Kinase Targeting Therapies in Chronic Lymphocytic Leukemia: Mechanisms of Acquired Ibrutinib Resistance and the Pre-Clinical Development of OSU-T315

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DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Ta-Ming Liu

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2014

Dissertation Committee:

John C. Byrd M.D., Co-advisor

Amy J. Johnson Ph.D., Co-advisor

Ching-Shih Chen Ph.D.

Gregory B. Lesinski Ph.D.

Ta-Ming Liu

2014

Abstract

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in western countries. Despite the improvement of treatment that is contributed by development of targeted therapy through antibodies and chemical agents to unique surface markers or critical kinases triggering survival signals in CLL, satisfactory remission remains deficient and requires improvement for this incurable disease. Given improved understanding of the etiology and prognosis in CLL biology, the development of therapeutic approaches shifts to targeting critical molecules relied on by CLL cells. Of note, B cell receptor (BCR) and phosphoinositide-3 kinase (PI3K)/AKT both appear to be the central axis triggering survival signals in this malignancy, and the majority of agents in clinical use suppress these pathways by focusing on inhibiting proximal kinases such as Bruton’s tyrosine kinase (BTK) and p110 δ subunits of PI3 Kinase, respectively.

Nowadays, the best characterized first-in-class agent inhibiting BTK and showing outstanding activities in CLL therapy is Ibrutinib. Despite the outstanding activity, no agent received as a monotherapy can achieve a complete remission; while relapse still occurs in patients receiving ibrutinib treatment, there is justification for developing new therapies.

The work presented herein focuses on two main projects, the identification of genetic lesions related to Ibrutinib resistance, and the evaluation of OSU-T315 as a CLL therapeutic. Chapter 1 begins with introductions of B cell and CLL biology, comprising

ii diagnostic and therapeutic strategies in this disease. The key survival signaling triggered by diverse kinases within the tumor environment will be outlined. Chapter 2 discusses the discovery of genetic lesions associated with Ibrutinib resistance. Using high-throughput sequencing, we uncover acquired genetic mutations in BTK, the direct target of Ibrutinib, or in the immediate downstream molecule of BTK, phospholipase C γ2 (PLCγ2), which drives CLL survival by antagonizing the therapeutic effect of Ibrutinib, leading to refractory disease. Functional studies show the mutations in BTK reverse inhibition of kinase activity, while gain-of function mutations in PLCγ2 propagate downstream survival signals such as AKT and extracellular signal-related kinase (ERK) after bypassing BTK inhibition by Ibrutinib. In chapter 3, we report a novel agent OSU-T315, originally designed by targeting the scaffold of the AKT docking site in integrin-linked kinase (ILK), represents better selectivity toward CLL cells compared to the alleviated cytotoxicity in normal B or T lymphocytes. In contrast to current kinase inhibitors targeting proximal molecules, OSU-T315 diminishes AKT phosphorylation by displacing

AKT translocation into lipid rafts, representing a unique strategy targeting the PI3K/AKT cascade. Further characterization reveals OSU-T315 abrogates both intrinsic and extrinsic stimuli-mediated survival signals, including BCR, CD40, toll-like receptor 9 (TLR9) and

CD49d, accompanied by the reduced level of anti-apoptotic molecules myeloid cell leukemia sequence 1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xl), thus leading to caspase-dependent apoptosis. The in vivo efficacy is supported by improved median survival in T cell leukemia/lymphoma 1 (TCL1) leukemia-engrafted mice. Pharmacology of this agent showed acceptable properties for eventual transition to clinical development,

iii although further modification to improve bioavailability and formulation may be required. Chapter 4 concludes our findings with perspective implications. Particularly, the chapter addresses the unanswered questions and proposes future directions.

Together, the work here provides first, novel mechanisms illustrating the genetic mutations associated with resistance in Ibrutinib therapy that may be relevant to further development for BCR signaling inhibitors; second, we also identify OSU-T315 as a

PI3K/AKT signaling blocker and apply the strategy in the lipid raft compartment for CLL and other related diseases.

Dedicated to my family

Acknowledgments

First, I would like to thank Dr. John Byrd and Dr. Amy Johnson for their mentorship and guidance in my PhD study. I joined the lab with little knowledge in pharmaceutics and clinical therapeutics. Both of you have provided me with invaluable inspiration and support throughout the graduate life, and strengthen my ability to conduct scientific research. Your patience and intellectual discussion in mentoring always encourage me when struggling in unidentified subjects. I feel so grateful to join this lab and receive exceptional learning experience. I would also like to acknowledge Dr. Tim

Huang, Dr. Ching-Shih Chen, and Dr. Greg Lesinski for your generous support and guidance in my study. I would also like to thank Priscilla Lee for enormous care and support. Although there are too many to list for your assistance and friendship to complete my research projects in the past few years, I would specifically like to thank

David Lucas and Jennifer Woyach for your counsel in article writing and research support. Thank you Yuh-Ying Yeh for your assistance and suggestions in my projects. To all lab members for the support and friendship in my daily life, thank you again

Rajeswaran Mani, Melanie Davis, Yo-Ting Tsai, Yiming Zhong, Jason Dubovsky,

Priscilla Do, Shuai Dong, Amber Gordon, Derek West, Michelle Grindley, Sue Scott, and

Chi-Ling Chiang.

Lastly, I want to thank my family for sharing experiences with me. No matter what my day might bring, you always stand next to me. Thank you for the love, care, and support.

vii

Vita

October 10th, 1980 ...... Born in Taipei, Taiwan

2003...... Bachelor of Science in Botany,

National Taiwan University

2005...... Master of Science in Immunology,

National Taiwan University

2009 to present ...... Graduate Research Associate, Program of

Molecular, Cellular, Developmental

Biology, The Ohio State University

viii

Publications

Liu TM, Ling Y, Woyach JA, Yeh YY, Hertlein E, Awan FT, Jones JA, Andritsos LA, Maddocks K , MacMurray J, Salunke SB, Chen CS, Phelps MA, Byrd JC, Johnson AJ. OSU-T315: A Novel Targeted Therapeutic that Antagonizes AKT Membrane Localization and Activation of Chronic Lymphocytic Leukemia Cells In preparation

Zhong Y, Liu TM, Johnson AJ. A Breakthrough Therapy in Relapsed Chronic Lymphocytic Leukemia: the Combination of Idelalisib and Rituximab. Journal of Postdoctoral Research 2014 2 (3): 41-43

Woyach JA*, Furman RR*, Liu TM*, Ozer HG*, Zapatka M, Ruppert AS, Xue L, Li DH, Steggerda SM, Versele M, Dave SS, Zhang J, Yilmaz AS, Jaglowski SM, Blum KA, Lozanski A, Lozanski G, James DF, Barrientos JC, Lichter P, Stilgenbauer S, Buggy JJ, Chang BY, Johnson AJ, Byrd JC. Resistance Mechanisms for the Bruton's Tyrosine Kinase Inhibitor Ibrutinib. N Engl J Med. 2014 May 28. [Epub ahead of print] * Co-first authors.

Dubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Zhong Y, Hessler JD, Liu TM, Chang BY, Larkin KM, Stefanovski MR, Chappell DL, Frissora FW, Smith LL, Smucker KA, Flynn JM, Jones JA, Andritsos LA, Maddocks K, Lehman AM, Furman R, Sharman J, Mishra A, Caligiuri MA, Satoskar AR, Buggy JJ, Muthusamy N, Johnson AJ, Byrd JC. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. 2013 Oct 10;122(15):2539-49.

Rodriguez BA, Weng YI, Liu TM, Zuo T, Hsu PY, Lin CH, Cheng AL, Cui H, Yan PS, Huang TH. Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells. Carcinogenesis. 2011 Jun;32(6):812-21.

Zuo T, Liu TM, Lan X, Weng YI, Shen R, Gu F, Huang YW, Liyanarachchi S, Deatherage DE, Hsu PY, Taslim C, Ramaswamy B, Shapiro CL, Lin HJ, Cheng AS, Jin VX, Huang TH. Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer. Cancer Res. 2011 Mar 1;71(5):1752-62.

Zuo T, Tycko B, Liu TM, Lin JJ, Huang TH. Methods in DNA methylation profiling. Epigenomics. 2009 Dec;1(2):331-45. Review.

Chen LS, Wei PC, Liu TM, Kao CH, Pai LM, Lee CK. STAT2 hypomorphic mutant mice display impaired dendritic cell development and antiviral response. J Biomed Sci. 2009 Feb 19;16:22.

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology

Table of Contents

Abstract ...... ii

Dedicated to my family ...... v

Acknowledgments...... vi

Vita ...... viii

Publications ...... ix

Fields of Study ...... x

Table of Contents ...... xi

List of Tables ...... xiii

List of Figures ...... xiv

Chapter 1: Introduction ...... 1

1.1 B cell development ...... 1

1.2 Chronic lymphocytic leukemia ...... 4

1.3 Molecular pathways in CLL ...... 7

1.4 CLL therapy ...... 9

1.5 Ibrutinib (PCI-32765 or ImbruvicaTM) ...... 12

1.6 PI3K/AKT in CLL ...... 13

1.7 Conclusions and hypothesis ...... 16

CHAPTER 2 Novel Recurring Resistance Mechanisms for the Irreversible Bruton’s

Tyrosine Kinase (BTK) Inhibitor Ibrutinib ...... 23

2.1 Introduction ...... 23

2.2 Materials and methods ...... 25

2.3 Results ...... 31

2.4 Discussion ...... 36

CHAPTER 3 OSU-T315: A Novel Targeted Therapeutic that Antagonizes AKT Membrane

Localization and Activation of Chronic Lymphocytic Leukemia Cells ...... 60

3.1 Introduction ...... 60

3.2 Materials and methods ...... 63

3.3 Results ...... 68

3.4 Discussion ...... 75

CHAPTER 4 Discussion ...... 100

4.1 Synopsis ...... 100

4.2 Future perspectives ...... 102

4.3 Future directions ...... 107

Appendix A OSU-T315 specifically targets PAK3 in class I PAK family kinases ...... 113

Appendix B The KINOMEScan results for OSU-T315 ...... 114

Reference list ...... 125

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List of Tables

Table 1: Rai and Binet staging systems for CLL diagnosis...... 19

Table 2: Characteristics of Ibrutinib Resistant Patients ...... 40

Table 3: All functional variants identified as acquired from baseline to relapse...... 41

Table 4: Alignment Statistics...... 43

Table 5: Variant frequency in the patient at relapse and 1 month post-relapse ...... 44

Table 6: Baseline data for patients deep sequenced with Ion Torrent ...... 45

Table 7: Biacore SPR binding assays...... 46

Table 8: Pharmacokinetic properties of OSU-T315 ...... 80

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List of Figures

Figure 1: The process of B cell development...... 20

Figure 2: B cell receptor signaling...... 21

Figure 3: Class ІA PI3K signaling pathway...... 22

Figure 4: Exome-Seq analysis pipeline...... 47

Figure 5: Copy number profile for all samples...... 48

Figure 6 Mutations identified and confirmed with DNA sequencing...... 49

Figure 7: Functional Characterization of C481S BTK...... 51

Figure 8: Functional Characterization of C481S BTK in BTK-deficient DT40 cells...... 53

Figure 9: Inhibition of BTK by ibrutinib is reversible in patients with C481S BTK mutation...... 54

Figure 10: Functional Characterization of R665W and L845F mutated PLCγ2...... 55

Figure 11: R665W and L845F reveal hyperactive Calcium influx...... 56

Figure 12: PLCG2 mutants display resistance to ibrutinib in downstream signaling...... 57

Figure 13: CLL with R665W PLCG2 bypasses BTK inhibition by ibrutinib...... 58

Figure 14: Ibrutinib resistance in CLL from the patient with both BTK and PLCG2 mutations ...... 59

Figure 15: OSU-T315 induces preferential cytotoxicity in CLL cells...... 81

Figure 16: OSU-T315 targets intrinsic AKT and ERK signals cascades in CLL cells. ... 82

Figure 17: OSU-T315 mediated cytotoxicity in CLL cells is ILK independent...... 84

xiv

Figure 18: OSU-T315 impairs AKT translocation to lipid raft subdomains in plasma membrane...... 86

Figure 19: OSU-T315 inhibits BCR, CD40L, and CpG induced survival signal...... 88

Figure 20: Downstream apoptotic machinery is induced upon T315 treatment in CLL cells.

...... 90

Figure 21: In vivo TCL1 leukemia progression is suppressed by OSU-T315...... 92

Figure 22: Mec-1 cells expressing Myr-flag-AKT...... 94

Figure 23: ILK expression is comparably low in CLL cells...... 95

Figure 24: OSU-T315 inhibits BCR mediated adhesion...... 96

Figure 25: OSU-T315 induces cytotoxicity regardless of overexpression of Mcl-1...... 97

Figure 26: OSU-T315 suppresses Bcl-xl upregulated by CD40 or TLR9 activation...... 98

Figure 27: Differential response of ERK status upon external stimuli after OSU-T315 treatment in CLL cells...... 99

Chapter 1: Introduction

1.1 B cell development

The principle cells of the adaptive immune system are lymphocytes, which specifically recognize and respond to foreign antigens, including bacteria, virus, and even tumor cells. While T cells are categorized as mediators of cellular immunity, B cells are considered to trigger humoral immunity due to their ability to secrete antibodies1. Upon activation, mature B cells proliferate and differentiate into plasma cells, secreting unique classes of antibodies (IgG, IgA, and IgE) to combat pathogens2. The potent antibodies conduct their neutralizing ability by binding to microbes and preventing them from infecting host cells. IgG antibodies also cover pathogens and target them for phagocytosis by neutrophils and macrophages, which destroy the microbes3. In addition to humoral response, B lymphocytes recognize soluble antigens by surface antibodies (IgM or B cell receptors), enable antigen internalization, and then serve the role of antigen-presenting cells (APCs) by interacting with major histocompatibility complexes II (MHCII) on T cells4. Most antibodies have a half-life of only a few days, though there are some long-lived IgG (up to 3 weeks). Some bone-marrow residing plasma cells serve as immediate protection against returning microbes, as these antibody-secreting cells live for years5. In healthy people, the percent of B cells in blood, lymph node, and spleen are

10-15%, 20-25%, and 40-45%, respectively. B lymphocytes were named because they were discovered in the bursa of Fabricius, an organ unique to birds. There is no anatomic 1 equivalent in human; however, human B cells originate from bone marrow progenitors.

The developmental process of B cells is tightly regulated6. The earliest stage of B cells after commitment into this lineage is called the pro-B cell (Figure 1). During this stage, the recombination of Ig genes in the heavy chain locus (H locus) occurs until the rearranged VDJ heavy chain is complete. Once the productive recombination at the H locus is generated, B cells differentiate into the pre-B stage, when they express the μ heavy chain, along with the λ5 surrogate light chain on the cell surface. The importance of pre-BCR mediated cell expansion is highlighted by studies of knockout mice at μ or λ5 genes, which resulted in significantly reduced mature B repertoire as those nonproductive

B cells underwent programmed cell death due to lack of pre-BCR survival signals. The immature B cells are termed by the expression of assembled IgM on cell surface after rearrangement at the κ or λ light chain locus to replace a surrogate light chain. During this stage, positive selection proceeds by identification of B cells bearing successfully rearranged antigen receptors7. Immature B cells receive constitutive tonic signals from

BCR independent of antigen and activate survival pathways, while accompanied by the shutoff of Rag gene expression to impede further rearrangement at Ig genes. The repertoire of BCR at the immature stage recognizes antigens with high avidity, including those self-reactive B cells, triggering the receptor editing process by reactivating Rag genes for additional recombination at the V-J locus of light chain. This process allows B cells to express distinct BCR without self-reactive ability. Contrasting to this, once receptor editing fails, the immature B cells will undergo apoptosis by the negative selection process, when B cells receive strong signals from autoreactive IgM. The

2 matured B repertoire consists of follicular and marginal zone B cells by the affinity of antigen receptors expressed on the B cells. Matured B cells primarily co-express both

IgM and IgD receptors via linkage of the VDJ complex with either Cμ or Cδ exons.

Follicular B cells circulate and migrate in the lymphoid tissues and can recognize foreign antigens with high affinity, then followed by antigen-induced B cell differentiation, leading to the generation of plasma cells that secrete unique immunoglobulins.

Contrasting to this, marginal zone B cells reside in the spleen, and can secrete natural antibodies with limited diversity to defend against polysaccharide antigens. In addition, a subset of B cells termed B-1 cells, which are distinct from conventional B-2 cells, originate from fetal liver derived progenitors8. B-1 cells reside in the peritoneum or mucosal sites, and express restricted repertoire of V segment. This lineage of B lymphocytes spontaneously secretes natural antibodies like marginal zone B cells to combat microbial antigens. Once mature B cells encounter antigens, a thymus-dependent, or T-dependent, response will occur in follicular B cells by stimulating and triggering B cell activation, resulting in substantial clonal expansion4. This differs from marginal zone

B cells or B-1 cells, which undergo a T-independent response to generate multivalent antibodies in mucosal or peritoneum tissues. Finally, activated B cells can further differentiate into long-lived plasma cells migrating into the bone marrow niche, or memory B cells which mount rapid response against pathogens2,9,10.

1.2 Chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in western countries. The criteria at diagnosis is defined as the presence of >5000/μl of malignant B cells in the peripheral blood11. Approximately 15,700 new cases are identified annually, with 4600 deaths from CLL estimated in the United States currently.

CLL accounts for one-third of total leukemia patients, with slightly higher risk in men than in women12. The average age at the time of diagnosis is about 72 years old, indicating the disease generally occurs in elderly people. CLL cells are characterized with expression of surface markers CD19, CD5, and CD23, and reduced levels of IgM, IgD, and CD79b13. Most CLL patients survive many years without symptoms, whereas some patients progress rapidly. In the past decades, intense studies identified novel biomarkers, contributing to predict prognosis and guide treatment schemes. The mutated status of the

V locus of the Ig heavy chain reflects an indolent course, while no mutations at V genes display a more progressive disease and are associated with short overall survival

(OS)14,15. The definition of mutated IgVH is <98% identity to corresponding germ line configuration. Extensive profiling reveals about 60% of patients harboring somatic hypermutations at the IgVH locus, whereas 40% of CLL patients are devoid of mutated

IgVH16,17. In addition to the IgVH mutation, zeta-chain-associated protein kinase 70

(ZAP70) and CD38 are two remarkable factors stratifying poor prognosis15,18-23. While

CD38 is a transmembrane glycoprotein expressed in precursor B cells, germinal B cells, and plasma cells, ZAP70 is an intracellular protein normally expressed in T cells.

ZAP70 is structurally homologous to spleen tyrosine kinase (SYK), the molecule that is

4 important to mediate BCR signaling. Given the rare presence of ZAP70 in normal B cells, it has been demonstrated that ZAP70 expression in CLL facilitates downstream signaling after BCR engagement. Cytoplasmic ZAP7024,25 expression in more than 20% and surface CD38 expression in more than 30% of patients anticipated unfavorable clinical courses.

Additional prognostic biomarkers are revealed when efforts to study the cytogenetic landscape in CLL cells identify novel genetic lesions. The recurrent common genomic abnormalities include deletions at 11q, 13q, and 17p, as well as trisomy 1226. These genomic aberrations provide information about disease progression and therapeutic decisions. Evidence suggests that the deleted region of 11q22-23 encodes tumor suppressor gene ataxia–telangiectasia mutated (ATM), regulates cell cycle, and confers resistance to chemotherapy27,28. Of note, recent studies identify the mutations of baculoviral IAP repeat-containing 3 (BIRC3), which is also positioned in the 11q22 region, correlate with fludarabine refractory, implicating the other candidate within

11q22 with inferior clinical course29. The most frequent alteration regarding genetic lesions is 13q14 deletion, occurring in 35-40% patients30,31. The region harbors deleted in lymphocytic leukemia 1 (DLEU1) and DLEU2 long non-coding RNAs as well as

MIR15A/16A microRNAs, in part contributing to CLL proliferation32-34. Despite the favorable outcome compared to del11q, del17p or trisomy 12, deletion of 13q14 leads to abnormal increase of B-cell lymphoma 2 (BCL2) and cyclin D1 (CCND1) and CCND3 expression, which drive CLL survival and proliferation in part due to the absence of mir15a/16a35. Approximately 3-7% of CLL patients bearing del17p13 involves lesion of

5 the tumor protein P53 (TP53) gene36. Since TP53 is the critical regulator for cell cycle arrest upon DNA damage, genomic complexity is expected in patients with this abnormality. Additionally, TP53 deletion or mutation contributes to poor clinical outcome with hard to treat or fatal disease course37-39. The aberration of trisomy 12 is detected in 11-16% CLL patients at diagnosis40. Although trisomy 12 is classically correlated with intermediate prognosis, emerging evidence suggests that NOTCH1 mutation is frequently associated with trisomy 12 (30%), conferring inferior prognosis41.

Given CLL is a heterogeneous disease and the majority of patients have an indolent clinical course with years until increased progression, standard definitions for disease progression at diagnosis are required. Rai42 and Binet43 are the current prognostic systems in clinical management to stratify CLL patients into different risk groups. The Rai system uses lymphadenopathy, organomegaly, and cytopenias to categorize patients into five groups and estimate the clinical outcome and medium survival. Comparing to the Rai system, the Binet system measures the number of involved nodal areas and cytopenias to classify patients into three groups. Both systems are useful in predicting prognosis and guide the initiation of treatment. Accordingly, symptomatic patients with Rai stage I-II and Binet B, or patients assigned with Rai stage III/IV and Binet stage C meet the criteria for treatment (Table 1)44.

1.3 Molecular pathways in CLL

Emerging evidence suggests that CLL cells escape apoptosis to survive and proliferate by external stimuli. Various signals within the lymph nodes (LNs) and bone marrow (BM) collectively support apoptotic resistance in CLL. Among these noted, BCR signaling appears to be crucial in CLL progression (Figure 2)45,46. It has been demonstrated that stereotyped BCR is expressed in 30% of CLL with the characteristic of heavy chain complementarity-determining region 3 (HCDR3) amino acid sequence, thereby suggesting that the subset of CLL cells recognize unique antigens14,47,48. On the other hand, cases responsive to in vitro anti-IgM engagement carry unmutated IgVH genes49, as well as high expression of ZAP7023 and CD3815. Abundant publications suggest the critical importance of BCR in activating survival signals by downstream Lyn,

Syk, and BTK activation, followed by PLCγ2 phosphorylation, which leads to subsequent calcium influx, as well as the activation of ERK1/2. In parallel, the PI3K cascade triggers AKT phosphorylation, enforcing downstream anti-apoptotic pathways in CLL50.

BCR activation also accompanies activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, triggering upregulation of Bcl-2 family members including Bcl-xL, Mcl-1, and BCL2-related protein A1 (Bfl-1) in the LNs46,51. NF-κB activation comprises two different pathways52. Both canonical and alternative NF-κB signaling are activated by the regulation of IκB kinase (IKK) complex. IKK activation is triggered by proteasomal degradation of IκB inhibitor, thereby releasing the NF-κB complex and facilitating subsequent translocation into the nucleus. Once the canonical pathway is activated, the NF-κB dimers consisting of V-Rel avian reticuloendotheliosis

7 viral oncogene homolog A (RelA), c-Rel, RelB, and p50 can be formed. In contrast, alternative NF-κB is processed by NF-κB-inducing kinase (NIK), which in turn induces

IKK activation, leading to the processing of p100 and releasing of the p52/RelB complex.

Dysregulation of NF-κB signaling in CLL cells can also be promoted by TNF super family members53. The ligands present for CLL development in the tumor environment include CD40L, B-cell activating factor (BAFF), and a proliferation-inducing ligand

(APRIL)54,55. The BM and lymphoid organs are the major microenvironment supporting

CLL survival and proliferation. Numerous studies indicate CLL cells are attracted toward the niche, feeding on the bone marrow derived stromal cells (BMSCs) in BM or nurse like cells (NLC) in lymphoid tissues56-58, which secrete chemokines C-X-C motif chemokine 12 (CXCL12), CXCL13, BAFF, and APRIL. Within the microenvironment,

CLL cells also encounter CD4 T cells, which express CD40L in addition to other critical cytokines such as interleukin-10 (IL-10), IL-4 or IL-21, protecting CLL from apoptosis by triggering NF-κB activation59,60. Upon receptor activation by tumor necrosis factor

(TNF) family members, multiple pathways including PI3K/AKT and mitogen-activated protein kinase (MAPK) cascades are triggered as well61. Furthermore, besides these receptor tyrosine kinases (RTK) triggered signaling by growth factors surrounded within tumor environment, the interaction between CLL cells and the extracellular matrix also induce survival signals, which specifically activate the α4β1-integrin very late antigen-4

(VLA-4) (CD49d/CD29)62. While VLA-4 expression level is demonstrated to correlate with the cytogenetic aberration trisomy 12 and contributes to prognostic impact63, it has been reported that treatment of fibronectin or vascular cell adhesion molecule-1

(VCAM-1), the ligands for CD49d, facilitates CLL migration toward BM or LNs, thereby activating downstream survival signals64. Collectively, advancing knowledge on signaling cascades in CLL cells illustrates the orchestrated mode of action driving leukemia progression, providing novel therapeutic candidates for clinical practice.

1.4 CLL therapy

CLL remains an incurable disease with extremely diverse clinical courses. Patients with advanced stages according to Rai or Binet definitions (Rai III-IV; Binet C) or symptomatic Rai I-II and Binet B require initiated treatment (Table 1)44. In past decades,

CLL therapy has improved owing to the new findings on critical molecules contributing to the etiology of CLL, providing information of discrete targets that leads to promising clinical activity. Historically, monotherapy with alkylating agents provided the frontline standard treatment to maintain disease progression until the combination therapy showed better activity65. Current purine analogs used in clinic include fludarabine, pentostatin, and cladribine (2-CdA). Fludarabine remains to be the most frequently practiced agent by far according to its well-studied trials66. Most recently, chemoimmunotherapy combined with monoclonal antibodies targeting antigens expressed on CLL cells suggests outstanding activity. Phase 2 studies of fludarabine/Cyclophosphamide plus rituximab

(FCR), the anti-CD20 antibody, achieve an overall response rate (ORR) of 95% with a complete response (CR) of 72%67. Further randomized trials demonstrate FCR improves the OS compared to the FC treated group (the medium OS: none vs. 86 months; p=0.001), designating FCR as the initial, gold standard therapy for non-treated patients68.

Similarly, a milder chemotherapeutic agent Chlorambucil (CLB) is also introduced to combine with anti-CD20 antibodies in previously untreated patients with comorbidities69.

According to the encouraging results, CLB combined with either rituximab or obinutuzumab (GA-101), an anti-CD20 antibody, reveals potent efficacy with increased

CR (22.3% vs 7.3% vs 0%; GCLB vs RCLB vs CLB alone) and prolong the progression-free survival (PFS) from 11.1 months (CLB) to 16.3 and 26.7 months (RCLB and GCLB), respectively, suggesting an alternative treatment scheme to alleviate toxicity in patients with comorbidities69. Contrasting to the patients lacking del (17p13.1) responding better to the current chemotherapeutic scheme, patients with del (17p13.1) or

TP53 mutations by far gain no advantage from traditional chemotherapy or chemoimmunotherapy. Despite the responsive rate above 50%, however, medium survival of patients with del17p or TP53 mutations received FCR is below 2-3 years due to poor response and tendency to relapse37,70. Current investigations with clinical trials focus on targeted therapy by either small compounds or monoclonal antibodies, from which contributing ORR is between 30-50% in patients with del (17p13.1) or TP53 mutations. Ideally, allogeneic stem cell transplantation (Allo-SCT) is the priority for patients with del (17p13.1) or TP53 mutations. Most recently suggested treatment scheme alternatives to Allo-SCT for that subset of patients include Alemtuzumab71 or

Ofatumumab72, or directly enrolled in clinical trials with novel agents.

Numerous targeted therapies aimed at critical survival pathways are under development and reveal promising clinical activity. Given that BCR is demonstrated as the central axis driving CLL, agents targeting BCR related kinases are promising73.

Antigen engagement or cell autologous interaction to BCR induces phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic regions of co-receptor CD79a/CD79b, subsequently triggering activation of proximal kinases Lyn and Syk. Besides the role of Syk phosphorylation, Lyn also functions as the signal mediator between ITAMs and the CD19 receptor in modulating BCR signaling, while

Syk amplifies the signals from ITAMs and is responsible for activating Ras, PI3K p110

δ, and PLCγ2, resulting in activation of MAPK, AKT, and NF-κB cascades. The reversible inhibitor dasatinib was originally designed as treatment in chronic myeloid leukemia (CML) by inhibiting the BCR-Abl kinase. Besides BCR-Abl, dasatinib inhibits

Src family kinases including Lyn and BTK and leads to therapeutic benefits in CLL treatment. A phase 2 trial in relapse/refractory patients (n=15) administrated with dasatinib monotherapy exhibited an OR of 20%, accompanied with reduction of lymphadenopathy in nine patients, and more than 50% reduction was shown in four out of nine patients74. Fostamatinib, a pro-drug of active metabolite R406, is previously documented Syk inhibitor in trials besides GS-9973, although it inhibits several other kinases as well. A phase 1/2 study, which includes 11 CLL patients, revealed an ORR of

55% (6/11). Nevertheless, the dose limiting toxicity observed from symptoms of diarrhea, fatigue, and neutropenia restricts the further evaluation of this agent. In addition to inhibitors targeting proximal kinases Lyn and Syk, Btk recently emerged as a novel target according to the encouraging clinical outcome. BTK is a member of the Tec protein tyrosine kinase (TEC) kinase family including TEC, IL2-inducible T cell kinase (ITK), and BMX non-receptor tyrosine kinase (BMX)/ETK. Previous studies demonstrated the

11 absence of BTK causes X-linked agammaglobulinemia75,76. Upon BCR activation, Btk is recruited toward the plasma membrane by its pleckstrin homolog (PH) domain and attached to phosphatidylinositol (3,4,5)-trisphosphate (PIP3)77. Btk appears to be the

B-lineage specific kinase for downstream calcium release and subsequent NF-κB activation. The best characterized BTK inhibitor is Ibrutinib (PCI-32765), showing outstanding clinical activity. The next section will describe the properties and efficacy of this novel agent in detail. Alternatively, CC-292, ACP-196 and GDC-0853 are other BTK inhibitors undergoing clinical evaluations50.

1.5 Ibrutinib (PCI-32765 or ImbruvicaTM)

Ibrutinib is the first-in-class Btk inhibitor in treatment of B cell malignancy, covalently binding to Cys-481 residue, thereby resulting in irreversible inhibition. The agent is currently under development for clinical practice in CLL, mantle cell lymphoma

(MCL), diffuse large B cell lymphoma (DLBCL), multiple myeloma (MM), follicular lymphoma (FL), and Waldenstrom’s macroglobulinemia (WM). Thus far, the FDA has approved ibrutinib for treatment in CLL and MCL78. The pre-clinical activity was documented in canine lymphoma by complete occupation of Btk79, and then followed by preliminary results in CLL showing its potency in abrogating BCR, CD40, BAFF, and

TLR induced AKT, ERK and NF-κB signaling80, as well as integrin-mediated adhesion and migration in CLL cells81. Previous reports indicate that ibrutinib does not show a toxic effect in normal T cells while modulating the Th2 response by alternatively targeting ITK in T cells82. A phase 1b/2 study for treatment naïve (n=31) CLL patients

12 exhibits high response rate (ORR=71%), with 96% of estimated PFS or OS in 22 months83. Further studies regarding a total of 85 relapsed or refractory CLL patients previously treated with purine analogues exhibits high response rate (71%) despite having unfavorable prognoses of unmutated IgVH or 17p deletion84. No difference in response rate was noticed in patients taking either 420mg or 840mg daily. Overall, patients bearing 17p deletion exhibit similar ORR compared to those without this high-risk aberration (68% v.s. 71%). Patients with unmutated IgVH reveal significantly improved difference of ORR (complete response plus partial response) to those with mutated status (77% v.s. 33%; p=0.005), whereas no difference was noticed in combined

ORR (including PR with lymphocytosis)84. Furthermore, a recent study demonstrates ibrutinib significantly prolonged PFS and ORR (42.6% v.s. 4.1%) compared to ofatumumab in previously treated CLL, regardless the presence of chromosome 17p13.1 deletion85. This study suggests an outstanding potency in high-risk patients, indicating the success of ibrutinib in therapeutic efficacy for patients with relapsed setting.

1.6 PI3K/AKT in CLL

Aberrant activation of PI3K/AKT pathways by various stimuli is implicated in CLL progression86,87, providing the justifications for developing agents abrogating this signaling axis (Figure 3). Accumulating evidence demonstrates that transgenic mice expressing constitutively active AKT rapidly develop aggressive B cell lymphoma88,89.

Moreover, transgenic mice expressing TCL1 develop a CLL-like disease in part due to the activation of AKT by Tcl190, suggesting the crucial effect of AKT activation in CLL

13 development. The PI3K family kinase generates intracellular lipid messenger PIP3 by phosphorylating the 3’-OH group of phosphatidylinositols. Three classes of PI3K are defined according to their substrate specificity and distinct lipid products. The Class I

PI3K consists of a p110 catalytic subunit and a p85 or p101 regulatory subunit, and is the class most often demonstrated involved in cancer survival and proliferation91. The p110α, p110β, and p110δ isoforms interact with the p85 subunit and are activated by RTKs, whereas p110γ interacts with p101 and is linked with Gβγ subunits released from G protein-coupled receptors (GPCRs). The formation of PIP3 from PIP2 at the plasma membrane by PI3K serves as the bait, recruiting critical kinases by interacting with the

PH domain of AKT and PDK1. The serine/threonine kinase AKT is subsequently phosphorylated by phosphoinositide-dependent kinase-1 (PDK1) at Thr308 in the activation loop of the catalytic domain, and the so-called PDK2 can activate Ser473 in the C-terminal hydrophobic motif to acquire fully activation. Once AKT is activated, it promotes CLL survival through activation of key components in apoptotic cascade such as the upregulation of Mcl-1 and Bcl-xl86. Glycogen synthase kinase 3 beta (GSK3β), the direct downstream molecule, is inhibited by AKT, contributing to prolonged half-life of

Mcl-192. Diverse stimuli have been reported to drive AKT activation in CLL cells, including BCR, CD40, TLR9, chemokine CXCL12, CXCL13, and integrin activation via

CD49d engagement13. The therapeutic advances in CLL in the past decade partially benefit from the development of agents targeting this pathway. Previous studies have identified p110δ isoform as the critical PI3K member highly expressed in CLL cells93.

Given that p110δ is restrictedly expressed in hematopoietic lineages compared to other

14 ubiquitous isoforms p110α and p110β, and mice with deleted or mutated p110δ are viable with reduced mature B cells population while B1 subset is absent, the evidence suggests the therapeutic promise in targeting it94-96. Idelalisib (GS-1101; formally CAL-101) is the most distinguished reversible inhibitor of P110δ currently under development in CLL treatment. The first-in-class p110δ inhibitor was pre-clinically proved to suppress

BCR-induced survival signals beside a variety of other environmental stimuli93. A phase

1 study in previously treated relapse/refractory patients exhibited an ORR of 39% along with 81% of nodal response (NR) 97. The majority of patients also reveal peripheral blood lymphocytosis and the PFS is improved from 17 to 29 months when they take 150mg twice a day. Although the dose-limiting toxicity was not observed, the study was discontinued in 7% of patients who displayed treatment-related adverse symptoms in part due to on-target effect to regulatory T cells by suppressing p110δ98. Remarkably, recent studies indicate the overwhelming efficacy regarding PFS (none v.s. 5.5 months, p<0.001), OS (81% v.s. 13%, p<0.001), and overall survival at 12 months (92% v.s. 80%, p=0.02) of Idelalisib combined with rituximab in relapsed patients when compared to rituximab alone99. IPI-145 is another novel agent under clinical evaluation. The agent was developed by targeting both p110δ and p110γ. The dosage of 25mg twice daily in clinical use can inhibit p110δ alone, while 75mg twice daily is indicated to suppress both isoforms. A phase 1 study enrolled relapse/refractory patients treated with escalating dose from 8 to 100mg twice daily exhibits the ORR of 55% with 87% of NR, similar to idelalisib. Regarding to the suppressive effect on p110γ, the agent potentially results in more pneumonia infections owing to the significant immune suppression100. Overall,

15 suppression of PI3K signaling provides an effective therapeutic approach besides chemotherapy and other novel targeting agents, and could also benefit treatment schemes when combined with other currently used agents.

1.7 Conclusions and hypothesis

In the face of the heterogeneous properties of CLL, recent discoveries in CLL biology have substantial impacts in improving the treatment approaches. Given the outstanding response by traditional chemoimmunotherapy in patients without del17p aberrations, the gold standard method is still administrated as the first line treatment. The evolved treatment paradigm in targeting critical kinases further provides alternative therapies for the relapse, refractory, and high-risk patients. Nevertheless, regardless of treatment as monotherapy or combined treatments with others, complete remission is still rare in the majority of patients requiring therapy, providing justifications to develop novel agents according to current knowledge. Present focus in clinical evaluations of novel agents suggests the promising efficacy by targeting the BCR signaling pathway, as well as critical survival signals relied by CLL cells.

The clinical achievement in PI3K inhibitors through suppressing AKT survival signal leads to the hypothesis that novel agents targeting this axis will benefit therapy.

Indeed, pharmaceutical companies are developing a multitude of compounds, including idelalisib, IPI-145, GS-9820, AMG-319, etc. The common principle for these agents is designed as proximal PI3K inhibitors for abrogating AKT activation. Interestingly, there is an absence of studies demonstrating promising activity directly targeting downstream

AKT activation apart from the ongoing trial for MK2206, an allosteric AKT inhibitor.

Accordingly, we aim to develop agents that effectively suppress AKT activation bypassing the proximal kinases, providing another agent utilizing different strategies in clinical practice. In collaboration with colleagues in the Ohio State University, we identify the novel compound OSU-T315, selectively targeting CLL cells by displacing

AKT from raft nodal, revealing promising activity in both in vitro and in vivo studies.

While several BTK inhibitors are undergoing development as noted by CC-292 or

GDC-0853, in the setting of BCR signaling inhibitors, ibrutinib still appears to be the most superior agent. Owing to the outstanding activity in relapsed, refractory, or high-risk patients with del17p, ibrutinib was approved by the Food and Drug

Administration (FDA) in 2014 for patients who have received at least one previous therapy. The advance provides alternative treatment in addition to allogeneic stem cells transplantation, the current treatment guideline recommended for aggressive relapse patients. While ibrutinib is emerging as a breakthrough in CLL therapeutics and patients will increasingly be expected to receive this agent, a small fraction of patients has shown resistance to prolonged treatment. Little data has been reported to clarify the underlying mechanisms of ibrutinib resistance, which may be contributed from either germ-line genetic lesions or acquired aberrations under intensive and continuous exposure of ibrutinib. Therefore, we aim to investigate the mechanisms in relapse patients after ibrutinib treatment. We speculate genetic mutations within critical kinases that related to

BCR signaling may hamper the efficacy of ibrutinib. Consequently, genome-wide exon sequencing was performed in relapsed patients and novel mutations within BTK and

PLCG2 genes were identified. Our functional studies verify the gain of function mutations in C481S at BTK, which results in both reversible inhibition and retained kinase activity, as well as R665W, L845F mutations at PLCG2, contributing to enhanced survival signals bypassing the ibrutinib inhibition function. Our findings provide insights explaining the causes of resistant outcome to ibrutinib, verifying the acquired mutations instead of naïve aberrations that could impede efficacy ultimately.

TableⅠCLL clinical staging system System Rai staging (1975) Binet staging (1981) Staging 0 Ⅰ Ⅱ Ⅲ Ⅳ A B C Lymphademopathy None >1 Hepatomegaly or splenomegaly None None >1 Hemoglobin (g/dL) >11 >11 >11 <11 >10 >10 <10 platelet (per μl) >10,000 >10,000 >10,000 >10,000 <10,000 >10,000 >10,000 <10,000 Number of involved area <3 >3 median survival 11.5 10 7.8 5.3 7 11.5 8.6 7 Table 1: Rai and Binet staging systems for CLL diagnosis. The clinical criteria for defining the staging of CLL progression are summarized42,43

Figure 1: The process of B cell development. The figure summarizes the events and characteristics in B cell development and differentiation. Briefly, B linage is committed as pro-B cells in bone marrow, and subsequently develops as pre-B cells after the appearance of pre-BCR by rearrangement of IgμH and assembled with surrogate Vλ5 light chain. At the process of immature B stage, the B cell receptor expressed in immature B stage undergoes negative selection to deplete self-reactive B repertoire. In periphery, transitional B cells eventually develop into mature follicular or marginal-zone B cells co-expressed both IgM and IgD. After encountering antigens, the activated mature B cells can further differentiate into antibodies secreting plasma cells or memory B cells. (Nature Reviews Immunology 7, 633-643)

Figure 2: B cell receptor signaling. The BCR is a transmembrane protein consisting of immunoglobulins and hetero-dimers of CD79a and CD79b. The cytoplasmic region of CD79 bears an immunoreceptor tyrosine-based activation motif (ITAM) (A). Phosphorylation of ITAM recruits signalosome toward membrane and trigger downstream signaling (B). BTK can be activated by itself, and cooperates with Syk to trigger PLCγ2 activation. Once BTK is activated, downstream PI3K and NF-κB signaling are propagated. PLCγ2 leads to subsequent protein kinase C activation by generating IP3 and calcium flux (C). (BLOOD, 9 AUGUST 2012, VOLUME 120, NUMBER 6)

Figure 3: Class ІA PI3K signaling pathway.

Class ІA PI3Ks phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to PIP3 on the plasma membrane. The PI3Ks consists of p110 and p85 subunits and are generally activated by receptor tyrosine kinases (RTKs) or Ras. 3’-phsphatase PTEN is responsible for reversing the activated PIP3 into PIP2. Accumulated PIP3 result in recruitment of AKT and PDK1 bearing pleckstrin homology (PH) domains. The activation of AKT at Thr308 by PDK1 and Ser473 by PDK2 in the case of mTOR complex in this figure leads to fully activation of this Ser/Thr kinase. AKT activation leads to subsequent phosphorylation of GSK3β, forkhead box O, BIM, and BAD, contributing to survival of malignant cells. Nature Reviews Cancer 9, 550-562 (August 2009)

CHAPTER 2 Novel Recurring Resistance Mechanisms for the Irreversible Bruton’s

Tyrosine Kinase (BTK) Inhibitor Ibrutinib

2.1 Introduction

The development of B-cell receptor (BCR) antagonists has been a major therapeutic advance in chronic lymphocytic leukemia (CLL). While BCR ligation in normal cells induces proliferation, apoptosis, or anergy,101 pathway dysregulation in CLL results in the propagation of proliferative and pro-survival signals.102,103 Several agents targeting the BCR pathway are in development, including the Bruton’s Tyrosine Kinase

(BTK) inhibitor ibrutinib. While BTK is not recurrently mutated in CLL,104,105 it is upregulated at the transcript level and is constitutively active.106 79 Ibrutinib irreversibly binds BTK at the C481 residue, rendering it kinase-inactive, inducing modest CLL cell apoptosis, and abolishing proliferation and BCR signaling in vitro.106,107 Ibrutinib has demonstrated significant activity in patients with relapsed CLL with a 71% overall response rate and an additional 15-20% attaining a partial response except for persistent lymphocytosis. At 26 months, the estimated progression free survival is 75%.108 While few patients have relapsed at this time, as more patients are treated with ibrutinib it becomes increasingly important to identify mechanisms of acquired resistance in order to offer effective salvage therapies. Additionally, characterizing whether persistent lymphocytosis bears similar resistant features could impact treatment choices for these patients.

The model for kinase inhibition in hematologic malignancies is the BCR-ABL inhibitor imatinib, which transformed chronic myelogenous leukemia (CML) therapy.109

The most common mechanisms of acquired resistance to imatinib are point mutations in the kinase domain of ABL. While the T315I mutation is the most common,110,111 more than 100 resistance mutations have been identified that prevent imatinib binding through binding site configuration or destabilization of the inactive conformation of ABL.112

Since BTK has not been identified as a mutated gene in CLL whereas BCR-ABL has been shown to be a mutational hotspot113 it is uncertain whether this mechanism will be relevant to CLL. Additionally, ibrutinib is an irreversible inhibitor of BTK by its ability to bind to the C481 site distinguishing it from imatinib and other reversible kinase inhibitors that have been studied in cancer to date. How cancer cells including CLL will develop resistance to ibrutinib or other irreversible inhibitors is yet unknown. The development of mutations in genes which re-activate downstream BCR signaling or other pathways is certainly possible, as clonal evolution is common in previously treated

CLL.114

Here we report for the first time the identification of mutations in BTK and

PLCγ2 in patients with acquired resistance to ibrutinib. We perform a functional analysis of these mutants and demonstrate relevant in vivo signaling alterations. Finally, we show that these mutations are not present in patients who have persistent lymphocytosis on ibrutinib without other signs of disease progression. We have therefore identified a novel pattern of resistance distinct from reversible kinase inhibitors

24 that may be relevant to further BCR pathway inhibitor development and other potent irreversible inhibitors.

2.2 Materials and methods

DNA Sequencing

Blood was obtained from patients enrolled on Institutional Review Board approved trials of ibrutinib. Tumor DNA was isolated from blood mononuclear cells using AllPrep DNA/RNA Mini kit (Qiagen). Sample preparation and whole-exome sequencing using Agilent SureSelect Human All Exon V4 and Illumina HiSeq2000 technology was performed by Expression Analysis (Durham, NC).

Data Analysis Workflow

Exome-Seq analysis pipeline can be found in Figure 4. Sequencing reads were aligned to the human reference genome (1000 Genomes Project human assembly/GRCh37) with BWA (v0.7.5)115. After potential PCR/optical duplicates were marked with Picard (v1.94, picard.sourceforge.net), local realignment around indels were performed with the Genome Analysis Toolkit (GATK v2.8.1)116, relapse-specific single point mutations and indels were detected with MuTect (v1.1.4)117 and GATK Somatic

Indel Detector, respectively. After filtering out variants previously reported in dbSNP

(build 137), variants were annotated and their potential mutational effects predicted with

SnpEff (v3.4,)118. Finally, newly acquired relapse-specific high quality nonsynonymous mutations were verified by Sanger or Ion Torrent sequencing.

Ion Torrent Analysis

DNA was extracted from cryopreserved cells using QIAmp DNA Mini kit

(Qiagen; Hilden, Germany). BTK and PLCγ2 genes were analyzed using the Ion

Torrent platform from Life Technologies (Carlsbad, CA). Library was prepared with

Ion AmpliSeq Library kit2.0 (4475345) with custom designed panel of AmpliSeq primers that covers the entire coding sequence and intronic splice acceptor and donor sites for both genes and IonExpress barcode adapters (kit#4471250). DNA was amplified on

GeneAmp PCR system 9700 Dual 96-well thermal cycler from Applied Biosystems. PCR product was purified with Agencourt AMPure XP kit (A63881 Beckman Coulter,

Indianapolis, Indiana). Library was quantified using real time PCR with Ion Library

TAQMAN Quantitation kit 44688022 on (Applied Biosystems ViiA7 Real Time PCR

System) instrument to allow for optimal final dilution of library for template preparation on One Touch DL version instrument with Ion One Touch 200Template Kit v2DL(4480285). The ISPs enrichment and purification was performed on One Touch ES using One Touch 200Template Kit v2DL (4480285). Purified ISPs were analyzed on Ion

Torrent personal Genome Machine using IonPGM 200 Sequencing kit (4474004) and

316chips (4466616). Data was collected and analyzed using Torrent Server (4462616) with Torrent Suite 3.6.2. Final analysis of sequence data was performed using combination of software: Variant Caller v.3.6.63335, IonTorrent IGV3.6.033 and

IonReporterUploader v.3.6.2-r62834. The following reference sequences were used for analysis; for BTK NM000061.2 and for PLCG2 NM002661.3. The entire length of

26 sequences was reviewed manually using these programs to assess for deviation from reference sequence and to evaluate the quality of sequence and the depth of coverage.

DNA Constructs and Cell Culture

The PiggyBac Transposon Vector System (System Biosciences, Mountain View,

CA) was used to generate DNA constructs of BTK with both GFP and puromycin selection markers, and protein expression is controlled by chicken β actin promoter. The chicken DT40 cell lines were kind gifts from Tomohiro Kurosaki (Kansai Medical

University, Japan). Cells were maintained in RPMI 1640 (Life Technologies, Grand

Island, NY) with 2 mM L-glutamine, 10% fetal calf serum, 1% chicken serum, 50 μM

β-mercaptoethanol, and 100 μg/ml primocin (Life Technologies, Grand Island, NY).

DT40 (BTK-/-) cells were transfected using Amaxa Nucleofector technology (Lonza,

Basel, Switzerland). Stably transfected cells were selected and maintained by adding puromycin (0.5 μg/mL) into the cell culture medium.

To generate DT40 expressing mutant PLCγ2, the pRetro-X Tet-On (Clontech,

Mountainview, CA) or pBABE vectors were used to generate DNA constructs of PLCγ2 that were introduced through retroviral infection. R665W or L845F mutated PLCγ2 was derived by site-directed mutagenesis (QuikChange II, Stratagene-Agilent Technologies,

Santa Clara, CA). Both constructs have puromycin selection markers. The expression of wild-type or mutant PLCγ2 is controlled by promotors of either CMV in pRetro-X tet-On or SV40 promotor in pBABE vectors. Cells were maintained in RPMI 1640 (Life

Technologies, Grand Island, NY) with 2 mM L-glutamine and 10% fetal bovine serum in

27 addition to Penicillin/Streptomycin antibiotics. Stably infected cells were selected and maintained by adding puromycin (1.0 μg/mL) into the cell culture medium.

In Vitro Kinase Assay

Full-length His6 and Strep-tagged BTK and BTK C481S were expressed in mammalian HEK-EBNA cells and purified by affinity chromatography. Purified proteins were used at 100ng/ml in a LanceUltra kinase assay, using 100nM Ulight-polyGT as a substrate and 200 μM ATP. Compounds were pre-incubated at room temperature with enzyme prior to the kinase reaction. After 45 minutes of kinase reaction, excess EDTA was added, and an EU-labeled anti-poly-GT antibody was incubated prior to signal measurement in a dual-laser Envision (Ex 320nm- Em 615-665nm).

Phosphoflow and Immunoblot Assays

HEK293T cells were transiently transfected with the indicated expression constructs, treated with ibrutinib for 1 hour, and fixed with paraformaldehyde or washed into fresh media and then fixed. Cells were permeabilized, stained, and analyzed on a BD

FACS Canto II. For immunoblots, whole cell lysates were prepared and equivalent amounts of protein were separated on polyacrylamide gels and transferred onto nitrocellulose membranes. After antibody incubations, proteins were detected with chemiluminescent substrate (SuperSignal; Pierce). Antibodies against phospho-BTK

(Tyr223), phospho-AKT (Ser473), AKT, ERK1/2, phospho-PLCγ2 (Tyr759; Tyr1217) were obtained from Cell Signaling Technologies (Danvers, MA). Antibodies against

Phospho-Erk (Thr202/Tyr204) and total PLCγ2 were obtained from Cell Signaling

Technologies or Santa Cruz Biotechnology (Santa Cruz, CA). The Antibody against

BTK was obtained from BD Biosciences (San Jose, CA) or AbCam (Cambridge, MD).

Anti-tubulin was obtained from Abcam, and anti-actin was obtained from Santa Cruz

Biotechnologies.

Primary CLL cells

For primary CLL cell experiments, peripheral blood mononuclear cells were obtained using Ficoll density gradient centrifugation. B cells were not specifically selected, but at the time of blood acquisition, clinical flow cytometry revealed 85-98% B cells as a percentage of total blood lymphocytes.

Calcium Flux Assays

DT40 cells stably expressing either wild-type or mutant BTK were loaded with 1

μg/ml Indo 1-AM (Life Technologies, Grand Island, NY) at 37°C for 30 min. After two washes, cells were treated with either DMSO or ibrutinib at 37°C for 5 min and the calcium mobilization was measured in a Fluoroskan Ascent FL (Thermo Scientific,

Waltham, MA). After 120 seconds of acquisition to determine the baseline, 5 μg/ml anti-chicken IgM (SouthernBiotech, Birmingham, AL) was added to stimulate the cells.

DT40 cells stably expressing either wild-type or mutated PLCγ2 were treated with

DMSO or 1μM ibrutinib at 37°C for 30 min. The intracellular calcium level was detected by Calcium Assay Kit (BD Biosciences, San Diego, CA) and measured by Beckman

Coulter DTX880 microplate reader. After 195 seconds of acquisition to determine the baseline, 3 μg/ml α-chicken IgM (SouthernBiotech, Birmingham, AL) was added to stimulate the cells.

Biacore Kinase Binding Assay

Full-length His6 and Strep-tagged BTK and BTK C481S were expressed in mammalian HEK-EBNA cells and purified by affinity and IEX chromatography. For

BTK protein immobilization, the ligands were captured with Ni2+ followed by covalent binding. The NTA chip was charged with Ni2+ followed by activation with EDC/NHS.

The ligands, BTK-WT and BTK-C481S, were diluted at a concentration of 3 μg/ml in immobilization buffer (HBS-P+) and injected for 200 sec at a flow rate of 10 µl/min on flow cell 2 and 4, respectively. Immobilization levels were approximately 1500 to 3000

RU on flow cell 2 and 4, respectively. Flow cell 1 and 3 were immobilized with an unrelated His-tagged protein at similar immobilization levels to serve as a reference surface. All surfaces were blocked with ethanolamine, pH 8.0. Temperature sample compartment was set at 15°C and the chip compartment at 25°C. After immobilization the buffer was exchanged to running buffer (HBS-P+ supplemented with 10 mM MgCl2,

2 mM DTT, 3% DMSO). To collect kinetic binding data, single cycle kinetics was used. The analyte (dasatinib or ibrutinib) diluted in running buffer, was injected over the four flow cells at concentrations of 4.7, 18.8, 75 and 300 nM, and a blank sample (0 nM) was used as reference. A flow rate of 90 μl per minute and a temperature of 25°C were used. The complex was allowed to associate and dissociate for 100 and 2000 sec, respectively. The surfaces were regenerated with two buffer injections of 60 sec each.

The sensograms were analyzed using Biacore T100 evaluation software V2.0.3, the data were fitted to a single site interaction model (1:1 (Langmuir) binding).

Statistical Methods

Linear mixed models with fixed and random effects were used to model all data from different experiments. In experiments designed to determine if autophosphorylation was inhibited in mutated versus wild-type cells and if this inhibition was different under treatment with ibrutinib or dasatinib, interaction contrasts at each concentration of interest were used to directly test the inhibitory hypotheses, including random effects associated with these contrasts. In the experiments testing if the increase in calcium flux over time and following stimulation was different in mutated cell lines treated with ibrutinib or vehicle control, models were fit with treatment and time as fixed effects, allowing for random intercepts and slopes for each condition and replicate. Only data from time points where the effects of stimulation had been observed were included (i.e. time > 39 seconds). Lastly, the experiment comparing maximum calcium flux values following stimulation between mutated and wild-type BTK cells was repeated three different times and all data were included in a model with cell type and concentration as fixed effects, and experiment replicate as a random effect. Statistical significance was declared at α = .05. All analyses were performed using SAS 9.3 (SAS Institute, Cary

NC).

2.3 Results

Whole exome sequencing reveals mutations in BTK and PLCγ2

Peripheral blood samples were available from 6 patients with progressive CLL at baseline and at the time of relapse. Whole exome sequencing (WES) was performed on

31 each sample and the analytical pipeline is outlined in Figure 4. Clinical characteristics and new mutations identified at relapse in the patients with matched samples are found in

Table 2 and 3. Alignment statistics can be found in Table 4. Copy number analysis was performed to eliminate potential bias confounding these data (Figure 5). All patients possess high-risk cytogenetic features including del(11q22.3), del(17p13.1), or complex karyotype. In patients 1-5, the relapse sample revealed a cysteine to serine mutation in

BTK at position 481 (C481S; Figure 6A), and in patient number 6, an arginine to tryptophan mutation in PLCγ2 at position 665 (R665W; Figure 6B). One patient with a low-frequency C481S BTK mutation also had three distinct PLCγ2 mutations: the

R665W mutation, a leucine to phenylalanine mutation at position 845 (L845F; Figure

6C), and a serine to tyrosine mutation at position 707 that was previously reported as an activating mutation in a dominantly inherited inflammatory disease.119 In this patient,

BTK C481S and PLCγ2 L845F were found by WES and other mutations were identified by Ion Torrent Sequencing. To verify these clones, Ion Torrent sequencing was performed again at a sample 1 month following relapse and all were still present (Table

5). All mutations identified by WES were confirmed by Sanger sequencing and/or Ion

Torrent deep sequencing. At baseline, no patient had evidence of mutations in either

BTK or PLCγ2 by WES. In patients 3, 5, and 6, Ion Torrent sequencing was performed, and no mutation was >0.5% of reads (Table 6). All patients at relapse were WT for other kinases containing a cysteine residue homologous to C481 in BTK that are irreversibly inhibited by ibrutinib (ITK, BMX, TEC, EGFR, JAK3, HER2, HER4, and

BLK).79,82 No other high-confidence recurrent mutation was noted in any of these six patients examined from diagnosis to relapse.

BTK C481S mutant is reversibly inhibited by ibrutinib in vitro and in vivo

We next performed functional characterization of WT and C481S BTK.

Intrinsic affinity of ibrutinib for WT BTK is significantly higher than for C481S (KD

0.2nM vs 10nM; Table 7), and while both WT and C481S BTK were inhibited by ibrutinib, the EC50 for C481S BTK was significantly higher than for WT (Figure 7A).

In a cellular model, ibrutinib was significantly less effective at blocking BTK autophosphorylation (Figure 7B) and downstream signaling (pERK, Figure 7C) in cells with C481S mutated BTK than WT, while dasatinib, a reversible inhibitor of BTK120 inhibited both WT and C481S BTK with similar efficacy. Ibrutinib washout experiments demonstrated that the C481S mutation allows reversible, but not irreversible inhibition of

BTK by ibrutinib, which was confirmed by kinase binding assays (Figure 7D; Table 7).

Next, we compared the function of WT and C481S BTK by stably transfecting constructs into DT40 (BTK-/-) cells. Following activation of the BCR, signaling downstream of

BTK was inhibited by ibrutinib, demonstrated by decreased downstream signaling

(PLCγ2, ERK, and AKT) (Figure 8A) and calcium flux (Figure 8B); this inhibition was diminished in C481S as compared to WT BTK. These data clearly demonstrate that

C481S mutation of BTK confers relative resistance to ibrutinib by preventing irreversible binding, confirming this as a functionally relevant resistance mutation.

We next obtained cells at baseline and relapse from a patient who developed a

BTK C481S mutation. At baseline, BTK phosphorylation is completely abrogated by

33 ibrutinib with either washout or continuous exposure to ibrutinib, however, at relapse ibrutinib can inhibit BTK phosphorylation with continuous exposure only, showing that the drug indeed binds reversibly in patients (Figure 9).

Identified mutations of PLCγ2 are potentially gain of function in the presence of

BCR stimulation and represent resistance mechanisms in patients

It has been shown that the S707Y mutation in PLCγ2 is gain of function due to disruption of an autoinhibitory SH2 domain.119 We chose, therefore, to focus on functional characterization of the R665W and L845F mutations. We stably transfected

WT PLCγ2, L845F PLCγ2, or R665W PLCγ2 into 293 cells and DT40 cells which lack endogenous PLCγ2 expression (Figure 10). We examined calcium flux in DT40 cells after anti-IgM stimulation in the presence of WT or mutant PLCγ2. In contrast to the

WT PLCγ2 where calcium flux was modestly inhibited by ibrutinib, both PLCγ2 mutants show enhanced IgM-mediated calcium flux that is not inhibited by ibrutinib (Figure 11).

This shows that these mutations allow for BCR-mediated signaling which is independent of BTK. Similarly, we see that after stimulation with anti-IgM, cells with either R665W or L845F PLCγ2 show less inhibition in the presence of ibrutinib than WT cells as measured by phosphorylation of ERK and AKT (Figure 12). These data demonstrate that R665W and L845F PLCγ2 are potentially gain of function mutations in the presence of BCR stimulation and could be relevant resistance mutations to ibrutinib in patients.

Finally, we examined CLL cells at baseline and at the time of relapse from patients #5 and 6. For patient 6, at the time of relapse while the patient was still taking ibrutinib, pBTK was decreased to a similar degree as at 11 months of therapy when the

34 patient was responding to drug (Figure 13A), whereas PLCγ2 showed enhanced phosphorylation at the time of relapse (Figure 13B). After ibrutinib was discontinued, ibrutinib still inhibited BTK phosphorylation (Figure 13C), but not PLCγ2 phosphorylation (Figure 13D). In patient 5 who possessed mutations in both BTK and

PLCγ2, in vitro ibrutinib did not inhibit either BTK or PLCγ2 phosphorylation (Figure

14). These data suggest that the gain of function phenotype seen in vitro is also relevant in patients.

Patients with prolonged lymphocytosis on ibrutinib do not have BTK or PLCγ2 mutations

Patients treated with ibrutinib develop a characteristic lymphocytosis as CLL cells are mobilized from lymph nodes and spleen. While most patients resolve their lymphocytosis within 8 months, a subset of patients have lymphocytosis that lasts >12 months in the presence of continued response to ibrutinib.121 To determine whether these patients developed new mutations in BTK or PLCγ2 and may therefore be at risk for relapse, we sequenced these two genes using Ion Torrent technology on 9 patients with at least 12 months of lymphocytosis at 12 months after the initiation of ibrutinib.

Sequencing depth for BTK at C481 and PLCγ2 at R665 and S707 was >700x, and for

PLCγ2 at L845 was >100x. No patient had evidence of any mutation of BTK or PLCγ2

(data not shown). This suggests that known resistance mutations are not present in patients with persistent lymphocytosis.

2.4 Discussion

Here we provide the first report of acquired resistance to ibrutinib due to recurrent mutations in BTK and PLCγ2. Our functional studies suggest that the C481S BTK mutation confers resistance to ibrutinib by preventing irreversible drug binding, the first such description for this class of drugs and irreversible kinase inhibitors in general.

S707Y, R665W, and L845F PLCγ2 mutations are all potentially gain of function mutations that allow BCR-mediated activation independent of BTK. Calcium flux assays demonstrate enhanced and sustained activation after anti-IgM stimulation which likely reflects the chronic antigen exposure experienced by CLL cells in patients.

Importantly, we show that none of these mutations are present in prior to drug exposure.

The significance of very low variant reads on Ion Torrent sequencing for the PLCγ2

R665W mutation at baseline is unknown, and more patients will need to be evaluated to determine whether this represents patients at risk for relapse. We also show that patients with persistent lymphocytosis during ibrutinib do not have evidence of these resistance mutations on deep sequencing. While we know that BTK and PLCγ2 are inhibited in these patients by immunoblot,121 these data confirm that these patients do not have small clones of resistant cells which would put them at high risk for relapse.

Tyrosine kinase resistance through mutations which alter drug binding are seen with other targets and other cancers, including BCR-ABL in CML,110 EGFR122,123 and

ALK124 in lung cancer, KIT in gastrointestinal stromal tumor,125 and FLT3 in acute myeloid leukemia.126,127 Distinguishing BTK from this group, however, is that rather than a second mutation in a previously mutated gene, the C481S mutation is a primary

36 mutation in a gene that is not recurrently mutated in CLL at the amino acid required for drug binding. Importantly, this mutation is not known to cause the X-linked agammaglobulinemia phenotype,128 but instead results in retained catalytic activity. Our identification of a previously identified activating PLCγ2 mutation119 and functional characterization of the two additional mutations demonstrate that the activation of BCR signaling distal to BTK can also confer resistance. This pattern of mutation localized to the irreversible binding site of ibrutinib and the immediate down-stream kinase is not commonly seen with reversible kinase inhibitors, and may be a result of continuous versus intermittent pressure on the drug target. As other potent irreversible kinase inhibitors are developed for key signaling pathways in cancer, it will be of interest to determine whether this pattern of resistance is generalized. Studies of the stability of these mutated RNA and proteins will also be important and is ongoing.

While only a small proportion of patients have developed acquired resistance to ibrutinib, these data suggest that patients with more genomic instability, including those with del(17p13.1), del(11q22.3), and complex karyotype, may be at risk for relapse. As all of the patients that we have studied have been previously treated with chemotherapy and del(17p13.1) and complex karyotype are much more common in relapsed disease, it is difficult to know whether loss of p53 function or other alterations in disease biology acquired through karyotypic evolution increase the risk of resistance or whether patients who are more likely to develop del(17p13.1) and complex karyotype as their disease progresses are also those who are more likely to develop ibrutinib resistance. This will likely become clear as additional patients with treatment-naïve disease are treated with

37 ibrutinib on clinical trials. Based upon data available, this group with del(17p13.1) or complex karyotype seem to be the most rationale to pursue combination therapies to avoid development of resistance.

While this series of patients demonstrates uniform presence of BTK and/or

PLCγ2 mutations, it is clear that other mechanisms of resistance are also present in a subset of patients. A recent preliminary report sequenced three ibrutinib resistant patients, demonstrating the PLCγ2 S707Y mutation seen in patient 6, but no BTK mutations129. In patients without BCR pathway mutations resistance may be mediated through mutations in other coding genes providing alternative survival signals not inhibited by ibrutinib, non-coding RNA, epigenetic activation or silencing, or select gene amplification. As well, other mutations may cooperate with BTK or PLCγ2 to drive resistance, such as the additional driver mutations outlined in Table 2.2. Work is ongoing to identify alternative mechanisms of resistance and also to determine the role, if any, of other identified mutations.

These data represent the first genetic proof in humans that BTK is the most clinically relevant target of ibrutinib. While ibrutinib inhibits similar kinases with C481 including ITK and BLK79,82, mutations have not been observed in these, confirming that

BTK is indeed the critical target that drives therapeutic benefit. This suggests that strategies to treat relapsed disease with acquired C481S mutation could involve novel

BTK inhibitors with alternative binding sites. As well, knowledge of downstream mediators of resistance will lead to the development of rational combinations that may prevent or treat resistant disease. Finally, the mutations that we have seen thus far are

38 critically important because they underscore the importance of the BCR signaling pathway in CLL. This knowledge may lead to further insights into the pathogenesis and progression of CLL and strategies to prevent or overcome ibrutinib resistance.

Table 2: Characteristics of Ibrutinib Resistant Patients *Includes FISH for del(17p13.1), del(11q22.3), centromere 12, and del(13q14.3) and complexity determined by stimulated banded metaphase analysis.

Chr Position Gene Amino Acid Change Variant Reference Alternate Coverage_P1_relapse Allelic Frequency_P1_relapse Coverage_P1_primary Allelic Frequency_P1_primary Coverage_P2_relapse Allelic Frequency_P2_relapse Coverage_P2_primary Allelic Frequency_P2_primary Coverage_P3_relapse Allelic Frequency_P3_relapse Coverage_P3_primary Allelic Frequency_P3_primary Coverage_P4_relapse Allelic Frequency_P4_relapse Coverage_P4_primary Allelic Frequency_P4_primary Coverage_P5_relapse Allelic Frequency_P5_relapse Coverage_P5_primary Allelic Frequency_P5_primary Coverage_P6_relapse Allelic Frequency_P6_relapse Coverage_P6_primary Allelic Frequency_P6_primary

1 110466417 CSF1 A392S snp G T 110 0.091 81 0.012

1 57481101 DAB1 Y333S snp T G 16 0.188 15 0

1 44401862 ARTN G70A snp G C 16 0.313 11 0.091

1 36563769 COL8A2 P505A snp G C 47 0.149 35 0.029

1 25883694 LDLRAP1 I132T snp T C 117 0.103 140 0

2 240982363 PRR21 Ins-FS ins GCCGTGGATGAAGA 6 0.33 6 6

2 240961743 NDUFA10 S30R snp G T 24 0.208 45 0.044

2 220379718 ASIC4 G218D snp G A 89 0.393 89 0.011

2 132021721 POTEE Y898C snp A G 33 0.091 30 0

2 61719838 XPO1 T477I snp G A 162 0.037 123 0

3 133553464 RAB6B A173T snp C T 42 0.071 47 0

3 111799804 TMPRSS7 G802E snp G A 37 0.081 49 0

3 53779715 CACNA1D L1044P snp T C 89 0.213 72 0.014

4 156726342 GUCY1B3 M621I snp G A 57 0.193 62 0

4 140811082 MAML3 Ins-Inframe ins TGT 56 0.321 63 0

4 79460515 FRAS1 S3789F snp C T 122 0.049 134 0

4 5586403 EVC2 S1002T snp A T 30 0.433 40 0

5 170827910 NPM1 S217L snp C T 17 0.176 22 0

5 151179815 G3BP1 R331Q snp G A 28 0.286 26 0

5 134688685 H2AFY E213D snp C A 97 0.062 72 0

5 41004968 HEATR7B2 L1307M snp A T 85 0.071 89 0

5 33684002 ADAMTS12 E265K snp C T 75 0.24 76 0

6 138629884 KIAA1244 Q1328K snp C A 104 0.068 104 0

6 132198149 ENPP1 P581S snp C T 89 0.067 77 0

6 124979357 NKAIN2 N100I snp A T 60 0.1 43 0

6 111804013 REV3L Ins-FS ins G 6 0.33 11 0

6 75797350 COL12A1 G3042* snp C A 68 0.074 79 0

6 394954 IRF4 D117V snp A T 74 0.311 74 0.014

7 150093654 ZNF775 T29A snp A G 8 0.625 14 0

7 149515191 SSPO Del-FS del C 6 0.66 8 0

7 149129529 ZNF777 N612Y snp T A 71 0.127 58 0

7 48550695 ABCA13 S4514P snp T C 191 0.084 203 0

8 116631533 TRPS1 H264Q snp A C 39 0.077 56 0

9 139975238 UAP1L1 A426T snp G A 36 0.361 39 0

9 139581759 AGPAT2 Ins-Inframe ins CAG 8 0.5 5 0

9 139272597 SNAPC4 T1228A snp T C 90 0.067 112 0

9 129870367 RALGPS1 Q215P snp T G 34 0.147 28 0.036

9 80537112 GNAQ T96S snp T A 80 0.063 85 0.012 10 98363776 PIK3AP1 S734I snp C A 31 0.258 20 0 Table 3: All functional variants identified as acquired from baseline to relapse. The table indicates all functional mutations identified on WES at relapse from ibrutinib that were not present at baseline. Coverage and allelic frequency at baseline and relapse are included as well.

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Table 3: Continued

10 64573332 EGR2 E356K snp C T 125 0.336 109 0.0092

10 33469096 NRP1 S894P snp A G 239 0.034 245 0

11 71259920 KRTAP5-9 G73S snp G A 29 0.138 30 0

11 64953655 CAPN1 Y202S snp A C 52 0.25 41 0.024

11 1092947 MUC2 T1589N snp C A 32 0.094 55 0.016

12 63964599 DPY19L2 I647V snp T C 25 0.16 41 0.024

12 53004498 KRT73 A411V snp G A 46 0.065 53 0

12 51384723 SLC11A2 R506L snp C A 37 0.083 20 0

12 49420563 MLL2 C5062W snp A C 60 0.233 86 0

12 33579224 SYT10 E120K snp C T 40 0.1 42 0.024

12 31270081 OVOS2 Ins-Inframe ins AAA 14 0.643 10 0

13 38248396 TRPC4 T448I snp G A 98 0.062 128 0.008

14 70634089 SLC8A3 R351C snp G A 47 0.085 56 0

15 91292908 BLM K137T snp A C 140 0.036 147 0

15 40660012 DISP2 R567G snp C G 109 0.128 86 0

15 34646791 C15orf55 V407G snp T G 48 0.271 36 0.057

16 81962183 PLCG2 L845F snp A T 56 0.286 60 0

16 81946260 PLCG2 R665W snp C T 21 0.381 13 0

16 3109044 MMP25 A545D snp C A 232 0.216 240 0.004

16 1797256 MAPK8IP3 E324G snp A G 47 0.085 58 0

17 73569701 LLGL2 Ins-FS ins G 14 0.5 13 0

17 39389215 KRTAP9-3 C154* snp C A 148 0.041 120 0

17 27071252 TRAF4 T41I snp C T 89 0.225 70 0

17 16256687 CENPV Ins-Inframe ins CGGAGGCCC 9 0.33 4 0

17 7577580 TP53 Y234C snp T C 11 0.364 10 0

19 51986333 CEACAM18 E307Q snp G C 122 0.475 100 0

19 50925782 SPIB P35L snp C T 84 0.048 75 0

19 48305639 TPRX1 Del-Inframe del GGGCCTGGGATC 14 0.643 9 0

19 36002422 DMKN Ins-Inframe ins CTGCTGCTG 17 0.647 21 0

19 18423421 LSM4 T46M snp G A 103 0.301 79 0

19 13318713 CACNA1A Ins-FS ins T 6 0.66 9 0

19 11541554 CCDC151 W137G snp A C 64 0.188 44 0.045

19 5705879 LONP1 R424L snp C A 24 0.125 33 0

19 4327156 STAP2 P243Q snp G T 31 0.097 48 0

21 46012220 TSPEAR Ins-Inframe ins GGGGCGCAGCAGCTG 6 0.66 5 0

21 39671581 KCNJ15 R133H snp G A 124 0.274 160 0

21 38862711 DYRK1A R300P snp G C 49 0.327 41 0.025

21 34721796 IFNAR1 D364N snp G A 55 0.309 82 0.012

22 32635017 SLC5A4 A180T snp C T 43 0.093 41 0

22 19365576 HIRA A477T snp C T 15 0.2 19 0

X 114249118 IL13RA2 L89Q snp A T 121 0.438 141 0

X 100611165 BTK C481S snp A T 63 0.413 47 0

X 100611164 BTK C481S snp C G 36 0.306 62 0 27 0.222 15 0 35 0.6 26 0 37 0.167 32 0

X 75650452 MAGEE1 Del-FS del G 6 0.33 5 0

X 50350729 SHROOM4 Ins-Inframe ins TCC 10 0.25 5 0 X 41332825 NYX Ins-FS ins T 6 0.66 7 0

Table 4: Alignment Statistics. On average 99 million reads were generated for each sample. While 98% mapped to the reference genome, on average 78% of them mapped to unique chromosomal positions and used for further analysis. These reads provide approximately 60X coverage of exonic regions.

Table 5: Variant frequency in the patient at relapse and 1 month post-relapse

Table 6: Baseline data for patients deep sequenced with Ion Torrent

Residence -1 -1 5 -1 -5 -9 kon[M s ](x10 ) koff[s ](x10 ) KD[M](x10 ) time [min]

BTK-WT 2.4±0.6 5.7±3.2* 0.24±1,5 294±156 Ibrutinib BTK-C481S 3.5±3,3 233±106 10.4±9,6 7±5

BTK-WT 81±2,6 733±463 1.1±0,74 2±2 Dasatinib BTK-C481S 29±7,3 278±130 0.98±39 6±3

* Koff is approaching the limits of the system.

Table 7: Biacore SPR binding assays. The same BTK and BTKC481S protein preps as used in the biochemical kinase assay, reveal that the affinity of ibrutinib toward BTK is approximately 40-fold higher than C481S BTK and that the C481S mutation results in reversible inhibition (reduction of residence time from ~5h to ~7min). This is in contrast to dasatinib, a fully reversible BTK inhibitor which has a similar affinity and residence time in wild type and in C481S mutated BTK. Data is from 3 independent runs. Kon refers to time to association, Koff time to dissociation after washout, and KD is affinity.

Figure 4: Exome-Seq analysis pipeline

Figure 5: Copy number profile for all samples. Data were plotted using DNAcopy package of BioConductor.

Figure 6 Mutations identified and confirmed with DNA sequencing. Partial chromatographs generated by chain-termination DNA sequencing of BTK and PLCg2 from peripheral blood mononuclear cells (PBMC) of patients at relapse. For patient 1, the T1634A mutation was observed that results in alteration of cysteine to serine at position 481(1A). For patients 2, 3, and 4 sequencing revealed the G1635C mutation, which also results in a cysteine to serine substitution at position 481. Patient 6 had wild-type BTK, but had the C2120T mutation in PLCγ2 that results in a Arginine to Tryptophan alteration at position 665 (1B). Patient 5 had the G1635 mutation in BTK and A149285T mutation in PLCγ2 that results in a Leucine to Phenylalanine substitution. This clone was very small on Sanger sequencing.

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Figure 6: Continued

Figure 7: Functional Characterization of C481S BTK. Kinase assay of recombinant wild type BTK versus C481S mutated BTK shows that the mutant form has enhanced kinase activity which can be inhibited by ibrutinib, though at a much higher EC50 than wild type (A). These data reflect two independent experiments with triplicate samples for each treatment with error bars indicating standard error of the mean (SEM). After transfection into HEK293T cells, administration of ibrutinib does not inhibit BCR signaling in cells with C481S to the same degree as wild type BTK. BTK (B) or ERK (C) phosphorylation is significantly more inhibited in cells with WT versus C481S mutated BTK at 0.01μM, 0.1μM, and 1μM ibrutinib (p<0.0001), and the difference in inhibition with WT vs mutated BTK is significantly greater for ibrutinib than for dasatinib (p<0.0001). The C481S mutant reverts ibrutinib to a reversible inhibitor, as cells transfected with C481S mutated BTK show inhibited BCR signaling when drug is in culture, but inhibition is removed when drug is washed out (D). Phosphorylation of both genes significantly increases with washout in mutated cells. This effect is significantly different than that observed in the wild-type cells, where increase in phosphorylation with washout did not occur (p<0.0001 for both genes). In this experiment, cells were exposed to ibrutinib for 45 minutes and then either fixed immediately or washed twice and then fixed. Error bars in both figures represent SEM.

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Figure 7: Continued

Figure 8: Functional Characterization of C481S BTK in BTK-deficient DT40 cells. After transfection of wild type or C481S mutated BTK into DT40 BTK -/- cells, BCR signaling (A) was evaluated. BCR signaling is inhibited by ibrutinib in cells with wild type BTK to a greater degree than those cells with C481S mutated BTK. The figure reflects at least 3 independent experiments. After transfection of wild type or C481S mutated BTK into DT40 BTK -/- cells, there was a significant difference in the maximum calcium flux between mutated and wild-type cells after treatment with ibrutinib, where on average the maximum calcium flux was higher in the mutated cells controlling for concentration (p = 0.0005). This is a representative figure depicting one of 3 independent experiments where statistical analysis includes all data. (B) 53

Figure 9: Inhibition of BTK by ibrutinib is reversible in patients with C481S BTK mutation. Cells were obtained at baseline and relapse from a patient who developed a C481S BTK mutation. Cells were treated in vitro with 1uM ibrutinib for 1 hour followed by either washed out or not. Cells were then rested for 1 hour and then stimulated with plate-immobilized IgM for 15 minutes. BTK phosphorylation at baseline is inhibited by either washout or non-washout condition, however, in the presence of C481S BTK mutation, BTK phosphorylation is only inhibited when ibrutinib is not washed out.

Figure 10: Functional Characterization of R665W and L845F mutated PLCγ2. pRetro X Tet-on Constructs containing wild-type PLCγ2 or mutations of either R665W or L845F were transfected or retro-virally delivered into 293 and PLCγ2 -/- DT40 cells. After transfection, PLCγ2 is present in these cells, and Y1217 phosphorylation can be detected in 293 cells.

Figure 11: R665W and L845F reveal hyperactive Calcium influx. After stimulation of DT40 cells with anti-IgM antibody, calcium flux assays showed calcium release in the cells with nonmutant PLCγ2 that can be completely inhibited by ibrutinib. Cells bearing either the R665W mutation or the L845F mutation showed calcium release that is not inhibited by 1 μM ibrutinib (P = 0.62 for R665W and P = 0.43 for L845F). Error bars represent standard errors. RFU denotes relative fluorescence units.

Figure 12: PLCG2 mutants display resistance to ibrutinib in downstream signaling. PLCγ2 -/- DT40 cells stably expressing either wild-type or mutated pRetro-PLCγ2 was treated with vehicle or 1μM ibrutinib for 30 minutes followed by stimulation for 15 minutes with 5μg/ml anti-IgM and then lysed. Immunoblot analysis shows that downstream BCR signaling as evidenced by phosphorylated AKT and ERK are intact in these cells, and in cells with the R665W or L845F mutations, these downstream signals are diminished to a lesser degree by ibrutinib after anti-IgM stimulation (B). All figures are representative and are reflective of at least 3 independent experiments.

Figure 13: CLL with R665W PLCG2 bypasses BTK inhibition by ibrutinib. One patient who was found to have a R665W mutation in PLCγ2 had sample available for analysis by immunoblot at baseline and relapse. Prior to drug discontinuation at relapse, sample was obtained for immunoblot analysis. Compared with samples taken at baseline and at a time of response to ibrutinib, BTK remained inhibited at the time of relapse (A). However, pPLCγ2 shows evidence of sustained activation at this timepoint (B) suggesting that the R665W mutation is gain of functionin patients. At the time of relapse after drug had been discontinued, fresh cells were treated with vehicle, plate-immobilized anti-IgM, 1μM ibrutinib, or ibrutinib + anti-IgM. BTK phosphorylation is able to be inhibited by ibrutinib (C), however, PLCγ2 phosphorylation is not (D), showing that the R665W mutation is not sensitive to ibrutinib in vitro. Densitometry values are located under each lane and reflect phosphorylated protein/total protein except plot A, which reflects phosphorylated protein/loading control as total BTK protein expression can change over time during ibrutinib therapy.

Figure 14: Ibrutinib resistance in CLL from the patient with both BTK and PLCG2 mutations One patient who was found to have both BTK and PLCγ2 mutations had sample available for analysis by immunoblot at relapse. At the time of relapse after drug had been discontinued, fresh cells were treated with vehicle, plate-immobilized anti-IgM, 1μM ibrutinib, or ibrutinib + anti-IgM. Phosphorylation of BTK (A), PLCγ2 (B), and ERK (C) are not inhibited by ibrutinib.

CHAPTER 3 OSU-T315: A Novel Targeted Therapeutic that Antagonizes AKT

Membrane Localization and Activation of Chronic Lymphocytic Leukemia Cells

3.1 Introduction

Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in adults, and remains incurable despite the introduction of targeted agents. CLL also has an uncertain etiology130,131, although current data support that CLL originates from antigen-experienced, post-germinal center B cells132. CLL has multiple recurrent cytogenetic abnormalities including del(13)(q14), trisomy 12, del(11)(q22.3) and del(17)(p13.1), of which the latter two portend a more rapid disease progression and shorter survival from diagnosis133. Approximately 60-65% of CLL cases exhibit somatic hypermutation in immunoglobulin heavy chain variable (IGHV) genes (M-CLL), while

35-40% of CLL cases are categorized with unmutated IGHV status (U-CLL), which is associated with poor prognosis15,49. The U-CLL patient subset also has a high proportion of ZAP-70 expression23, enhanced B-cell receptor signaling, and disproportionate number of del(11)(q22.3) and del(17)(p13.1) cases. Recently, comprehensive genome-wide sequencing unveiled numerous common mutations in NOTCH1, SF3B1, MYD88 and

BIRC3134-137, which are also likely to be useful in prognostication138. Overall, identification of biological markers associated with clinical outcome facilitates the identification of therapies targeted toward aberrant signaling pathways.

The current initial therapy for CLL patients lacking del(17p13.1) typically includes fludarabine and cyclophosphamide plus rituximab (FCR) for younger, fit patients68, whereas for older infirm patients, chlorambucil plus obinutuzumab139 is most appropriate.

Patients with del(17p13.1) do not benefit in terms of PFS and OS with chemoimmunotherapy26,140. Despite chemoimmunotherapy prolonging survival, this treatment is not curative. A proposed reason that available CLL therapies are incompletely effective is the increased proliferation and acquisition of tumor cell resistance to apoptosis as a result of stimuli within microenvironments in lymphoid tissues. Following recent advances in our understanding of CLL disease biology, efforts have focused on antagonizing oncogenic signaling initiated from the tumor microenvironment.56 Key pro-survival signals in CLL include BCR activation46,48,141, the TNF-receptor family molecules CD40L, BAFF, and APRIL54,142, and the chemokines CCL3, CCL4143,

CXCL12144, and CXCL13145, all of which augment downstream activation of AKT and/or

ERK signaling in CLL cells and contribute to CLL survival and proliferation86. To date, the best success in targeting the pathways activated by these signals has been through the use of agents inhibiting proximal or distal BCR signaling, such as the Syk inhibitor fostamatinib146,147, the PI3K p110δ inhibitor idelalisib148, and the BTK inhibitor ibrutinib80. Despite a high frequency of durable partial responses with the latter two agents in CLL patients, complete remissions are infrequent. Indeed, none of these agents sufficiently overcomes AKT and/or ERK signaling pathways concurrently in the presence of multiple survival stimuli. Agents that inhibit AKT and/or ERK pathways in a novel manner therefore represent an exciting strategy for this disease.

The activation of the PI3K/AKT pathway is initiated at the plasma membrane, where phosphatidylinositol (3,4,5) trisphosphate (PIP3) generated by PI3 kinase recruits AKT to the unique membrane compartments termed lipid rafts upon interaction via PH domains, leading to its subsequent phosphorylation and activation by PDK1/2149. Recent studies show that these glycosphingolipid- and cholesterol-rich rafts serve as platforms for the initiation of a variety of signaling pathways150. These include the PI3K/AKT151, CD40L152 and BCR153 signaling pathways, each of which are associated with CLL tumor cell survival and disease progression. Notably, disruption of lipid rafts by cholesterol sequestration using saponin, cholesterol depletion by methyl-β-cyclodextrin154, or inhibition of cholesterol biosynthesis by simvastatin155 results in ablation of AKT phosphorylation and induces preferential cytotoxicity toward malignant cells in prostate cancer156. Likewise, investigations using alkyl-lysophospholipid (ALP) analogues further support the anti-tumor potential of targeting lipid rafts in mantle cell lymphoma (MCL) and CLL via the recruitment of Fas/CD95157. Building upon these promising results, we herein report that the novel agent OSU-T315, modified from the scaffold of AKT binding site at ILK, employs an analogous mechanism of anti-tumor activity in CLL by displacing AKT from lipid rafts.

Integrin-linked kinase (ILK) is a master regulator of intracellular signaling that controls cell proliferation, survival, migration, invasion and angiogenesis158. The crucial function of ILK in tumorigenesis involves downstream activation of AKT via Ser473 phosphorylation, suggesting ILK as a promising target in malignancies that rely on the

PI3K pathway159. Similarly, overexpression of ILK in myoblasts results in enhanced ERK 62 activation, supporting the involvement of ILK in the MAPK pathway160. Recently, generation of the novel ILK inhibitor OSU-T315 was reported161. Given the justification that targeted therapy directed at ILK would be effective in CLL through suppressing AKT and/or ERK activation, we pursued pre-clinical in vitro and in vivo studies to determine the activity of OSU-T315 toward primary CLL cells. Concurrently, we investigated whether this agent caused disruption of PI3K and MAPK signaling promoted by BCR or integrin engagement, besides specific inhibition of PI3K axis mediated by CD40 and TLR9.

Remarkably, we demonstrate that these effects are not through inhibition of ILK as expected, but rather by the prevention of AKT recruitment to lipid rafts. This surprising finding introduces a novel mechanism to therapeutically target CLL.

3.2 Materials and methods

Reagents and antibodies

OSU-T315 was synthesized as previously described161. Stock solutions were prepared in

DMSO. The commercial sources of antibodies used are: p-AKT Ser473, p-AKT Thr308,

AKT, p-ERK Thr202/Tyr204, ERK, p-GSK3β S9, GSK3β, p-CRAF Ser338, BTK, p-FAK

Tyr925, p-p38 Thr180/Tyr182, p38 (Cell Signaling Technologies), p-BTK Tyr223

(Abcam), p-FAK Tyr397 (BD Transduction Laboratories), and FAK, Raf-1 (Santa Cruz

Biotechnologies). Other reagents include: recombinant Human VCAM-1 and CD40L

(PEPROTECH); α-IgM μ-specific polyclonal antibody (MP Biomedicals); CpG ODN

(Invivogen); BioCoat™ fibronectin plate (BD); Z-VAD-FMK (Santa Cruz); okadaic acid

63 and Active Ras Detection Kit (Cell Signaling); and PI3 Kinase Activity/Inhibitor ELISA

(Millipore).

CLL, normal B-Cell, and T-Cell Isolation

Peripheral blood samples were obtained from CLL patients after written informed consent was provided on an OSU institutional review board approved protocol. Human CLL and normal B or T cells were isolated and cultured as previously described80. Briefly, whole blood from patients or healthy donors was negatively selected using Rosette-Sep kits for isolating human B and T cells (StemCell Technologies), followed by Ficoll separation to enrich viable CLL, B or T cells. The purity after selection was examined indicating >90%

CD19+ B cells or CD3+ T cells. Purified cells were cultured in complete RPMI 1640 with

10% fetal bovine serum. Human CLL cell line MEC-1 (ACC 497) and B cell precursor leukemia 697 cell line (ACC 42) were purchased from DMSZ; OSU-CLL cell line was derived as previously described162. All lines were cultured in complete RPMI plus 10%

FBS.

Cell lysis and Immunoblot

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce). Proteins were quantified by BCA protein assay kit (Pierce) and separated by SDS-PAGE. The blots were probed with the appropriate primary and HRP-conjugated secondary antibodies and developed with chemiluminescent substrate (Pierce), followed by detection using X-ray film and quantification using ImageJ software.

Assessment of cell death

Cell viability was assessed using annexin-V and propidium iodide (PI) double staining

64 followed by analysis on an EPICS XL flow cytometer (Beckman-Coulter). Mitochondrial activity was measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay

(Promega) according to the manufacturer’s instructions.

Animal studies

CD19+ cells were obtained from spleens of TCL1 transgenic mice with CLL-like leukemia and splenomegaly, and 1 x 106 cells were injected intravenously (IV) into a C57BL/6 mouse as previously described163. Peripheral blood lymphocytes (PBL) from implanted mice were assessed weekly, and treatment was initiated when the CD19+ CD5+ cells exceeded 10% of the PBL population. Mice were treated once daily by oral gavage with vehicle or 50 mg/kg OSU-T315 in sterile water containing 0.5% methylcellulose (w/v) +

0.1% Tween-80 (v/v). Disease progression was monitored as previously described163.

Mice were sacrificed when PBL counts exceeded 20,000 cells/μl and the presence of leukemia produced discomfort. All experiments were carried out under protocols approved by The Ohio State University Institutional Animal Care and Use Committee.

Pharmacokinetics

Ten-week old C57BL/6 mice (body weight range 18-21g) received 50 mg/kg OSU-T315 by tail vein (IV) or oral gavage (P.O.) delivery. For PO administration, the vehicle was

0.5% methylcellulose + 0.1% Tween-80 formulated sterile water. For IV administration,

10% DMSO + 10% Cremophor EL formulated saline was used to improve the solubility.

Six mice in each group were sacrificed at 5, 15, 30, 45, 60 min, and 2, 4, 8, 24 hr after IV dosing. PO dosed mice were sacrificed at 15, 30, 45, 60 min, and 2, 4, 6, 8, 24 hr post-treatment. Blood samples were collected by cardiac puncture and transferred into

65 lithium heparin additive green-top tubes (Becton, Dickinson and Company). After centrifuging for 2 minutes at 6000g, plasma was collected and stored at -80oC. OSU-T315 was quantified by a validated liquid chromatography-mass spectrometry (LC-MS) assay.

The LC-MS system consists of a Finnigan TSQ Quantum EMR Triple Quadrupole mass spectrometer (Thermo Fisher Scientific Corporation, San Jose, CA) and a Shimadzu

Prominence HPLC system (Shimadzu, Columbia, MD). Both OSU-T315 and internal standard OSU-Arg were separated on a Betabasic-8 column (Thermo Scientific Corp,

50x2.1mm ID, 5µm) by isocratic elution with 55% acetonitrile containing 0.5% acetic acid. Two parent and product ion pairs 534.10>503.10 and 446.15>429.19 were selected to monitor OSU-T315 and OSU-Arg in multiple reaction monitoring (MRM) mode respectively. The plasma concentrations of OSU-T315 were calculated against a calibration curve of OSU-T315 spiked in mouse plasma prior to extraction with ethyl acetate. The dynamic range of the calibration curve of OSU-T315 was from 0.5 ng/mL to

1000 ng/mL with regression coefficiency exceeding 0.99. Sample analysis was accompanied by quality controls with acceptable precision and accuracy values. shRNA knockdown

Lentiviral particles were generated in 293 cells as previously described164. GIPZ lentiviral based shRNA (Thermo Scientific RHS4531-EG3611) for ILK specific knockdown was transduced into Mec-1 or 697 cells by spin-down infection. ILK shRNA expressing cells were selectively grown in the presence of puromycin (1ug/ml), and more than 90% of cells were GFP-positive after 5 days.

Lipid raft extraction

Mec-1 cells (1x108) cells stably expressing pBabe-Puro-Myr-Flag-AKT1 (Addgene

15294) were harvested and resuspended in 1ml of ice-cold MBS (25 mM MES, 150 mM

NaCl [pH6.5]), 1% Triton X-100 supplemented with 1 mM PMSF, and 1:100 diluted protease inhibitor cocktail (Sigma P8340). Following 30 min incubation on ice, the lysate was subjected to chamber-based sonication for 10 seconds, then gently mixed with an equal volume of ice-cold 80% sucrose (w/v) in 1 x MBS, and loaded into a centrifuge tube.

The sample was overlaid with 2 ml of 35% sucrose and 1 ml of 5% sucrose (w/v), both prepared in 1 x MBS. The sucrose gradient samples were spun at 43,600 rpm (180,000 x g) in a SW55Ti rotor (Beckman-Coulter) in an ultracentrifuge at 4°C for 20 hours.

Glycolipid-enriched domains (DIGs) were harvested by collecting 0.5ml fractions from the top of gradients.

Confocal immunofluorescent microscopy

Cells were first labeled with Alexa Fluor® 488 conjugated Cholera Toxin Subunit B

(CT-B) according to the manufacturer’s specification (Life Technologies). Labeled cells were positioned on the microscope slide using Cytospin3 centrifuge (Thermo, Asheville,

NC) and stained with fluorescence-conjugated antibodies. Briefly, cells were fixed with

4% paraformaldehyde and followed by permeabilization with 0.5% Triton X-100 in PBS.

After washing and incubating with blocking solution (2% BSA in PBS), cells were stained with anti-AKT antibodies overnight at 4o, followed by incubation with secondary antibody conjugated with Alexa Fluor® 594 (Life Technologies). Nuclei were stained blue with

DAPI (Vector Laboratories, Burlingame, CA). Images were observed and quantified using

67 a 60X objective and 10X digital zoom with Olympus FV1000-Filter Confocal System at the Ohio State University Campus Microscopy and Imaging Facility.

Statistical analysis

Percent viability of CLL compared to B or T cells upon treatment was outlined by means with SEM and analyzed based on unpaired Student t tests. The ratios of phospho-protein were measured after normalization to GAPDH expression. Differences between treatments were calculated by unpaired Student t tests. Overlap index of AKT content with lipid rafts was analyzed using Olympus FluoView viewer. For mouse models, overall survival (OS) was obtained using the Kaplan-Meier method. All tests were two-sided, and statistical significance was declared for P < .05.

3.3 Results

OSU-T315 Induces Selective Cytotoxicity toward CLL cells

We first sought to assess the efficacy of OSU-T315 toward primary CLL cells. Our data indicate dose dependent cytotoxicity toward CLL cells. The LC50 after 24 hour incubation of primary CLL (n=15) cells was 4-5μM. Contrasting with this, the cytotoxicity of OSU-T315 in normal B (n=7) or T cells (n=8) was significantly lower (IC50> 8μM; p<0.0001 for each) (Figure 15A and 15B), suggesting a therapeutic index of OSU-T315.

We next examined the activity of T315 toward two CLL-derived cell lines, Mec-1 (Figure

15C) and OSU-CLL (Figure 15C) and found similar dose-dependent cytotoxicity (LC50

2-3μM in both) after 24 hour treatment.

OSU-T315 inhibits AKT Signaling Independent of ILK

Activation of the PI3K/AKT pathway is essential for CLL survival and proliferation86. Given that OSU-T315 has been shown to inhibit activation of AKT and

ERK through targeting ILK in solid tumors161, components of key signaling pathways were evaluated following OSU-T315 treatment in Mec-1 and OSU-CLL cells (Figure 16D).

Consistent with the previous report, this cellular profiling demonstrates notably diminished

AKT and ERK phosphorylation upon treatment, along with dephosphorylation of GSK-3β, a direct downstream target of AKT165. Phosphorylation of AKT initially occurs at the

Thr308 site in the activation loop by PDK1, with full activation occurring following phosphorylation of Ser473 in the C-terminal hydrophobic domain by the putative PDK2 kinase. Several candidates for PDK2 have been proposed, including RICTOR-mTOR

(TORC2)166, ILK159, DNA-dependent protein kinase (DNA-PK)167, ataxia telangiectasia mutated (ATM)168, and protein kinase C (PKC)169, although no consensus has been reached. To elucidate the role of ILK in phosphorylating Ser473 site of AKT, we silenced

ILK by shRNA. Despite efficient knock down of ILK (Figure 17A), the proliferation rate in Mec-1 cells was not altered (Figure 17B). Furthermore, ILK-depleted cells showed no differences in phosphorylation of AKT Ser473 or ERK, either at baseline or following anti-IgM (Figure 17C) or fibronectin (Figure 17D) stimulation. ILK down-regulation also did not abrogate T315-mediated killing (Figure 17E). Together, these results suggest a cytotoxic role of T315 independent of ILK in lymphoid cells.

OSU-T315 Does not Influence Proximal Membrane Signaling of AKT

Given the localization of PDK1 to plasma membrane is essential to phosphorylate

AKT, and the generation of PIP3 via PI3-kinase is essential for this recruitment, we hypothesized that OSU-T315 may interfere with PDK1 activity or even proximal

PI3-kinase. This activity could then disrupt the generation of PIP3, leading to failure of

PDK1 (or PDK2 candidate) membrane localization and inhibition of AKT Thr308 and

Ser473 phosphorylation. To investigate these possibilities, we examined the effects of

OSU-T315 on AKT-related signaling events. OSU-T315 treatment failed to disrupt PDK1 phosphorylation170 (Figure 16A), PI3 kinase activity (Figure 16B) or Ras interaction with c-RAF (Figure 16C) under conditions in which AKT phosphorylation is inhibited. Other membrane-associated kinases including focal adhesion kinase (FAK) and BTK, a key component of the BCR signaling pathway, were also not affected by OSU-T315 (Figure

16D). Similarly, the activation of phosphatase PP2A, which has been known to negatively regulate AKT activity, was not influenced by T315 as evidenced by continued dephosphorylation of AKT in the presence of okadaic acid, the PP2A inhibitor (Figure

16E). Together, these findings indicate that OSU-T315 induces cytotoxicity in CLL cells by suppressing intrinsic AKT activation without influencing proximal kinases or relevant phosphatases involved in regulation of this important pathway.

OSU-T315 Antagonizes AKT Membrane Localization

It is now evident that lipid rafts are highly important for the interactions and regulations of membrane-localized kinases, crucially mediating AKT translocation and downstream activation. Lipid rafts are plasma membrane microdomains enriched in

70 cholesterol, sphingolipids, as well as external and internal signaling proteins. PI3K/AKT signaling originates from these rafts following the recruitment of AKT via the interaction of the PH domain of AKT with phosphatidylinositol (3,4,5)-trisphosphate (PIP3) or phosphatidylinositol (3,4)-bisphosphate (PIP2)155,156. In B cells, Src-family kinases including Lyn, as well as the CD40 receptor, have been shown to be stably anchored within lipid rafts. Certain proteins, including AKT, are also accumulated in rafts in part due to myristoylation. As OSU-T315 inhibits AKT phosphorylation without affecting the activity of PI3K, PDK1, RAS, or PP2A, we postulated that OSU-T315 may elicit its effects through altering recruitment of AKT to lipid rafts. To determine if OSU-T315 impedes the recruitment of AKT, myristoylated-AKT (myr-AKT) that spontaneously translocates to lipid rafts171, was introduced into Mec-1 cells to reinforce membrane docking of AKT in resting cells. The diminished phosphorylation of myr-AKT was observed following

OSU-T315 treatment (Figure 22A and B), followed by reduced content of myr-AKT within the lipid raft fraction (Figure 18A and B), while retaining equivalent amounts of PI3 kinases, PTEN, and BCR-associated molecules Lyn and BTK. These data demonstrate that

OSU-T315 inhibits AKT translocation into lipid rafts without affecting raft integrity.

Furthermore, confocal microscopy to examine protein distribution shows reduced co-localization of myr-AKT with lipid rafts upon OSU-T315 treatment (Figure 18C and

D). Together, these findings confirm that OSU-T315 inhibits AKT activation by preventing its localization into lipid rafts.

B cell receptor, CD40, and TLR9 induced survival signals are abrogated by

OSU-T315.

Diverse stimuli within tumor environments provoke ATK activation, presumably supported by CD154 (CD40 ligand), CpG (Toll-like receptor 9 ligand), chemokines or cytokines such as SDF-1 and BAFF172. Numerous studies indicate that the BCR is a key transmitter of survival signals in CLL cells141. Upon engagement of BCR, PI3K and

MAPK cascades are activated, from which CLL acquires the competency to evade apoptosis. To assess whether OSU-T315 can overcome external survival stimuli, CLL cells stimulated with immobilized α-IgM, CD40L, or CpG were treated with different concentrations of OSU-T315. The results suggest the potency of OSU-T315 suppressing downstream AKT activation triggered by external factors (Figure 19A and C). The protection of CLL cells mediated by these external stimuli was abrogated by increasing doses of OSU-T315 (Figure 19E-G).

CD49d mediated survival signals are abolished by OSU-T315

CLL cells traffic and interact with stromal or nurse-like cells within lymphoid tissues through the VLA-4 (α4β1, CD49d/CD29) complex, from which survival signals originate to prevent spontaneous or drug-induced apoptosis in part resulting from AKT activation57,173. Notably, CD49d/α4 integrin expression has been identified as an unfavorable prognostic factor for overall survival in CLL, and proportionally coexists with

CD38 and ZAP7062, other poor prognostic factors in CLL. CD49d interacts with extracellular matrix components by binding to fibronectin or vascular cell adhesion molecular-1 (VCAM-1)174, facilitating integrin-mediated adhesion. Given that OSU-T315

72 interferes with integrin signaling as evidenced by inhibition of fibronectin-induced AKT phosphorylation, we examined the potential of OSU-T315 to block CD49d-mediated protection of CLL cells. Our results demonstrate CD49d activation via interaction with fibronectin or VCAM-1 can promote both AKT and ERK signals, and these effects are suppressed by OSU-T315 (Figure 19B and 19D).

OSU-T315 reduces Mcl-1 and Bcl-xl levels to trigger Caspase activation in CLL cells.

Bcl-2 family proteins are known to modulate apoptosis in CLL. Among these family members, Mcl-151,86 appears to play a particularly important role in CLL cell survival, and can be upregulated by BCR activation175. Despite the current lack of a Mcl-1 inhibitor for clinical use, numerous agents are under development to target Mcl-1 expression or function, as this is known to inversely correlate with responsiveness of CLL to chemotherapy176. To characterize the effect of OSU-T315 on Bcl-2 family proteins, Mcl-1,

Bcl-2, Bad, and Bim were examined. While expression levels of Bcl-2, Bad and Bim were unaffected, Mcl-1 levels were increased upon BCR, CD40, TLR9, and CD49d activation, and OSU-T315 treatment was able to completely reverse this effect (Figure 20A-D).

Remarkably, Mcl-1 overexpressing cells, which show resistance to fludarabine, were similarly sensitive to OSU-T315 mediated cytotoxicity as the empty vector control cells, indicating that OSU-T315 activity appears to be independent of Mcl-1, unlike the majority of currently available CLL agents (Figure 25). Interestingly, Bcl-xl upregulation mediated by CpG, and to a lesser extent, CD40L, was strongly repressed by OSU-T315 (Figure 26).

Since Mcl-1 or Bcl-xl is involved in antagonizing apoptosis in CLL86,175, our results further

73 suggests that caspase 3/7 activity is significantly augmented when increasing concentrations of OSU-T315 are administrated in both Mec-1 and OSU-CLL (Figure 20E).

The importance of caspase activation in OSU-T315-mediated apoptosis was verified using

Z-VAD-FMK, a pan-caspase inhibitor (Figure 20F). These data demonstrate that

OSU-T315 mediates cytotoxicity in CLL cells in a caspase-dependent manner, but independent of Mcl-1 expression.

OSU-T315 Has In Vivo Activity in the TCL1 Mouse Model of Human CLL

Eμ-TCL1 adult mice develop clonal B cell leukemia with surface expression of

IgM+/CD19+/CD5+177, mimicking the characteristics of primary CLL cells. TCL1 leukemia also exhibits features similar to human CLL cells with regards to activation of AKT signaling, in part resulting from the interaction of TCL1 with AKT and subsequent enhancement of its kinase activity90. Thus, leukemia cells derived from Eu-TCL1 mice were transplanted into C57BL/6 mice as a model to assess the in vivo efficacy of

OSU-T315. Mice that developed leukemia in the bloodstream (at least 10% CD5+/CD19+ cells) were treated orally with vehicle or OSU-T315 (50 mg/kg daily). Mice that received

OSU-T315 show significantly reduced white blood cell (WBC) counts after 4 weeks of treatment (p=0.036; n=6) (Figure 21B). This improvement also correlated with prolonged overall survival in the OSU-T315 treated group (p=0.037; n=6) (Figure 21C). Improved in vivo efficacy of OSU-T315 was further observed by intraperitoneal delivery approach

(p=0.012; n=9) (Figure 21D). Collectively, these findings show that OSU-T315 displays the capacity to delay leukemia progression and enhance median survival in mice bearing

TCL1 leukemia cells.

Pharmacokinetic properties of OSU-T315

Previous studies verified the efficacy of orally administered OSU-T315 in mice xenografted with prostate cancer cells161. While our data also confirms the in vivo efficacy of OSU-T315 in leukemia, pharmacokinetic studies on OSU-T315 remain necessary to optimize the treatment regimen. Therefore, plasma from mice treated with OSU-T315 by oral gavage (PO), intravenous (IV) or intraperitoneal (IP) injection was analyzed (Figure

21A and Table 8). Results indicate similar elimination half-life by these delivery approaches. While IV administration achieves peak concentration of OSU-T315 within 5 min, either IP or PO administration requires 2 hours to reach maximum levels in the bloodstream, and these maximum levels are substantially lower than the value achieved via the IV route. Importantly, the low bioavailability (5.8%) via the oral route may in part have contributed to the relatively modest survival benefit of this agent, and strategies are being explored to address this obstacle.

3.4 Discussion

The data report herein characterizes a novel mechanism of action of OSU-T315.

Specifically, we demonstrate that OSU-T315 displaces AKT from lipid rafts, thus impairing AKT phosphorylation induced by a variety of pathways including BCR, CD40L,

TLR9, and integrin. The downstream consequences of this AKT inhibition include down-regulation of Mcl-1 or Bcl-xl, and leading to caspase-dependent apoptosis.

Remarkably, we further clarify in vivo efficacy showing improved median survival in a

TCL-1 transplant model, along with selective targeting of CLL cells relative to normal B or

T lymphocytes.

Targeting BCR-associated kinases with small compounds has emerged as an exciting new treatment paradigm in CLL178. The shift of focus toward BCR-associated kinases is owed in part to a better understanding of the essential role of the BCR in CLL pathogenesis and the promising clinical results achieved with continuous dosing of BTK and PI3K inhibitors. Notably, all agents effective in targeting BCR signaling suppress the PI3K/AKT signaling cascade. Unusual activation of AKT drives CLL expansion and evades apoptotic mechanisms, critically contributing to this lymphoproliferative disorder179. Previous studies identified the importance of the PI3K delta subunit in provoking AKT activation in

CLL cells, thus driving the development of Idelalisib (GS-1101)93 and IPI-145

(INK-1147)100 that both show significant clinical activity through abrogating the

PI3K/AKT axis. In contrast to existing agents targeting upstream kinases, OSU-T315 impedes the recruitment of AKT to its signalosome in the lipid raft. The characteristics of

OSU-T315 provide the following advantages: (1) Shortcomings of recent strategies in developing targeted therapeutic arise from the balancing between specificity and efficiency. As multiple stimuli lead to AKT activation, ubiquitously inhibition of this survival signal is challenging. The effect of OSU-T315 via AKT regardless of upstream pathways activity may improve efficacy. Contrary to current AKT inhibitors that abrogate phosphorylation with selectivity, OSU-T315 universally prevents phosphorylation of AKT isoforms, rather than showing selectivity toward a particular isoform. (2) The functional redundancy of different PI3 Kinase may render the efficacy and drive drug resistance for

76 long term treatment. Compared to existing PI3K inhibitors in CLL clinical practice,

OSU-T315 shows the potential to surpass the issue. (3) The preferential cytotoxicity of

OSU-T315 toward CLL cells compared to normal B and T cells suggests a beneficial therapeutic window.

Our observations suggest that OSU-T315 impairs the ability of AKT to be retained in cholesterol-enriched lipid rafts as previously described151, resembling the effect of cholesterol inhibitors such as simvastatin that reduce the cholesterol content of lipid rafts, thus destabilizing their integrity to inhibit AKT phosphorylation155,156. This effect was also observed with synthetic antitumor lipids (ATLs), a family of compounds widely referred to as alkyl-lysophospholipid analogs, which show selective anti-tumor activity in CLL, mantle cell lymphoma (MCL)157, and multiple myeloma (MM)180. Notably, the effect of

AKT displacement from lipid rafts upon ATL treatment in MCL cells181 indicates that

OSU-T315 may share a related mechanism to impact the structure of the raft compartment.

Furthermore, consistent with the assertions that lipid rafts are critical for AKT activation and survival signaling in tumor cells, both ATLs182,183 and cholesterol inhibitors155 exhibit selective cytotoxicity toward malignant cells, similar to OSU-T315. These combined observations suggest that hypercholesterolemia might potentiate CLL development184, and also that our novel lipid raft-targeting pathway could show significant clinical activity in

CLL. In contrast to ATLs that impact diverse molecules in lipid rafts such as PI3K, PDK1, and mTOR in addition to AKT51, OSU-T315 appears to target AKT alone, indicating that this agent uniquely blocks a component essential for AKT docking. We are currently investigating the underlying candidates by OSU-T315 using proteomic approaches.

OSU-T315 was originally designed to specifically disrupt the interaction of AKT with its binding site on ILK, a putative PDK2 anticipated being a promising therapeutic target in several cancers. Although the role of ILK as a true kinase has been questioned due to its lack of a defined kinase domain, functional studies verified its critical role promoting solid tumor development by driving AKT activation. However, studies assessing the function of ILK in CLL or other leukemias are lacking. Our data showed slightly lower amounts of ILK in CLL compared to normal cells (Figure 23 A and B), and ILK knockdown could not recapitulate cytotoxicity by OSU-T315. Additionally, OSU-T315 did not appear to affect the interaction of AKT with ILK (Figure 22C). Together, these data do not support a strong role for ILK in CLL development and progression. To further elucidate the functional role of ILK in CLL pathogenesis, a mouse model is being generated in our laboratory that lacks ILK expression in mature B cells. This mouse will be crossed with Eu-TCL1 mice to examine the impact of ILK on the development and progression of TCL1 leukemia. Given that OSU-T315 disrupts function of ILK besides abrogating PI3K/AKT cascade, we speculate that OSU-T315 may interrupt integrin-mediated adhesion or trafficking toward lymphoid tissues or bone marrow.

Indeed, OSU-T315 remarkably impaired BCR-mediated adhesion (Figure 24), supporting the postulation that the agent may attenuate retention and homing of CLL cells mediated by

BCR signaling or chemokines. Besides being activated by PLCγ2/PKC-β through the BCR pathway, ERK1/2 is also provoked via diverse external signals. This subsequently activate

FOS, JUN and ETS family members, leading to CLL proliferation and survival50.

Interestingly, despite the comprehensive profiling by immunoblotting showing reduction

78 of tonic p-AKT and p-ERK upon OSU-T315 treatment, only BCR- or CD49d-mediated

ERK activation was consistently abrogated by OSU-T315. Conversely, CD40L or CpG induced signaling exhibited differential response in ERK status (Figure 27), suggesting that OSU-T315 particularly impacts PI3K/AKT signaling. Finally, our in vivo study shows that, despite being hampered by low oral bioavailability, OSU-T315 significantly prolonged survival of leukemic mice. These promising data support the development of improved formulation, delivery strategies, and/or derivatives, which are being actively pursued by our group. Together, these findings provide an alternative strategy to target

CLL cell survival by disrupting AKT recruitment to lipid rafts, and introduce an outstanding candidate for further investigation and development in CLL and potentially other B-cell malignancies.

PK parameters I.V.- 50mg/kg P.O.- 50mg/kg I.P.- 25mg/kg Cmax (nM) 48764 369.09 3604 Tmax (min) 5 120 120 T1/2 (hr) 5.51 5.07 6.08 Bioavailability (%) N.A. 5.8 58.6 Table 8: Pharmacokinetic properties of OSU-T315

Figure 15: OSU-T315 induces preferential cytotoxicity in CLL cells. 1E7/ml primary CLL cells from patients; healthy B cells (CD19+) (A) and T cells (CD3+) (B) from leukopaks purified by CD19+ or CD3+ enrichment kit respectively were incubated in complete RPMI with 10% FBS when treating increasing dose of OSU-T315. Cells viability was analyzed by flow cytometry at 24hrs. 1E6/ml cells in complete RPMI with 10% FBS was incubated and treated with increasing concentration of OSU-T315. Cells viability upon treatment in Mec-1 (C) or OSU-CLL (D) cells was examined at 24hr by Annexin V/ PI staining.

Figure 16: OSU-T315 targets intrinsic AKT and ERK signals cascades in CLL cells. Lysate from primary CLL cells treated with OSU-T315 were subjected to analyze PDK1 (Ser241) and subsequent AKT (Thr308) activation (A). In vitro kinase activity of class I PI3K is evaluated by PI3 Kinase Activity/Inhibitor Assay Kit (Millipore) according to the instruction manual. 100 nM Wortmannin was applied as positive control. The Biotinylated-PIP3 was set as 100%. The kinase reactions with vehicle or OSU-T315 were referenced to the Biotinylated-PIP3 signal to have the relative percentage (B). RAS activity in 697 cells upon treatments was measured by Active Ras Detection Kit (Cell signaling) according to instruction manual. GTPγS (positive control) and GDP (negative control) ensure the immunoprecipitation procedures worked properly (C). Mec-1 and OSU-CLL cells treated with serial concentrations of OSU-T315 for 15 minutes are subjected to western blot analysis (D). Mec-1 and OSU-CLL cells pretreated with Okadaic acid (1μM) were incubated with either OSU-T315 (4μM) or OSU-03012 (5μM), the PDK1 inhibior, and the total lysate are subject to immunoblotting to verify downstream signaling (E).

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Figure 16: Continued

Figure 17: OSU-T315 mediated cytotoxicity in CLL cells is ILK independent. Mec-1 or 697 cells were lenti-virally transduced with vector or shRNA for ILK. The knockdown efficiency was verified (A). Expansion of Mec-1 cells bearing the vector alone or ILK shRNA was examined by MTS assay (B). 697 cells are treated with fibronectin-coated plate (C) or 10μg/ml α-IgM stimulation (D) in vector and ILK shRNA incorporated cells for 15 minutes and the total lysate were subject to western blot. 697 cells bearing vector alone or ILK shRNA treated with indicated dose of OSU-T315 for 48hrs were stained with propidium iodide (PI). The viable cells were defined as GFP+ (shRNA expressing marker) and PI negative (E).

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Figure 17: Continued

Figure 18: OSU-T315 impairs AKT translocation to lipid raft subdomains in plasma membrane. Lipid raft from Mec-1 cells expressing Myr-flag-AKT was purified after vehicle or OSU-T315 (4uM) treatment by ultracentrifugation approach. The raft and non-raft fraction were analyzed for AKT, BTK, LYN content, and Flotillin-1 serves as lipid raft markers (A). The three independent studies were quantified (B). Mec-1 cells with Myr-flag-AKT were subject to immunofluorescence staining with antibodies of anti-AKT (Alexa 594) and anti-CT-B (Alexa 488) (C). The co-localization index was analyzed double-blindly (D).

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Figure 18: Continued

Figure 19: OSU-T315 inhibits BCR, CD40L, and CpG induced survival signal. CLL cells were treated with either plate-bounded α-IgM, 0.5ug/ml CD40L, 3.2uM CpG-ODN, plate-bound fibronectin, or 1ug/ml VCAM-1 coupled with vehicle or 4uM OSU-T315 for 15 minutes. The total lysate is subjected to immunoblotting (A and B). Data from individual patients was quantified and normalized to GAPDH (C and D). Cell viability upon treatment by plate-bounded α-IgM, or 0.5ug/ml CD40L, or 3.2uM CpG-ODN along with vehicle or 4uM OSU-T315 was examined after 24 hours incubation (E-G).

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Figure 19: Continued

Figure 20: Downstream apoptotic machinery is induced upon T315 treatment in CLL cells. CLL cells were incubated in α-IgM coated plates, 0.5ug/ml CD40L or 3.2uM CpG-ODN and treated 4uM OSU-T315 for 16hrs (A-B). CLL cells were treated with fibronectin-coated plates or 1ug/ml VCAM-1 accompanied with 4μM OSU-T315 treatment for 16hr (C) and (D). Protein lysate was analyzed by immunoblotting and normalized to GAPDH. Caspase 3/7 activity was accessed by Caspase-Glo® 3/7 Assay Systems (Promega) and normalized to vehicle after 16hr treatment (E). Mec-1 or OSU-CLL cells pretreated with vehicle or 20uM z-VAD-FMK prior 4uM OSU-T315 treatment were analyzed by Annexin V / PI staining after 24 hours (F).

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Figure 20: Continued

Figure 21: In vivo TCL1 leukemia progression is suppressed by OSU-T315. Data represents pharmacokinetic of OSU-T315 in plasma after intravenous, Intraperitoneal, or oral dosing (n=6) (A). C57BL/6 mouse engrafted with TCL1 leukemia cells were treated orally with vehicle or 50mg/kg OSU-T315 daily after appearance of 10% leukemia cells in peripheral blood. White blood counts (WBC) were monitored by blood smear slides weekly until sacrifice was required. Data represents WBC counts after 4-weeks treatment (B). Overall survival was analyzed after treatment starts (n=6) (C). C57BL/6 mouse engrafted with TCL1 leukemia cells were treated by intraperitoneal injection with vehicle or 25mg/kg OSU-T315 once daily for 2 weeks after appearance of 5% CD19+, CD5+ leukemia cells among CD45+ peripheral blood mononuclear cells. OSU-T315 was formulated in PBS containing 10 % Cremophor EL (Sigma). After 2-weeks daily scheme, leukemic mice were treated every other day with vehicle or 25mg/kg OSU-T315 to prevent weight loss. Percent of leukemic cells was monitored weekly by flow cytometry until sacrifice was required. Overall survival was analyzed (n=9 for each group). (D)

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Figure 21: Continued

Figure 22: Mec-1 cells expressing Myr-flag-AKT. Mec-1 cells were retro-virally transduced with pBABE-Myr-flag-AKT vector (Addgene). The total lysate are subjected to western blot to verify the myristoylated AKT expression (A). Mec-1 expressing Myr-flag-AKT was treated with increasing concentration of OSU-T315. Total lysate was analyzed by immunoblotting (B). Cell lysate from Mec-1 with Myr-flag-AKT was subjected to immunoprecipitation procedure by α-flag antibody. After extensive wash steps, the pull-down content was further analyzed by ILK or AKT antibody (C).

Figure 23: ILK expression is comparably low in CLL cells. Total cell lysate extracted from purified healthy B or CLL cells by CD19 enrichment kit was subjected to western blot for detection of ILK (A), and the quantitative values is obtained by GAPDH normalization in panel (B).

Figure 24: OSU-T315 inhibits BCR mediated adhesion. Cell adhesion upon BCR engagement or CD49d activation is assessed. Briefly, 3e5 CLL cells pretreated with either vehicle or indicated concentrations of OSU-T315 were incubated in pre-coated 96-well plate by either 10ug/ml α-IgM or 500ng/ml VCAM-1 for 30 minutes at 37oC. After extensive washing and fixation with 4% para-formaldehyde in PBS for 10 minutes, followed by crystal violet (CV) staining for 30 minutes, the retained CV was dissolved in methanol and measured the absorbance at 562nm on the plate reader. Maximal adhesion (100%) is determined by the value of α-IgM incubated sample. Other samples are normalized to 100%.

Figure 25: OSU-T315 induces cytotoxicity regardless of overexpression of Mcl-1. 697 cells overexpressing Mcl-1 were treated with indicated dose of OSU-T315 or 5μM fludarabine. Cells viability after 24 hours incubation was examined by Annexin V and PI double staining. The percentage of live cells was normalized to vehicle treated samples (n=3).

Figure 26: OSU-T315 suppresses Bcl-xl upregulated by CD40 or TLR9 activation. 3e7 CLL cells from patients were incubated with 0.5μg/ml CD40L or 3.2μM CpG combined with or without 4μM OSU-T315 for 16hrs. Total cell lysate was subjected to immunoblotting.

Figure 27: Differential response of ERK status upon external stimuli after OSU-T315 treatment in CLL cells. 2e7 purified CLL cells were treated with either plate-bound α-IgM, 0.5ug/ml CD40L, 3.2uM CpG, fibronectin-coated plate, or 0.5ug/ml VCAM-1 for 16 hours and the total lysate was subjected to immunoblotting. The quantitative value is normalized to GAPDH.

CHAPTER 4 Discussion

4.1 Synopsis

Chronic lymphocytic leukemia is the heterogeneous malignancy with unclear etiology. The standard therapy has been evolving in the past decades according to the promising outcome in clinical trials. The most proper approach so far for initial administration in CLL patients from diagnosis of symptomatic Rai I/II and Binet B, or Rai

III-IV and Binet C is fludarabine plus cyclophosphamide combined with rituximab

(FCR). The infirmed patients without physically fit are expected to be alternatively treated with chlorambucil combined with either rituximab or obinutuzumab (GA-101) regimen with reduced toxicity. Unfortunately, a subset of patients that are defined as the high-risk group bearing del17p or p53 mutations do not benefit from traditional chemotherapy. Additionally, numerous relapsed or refractory patients occur after receiving the standard chemotherapy. Therefore, new therapies are urgently required for this incurable disease. The focus of targeted therapies emulates from translating our new knowledge in molecular pathogenesis of CLL. Of noted, BCR is recognized as the central determinant in CLL pathogenesis given its potency inducing survival signals, thereby focusing the development of agents targeting this axis. Remarkably, ibrutinib, the first-in-class BTK inhibitor, reveals outstanding activity in the clinical practice against this malignancy, and has been demonstrated to achieve favorable outcome in relapsed or high risk patients, signifying a significant breakthrough in the CLL therapy. 100

Given the efficacy of ibrutinib in high risk patients, patients bearing abnormalities related to TP53 gene or refractory ones would be suggested to receive this treatment.

Unfortunately, relapses in a small subset of patients receiving ibrutinib have been reported, raising the issue that identification of the cause for ibrutinib resistance is necessary to benefit future diagnosis, and providing alternative therapeutics to overcome disease progression. We therefore investigate the mechanisms using next generation sequencing to examine whole exons derived from relapsed patients receiving ibrutinib, and identify the novel recurrent mutations at BTK and PLCG2. We further perform functional validations to verify that the C481S mutation at BTK is less sensitive toward ibrutinib, leading to sustained kinase activity and only reversible inhibition. In parallel, we also reveal the gain of function mutations occurring at PLCG2 (R665W and L845F), from which augment the response of BCR engagement regarding enhanced calcium influx and downstream AKT and ERK activations bypassing BTK inhibition. The findings are the first indication in mechanisms of ibrutinib resistance, identifying the genetic aberrations-mediated drug resistance.

In addition to novel agents targeting the B cell receptor pathway, therapeutics directly suppressing critical survival pathways reveal promising activity in clinical trials.

PI3K/AKT has been documented to promote CLL survival by diverse environmental stimuli. Agents antagonize this pathway have been shown clinical efficacy. In terms of

PI3K inhibitors, the selective p110δ inhibitors are noteworthy (ex: Idelalisib, IPI-145) as this subunit is restrictedly expressed in hematopoietic linage and increased in CLL cells. Nevertheless, lack of agent targeting downstream AKT is clinically used. We

101 therefore aim to develop the novel agent suppressing this signaling, thereby reporting

OSU-T315, a reported ILK inhibitor in solid tumors, acquiring therapeutic potency in

CLL. While the effects by OSU-T315 are independent of targeting ILK in lymphoid cells, our findings suggest OSU-T315 a potent AKT inhibitor, displacing this molecule from lipid rafts and triggering caspase-dependent apoptosis in CLL cells. The agent displays a favorable therapeutic window by targeting CLL rather than normal B or T lymphocytes. Subsequently studies verify the potency of OSU-T315 in abrogating diverse environmental stimuli-mediated protection (α-IgM, CD40L, CpG, fibronectin or

VCAM-1). In vivo efficacy is validated by extended median survival in Tcl-1 leukemia engrafted mice receiving OSU-T315. Together, the findings provide an alternative targeting strategy in CLL therapeutics, by which impeding AKT docking into lipid rafts and leading to its deactivation. The agent further reveals both in vitro and in vivo efficacy, implying the potential therapeutic in clinical practice.

4.2 Future perspectives

CLL originates with unknown causes and displays heterogeneous features in the clinical course. The allogeneic stem cells transplantation is the only option to cure this disease. Since ibrutinib is remarkably effective in the therapeutic scenario for naïve-treated or relapsed patients from traditional chemotherapy, it is essential to scrutinize the mechanisms of ibrutinib resistance. We here provide the first implications on how CLL cells escape and survive from ibrutinib. Given the acquired mutations are always identified in relapsed patients instead of germ-line aberrations, the prognosis will not likely to be

102 concerned in patients never received ibrutinib previously. Contrary to this, patients who are continuously administrated with this agent would expect to receive a simple examination periodically for the recurrent mutations at BTK or PLCG2. The assessment will determine when to discontinue ibrutinib and replace with alternative therapies in patients before relapse occurs. Interestingly, the highly coexistence of del(17p13.1) with the appearance of these mutations in relapsed patients implies the acquired mutations may contribute from complex genetic abnormalities due to the lack of functional p53, which facilitate the integrity of genome by halting the cell cycle in the presence of genomic aberrations. Accordingly, the aberrant lesions appear and enrich either under the selective pressure of ibrutinib or the survival advantage of these mutations. The in vitro affinity assay indicates reversible interaction between ibrutinib to BTK with C481S mutation, resulting in retained kinase activity at the IC50 concentrations of ibrutinib to wild type

BTK. The C481S mutation in DT40 can induce enhanced downstream signaling activation regarding p-BTK and p-ERK upon BCR engagement, indicating the mutation may gain the potency for downstream signaling activation or lose the sensitivity to negative regulation.

We further examine the effect of ibrutinib in the setting of α-IgM stimulation in the patient at base-line and after relapse. BTK bearing C481S mutation reverts the inhibition by ibrutinib after removal of this agent, confirming our in vitro kinase and affinity assays.

Therefore, we speculate the prominent activity of ibrutinib in CLL patients is moderated once the agent lost the capacity to covalently bind with Cysteine 481 residue. The concept in evaluating the efficacy may be relevant to new designed BTK targeting agents by assessing the affinity and half-life occupancy to BTK along with in vivo pharmacokinetic

103 properties. In addition to C481S mutation in BTK, we also report PLCG2 mutations as another mechanism of ibrutinib resistance. S707Y mutation at PLCG2 has been documented as the gain-of-function mutation regarding the elevated calcium influx and elevated ERK activation upon stimulation. We identified two acquired mutations, R665W and L845F in relapse patients and verified the hyperactive response similar to S707Y upon

BCR engagement. Both mutations are resistant to ibrutinib regarding BCR-induced calcium response and subsequent downstream survival signaling. Given S707Y is located in the auto-inhibitory SH2 domain and results in gain-of-function phenotype, the mutation at R665W is expected to cause equivalent outcome as both harbored closely in the same region. Contrasting to this, L845F is located in the linker region flanked by SH3 and PH domains. Interestingly, all these mutations are in close proximity of identified phosphorylation sites, Tyr753 and Tyr 759, raising the possibility that the mutations may lead to conformation change of PLCG2, thereby facilitating the activation of these residues. Notably, R665W or L845F in PLCG2 does not elevate the kinase activity regarding phosphorylation status at Tyr759 or Tyr1217, suggesting the mutations mediated hyper-responses are independent of PLCG2 activation status. The mechanism on how mutated PLCG2 maintains activity bypassing BTK inhibition is elusive. Previous studies demonstrate SYK185 or BLNK186, besides BTK, reveals capability to activate PLCG2. Our findings indicate mutant PLCG2 acquires activity regardless of BTK inhibition, suggesting other kinases, such as SYK and BLNK, may involve in this process. The genetic technique by knocking out of these candidates while introducing mutated PLCG2 will validate the dependency of these upstream molecules in mutant PLCG2. Given SYK has also been

104 reported to phosphorylate BLNK and subsequently facilitate PLCG2 docking via interaction with this adaptor protein187, raising the other possibility that PLCG2 bearing mutations may enforce the interaction with BLNK and then trigger downstream response.

PI3K/AKT pathway is renowned involving in the survival of diverse malignancies and also recognized as the therapeutic target in CLL. In clinical practice, agents specifically targeting this pathway focus on suppressing upstream PI3 kinases. Here we provide the first evidence that targeting downstream AKT through modulating AKT content in the raft compartment by OSU-T315 revealing an effective therapy in CLL treatment. Evidence from previous studies demonstrated ablation of AKT by cholesterol depletion or inhibition of cholesterol biosynthesis shows therapeutic potency and preferential cytotoxicity toward tumor cells. Similar to this, OSU-T315 diminishes AKT activation on lipid raft, and selectively targets CLL cells. Prominently, the activities and localizations of proximal kinases (PI3 Kinases, PTEN, PDK1, RAS, PP2A) are unaltered, suggesting the disability of AKT translocation is not a consequence of kinase inhibition.

Recent studies identify a recurrent somatic mutation of AKT1 (E17K) in human breast, colorectal, and ovarian cancers188. The missense mutation occurring within PH domain activates downstream signaling by constitutively forcing the AKT1 PH domain to the plasma membrane, suggesting the oncogenic role in tumor development. Although the presence of this mutation in CLL is rejected189, it is still possible that other gain-of-function abnormalities at AKT, originating from somatic mutation or clonal selection through continuous therapy, may render the effect of inhibitors targeting upstream PI3Ks.

Contrasting to targeting p110δ subunit of PI3 kinase in CLL therapy, the novel strategy by

105 displacing AKT from the raft compartment obtains advantages in the case of oncogenic mutations occurring at AKT. Moreover, previous findings suggest alterations of cholesterol content at rafts of tumor cells are critical for modulating AKT signaling.

Interestingly, accumulative studies have linked hyperlipidemia with increased risk of cancers. Consistent with the concept, the overall relative risk of CLL on the basis of overweight and obesity, as measured by body mass index (BMI) is 1.26 (95% CI,

1.09-1.46)184, suggesting a correlation of hyperlipidemia with increased risk for CLL development. Given OSU-T315 efficiently suppresses AKT activation from happening within raft compartment, we speculate the agent will show substantial efficacy in CLL patients. Besides AKT ablation, OSU-T315 also inhibits Ras-Raf-MAPK cascade according to the results of cellular profiling. While Ras activity is not altered based on the in vitro binding assay, Raf activation is significantly reduced, thereby leading to downstream ERK dephosphorylation. Numerous studies indicate the cross-talk between both signaling pathways; nevertheless, the clinical agent acquires the potency concurrently inhibiting both signaling is rare, suggesting the potential of OSU-T315 with improved outcome compared to mono-targeting therapy toward either pathway. Despite the variable consequence of ERK deactivation among diverse stimuli in our results, the B cell receptor induced ERK activation is significantly reduced by OSU-T315. Previous studies demonstrate that the cross-talk between AKT and ERK signaling is dynamic, which is regulated in a concentration- and ligand- dependent manner. While high doses of insulin-like growth factor (IGF-1) can strongly activate AKT, whereas Raf activity is suppressed in this condition. The concept may explain why we observe inconsistent

106 outcome regarding the status of ERK activation in treatment of OSU-T315 upon different stimuli. Intriguingly, the target mediating suppression of both AKT and ERK by

OSU-T315 is still elusive. Since our findings implicate OSU-T315 elicits the effects at plasma membrane, the putative target of OSU-T315 should be harbored proximally at this region. To clarify this question, we perform the KINOME screening for 451 currently identified kinases (Appendix I and II), and PAK3 appears as the only target significantly inhibited by OSU-T315, while PAK1 and PAK2, the two other class I PAK family members are not affected. PAK3 has been reported to directly induce Raf-1 activation at

Ser338190, and coordinates downstream signaling by interaction with PAK1191, the other member of group I of PAK family that serves as the adaptor for AKT activation192. Despite the linage-specific expression of PAK3 restrictedly in neuron tissue, the functional investigations for PAK3 in malignant cells will be necessary to expand the current knowledge in tumorigenesis.

4.3 Future directions

Here we provide the mechanisms of ibrutinib resistance in part from acquired mutations that identified through genome-wide sequencing, and illustrate the functional properties between wild type and mutant BTK or PLCG2. Accordingly, the potency of ibrutinib is diminished once the covalent binding at Cysteine 481 is disrupted. The result raises the question of how C481S, which is absent in the genome at baseline, appears after continuous pressure under ibrutinib. The possible explanations include clonal evolution by selective pressure under long-term treatment, especially in patients with p53 aberrations.

The concept could be validated by statistical analysis for correlations in both incidents

107 when accumulating relapsed cases identified. Alternatively, since the sequencing was performed in CLL collected from peripheral blood, it is possible that BTK with C481S may exist in a small portion of CLL residing within lymphoid tissues that could not be detected at baseline, and ibrutinib releases the subset, or selectively enriches this population.

Further analysis in CLL derived from either lymph node biopsy or bone marrow aspirate before and after ibrutinib treatment could clarify the origin of this mutation.

While only a small portion of copy number under sequencing reveals variant calling in relapsed samples, the refractory effects resulting from this minor population is still obscure. One possibility is that resistant CLL bearing identified mutations may accumulate and enrich in lymphoid tissues. Examinations from these organs may better explicate the consequence. On the other hand, it is questionable whether the mutations mediated resistance is an autonomous effect. Recent studies suggest that exosome secretions by malignant cells could transfer functional molecules including microRNAs and peptides toward adjacent cells. Thus, CLL with BTK or PLCγ2 mutations may shuttle the mutated BTK and PLCγ2, or microRNAs that are absent in most CLL, then triggering the resistant effects in CLL without mutations. Moreover, it could not exclude the possibility that CLL bearing mutations may release critical chemokines or cytokines that promote the survival of CLL systemically. To examine the concept, genetic manipulations in vitro to introduce mutated BTK or PLCG2 in leukemia cell lines deficient of WT BTK or PLCγ2 and followed by the collections of culturing media to profile cytokines and chemokines secretions, or assess the elevated microRNAs in exosomes, can identify candidates mediating ibrutinib resistance. The in vivo study in mice engrafted with

108 heterogeneous leukemia cells bearing either WT or mutant genes, along with the expression of different luciferase markers will be useful. By monitoring the disease progression under ibrutinib treatment, the sensitivity toward ibrutinib can be exhibited by assessing the distribution and the ratio of different bioluminescent markers.

As BTK targeting displays outstanding activity in the first-in class agent while the refractory patients received ibrutinib are reported, the development of next-generation inhibitors targeting BTK is urgent. The importance for new inhibitors may substantiate in the outstanding clinical activity regardless of the interaction at Cysteine 481 residue as the

C481S will render the effect in relapse patients. Besides, alternative therapy that indirectly reduce BTK activity may show therapeutic potential to treat in relapsed patient. BTK belongs to TEC family kinases with SH3 and SH2 domains followed by the catalytic domain. Unlike Src family kinases, BTK also contains pleckstrin homology (PH) domain that triggers the association with PIP3 and docking at plasma membrane for activation.

BTK activation is initiated at plasma membrane when Tyrosine 551 residue is phosphorylated by Src family kinases or Syk. Given the efficacy of ibrutinib is subsided due to the enhanced kinase activity in the presence of C481S mutation, ibrutinib resistance may be overcome when combining other treatments that antagonize the upstream kinases or impede the recruitment BTK toward plasma membrane. Regarding this, PI3K or Syk inhibitors could therefore show therapeutic activity when introduced in relapsed patients receiving ibrutinib.

Despite of the lack of PLCγ2 inhibitor available for clinical practice, previous studies have been demonstrated that Syk also involves in PLCγ2 activation in addition to

109 direct upstream effector BTK. It raises the possibility that gain of function mutations in

PLCG2 may be alternatively activated by Syk while BTK is inhibited by ibrutinib. On the other hand, the activation of PLCG2 also requires the localization at plasma membrane via interaction of the PH domain with PIP3 or PIP2. To reverse the resistance in the setting of gain-of-function PLCγ2, administration of Syk or PI3K inhibitors with ibrutinib may improve the outcome in relapsed patients. The other rationale for combining Syk inhibitors stems from the potency concurrently targeting downstream molecules BTK and PLCγ2, as well as BLNK that is demonstrated to facilitate PLCγ2 docking for activation, thereby reinforces the therapeutic activity in addition to ibrutinib. Moreover, several growth factors are reported to activate PLCγ2 including Epidermal growth factor (EGF) and

Platelet-derived growth factor (PDGF). Identification of alternative signaling pathway besides BCR may contribute the understanding of survival in relapsed CLL bearing mutated PLCG2.

Lastly, recent studies in mantle cell lymphoma (MCL) indicate that elevated non- canonical NF-κB pathway could diminish the sensitivity of MCL cells toward ibrutinib.

The phenomenon is validated in MCL cell lines with increased NIK and downstream p52 activation193. Thus, the concept raises the possibility that in addition to current identified mutations contributing to ibrutinib resistance, other aberrant survival signaling may involve in CLL refractory. To overcome ibrutinib resistance, investigations for critical signaling are necessary. Since AKT, ERK, and NF-κB are the fundamental survival signals in CLL, we speculate elevated survival signals in any of these settings could be observed upon ibrutinib treatment. To verify this, the phospho-kinase array to extensively examine

110 upregulated signaling is cooperative to elucidate alternative mechanisms behind ibrutinib resistance.

OSU-T315 has been pre-clinically validated to show in vitro and in vivo efficacy in

CLL depletion. However, despite of the defined mechanisms through targeting AKT from rafts and the functional properties by this agent, the specific target is still elusive. Since

OSU-T315 reveals a superior therapeutic window, identification of this agent’s target will facilitate the understanding of CLL biology and the development of therapeutic approaches in this disease. The KINOME screening data unveils PAK3 as the only kinase target, nevertheless, it does not exclude the possibility that OSU-T315 may interact with other uncategorized kinases, lipid, or protein molecules. Owing to the restriction for tagging chemical compound by either biotinylation, fluorescent moiety, or other labeling structure as these modifications often abolish agents’ properties, the proposed idea to achieve this aim is oppositely by introducing Myc- or His-tagged AKT into leukemic cells and following by comprehensive analysis for the interatomic complex with tagged AKT using

Matrix-assisted laser desorption/ionization (MALDI). In comparison to samples treated

OSU-T315, the missed binding partner can be uncovered. The approach may provide more information regarding the process of AKT activation upon OSU-T315.

The pharmacokinetic study on OSU-T315 reveals poor oral bioavailability compared to I.V. or I.P. dosing. Given the hydrophobic property of OSU-T315 which may elicit the effects by modulating lipid-associated signaling event, we speculate the agent may be restricted and deposited within hydrophobic layers once being absorbed.

Accordingly, a comprehensive pharmacokinetic analysis in different organs will be useful

111 to determine the distribution of OSU-T315. Moreover, to improve the efficiency of

OSU-T315 targeting leukemia cells, the development of derivatives or alternative formulations is desired to optimize the bioavailability. Targeted delivery by encapsulation of chemical compound into nonporous particle-supported lipid bilayers194 is the novel approach to transfer effective material into malignant cells. The concept of capsulizing

OSU-T315 into nano-particles could useful to overcome the challenge in absorption and enhance the specificity.

Finally, since ibrutinib resistance is recently reported and the compensatory therapy for this group of patients is still lack, we speculate OSU-T315 may show efficacy by overcoming critical survival signals. Given the potential of OSU-T315 in abolishing

BCR mediated downstream AKT and ERK signaling, we postulate the combination of

OSU-T315 with ibrutinib in treatment of relapse patients can show promising outcome.

The functional characterizations in vitro and in the mouse model will be required to verify this.

112

Appendix A OSU-T315 specifically targets PAK3 in class I PAK family kinases

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Appendix B The KINOMEScan results for OSU-T315

Entrez Gene KINOMEscan Gene Symbol Symbol % of Control 2uM % of Control 5uM PAK3 PAK3 36 13 GSK3A GSK3A 61 60 CTK MATK 66 62 DCAMKL1 DCLK1 67 57 MLCK MYLK3 69 43 PKN2 PKN2 71 100 AKT1 AKT1 75 87 SNRK SNRK 76 94 SRPK2 SRPK2 76 97 AKT3 AKT3 76 100 ERN1 ERN1 77 66 PFTAIRE2 CDK15 77 81 YANK2 STK32B 77 91 SRC SRC 77 95 ASK2 MAP3K6 78 57 PKAC-alpha PRKACA 78 86 RPS6KA5(Kin.Dom.1-N-terminal) RPS6KA5 78 100 ERK3 MAPK6 79 85 MARK3 MARK3 79 85 TRPM6 TRPM6 79 98 SBK1 SBK1 81 87 DAPK3 DAPK3 81 89 DMPK DMPK 81 100 LIMK2 LIMK2 82 80 MELK MELK 82 100 DYRK1B DYRK1B 83 79 AMPK-alpha2 PRKAA2 83 100 CDKL3 CDKL3 83 100 PDGFRA PDGFRA 83 100 DMPK2 CDC42BPG 84 79 DYRK2 DYRK2 84 90 CDK8 CDK8 84 96 CDK11 CDK19 84 100 CLK4 CLK4 84 100 PIP5K2C PIP4K2C 84 100 VEGFR2 KDR 84 100 114 (Continue)

CDKL5 CDKL5 85 100 PFTK1 CDK14 85 100 PIP5K1A PIP5K1A 86 64 MAP4K5 MAP4K5 86 78 TTK TTK 86 81 MAP4K4 MAP4K4 86 91 SRPK1 SRPK1 87 82 NEK5 NEK5 87 97 KIT-autoinhibited KIT 87 100 GRK4 GRK4 88 62 PDPK1 PDPK1 88 99 DAPK1 DAPK1 88 100 ERBB3 ERBB3 88 100 EGFR(L861Q) EGFR 89 79 RIOK1 RIOK1 89 85 ERK8 MAPK15 89 87 DAPK2 DAPK2 89 94 CDC2L2 CDC2L2 90 100 CDC2L1 CDK11B 91 83 CDK9 CDK9 91 87 DRAK2 STK17B 91 97 CAMKK1 CAMKK1 91 98 ACVR1 ACVR1 91 100 CAMKK2 CAMKK2 91 100 DYRK1A DYRK1A 91 100 RPS6KA4(Kin.Dom.1-N-terminal) RPS6KA4 91 100 CAMK1 CAMK1 92 90 PRKX PRKX 92 90 EIF2AK1 EIF2AK1 92 92 PRKD1 PRKD1 92 96 GSK3B GSK3B 92 100 PIK3CA(H1047Y) PIK3CA 92 100 RIPK1 RIPK1 93 90 MYO3B MYO3B 93 94 GRK7 GRK7 93 96 CDKL2 CDKL2 93 100 IRAK1 IRAK1 93 100 ERBB2 ERBB2 94 78 YANK1 STK32A 94 83 KIT(V559D,T670I) KIT 94 86 ACVR1B ACVR1B 94 87 ERK2 MAPK1 94 90 JAK1(JH1domain-catalytic) JAK1 94 90 PRKD2 PRKD2 94 92 (Continue) 115

BLK BLK 94 95 DRAK1 STK17A 94 100 FLT3-autoinhibited FLT3 94 100 MAP3K4 MAP3K4 94 100 p38-gamma MAPK12 94 100 TNIK TNIK 94 100 TYRO3 TYRO3 94 100 EGFR(L747-T751del,Sins) EGFR 95 80 ERBB4 ERBB4 95 86 ABL1(F317I)-nonphosphorylated ABL1 95 92 FRK FRK 95 95 STK36 STK36 95 97 ARK5 NUAK1 95 100 CAMK2G CAMK2G 95 100 PIP5K1C PIP5K1C 95 100 WNK1 WNK1 95 100 EGFR(S752-I759del) EGFR 96 82 CASK CASK 96 84 LKB1 STK11 96 88 PAK6 PAK6 96 88 KIT(V559D,V654A) KIT 96 89 CLK2 CLK2 96 94 RSK2(Kin.Dom.1-N-terminal) RPS6KA3 96 95 CAMK2B CAMK2B 96 96 BRSK1 BRSK1 96 100 CDK3 CDK3 96 100 EPHB3 EPHB3 96 100 MAP3K1 MAP3K1 96 100 PIK3CA(E542K) PIK3CA 96 100 PRKCI PRKCI 96 100 SLK SLK 96 100 BMPR1A BMPR1A 97 68 SRPK3 SRPK3 97 70 EGFR(G719S) EGFR 97 80 MAPKAPK2 MAPKAPK2 97 84 RIPK2 RIPK2 97 87 PIK3CD PIK3CD 97 91 ADCK4 ADCK4 97 93 MRCKA CDC42BPA 97 93 PRKR EIF2AK2 97 93 RPS6KA5(Kin.Dom.2-C-terminal) RPS6KA5 97 95 MST4 MST4 97 96 ABL1(Y253F)-phosphorylated ABL1 97 100 CAMK1D CAMK1D 97 100

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FLT3(ITD) FLT3 97 100 JAK1(JH2domain-pseudokinase) JAK1 97 100 MARK2 MARK2 97 100 MLK3 MAP3K11 97 100 NEK2 NEK2 97 100 PIK3CA(C420R) PIK3CA 97 100 TYK2(JH2domain-pseudokinase) TYK2 97 100 YSK4 YSK4 97 100 FLT3(K663Q) FLT3 98 96 LCK LCK 98 99 AAK1 AAK1 98 100 ABL1(Q252H)-phosphorylated ABL1 98 100 BUB1 BUB1 98 100 CAMK1G CAMK1G 98 100 CSK CSK 98 100 GCN2(Kin.Dom.2,S808G) EIF2AK4 98 100 NEK11 NEK11 98 100 PIK3C2G PIK3C2G 98 100 TSSK1B TSSK1B 98 100 RSK3(Kin.Dom.2-C-terminal) RPS6KA2 99 76 ACVRL1 ACVRL1 99 85 EGFR(L747-E749del, A750P) EGFR 99 88 MYLK4 MYLK4 99 90 CSNK1A1L CSNK1A1L 99 95 CDC2L5 CDK13 99 96 EPHA4 EPHA4 99 99 LRRK2 LRRK2 99 99 AURKB AURKB 99 100 CAMK2D CAMK2D 99 100 NEK9 NEK9 99 100 PLK2 PLK2 100 56 CSNK1G2 CSNK1G2 100 73 EGFR(G719C) EGFR 100 73 PLK3 PLK3 100 77 CSNK1G3 CSNK1G3 100 78 PRP4 PRPF4B 100 80 ABL1(F317I)-phosphorylated ABL1 100 81 STK33 STK33 100 82 ASK1 MAP3K5 100 83 EGFR(L858R,T790M) EGFR 100 84 HUNK HUNK 100 84 KIT(V559D) KIT 100 84 TGFBR2 TGFBR2 100 84 YANK3 STK32C 100 84

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ACVR2A ACVR2A 100 86 AURKC AURKC 100 86 EGFR EGFR 100 86 MET MET 100 86 RIPK5 DSTYK 100 86 MAP3K15 MAP3K15 100 87 CLK1 CLK1 100 88 ERK1 MAPK3 100 88 BIKE BMP2K 100 89 CAMK2A CAMK2A 100 89 KIT KIT 100 89 TNNI3K TNNI3K 100 89 AMPK-alpha1 PRKAA1 100 90 CSNK1D CSNK1D 100 90 EPHA5 EPHA5 100 90 IGF1R IGF1R 100 90 MST1 STK4 100 90 PRKG2 PRKG2 100 90 CDK5 CDK5 100 91 CHEK1 CHEK1 100 91 PIP5K2B PIP4K2B 100 91 CSNK1E CSNK1E 100 92 QSK KIAA0999 100 92 EPHA6 EPHA6 100 93 RIOK3 RIOK3 100 93 WEE1 WEE1 100 93 ABL1-nonphosphorylated ABL1 100 94 BRSK2 BRSK2 100 94 BRK PTK6 100 95 p38-beta MAPK11 100 95 PHKG2 PHKG2 100 95 MEK6 MAP2K6 100 96 PKAC-beta PRKACB 100 96 RSK1(Kin.Dom.2-C-terminal) RPS6KA1 100 96 SIK2 SIK2 100 96 TIE2 TEK 100 96 ABL1(M351T)-phosphorylated ABL1 100 97 CLK3 CLK3 100 97 FLT1 FLT1 100 97 MARK1 MARK1 100 97 MET(Y1235D) MET 100 97 MYLK MYLK 100 97 PCTK2 CDK17 100 97 TAK1 MAP3K7 100 97

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TESK1 TESK1 100 97 TGFBR1 TGFBR1 100 97 MRCKB CDC42BPB 100 98 SIK SIK1 100 98 STK16 STK16 100 98 ABL1-phosphorylated ABL1 100 99 ACVR2B ACVR2B 100 99 FGFR3 FGFR3 100 99 PHKG1 PHKG1 100 99 PYK2 PTK2B 100 99 WEE2 WEE2 100 99 ABL1(E255K)-phosphorylated ABL1 100 100 ABL1(F317L)-nonphosphorylated ABL1 100 100 ABL1(F317L)-phosphorylated ABL1 100 100 ABL1(H396P)-nonphosphorylated ABL1 100 100 ABL1(H396P)-phosphorylated ABL1 100 100 ABL1(Q252H)-nonphosphorylated ABL1 100 100 ABL1(T315I)-nonphosphorylated ABL1 100 100 ABL1(T315I)-phosphorylated ABL1 100 100 ABL2 ABL2 100 100 ADCK3 CABC1 100 100 AKT2 AKT2 100 100 ALK ALK 100 100 ANKK1 ANKK1 100 100 AURKA AURKA 100 100 AXL AXL 100 100 BMPR1B BMPR1B 100 100 BMPR2 BMPR2 100 100 BMX BMX 100 100 BRAF BRAF 100 100 BRAF(V600E) BRAF 100 100 BTK BTK 100 100 CAMK4 CAMK4 100 100 CDK2 CDK2 100 100 CDK4-cyclinD1 CDK4 100 100 CDK4-cyclinD3 CDK4 100 100 CDK7 CDK7 100 100 CDKL1 CDKL1 100 100 CHEK2 CHEK2 100 100 CIT CIT 100 100 CSF1R CSF1R 100 100 CSF1R-autoinhibited CSF1R 100 100 CSNK1A1 CSNK1A1 100 100 CSNK1G1 CSNK1G1 100 100

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CSNK2A1 CSNK2A1 100 100 CSNK2A2 CSNK2A2 100 100 DCAMKL2 DCLK2 100 100 DCAMKL3 DCLK3 100 100 DDR1 DDR1 100 100 DDR2 DDR2 100 100 DLK MAP3K12 100 100 EGFR(E746-A750del) EGFR 100 100 EGFR(L747-S752del, P753S) EGFR 100 100 EGFR(L858R) EGFR 100 100 EGFR(T790M) EGFR 100 100 EPHA1 EPHA1 100 100 EPHA2 EPHA2 100 100 EPHA3 EPHA3 100 100 EPHA7 EPHA7 100 100 EPHA8 EPHA8 100 100 EPHB1 EPHB1 100 100 EPHB2 EPHB2 100 100 EPHB4 EPHB4 100 100 EPHB6 EPHB6 100 100 ERK4 MAPK4 100 100 ERK5 MAPK7 100 100 FAK PTK2 100 100 FER FER 100 100 FES FES 100 100 FGFR1 FGFR1 100 100 FGFR2 FGFR2 100 100 FGFR3(G697C) FGFR3 100 100 FGFR4 FGFR4 100 100 FGR FGR 100 100 FLT3 FLT3 100 100 FLT3(D835H) FLT3 100 100 FLT3(D835Y) FLT3 100 100 FLT3(N841I) FLT3 100 100 FLT3(R834Q) FLT3 100 100 FLT4 FLT4 100 100 FYN FYN 100 100 GAK GAK 100 100 GRK1 GRK1 100 100 HASPIN GSG2 100 100 HCK HCK 100 100 HIPK1 HIPK1 100 100 HIPK2 HIPK2 100 100 HIPK3 HIPK3 100 100

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HIPK4 HIPK4 100 100 HPK1 MAP4K1 100 100 ICK ICK 100 100 IKK-alpha CHUK 100 100 IKK-beta IKBKB 100 100 IKK-epsilon IKBKE 100 100 INSR INSR 100 100 INSRR INSRR 100 100 IRAK3 IRAK3 100 100 IRAK4 IRAK4 100 100 ITK ITK 100 100 JAK2(JH1domain-catalytic) JAK2 100 100 JAK3(JH1domain-catalytic) JAK3 100 100 JNK1 MAPK8 100 100 JNK2 MAPK9 100 100 JNK3 MAPK10 100 100 KIT(A829P) KIT 100 100 KIT(D816H) KIT 100 100 KIT(D816V) KIT 100 100 KIT(L576P) KIT 100 100 LATS1 LATS1 100 100 LATS2 LATS2 100 100 LIMK1 LIMK1 100 100 LOK STK10 100 100 LRRK2(G2019S) LRRK2 100 100 LTK LTK 100 100 LYN LYN 100 100 LZK MAP3K13 100 100 MAK MAK 100 100 MAP3K2 MAP3K2 100 100 MAP3K3 MAP3K3 100 100 MAP4K2 MAP4K2 100 100 MAP4K3 MAP4K3 100 100 MAPKAPK5 MAPKAPK5 100 100 MARK4 MARK4 100 100 MAST1 MAST1 100 100 MEK1 MAP2K1 100 100 MEK2 MAP2K2 100 100 MEK3 MAP2K3 100 100 MEK4 MAP2K4 100 100 MEK5 MAP2K5 100 100 MERTK MERTK 100 100 MET(M1250T) MET 100 100 MINK MINK1 100 100

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MKK7 MAP2K7 100 100 MKNK1 MKNK1 100 100 MKNK2 MKNK2 100 100 MLK1 MAP3K9 100 100 MLK2 MAP3K10 100 100 MST1R MST1R 100 100 MST2 STK3 100 100 MST3 STK24 100 100 MTOR MTOR 100 100 MUSK MUSK 100 100 MYLK2 MYLK2 100 100 MYO3A MYO3A 100 100 NDR1 STK38 100 100 NDR2 STK38L 100 100 NEK1 NEK1 100 100 NEK3 NEK3 100 100 NEK4 NEK4 100 100 NEK6 NEK6 100 100 NEK7 NEK7 100 100 NIM1 MGC42105 100 100 NLK NLK 100 100 OSR1 OXSR1 100 100 p38-alpha MAPK14 100 100 p38-delta MAPK13 100 100 PAK1 PAK1 100 100 PAK2 PAK2 100 100 PAK4 PAK4 100 100 PAK7 PAK7 100 100 PCTK1 CDK16 100 100 PCTK3 CDK18 100 100 PDGFRB PDGFRB 100 100 PFCDPK1(P.falciparum) CDPK1 100 100 PFPK5(P.falciparum) MAL13P1.279 100 100 PIK3C2B PIK3C2B 100 100 PIK3CA PIK3CA 100 100 PIK3CA(E545A) PIK3CA 100 100 PIK3CA(E545K) PIK3CA 100 100 PIK3CA(H1047L) PIK3CA 100 100 PIK3CA(I800L) PIK3CA 100 100 PIK3CA(M1043I) PIK3CA 100 100 PIK3CA(Q546K) PIK3CA 100 100 PIK3CB PIK3CB 100 100 PIK3CG PIK3CG 100 100 PIK4CB PI4KB 100 100

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PIM1 PIM1 100 100 PIM2 PIM2 100 100 PIM3 PIM3 100 100 PKMYT1 PKMYT1 100 100 PKN1 PKN1 100 100 PKNB(M.tuberculosis) pknB 100 100 PLK1 PLK1 100 100 PLK4 PLK4 100 100 PRKCD PRKCD 100 100 PRKCE PRKCE 100 100 PRKCH PRKCH 100 100 PRKCQ PRKCQ 100 100 PRKD3 PRKD3 100 100 PRKG1 PRKG1 100 100 RAF1 RAF1 100 100 RET RET 100 100 RET(M918T) RET 100 100 RET(V804L) RET 100 100 RET(V804M) RET 100 100 RIOK2 RIOK2 100 100 RIPK4 RIPK4 100 100 ROCK1 ROCK1 100 100 ROCK2 ROCK2 100 100 ROS1 ROS1 100 100 RPS6KA4(Kin.Dom.2-C-terminal) RPS6KA4 100 100 RSK1(Kin.Dom.1-N-terminal) RPS6KA1 100 100 RSK2(Kin.Dom.2-C-terminal) RPS6KA3 100 100 RSK3(Kin.Dom.1-N-terminal) RPS6KA2 100 100 RSK4(Kin.Dom.1-N-terminal) RPS6KA6 100 100 RSK4(Kin.Dom.2-C-terminal) RPS6KA6 100 100 S6K1 RPS6KB1 100 100 SGK SGK1 100 100 SgK110 SgK110 100 100 SGK3 SGK3 100 100 SNARK NUAK2 100 100 SRMS SRMS 100 100 STK35 STK35 100 100 STK39 STK39 100 100 SYK SYK 100 100 TAOK1 TAOK1 100 100 TAOK2 TAOK2 100 100 TAOK3 TAOK3 100 100 TBK1 TBK1 100 100 TEC TEC 100 100

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TIE1 TIE1 100 100 TLK1 TLK1 100 100 TLK2 TLK2 100 100 TNK1 TNK1 100 100 TNK2 TNK2 100 100 TRKA NTRK1 100 100 TRKB NTRK2 100 100 TRKC NTRK3 100 100 TXK TXK 100 100 TYK2(JH1domain-catalytic) TYK2 100 100 ULK1 ULK1 100 100 ULK2 ULK2 100 100 ULK3 ULK3 100 100 VRK2 VRK2 100 100 WNK3 WNK3 100 100 YES YES1 100 100 YSK1 STK25 100 100 ZAK ZAK 100 100 ZAP70 ZAP70 100 100

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Reference list

1. Parkin J, Cohen B. An overview of the immune system. Lancet. Jun 2 2001;357(9270):1777-1789. 2. Shapiro-Shelef M, Calame K. Regulation of plasma-cell development. Nature reviews. Immunology. Mar 2005;5(3):230-242. 3. Cerottini JC, Brunner KT. Cell-mediated cytotoxicity, allograft rejection, and tumor immunity. Advances in immunology. 1974;18:67-132. 4. Parker DC. T cell-dependent B cell activation. Annual review of immunology. 1993;11:331-360. 5. Kunkel EJ, Butcher EC. Plasma-cell homing. Nature reviews. Immunology. Oct 2003;3(10):822-829. 6. Hardy RR, Hayakawa K. B cell development pathways. Annual review of immunology. 2001;19:595-621. 7. Hardy RR, Li YS, Allman D, Asano M, Gui M, Hayakawa K. B-cell commitment, development and selection. Immunological reviews. Jun 2000;175:23-32. 8. Hardy RR. B-1 B cell development. J Immunol. Sep 1 2006;177(5):2749-2754. 9. McHeyzer-Williams LJ, McHeyzer-Williams MG. Antigen-specific memory B cell development. Annual review of immunology. 2005;23:487-513. 10. McHeyzer-Williams M, Okitsu S, Wang N, McHeyzer-Williams L. Molecular programming of B cell memory. Nature reviews. Immunology. Jan 2012;12(1):24-34. 11. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. Jun 15 2008;111(12):5446-5456. 12. Cantu ES, McGill JR, Stephenson CF, et al. Male-to-female sex ratios of abnormalities detected by fluorescence in situ hybridization in a population of chronic lymphocytic leukemia patients. Hematology reports. Jan 25 2013;5(1):13-17. 13. Gaidano G, Foa R, Dalla-Favera R. Molecular pathogenesis of chronic lymphocytic leukemia. The Journal of clinical investigation. Oct 1 2012;122(10):3432-3438. 14. Stamatopoulos K, Belessi C, Moreno C, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: Pathogenetic implications and clinical correlations. Blood. Jan 1 2007;109(1):259-270. 15. Damle RN, Wasil T, Fais F, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. Sep 15 1999;94(6):1840-1847. 16. Ghia P, Stamatopoulos K, Belessi C, et al. ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia. Leukemia. Jan 2007;21(1):1-3. 125

17. Langerak AW, Davi F, Ghia P, et al. Immunoglobulin sequence analysis and prognostication in CLL: guidelines from the ERIC review board for reliable interpretation of problematic cases. Leukemia. Jun 2011;25(6):979-984. 18. Del Poeta G, Maurillo L, Venditti A, et al. Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood. Nov 1 2001;98(9):2633-2639. 19. Malavasi F, Deaglio S, Damle R, Cutrona G, Ferrarini M, Chiorazzi N. CD38 and chronic lymphocytic leukemia: a decade later. Blood. Sep 29 2011;118(13):3470-3478. 20. Hamblin TJ, Orchard JA, Ibbotson RE, et al. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood. Feb 1 2002;99(3):1023-1029. 21. Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood. Jun 15 2003;101(12):4944-4951. 22. Del Principe MI, Del Poeta G, Buccisano F, et al. Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia. Blood. Aug 1 2006;108(3):853-861. 23. Crespo M, Bosch F, Villamor N, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. The New England journal of medicine. May 1 2003;348(18):1764-1775. 24. Chen L, Apgar J, Huynh L, et al. ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia. Blood. Mar 1 2005;105(5):2036-2041. 25. Gobessi S, Laurenti L, Longo PG, Sica S, Leone G, Efremov DG. ZAP-70 enhances B-cell-receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells. Blood. Mar 1 2007;109(5):2032-2039. 26. Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. The New England journal of medicine. Dec 28 2000;343(26):1910-1916. 27. Bullrich F, Rasio D, Kitada S, et al. ATM mutations in B-cell chronic lymphocytic leukemia. Cancer research. Jan 1 1999;59(1):24-27. 28. Schaffner C, Stilgenbauer S, Rappold GA, Dohner H, Lichter P. Somatic ATM mutations indicate a pathogenic role of ATM in B-cell chronic lymphocytic leukemia. Blood. Jul 15 1999;94(2):748-753. 29. Rossi D, Fangazio M, Rasi S, et al. Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia. Blood. Mar 22 2012;119(12):2854-2862. 30. Ouillette P, Erba H, Kujawski L, Kaminski M, Shedden K, Malek SN. Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14. Cancer research. Feb 15 2008;68(4):1012-1021. 31. Mosca L, Fabris S, Lionetti M, et al. Integrative genomics analyses reveal molecularly distinct subgroups of B-cell chronic lymphocytic leukemia patients with 13q14 deletion. Clinical cancer research : an official journal of the American Association for Cancer Research. Dec 1 2010;16(23):5641-5653.

126

32. Liu Y, Corcoran M, Rasool O, et al. Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome 13q14, frequently deleted in chronic lymphocytic leukemia. Oncogene. Nov 13 1997;15(20):2463-2473. 33. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America. Nov 26 2002;99(24):15524-15529. 34. Klein U, Lia M, Crespo M, et al. The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer cell. Jan 19 2010;17(1):28-40. 35. Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proceedings of the National Academy of Sciences of the United States of America. Sep 27 2005;102(39):13944-13949. 36. Forconi F, Rinaldi A, Kwee I, et al. Genome-wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion. British journal of haematology. Nov 2008;143(4):532-536. 37. Zenz T, Habe S, Denzel T, et al. Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood. Sep 24 2009;114(13):2589-2597. 38. Zenz T, Eichhorst B, Busch R, et al. TP53 mutation and survival in chronic lymphocytic leukemia. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Oct 10 2010;28(29):4473-4479. 39. Malcikova J, Smardova J, Rocnova L, et al. Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage. Blood. Dec 17 2009;114(26):5307-5314. 40. Losada AP, Wessman M, Tiainen M, et al. Trisomy 12 in chronic lymphocytic leukemia: an interphase cytogenetic study. Blood. Aug 1 1991;78(3):775-779. 41. Riches JC, O'Donovan CJ, Kingdon SJ, et al. Trisomy 12 chronic lymphocytic leukemia cells exhibit up-regulation of integrin signaling that is modulated by NOTCH1 mutations. Blood. May 14 2014. 42. Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood. Aug 1975;46(2):219-234. 43. Binet JL, Lepoprier M, Dighiero G, et al. A clinical staging system for chronic lymphocytic leukemia: prognostic significance. Cancer. Aug 1977;40(2):855-864. 44. Hallek M. Signaling the end of chronic lymphocytic leukemia: new frontline treatment strategies. Blood. Nov 28 2013;122(23):3723-3734. 45. Quiroga MP, Balakrishnan K, Kurtova AV, et al. B-cell antigen receptor signaling enhances chronic lymphocytic leukemia cell migration and survival: specific targeting with a novel spleen tyrosine kinase inhibitor, R406. Blood. Jul 30 2009;114(5):1029-1037. 46. Herishanu Y, Perez-Galan P, Liu D, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood. Jan 13 2011;117(2):563-574.

127

47. Murray F, Darzentas N, Hadzidimitriou A, et al. Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis. Blood. Feb 1 2008;111(3):1524-1533. 48. Agathangelidis A, Darzentas N, Hadzidimitriou A, et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood. May 10 2012;119(19):4467-4475. 49. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood. Sep 15 1999;94(6):1848-1854. 50. Woyach JA, Johnson AJ, Byrd JC. The B-cell receptor signaling pathway as a therapeutic target in CLL. Blood. Aug 9 2012;120(6):1175-1184. 51. Smit LA, Hallaert DY, Spijker R, et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood. Feb 15 2007;109(4):1660-1668. 52. Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes & development. Sep 15 2004;18(18):2195-2224. 53. Hertlein E, Byrd JC. CLL and activated NF-kappaB: living partnership. Blood. May 1 2008;111(9):4427. 54. Endo T, Nishio M, Enzler T, et al. BAFF and APRIL support chronic lymphocytic leukemia B-cell survival through activation of the canonical NF-kappaB pathway. Blood. Jan 15 2007;109(2):703-710. 55. Pascutti MF, Jak M, Tromp JM, et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood. Oct 24 2013;122(17):3010-3019. 56. Burger JA, Ghia P, Rosenwald A, Caligaris-Cappio F. The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood. Oct 15 2009;114(16):3367-3375. 57. Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell'Aquila M, Kipps TJ. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood. Oct 15 2000;96(8):2655-2663. 58. Burkle A, Niedermeier M, Schmitt-Graff A, Wierda WG, Keating MJ, Burger JA. Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood. Nov 1 2007;110(9):3316-3325. 59. Riches JC, Davies JK, McClanahan F, et al. T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood. Feb 28 2013;121(9):1612-1621. 60. Scielzo C, Apollonio B, Scarfo L, et al. The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia. Leukemia. Nov 2011;25(11):1760-1767. 61. Mainou-Fowler T, Miller S, Proctor SJ, Dickinson AM. The levels of TNF alpha, IL4 and IL10 production by T-cells in B-cell chronic lymphocytic leukaemia (B-CLL). Leukemia research. Feb 2001;25(2):157-163.

128

62. Gattei V, Bulian P, Del Principe MI, et al. Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood. Jan 15 2008;111(2):865-873. 63. Zucchetto A, Caldana C, Benedetti D, et al. CD49d is overexpressed by trisomy 12 chronic lymphocytic leukemia cells: evidence for a methylation-dependent regulation mechanism. Blood. Nov 7 2013;122(19):3317-3321. 64. Eksioglu-Demiralp E, Alpdogan O, Aktan M, et al. Variable expression of CD49d antigen in B cell chronic lymphocytic leukemia is related to disease stages. Leukemia. Aug 1996;10(8):1331-1339. 65. Chemotherapeutic options in chronic lymphocytic leukemia: a meta-analysis of the randomized trials. CLL Trialists' Collaborative Group. Journal of the National Cancer Institute. May 19 1999;91(10):861-868. 66. Keating MJ, O'Brien S, Lerner S, et al. Long-term follow-up of patients with chronic lymphocytic leukemia (CLL) receiving fludarabine regimens as initial therapy. Blood. Aug 15 1998;92(4):1165-1171. 67. Tam CS, O'Brien S, Wierda W, et al. Long-term results of the fludarabine, cyclophosphamide, and rituximab regimen as initial therapy of chronic lymphocytic leukemia. Blood. Aug 15 2008;112(4):975-980. 68. Hallek M, Fischer K, Fingerle-Rowson G, et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. Oct 2 2010;376(9747):1164-1174. 69. Goede V, Fischer K, Busch R, et al. Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. The New England journal of medicine. Mar 20 2014;370(12):1101-1110. 70. Zenz T, Krober A, Scherer K, et al. Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up. Blood. Oct 15 2008;112(8):3322-3329. 71. Stilgenbauer S, Dohner H. Campath-1H-induced complete remission of chronic lymphocytic leukemia despite p53 gene mutation and resistance to chemotherapy. The New England journal of medicine. Aug 8 2002;347(6):452-453. 72. Keating MJ, Dritselis A, Yasothan U, Kirkpatrick P. Ofatumumab. Nature reviews. Drug discovery. Feb 2010;9(2):101-102. 73. Chiorazzi N, Ferrarini M. B cell chronic lymphocytic leukemia: lessons learned from studies of the B cell antigen receptor. Annual review of immunology. 2003;21:841-894. 74. Amrein PC, Attar EC, Takvorian T, et al. Phase II study of dasatinib in relapsed or refractory chronic lymphocytic leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research. May 1 2011;17(9):2977-2986. 75. Tsukada S, Saffran DC, Rawlings DJ, et al. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell. Jan 29 1993;72(2):279-290. 76. Thomas JD, Sideras P, Smith CI, Vorechovsky I, Chapman V, Paul WE. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science. Jul 16 1993;261(5119):355-358.

129

77. Mohamed AJ, Yu L, Backesjo CM, et al. Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain. Immunological reviews. Mar 2009;228(1):58-73. 78. Cameron F, Sanford M. Ibrutinib: first global approval. Drugs. Feb 2014;74(2):263-271. 79. Honigberg LA, Smith AM, Sirisawad M, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proceedings of the National Academy of Sciences of the United States of America. Jul 20 2010;107(29):13075-13080. 80. Herman SE, Gordon AL, Hertlein E, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. Jun 9 2011;117(23):6287-6296. 81. de Rooij MF, Kuil A, Geest CR, et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood. Mar 15 2012;119(11):2590-2594. 82. Dubovsky JA, Beckwith KA, Natarajan G, et al. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. Oct 10 2013;122(15):2539-2549. 83. O'Brien S, Furman RR, Coutre SE, et al. Ibrutinib as initial therapy for elderly patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma: an open-label, multicentre, phase 1b/2 trial. The lancet oncology. Jan 2014;15(1):48-58. 84. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. The New England journal of medicine. Jul 4 2013;369(1):32-42. 85. Byrd JC, Brown JR, O'Brien S, et al. Ibrutinib versus Ofatumumab in Previously Treated Chronic Lymphoid Leukemia. The New England journal of medicine. May 31 2014. 86. Longo PG, Laurenti L, Gobessi S, Sica S, Leone G, Efremov DG. The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells. Blood. Jan 15 2008;111(2):846-855. 87. Barragan M, de Frias M, Iglesias-Serret D, et al. Regulation of Akt/PKB by phosphatidylinositol 3-kinase-dependent and -independent pathways in B-cell chronic lymphocytic leukemia cells: role of protein kinase C{beta}. Journal of leukocyte biology. Dec 2006;80(6):1473-1479. 88. Wendel HG, De Stanchina E, Fridman JS, et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature. Mar 18 2004;428(6980):332-337. 89. Carpten JD, Faber AL, Horn C, et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature. Jul 26 2007;448(7152):439-444. 90. Pekarsky Y, Koval A, Hallas C, et al. Tcl1 enhances Akt kinase activity and mediates its nuclear translocation. Proceedings of the National Academy of Sciences of the United States of America. Mar 28 2000;97(7):3028-3033. 91. Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nature reviews. Cancer. Aug 2009;9(8):550-562. 92. Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Molecular cell. Mar 17 2006;21(6):749-760. 93. Herman SE, Gordon AL, Wagner AJ, et al. Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by 130

antagonizing intrinsic and extrinsic cellular survival signals. Blood. Sep 23 2010;116(12):2078-2088. 94. Vanhaesebroeck B, Ali K, Bilancio A, Geering B, Foukas LC. Signalling by PI3K isoforms: insights from gene-targeted mice. Trends in biochemical sciences. Apr 2005;30(4):194-204. 95. Jou ST, Carpino N, Takahashi Y, et al. Essential, nonredundant role for the phosphoinositide 3-kinase p110delta in signaling by the B-cell receptor complex. Molecular and cellular biology. Dec 2002;22(24):8580-8591. 96. Okkenhaug K, Bilancio A, Farjot G, et al. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science. Aug 9 2002;297(5583):1031-1034. 97. Brown JR, Byrd JC, Coutre SE, et al. Idelalisib, an inhibitor of phosphatidylinositol 3 kinase p110delta, for relapsed/refractory chronic lymphocytic leukemia. Blood. Mar 10 2014. 98. Patton DT, Garden OA, Pearce WP, et al. Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells. J Immunol. Nov 15 2006;177(10):6598-6602. 99. Furman RR, Sharman JP, Coutre SE, et al. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. The New England journal of medicine. Mar 13 2014;370(11):997-1007. 100. IPI-145 shows promise in CLL patients. Cancer discovery. Feb 2014;4(2):136. 101. Dal Porto JM, Gauld SB, Merrell KT, Mills D, Pugh-Bernard AE, Cambier J. B cell antigen receptor signaling 101. Mol Immunol. Jul 2004;41(6-7):599-613. 102. Deglesne PA, Chevallier N, Letestu R, et al. Survival response to B-cell receptor ligation is restricted to progressive chronic lymphocytic leukemia cells irrespective of Zap70 expression. Cancer research. Jul 15 2006;66(14):7158-7166. 103. Bernal A, Pastore RD, Asgary Z, et al. Survival of leukemic B cells promoted by engagement of the antigen receptor. Blood. Nov 15 2001;98(10):3050-3057. 104. Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature. Jul 7;475(7354):101-105. 105. Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N Engl J Med. Dec 29;365(26):2497-2506. 106. Herman SE, Gordon AL, Hertlein E, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. Jun 9;117(23):6287-6296. 107. Ponader S, Chen SS, Buggy JJ, et al. The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo. Blood. Feb 2;119(5):1182-1189. 108. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with Ibrutinib in Relapsed Chronic Lymphocytic Leukemia. N Engl J Med. Jun 19. 109. O'Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. Mar 13 2003;348(11):994-1004. 110. Gorre ME, Mohammed M, Ellwood K, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science. Aug 3 2001;293(5531):876-880. 131

111. Cortes J, Jabbour E, Kantarjian H, et al. Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors. Blood. Dec 1 2007;110(12):4005-4011. 112. Quintas-Cardama A, Kantarjian HM, Cortes JE. Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer Control. Apr 2009;16(2):122-131. 113. Stoklosa T, Poplawski T, Koptyra M, et al. BCR/ABL inhibits mismatch repair to protect from apoptosis and induce point mutations. Cancer Res. Apr 15 2008;68(8):2576-2580. 114. Landau DA, Carter SL, Stojanov P, et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell. Feb 14;152(4):714-726. 115. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. Jul 15 2009;25(14):1754-1760. 116. McKenna A HM, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. . The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. . Genome research. 2010;20:1297-1303. 117. Cibulskis K LM, Carter SL, Sivachenko A, Jaffe D, Sougnez C, Gabriel S, Meyerson M, Lander ES, Getz G. . Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnology 2013;31:213-219. 118. Cingolani P PA, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012;6:80-92. 119. Zhou Q, Lee GS, Brady J, et al. A hypermorphic missense mutation in PLCG2, encoding phospholipase Cgamma2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am J Hum Genet. Oct 5;91(4):713-720. 120. Hantschel O, Rix U, Schmidt U, et al. The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. Proc Natl Acad Sci U S A. Aug 14 2007;104(33):13283-13288. 121. Woyach JA, Smucker K, Smith LL, et al. Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy. Blood. Jan 10. 122. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. Feb 24 2005;352(8):786-792. 123. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. Mar 2005;2(3):e73. 124. Awad MM, Katayama R, McTigue M, et al. Acquired resistance to crizotinib from a mutation in CD74-ROS1. N Engl J Med. Jun 20;368(25):2395-2401. 125. Antonescu CR, Besmer P, Guo T, et al. Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation. Clin Cancer Res. Jun 1 2005;11(11):4182-4190. 126. Smith CC, Wang Q, Chin CS, et al. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature. May 10;485(7397):260-263.

132

127. Heidel F, Solem FK, Breitenbuecher F, et al. Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood. Jan 1 2006;107(1):293-300. 128. Valiaho J, Smith CI, Vihinen M. BTKbase: the mutation database for X-linked agammaglobulinemia. Hum Mutat. Dec 2006;27(12):1209-1217. 129. Burger JA LD, Hoellenriegel J, Sougnez C, Schlesner M, Ishaque N, Brors B, Keating MJ, Wierda WJ, Cibulskis K, Kantarjian HM, O'Brien SM, Neuberg DS, Zenz T, Getz G, Wu CJ. Clonal Evolution In Patients With Chronic Lymphocytic Leukemia (CLL) Developing Resistance To BTK Inhibition. Blood (Abst 866). 2013. 130. Zenz T, Mertens D, Kuppers R, Dohner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer. Jan 2010;10(1):37-50. 131. Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. The New England journal of medicine. Feb 24 2005;352(8):804-815. 132. Klein U, Dalla-Favera R. Germinal centres: role in B-cell physiology and malignancy. Nature reviews. Immunology. Jan 2008;8(1):22-33. 133. Zenz T, Mertens D, Dohner H, Stilgenbauer S. Importance of genetics in chronic lymphocytic leukemia. Blood reviews. May 2011;25(3):131-137. 134. Fabbri G, Rasi S, Rossi D, et al. Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation. The Journal of experimental medicine. Jul 4 2011;208(7):1389-1401. 135. Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature. Jul 7 2011;475(7354):101-105. 136. Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. The New England journal of medicine. Dec 29 2011;365(26):2497-2506. 137. Quesada V, Conde L, Villamor N, et al. Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nat Genet. Jan 2012;44(1):47-52. 138. Rossi D, Rasi S, Spina V, et al. Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia. Blood. Feb 21 2013;121(8):1403-1412. 139. Robak T. GA-101, a third-generation, humanized and glyco-engineered anti-CD20 mAb for the treatment of B-cell lymphoid malignancies. Curr Opin Investig Drugs. Jun 2009;10(6):588-596. 140. Stephens DM, Ruppert AS, Jones JA, et al. Impact of targeted therapy on outcome of chronic lymphocytic leukemia patients with relapsed del(17p13.1) karyotype at a single center. Leukemia. Jan 23 2014. 141. Duhren-von Minden M, Ubelhart R, Schneider D, et al. Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling. Nature. Sep 13 2012;489(7415):309-312. 142. Rickert RC, Jellusova J, Miletic AV. Signaling by the tumor necrosis factor receptor superfamily in B-cell biology and disease. Immunol Rev. Nov 2011;244(1):115-133. 143. Burger JA, Quiroga MP, Hartmann E, et al. High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation. Blood. Mar 26 2009;113(13):3050-3058. 133

144. Mohle R, Failenschmid C, Bautz F, Kanz L. Overexpression of the chemokine receptor CXCR4 in B cell chronic lymphocytic leukemia is associated with increased functional response to stromal cell-derived factor-1 (SDF-1). Leukemia. Dec 1999;13(12):1954-1959. 145. Durig J, Schmucker U, Duhrsen U. Differential expression of chemokine receptors in B cell malignancies. Leukemia. May 2001;15(5):752-756. 146. Friedberg JW, Sharman J, Sweetenham J, et al. Inhibition of Syk with fostamatinib disodium has significant clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Blood. Apr 1 2010;115(13):2578-2585. 147. Suljagic M, Longo PG, Bennardo S, et al. The Syk inhibitor fostamatinib disodium (R788) inhibits tumor growth in the Emu- TCL1 transgenic mouse model of CLL by blocking antigen-dependent B-cell receptor signaling. Blood. Dec 2 2010;116(23):4894-4905. 148. Hoellenriegel J, Meadows SA, Sivina M, et al. The phosphoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood. Sep 29 2011;118(13):3603-3612. 149. Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. PI3K/Akt signalling pathway and cancer. Cancer treatment reviews. Apr 2004;30(2):193-204. 150. Simons K, Toomre D. Lipid rafts and signal transduction. Nature reviews. Molecular cell biology. Oct 2000;1(1):31-39. 151. Adam RM, Mukhopadhyay NK, Kim J, et al. Cholesterol sensitivity of endogenous and myristoylated Akt. Cancer research. Jul 1 2007;67(13):6238-6246. 152. Pham LV, Tamayo AT, Yoshimura LC, et al. A CD40 Signalosome anchored in lipid rafts leads to constitutive activation of NF-kappaB and autonomous cell growth in B cell lymphomas. Immunity. Jan 2002;16(1):37-50. 153. Pierce SK. Lipid rafts and B-cell activation. Nature reviews. Immunology. Feb 2002;2(2):96-105. 154. Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. The Journal of clinical investigation. Sep 2002;110(5):597-603. 155. Zhuang L, Kim J, Adam RM, Solomon KR, Freeman MR. Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts. The Journal of clinical investigation. Apr 2005;115(4):959-968. 156. Zhuang L, Lin J, Lu ML, Solomon KR, Freeman MR. Cholesterol-rich lipid rafts mediate akt-regulated survival in prostate cancer cells. Cancer research. Apr 15 2002;62(8):2227-2231. 157. Mollinedo F, de la Iglesia-Vicente J, Gajate C, et al. In vitro and In vivo selective antitumor activity of Edelfosine against mantle cell lymphoma and chronic lymphocytic leukemia involving lipid rafts. Clinical cancer research : an official journal of the American Association for Cancer Research. Apr 1 2010;16(7):2046-2054. 158. McDonald PC, Fielding AB, Dedhar S. Integrin-linked kinase--essential roles in physiology and cancer biology. Journal of cell science. Oct 1 2008;121(Pt 19):3121-3132. 159. Persad S, Attwell S, Gray V, et al. Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. The Journal of biological chemistry. Jul 20 2001;276(29):27462-27469. 134

160. Huang Y, Li J, Zhang Y, Wu C. The roles of integrin-linked kinase in the regulation of myogenic differentiation. The Journal of cell biology. Aug 21 2000;150(4):861-872. 161. Lee SL, Hsu EC, Chou CC, et al. Identification and characterization of a novel integrin-linked kinase inhibitor. J Med Chem. Sep 22 2011;54(18):6364-6374. 162. Hertlein E, Beckwith KA, Lozanski G, et al. Characterization of a new chronic lymphocytic leukemia cell line for mechanistic in vitro and in vivo studies relevant to disease. PloS one. 2013;8(10):e76607. 163. Woyach JA, Bojnik E, Ruppert AS, et al. Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL). Blood. Feb 20 2014;123(8):1207-1213. 164. Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proceedings of the National Academy of Sciences of the United States of America. Sep 15 1993;90(18):8392-8396. 165. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. Dec 21-28 1995;378(6559):785-789. 166. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. Feb 18 2005;307(5712):1098-1101. 167. Surucu B, Bozulic L, Hynx D, Parcellier A, Hemmings BA. In vivo analysis of protein kinase B (PKB)/Akt regulation in DNA-PKcs-null mice reveals a role for PKB/Akt in DNA damage response and tumorigenesis. The Journal of biological chemistry. Oct 31 2008;283(44):30025-30033. 168. Viniegra JG, Martinez N, Modirassari P, et al. Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. The Journal of biological chemistry. Feb 11 2005;280(6):4029-4036. 169. Partovian C, Simons M. Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells. Cellular signalling. Aug 2004;16(8):951-957. 170. Casamayor A, Morrice NA, Alessi DR. Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo. The Biochemical journal. Sep 1 1999;342 ( Pt 2):287-292. 171. Kohn AD, Takeuchi F, Roth RA. Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. The Journal of biological chemistry. Sep 6 1996;271(36):21920-21926. 172. de Weerdt I, Eldering E, van Oers MH, Kater AP. The biological rationale and clinical efficacy of inhibition of signaling kinases in chronic lymphocytic leukemia. Leuk Res. Apr 15 2013. 173. Kurtova AV, Balakrishnan K, Chen R, et al. Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance. Blood. Nov 12 2009;114(20):4441-4450. 174. Elices MJ, Osborn L, Takada Y, et al. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell. Feb 23 1990;60(4):577-584.

135

175. Petlickovski A, Laurenti L, Li X, et al. Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells. Blood. Jun 15 2005;105(12):4820-4827. 176. Pepper C, Lin TT, Pratt G, et al. Mcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers. Blood. Nov 1 2008;112(9):3807-3817. 177. Johnson AJ, Lucas DM, Muthusamy N, et al. Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia. Blood. Aug 15 2006;108(4):1334-1338. 178. Robak P, Robak T. A targeted therapy for protein and lipid kinases in chronic lymphocytic leukemia. Current medicinal chemistry. 2012;19(31):5294-5318. 179. Baracho GV, Miletic AV, Omori SA, Cato MH, Rickert RC. Emergence of the PI3-kinase pathway as a central modulator of normal and aberrant B cell differentiation. Current opinion in immunology. Apr 2011;23(2):178-183. 180. Mollinedo F, de la Iglesia-Vicente J, Gajate C, et al. Lipid raft-targeted therapy in multiple myeloma. Oncogene. Jul 1 2010;29(26):3748-3757. 181. Reis-Sobreiro M, Roue G, Moros A, et al. Lipid raft-mediated Akt signaling as a therapeutic target in mantle cell lymphoma. Blood cancer journal. 2013;3:e118. 182. Mollinedo F, Fernandez-Luna JL, Gajate C, et al. Selective induction of apoptosis in cancer cells by the ether lipid ET-18-OCH3 (Edelfosine): molecular structure requirements, cellular uptake, and protection by Bcl-2 and Bcl-X(L). Cancer research. Apr 1 1997;57(7):1320-1328. 183. Gajate C, Mollinedo F. Edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts. Blood. Jan 15 2007;109(2):711-719. 184. Larsson SC, Wolk A. Overweight and obesity and incidence of leukemia: a meta-analysis of cohort studies. International journal of cancer. Journal international du cancer. Mar 15 2008;122(6):1418-1421. 185. Mocsai A, Ruland J, Tybulewicz VL. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nature reviews. Immunology. Jun 2010;10(6):387-402. 186. Engelke M, Oellerich T, Dittmann K, et al. Cutting edge: feed-forward activation of phospholipase Cgamma2 via C2 domain-mediated binding to SLP65. J Immunol. Dec 1 2013;191(11):5354-5358. 187. Ishiai M, Kurosaki M, Pappu R, et al. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells. Immunity. Jan 1999;10(1):117-125. 188. Kim MS, Jeong EG, Yoo NJ, Lee SH. Mutational analysis of oncogenic AKT E17K mutation in common solid cancers and acute leukaemias. British journal of cancer. May 6 2008;98(9):1533-1535. 189. Mahmoud IS, Sughayer MA, Mohammad HA, Awidi AS, MS EL-K, Ismail SI. The transforming mutation E17K/AKT1 is not a major event in B-cell-derived lymphoid leukaemias. British journal of cancer. Aug 5 2008;99(3):488-490. 190. King AJ, Sun H, Diaz B, et al. The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature. Nov 12 1998;396(6707):180-183.

136

191. Combeau G, Kreis P, Domenichini F, et al. The p21-activated kinase PAK3 forms heterodimers with PAK1 in brain implementing trans-regulation of PAK3 activity. The Journal of biological chemistry. Aug 31 2012;287(36):30084-30096. 192. Higuchi M, Onishi K, Kikuchi C, Gotoh Y. Scaffolding function of PAK in the PDK1-Akt pathway. Nature cell biology. Nov 2008;10(11):1356-1364. 193. Rahal R, Frick M, Romero R, et al. Pharmacological and genomic profiling identifies NF-kappaB-targeted treatment strategies for mantle cell lymphoma. Nature medicine. Jan 2014;20(1):87-92. 194. Ashley CE, Carnes EC, Phillips GK, et al. The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers. Nature materials. May 2011;10(5):389-397.

137