcl-XL is overexpressed in both SCLC and NSCLC cells (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 development 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 targeting the BH3 domain of Bcl-XL suppress tumor growth through apoptosis by disrupting Bcl-XL/Bak association, activating Bak oligomeriazation and inducing 25

cytochrome c release (Figure 1-4). To reach this goal, we will screen novel Bcl-XL inhibitors (BXIs) using 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. 26

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 only (depressor) Bad, Bik, Puma, Noxa, others 27

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). 28

Figure 1-2. Intrinsic pathways in apoptosis. Intrinsic pathway is initiated by cytochrome c release into the cytosol from mitochondrial intermembrane space in response to a variety of pro-apoptotic stimuli such as DNA damage, hypoxia, growth factor withdrawal, cytotoxic agents and ionizing radiation. 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 Bad 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. 29

Figure 1-3. Expression level 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, respectively. 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. 30

Figure 1-4. Proposed Model. We hypothesized that small molecule drugs targeting 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. 31

CHAPTER 2 DISCOVERY AND CHARACTERIZATION OF NOVEL BCL-XL INHIBITORS (BXI) FOR TREATMENT OF LUNG CANCER Background Overexpression of anti-apoptotic Bcl2 family proteins such as Bcl2, Bcl-XL and Mcl-1 plays critical roles in resistance against apoptosis induced by chemotherapy or radiotherapy (Datta et al., 1995). Elevated expression levels 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 trials 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. 32

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 target Bcl-XL, a library containing approximately 300,000 small molecules from the NCI was used to dock the structural pocket of the BH3 domain (aa90-lreagdege-98; 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 lung 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 Bcl-XL inhibitor 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-737 in human lung cancer cells, A549, H157 and H1299 cells were treated with increasing concentrations (0, 0.5, 1, 2, 5, 10, 20 and 30µM) 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 optimized assay for the toxicity screening of compounds on 33

adherent cells in a 96-well format. Results indicate that BXI-61 and BXI-72 are superior to ABT-737 in suppressing lung cancer cell growth (Figure 2-2). BXI-72 showed the greatest potency based on its low IC 50 concentrations against the tested cell lines (i.e. H1299: 0.94 0.12 µm; A549: 0.68 0.08 µm; Figure 2-2B). Importantly, both BXI-61 and BXI-72 displayed selective cytotoxicity against lung cancer cells compared to transformed immortalized normal human bronchial epithelial cells (BEAS-2B) and nontransformed immortalized normal human bronchial epithelial cells (HBEC-3KT) (Figure 2-3), indicating that these compounds are relatively selective for tumor cells. To directly measure BXI/Bcl-XL binding, we used an in vitro fluorescence polarization assay with a fluorescent 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). We found that BXIs directly bind 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), indicating 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 according to the manufacture s instructions (Martin et al., 1995). As expected, BXI-61 and BXI-72 treatments significantly increased Annexin V positive cells in dose dependent manner (Figure 2-5). In addition, BXI-61 and BXI-72 more effectively inhibit growth of A549 and H1299 cells that ABT-737, ABT-263, ciplatin and erlotinib (Figure 2-6). 34

Interestingly, BXIs showed much higher cytotoxic effects on A549 cells compared to H460 cells and these effects were Bcl-XL expression level dependent manner (Figure 1-3 and Figure 2-7). The complete inhibition of colony formation was observed at 0.5µM of BXI-72 in A549 cells whereas 6.67% inhibition observed in H460 cells (Figure 2-7). There was over 40-fold difference in IC 50 of BXI-72 for H460 (>30µM) and A549 (0.68µM) (Figure 2-7A) determined by SRB assays and 100 times more concentration (10µM) of BXI-72 needed to induce 100% cell growth inhibition of H460 than that of A549 (0.5µM) in colony formation assays (Figure 2-7B). BXI-61 also showed significantly different cytotoxic effect on H460 (IC 50 >30µM) and H1299 (IC 50 =2.31µM) and required 15 times higher concentration (>15µM) 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 Figure 2-10). Up-regulation of Bcl-XL in H460 cells sensitized cells to the cell growth inhibition in response to BXI-61 (IC 50 =25.45µM) (Figure 2-9B) or BXI-72 (IC 50 =8.55µM) (Figure 2-9B) whereas control H460 cells were highly insensitive to BXI-61 and BXI-72 (IC 50 >50µM, BXI-61 and BXI-72; Figure 2-2E and 2-9B) determined by SRB assays. In addition, BXI-61 and BXI-72 inhibited colony formation of Bcl-XL-overexpressed H460 cells whereas control H460 cells were insensitive to BXI-61 and BXI-71 at indicated concentrations (Figure 2-9C). The 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 35

depletion of endogenous Bcl-XL significantly reduced the sensitivity of H1299 cells to BXI-61 and BXI-72 (Figure 2-10B, C), suggesting that Bcl-XL is necessary for the anticancer activity of BXIs. Treatment of human lung cancer cells with BXI results in disruption of Bcl-XL/Bak association, Bak oligomerization and Cyt c release. Bcl-XL forms a heterodimer with Bak through the BH3 domain and suppresses apoptosis (Sattler et al., 1997; Willis 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 homooligomerization 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. 0.1-5µM) for 24h. Co-immunoprecipitation experiments were performed using a Bcl-XL antibody. Results indicated that treatment of cells with BXI-72 resulted in a dose-dependent Bcl-XL/Bak dissociation (Figure 2-11A). To assess whether BXIdissociated 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 as described in Methods. 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 estimated to be multiples of ~28kDa, suggesting the formation of Bak homo-oligomers. It is known that formation of Bak oligomers can result in cyt 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 activation and PARP cleavage in lung cancer cells (Figure 2-11D and Figure 2-12). 36

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 was 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 therefore 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 described (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 cancer xenografts (Figure 2-14A). 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- 37

dependent apoptosis was observed in tumor tissues after BXI-72 treatment (Figure 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 2-15A). There were no significant increases in ALT, AST and BUN or decreases in WBC, RBC, Hb and PLT (Figure 2-15B). Histopathology of harvested 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 (Figure 2-15C). However, treatment of mice with doses of 40-50mg/kg/d for two weeks resulted in increases in ALT, AST and BUN, indicating renal and hepatic toxicities at these higher doses (Figure 2-15B). Elevated liver function test was associated with hepatocellular necrosis in mice treated with 50mg of BXI-72 (Figure 2-15C). These findings 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 xenografts 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 repressed 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 than 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 38

mitochondria-mediated apoptotic mechanism of actions and (v) effectively repress 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; Paoluzzi 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 BH3 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 extensively 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) domains (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 39

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 function of Bcl-XL using computerized UCSF 6.1 DOCK suite of programs. Approximately, 300,000 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 were 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 inhibitors of Bcl-XL (Huo et al., 2002). Here, we report that BXIs specifically bind to 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 previous 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 apoptosis in 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 40

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 tumor repression, TUNEL- and active casepase 3-positive cells 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 effects. 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. 41

Table 2-1. Molecular docking scores of small molecules targeting 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 172971-53.92231 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 34564-48.89887 50 38279-44.10612 42

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. 43

Figure 2-2. BXIs repress human lung cancer cell growth. A) and B) A549, H157 and H1299 cells were treated with increasing concentrations (0, 0.5, 1, 2, 5, 10, 20 or 30µM) 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. 44

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 1µM of BXI-61 or BXI-72 for 72h. Cell growth was analyzed by SRB assay. Error bars represent ± S.D. 45

Figure 2-4. BXI-61 and BXI-72 preferentially bind to Bcl-XL. Fluorescent Bak BH3 domain peptide (3nM) was incubated with purified human 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 fluorescence polarization assay as described in 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 in polarization (mp). 46

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 10µM) for 24h. Apoptotic positive cells are determined by Annexin V assays. Data are mean ± SD from three independent experiments. 47

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

Figure 2-7. BXI-72 shows 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 30µM) for 72 hours. The growth inhibitions of cells were determined using SRB assays. B) The complete inhibition of colony formation was observed at 0.5µM of BXI-72 in A549 cells whereas 6.67% inhibition observed in H460 cells. Data are mean ±SD from three independent experiments. 49

Figure 2-7. Continued. 50

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) Knockdown of Bcl-XL proteins in H1299 cells expressing high relatively high level of Bcl-XL. 51

Figure 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. A) Expression level changes of Bcl2 family proteins after overexpression of Bcl-XL in H460 cells. B) H460 control (closed circle or rectangle) and H460 Bcl-XL overexpression cells (open circle or rectangle) were treated with indicated concentrations of BXI-61 or BXI-72 (0, 0.5, 1, 2, 5, 10, or 30µM) for 72 hours. Cytotoxicity was determined using B) SRB assays or C) colony formation assays. Data are mean ± SD from three independent experiments. 52

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 transfected 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 determined by B) SRB assays or C) colony formation assays. Data are mean ± SD from three independent experiments. 53

Figure 2-11. Treatment of human lung cancer cells with BXI-72 results in Bcl-XL/Bak dissociation, oilgomerization of Bak and Cyt c release. A) H1299 cells were treated with increasing concentrations of BXI-72 for 24h. Coimmunoprecipitation (IP) was performed using a Bcl-XL antibody. Bcl-XLassociated Bak and Bcl-XL were analyzed by Western blot. Normal IgG was used as control for IP. B) and C) H1299 cells were treated as in A). Bak oligomerization and cyt c release were analyzed as described in Methods. Prohibitin was used as a mitochondrial marker. The purity of cytosol fractions was confirmed by non-detectable protein level of prohibitin. D) PARP 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. 54

Figure 2-11. Continued. 55

Figure 2-12. BXI-72 activates caspases and induces PARP cleavage. Treatment of human lung cancer cells with BXI-72 results in activation of caspases in H358 cells and cleavage of PARP in H69 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. 56

Figure 2-13. 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 50mg/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. 57

Figure 2-14. BXI-72 potently represses lung cancer growth in vivo. A) Nu/Nu mice with H1299 lung cancer xenografts 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 represents 10mm. B) Active caspase 3 was analyzed in tumor tissues at the end of experiments by IHC staining and quantified as described in Methods. C) TUNEL-positive cells were counted at 400X magnification from three different fields of three independent tumor samples. 58

Figure 2-14. continued. 59

Figure 2-15. 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. 60

Figure 2-15. continued. 61

Figure 2-16. BXI-61 represses 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 was 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 every other day during the treatment with BXI-61 (40mg/kg/d Size bar represents 10mm). 62

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 combination with surgery and/or chemotherapy (Lyng et al., 2005). Ionizing radiation (IR) induces cell cycle arrest and apoptotic 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 mutated (ATM) phosphorylates H2AX (i.e. γ-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 repaired at lower radiation does. At higher radiation doses, apoptotic cell death occurs. However, reiterative 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-apoptotic 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 machinery and Bcl- XL may be a therapeutically potential target protein for developing anti-cancer drugs treating human lung cancer. 63

Results BXI reverses radioresistance and restore radiation sensitivity of lung cancer cells. We established IR-resistant lung cancer 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 resistance to ionizing radiation (i.e. A549- IRR) as described in Chapter 5; Materials and Methods (Lee et al., 2010). Increased levels of Bcl-XL were observed in A549-IRR cells as compared to A549-P cells (Figure 3-1A). Similar findings were also observed in other lung cancer cells (i.e. H157-P/H157- IRR and H358-P/H358-IRR, Figure 3-1A). After multiple IR exposures to A549-P, H157-P, and H358-P cells at 2Gy, Bcl-XL protein was significantly up-regulated in A549-IRR (139.24%), H157-IRR (130.43%) and H358-IRR cells (218.33%), respectively. In addition to Bcl-XL, Bcl2 protein was also increased about 2-fold in H157-IRR and H358-IRR cells (Figure 3-1A). Importantly, parental A549 (A549-P) cells remained sensitive but A549-IRR became insensitive to IR (Figure 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 remained sensitive to BXI-61 and BXI-72 (Figure 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 whether 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 21) alone or in combination as indicated. Mice were treated with vehicle 64

(0.5% DMSO, 100µL/d, i.p.), IR (2Gy/every two days at a rate of 0.8Gy/min, 5times), BXI-72 (20mg/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 sensitive to IR treatment (Figure 3-1D). Consistent with the in vitro observation, BXI-72 repressed tumors 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 IRtreated 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 overcome 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 Bcl-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 protein (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. 65

Tumor repression was observed after 21-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 cells (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 cytotoxicity assays and in vivo animal models via apoptosis. 66

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 irradiation 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 assay after 72h. D) Mice bearing A549-P or A549-IRR lung cancer xenografts were treated with IR, BXI-72 or their 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 sacrificed and the tumors were removed and analyzed. 67

Figure 3-1. Continued. 68

Figure 3-2. Analysis of toxicity for combination of BXi-72 and IR in vivo. A) Body weight of mice with A549-P or A549-IRR xenografts was measured once every 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 histology of various organs after treatment with 0.5% DMSO (control), IR (2Gy 5), BXI-72 (20mg/kg/d) or their combination. H&E histological images are representative for 8 mice per group. 69

Figure 3-2. Continued. 70

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 (Chonghaile 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 that its mechanism of action may be predominantly through the Bcl2 family (Chonghaile and Letai, 2008; van Delft 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 caused 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 suite and a database of small molecules from the NCI as described (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 (Ki) values 71

at nanomolar levels (Figure 2-4). These two compounds exhibited significantly lower binding affinity for Bcl2 (Figure 2-4), indicating a more selective binding to Bcl-XL. This is especially important because Bcl-XL is more widely expressed in NSCLC and SCLC cells than Bcl2 (Figure 1-3) (Hann et al., 2008; Li et al., 2008). A relatively high dose of ABT-737 (i.e. Bcl2 inhibitor) 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 inhibitors (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 (Figure 2-2). Consistent with our discovery approach, BXI repression of lung cancer growth occurs in a Bcl-XL-dependent manner since depletion of Bcl-XL significantly reduces sensitivity of lung cancer cells to BXI (Figure 2-10). Importantly, BXI-72, which showed a stronger Bcl-XL binding affinity (Ki 0.9 0.15 nm), also displayed greater cytotoxicity against human lung cancer cells as compared to BXI-61 (Ki 14.8 1.54 nm) (Figure 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) domains (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 72

insertion of the BH3 domain of one Bak molecule into the groove of another and may produce 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 cyt c release (Figure 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, respectively. This can also help explain why BXI suppresses lung cancer growth in a Bcl-XL dependent manner (Figure 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 (Figure 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 (Figure 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 (Figure 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 tumor tissues was observed in the BXI treatment group, this suggests that repression of lung cancer by BXI occurs through induction of apoptosis in tumors (Figure 2-14B). Radiotherapy is a major therapeutic intervention for patients with lung cancer and is administered 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 73

radioresistance of lung cancer, we established acquired radiation-resistant lung cancer cell model systems (Figure 3-1). Elevated levels of the anti-apoptotic proteins 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) (Figure 3-1A), suggesting that this upregulation could, at least in part, contribute to radioresistance. 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 resistant to radiation (Figure 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 (Figure 3-2). In summary, we have 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. These 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 radioresistance 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 the treatment of lung cancer, including those patients who are resistant to radiotherapy, leading to long-term tumor-free survival. 74

CHAPTER 5 MATERIALS AND METHODS Materials Small molecules, including NSC354961 (BXI-61) and NSC334072 (BXI-72), were obtained 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/requestcompounds). 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 antibodies 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-GQVGRQLAIIGDDINR) 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, approximately 300,000 small molecules from the NCI database were docked into a structural pocket within the BH3 domain (aa 90-98) of Bcl-XL and two hundred candidates were identified using the DOCK programs (version 6.1, University of California, San Francisco, CA). Subsequently, these small molecules were screened on A549 and H1299 lung cancer 75

cells 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 lines (BEAS-2B and 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 37ºC 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) and 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 50µg/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 100µl/well in 96-well plates. After overnight incubation, cells were treated with BXI-61, BXI-72, ABT-737, or cisplatin at various concentrations for 72 hours. Cells 76

were fixed by adding 100µl cold 10% TCA for 60 min at 4ºC after discarding the medium. Plates were washed 5 times with deionized water and air-dried. 50µl 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. 100µl 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 510nm. 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. The medium was replaced with fresh medium containing the corresponding concentration of the potent compounds every 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 Apoptotic 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 membrane in apoptotic cells during early apoptosis according to the manufacture s instructions (Martin et al., 1995). The percentage of annexin V low cells (percentage of viable cells) or annexin V high cells (percentage of apoptotic cells) was determined by fluorescence activated cell sorter analysis (FACS). 77

Fluorescence polarization assays Fluorescent Bak BH3 domain peptide and Bcl-XL protein were purchased from NeoBioSci TM (Cambridge, MA). To measure 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 presence 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 for an additional 15 min. Polarization, defined as millipolarization units (mp), was measured at room temperature with a fluorescence microplate reader at 485/530nm (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)/range) 100%. Inhibitory constant (Ki) 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, 50mM NaF, 1mM β-mercaptoethanol, 0.5% NP-40, and 100x protease inhibitors (10µl/ml, added right before use)). Proteins were separated on 78

12% or 15% SDS-PAGE gels and transferred to nitrocellulose membranes (0.34µm, GE). Membranes were incubated overnight at 4ºC with primary antibodies. Protein expression analysis was performed using appropriate secondary horseradish peroxidase (HRP, Santa Cruz)-conjugated antibodies and enhanced chemoluminescence (ECL, GE Healthcare, Little Chalfont, UK). Membranes were 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 using 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 (Invitrogen, Carlsbad, CA) (overnight at 4ºC). 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 Transfection Kit (Novagen, Madison, WI) according to the manufacturer s protocol. 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 79

H460 cells using NanoJuice TM Transfection according to the manufacturer s protocol and the expression level of Bcl-XL was analyzed using western blotting to confirm Bcl- XL up-regulation. Human Bcl-XL cdna was kindly provided 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 centrifuged 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 Bis(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 sulfhydryl 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 using a Bak antibody to detect Bak oligomerization. 80

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. All animal treatments were undertaken in accordance 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 nude 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 (Oltersdorf 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, 100µL/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 tumors were measured by the digital caliper (Sigma) every two days. Tumor volume (V) was calculated with the following formula: V=LxW 2 /2 (L: length; W: width) as described (Vlahovic et al., 2007). After the 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 embedded tumor tissues were labeled using TumorTACS TM In Situ Apoptosis Detection Kit (Trevigen, Inc., Gaithersburg, MD) according to the manufacturer s protocol to detect TUNEL positive cells. TUNEL positive cells were counted at 400X 81

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 400X 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 (250µM) 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 (RBC), 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 (250µM) was centrifuged at 15,000g for 2 min to separated serum from cells and fibrin after collecting whole blood in EDTA-coated tubes. Establishment of ionizing radiation resistant (IRR) cell line We chose A549, H157, and H358To induce IR resistant cell lines, A549, H157, and H358 cell lines 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, and H358 cells (1x10 6 ) were serially irradiated with 2Gy of X-rays to a final dose of 100Gy using X-RAD 320 (Precision X-ray Inc., North Branford, CT) at a rate of 82

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 of 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 lung 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 Institute, 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-IRR cells in HBSS into the dorsal flank area of nude mice. Treatments were initiated when tumors reached between 200 and 300mm 3. Mice were randomized into eight groups (n=6 per group) and treated with vehicle (0.5% DMSO, 100µL/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 deliver 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 83

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 groups was analyzed with twosided unpaired student s t-test or Fisher s exact 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 standard deviation (S.D.). 84

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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 microbiology under Dr. Min-Woong Lee s mentoring for four years. And he earned his Bachelor of Science degree in Applied Biology 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 Sciences in College of Medicine at the University of Florida, Gainesville, FL in 2005. He had been training in Dr. Deng s lab as a graduate research assistant in Department of Medicine, Shands Cancer Center, University of Florida from November 2007 to August 2009. In 2009, Dr. Deng accepted new position in Winship Cancer Institute, Emory University, Atlanta, GA. To continue his Ph.D. dissertation project, he also joined Emory University with Dr. Deng started from August 2009. Following Dr. Deng s instructions, he has discovered 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 overcome 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 Sciences-Molecular Cell Biology in August of 2012. 96