Targeting Nuclear Export in Chronic Lymphocytic

DISSERTATION

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

By

Zachary Andrew Hing

Graduate Program in Integrated Biomedical Science Program

The Ohio State University

2018

Dissertation Committee:

John C. Byrd MD, Co-Advisor

Rosa Lapalombella PhD, Co-Advisor

Robert A. Baiocchi MD, PhD

Lynne Abruzzo MD, PhD

Guramrit Singh PhD

Copyright by

Zachary Andrew Hing

2018

Abstract

In the past decade there has been increased appreciation for the complexity and importance of post-transcriptional processes that control regulation and post- translational processes that control function in the cell. In particular, intense study has focused on how they become altered in . controls both of these processes through temporospatial separation of macromolecules. Alterations of nuclear export, particularly via dysregulation of the exportin 1/ region maintenance 1 (XPO1/CRM1) pathway, have recently been identified in many different human , including chronic lymphocytic leukemia (CLL), where XPO1 inactivates tumor suppressor and regulatory by sequestering them from the nucleus.

In CLL, recent therapies targeting components of the B cell receptor pathway such as inhibitors to Bruton tyrosine kinase (BTK) have produced dramatic response rates; however, complete responses are infrequent and resistance to BTK inhibition is a growing clinical concern. Therefore, restoring nuclear localization of tumor suppressor proteins via inhibition of XPO1 is a promising strategy to reestablishing their function.

Oral selective inhibitor of nuclear export (SINE) compounds block XPO1 function and represent a novel therapeutic approach in CLL. The lead SINE compound, , has shown promise in clinical trials, but is limited to twice weekly dosing.

Therefore, novel SINE compounds with enhanced tolerability may result in improved

ii

clinical efficacy. In addition, we hypothesized that combination of XPO1 blockade with

BTK inhibition may result in increased efficacy. Herein we describe the discovery and

characterization of a novel SINE compound and the identification of novel targeted

combination therapies using dual XPO1 and BTK inhibition. Using a combination of

cell-free, in vitro, and in vivo models we have established the efficacy of a novel XPO1 inhibitor, KPT-8602.

The identification of rational drug combinations in oncology holds promise as a strategy to increase durability of responses and prevent resistance to any single-agent. In

CLL, combination strategies that display synergy with ibrutinib are of intense interest to

address the growing issue of acquired drug resistance. Rational combination strategies

that include XPO1 blockade are particular appealing as a method of restoring function to

multiple tumor suppressor proteins and eliciting synergistic responses.

Here, we have described a novel combination therapy targeting XPO1 and BTK

that results in synergistic responses in CLL. We also demonstrate that XPO1 inhibition is

active in the growing setting of acquired ibrutinib resistance. Overall, our data indicate

that nuclear export represents a promising therapeutic target in CLL, particularly in the

setting of acquired resistance to ibrutinib. Moreover, combination therapies that

encompass XPO1 inhibition hold promise in CLL. These results may have relevance to

other cancers in which XPO1 overexpression is seen.

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Dedicated to my unbelievable mentors, Rosa and Dr. Byrd. Thank you for making me the luckiest student in the world. And to my wife, Kris, for her incredible support.

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Acknowledgments

First and foremost, I would like to thank my mentors, Drs. John Byrd and Rosa

Lapalombella. They are my role models in their dedication and excitement for science.

Throughout my professional journey I hope to embody the ideals and wisdom they have kindly imparted to me. Thank you both for helping me understand our most important mission as scientists, which is to pursue questions that are impactful for our patients.

Thank you for creating a stimulating laboratory environment that emphasizes team work, collaboration, and integrity. Thank you for helping cultivate the passion that I have for science and medicine. And thank you for letting me take risks and gain independence. I have learned a lot over the past years.

I would like to acknowledge the generous support of my committee members Drs.

Guramrit Singh, Lynne Abruzzo, and Robert Baiocchi. I am deeply grateful for their guidance and shared wisdom throughout my PhD training.

I would like to thank all past and present members of the Experimental

Hematology Laboratory. It has been humbling to work among people who inspire me daily. I would like to thank Ashley Bertran, Derek West, Sue Scott, and Amber Gordon for their kind administrative support. I thank Drs. Larry Schlesinger and Larry Kirschner for their ongoing commitment to my education.

v

During my thesis research, I have had the privilege to be mentored clinically by

Drs. John Byrd, Lapo Alinari, Jennifer Woyach, Robert Baiocchi, James Blachly, Kerry

Rogers, and Vijay Duggirala. I thank you all for your lessons and time.

I am grateful for fellowship support from the National Institutes of Health (F30).

I would like to thank my family for their lifelong inspiration and support.

Maintaining a positive attitude, work ethic, and teamwork all stem from your wonderful examples and early life lessons.

And finally, thank you to my wonderful wife, Kris. You support means the world to me.

vi

Vita

June 1989 ...... Born—St. Louis, Missouri

2011...... B.S. Chemistry, Magna Cum Laude,

Duke University

2011-2012 ...... Postbaccalaureate Intramural Research

Training Award (IRTA) Program, National Institutes of Health

2012 to present ...... Medical Scientist Training Program, The

Ohio State University

vii

Publications

Hatice Gulcin Ozer*, El-Gamal D*, Powell B*, Hing ZA, Blachly JS, Mitchell S,

Williams K, Zhang J, Ma Y, Zhang Y, Harrington BK, Alinari L, Baiocchi RA, Lindsey

Brinton, Cannon M, Brennan PJ, Goettl VM, Woyach JA, Sampath D, Jones JA,

Lehman AM, Yu L, Spevak W, Shi S, Shellooe R, Carias H, Tsang G, Dong K, Ewing T,

Marimuthu A, Tantoy C, Walters J, Sanftner L, Rezaei H, Nespi M, Matusow B, Habets

G, Ibrahim P, Zhang C, Gideon Bollag, Byrd JC, Lapalombella R. BRD4 profiling

identifies critical Chronic Lymphocytic Leukemia oncogenic circuits and reveals sensitivity to PLX51107, a novel structurally distinct BET inhibitor (2018). Cancer

Discovery, accepted.

Hing ZA, Fung HYJ, Ranganathan P, Mitchell S, El-Gamal D, Woyach JA, Williams K,

Goettl VM, Smith J, Yu X, Meng X, Sun Q, Cagatay T, Lehman AM, Lucas DM,

Baloglu E, Shacham S, Kauffman MG, Byrd JC, Chook YM, Garzon R and

Lapalombella R. Next generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematologic malignancies (2016). Leukemia, 30: 2364-2372.

Hing ZA, Mantel R, Beckwith KA, Guinn D, Williams E, Smith LL, Williams K,

Johnson AJ, Lehman AM, Byrd JC, Woyach JA, and Lapalombella R. Selinexor is

viii

effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic

lymphocytic leukemia (2015). Blood, 125(20): 3128-3132.

Kim, B*, Hing, ZA*, Wu, A*, Schiller, T, Struble, EB, Liuwantara, D, Kempert, PH,

Broxham, EJ, Edwards, NC, Marder, VJ, Simhadri, VL, Sauna, ZE, Howard, TE &

Kimchi‐Sarfaty, C. Single-nucleotide variations defining previously unreported

ADAMTS13 haplotypes are associated with differential expression and activity of the

VWF-cleaving protease in a salvadoran congenital thrombotic thrombocytopenic purpura family (2014). *Co-first author. British journal of haematology, 165(1): 154-158.

Hing, ZA, Schiller, T, Wu, A, Hamasaki‐Katagiri, N, Struble, EB, Russek-Cohen, E, &

Kimchi‐Sarfaty, C. Multiple in silico tools predict phenotypic manifestations in congenital thrombotic thrombocytopenic purpura (2013). British journal of haematology,

160(6): 825-837.

Edwards, NC*, Hing, ZA*, Perry, A, Blaisdell, A, Kopelman, DB, Fathke, R, Plum, W,

Newell, J, Allen, CE, Geetha, S, Shapiro, A, Okunji, C, Kosti, I, Shomron, N, Grigoryan,

V, Przytycka, TM, Sauna, ZE, Salari, R, Mandel-Gutfreund, Y, Komar, AA & Kimchi-

Sarfaty, C. Characterization of coding synonymous and non-synonymous variants in

ADAMTS13 using ex vivo and in silico approaches (2012). *Co-first author. PloS one,

7(6), e38864.

ix

Fields of Study

Major Field: Integrated Biomedical Science Program

Specialization: Genetics

x

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vii

List of Tables ...... xv

List of Figures ...... xvi

List of Abbreviations ...... xix

Chapter 1: Introduction ...... 1

1.1 Chronic lymphocytic leukemia ...... 1

1.1.1 Overview of CLL...... 1

1.1.2 Genomic aberrations in CLL ...... 2

1.1.3 Therapeutic landscape in CLL ...... 3

1.2 Nuclear transport in leukemia ...... 3

1.2.1 Nucleocytoplasmic transport—concepts and mechanisms...... 3

1.2.2 XPO1 Cargos ...... 5

1.2.3 Nuclear transport defects in hematologic malignancies including CLL ...... 7

1.2 Targeting nuclear export – theory to practice ...... 9

xi

1.3.1 Nuclear export inhibitory drugs ...... 9

1.3.2 SINEs: Preclinical, Clinical Development in hematologic malignancies ...... 11

1.3.3 Potential XPO1 dual targeting strategies in CLL ...... 13

1.4 Conclusions and hypothesis ...... 14

1.5 References ...... 16

Chapter 2: Novel therapeutics targeting exportin 1 in hematologic malignancies ...... 27

2.1 Introduction ...... 27

2.2 Materials and Methods ...... 30

2.2.1 Cloning, expression, and protein purification ...... 30

2.2.2 Assembly of the XPO1--Yrb1 complex, crystallization and X-ray data

collection ...... 31

2.2.3 X-ray structure determination and refinement ...... 32

2.2.4 In vitro inhibition assays...... 32

2.2.5 In vivo XPO1 degradation by SINE treatment ...... 33

2.2.6 Cell isolation and reagents ...... 34

2.2.7 Cell lysis and immunoblot ...... 34

2.2.8 Immunofluorescent Staining...... 35

2.2.9 Animal studies ...... 35

2.2.10 TCL1 transplant mouse model ...... 35

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2.2.11 MV4-11 xenograft mouse model ...... 36

2.2.12 Statistical analysis...... 36

2.3 Results ...... 37

2.3.1 KPT-8602 binds in the NES-binding groove of XPO1 ...... 37

2.3.2 KPT-8602 inhibits XPO1-cargo interactions ...... 38

2.3.3 KPT-8602 induces of primary CLL cells and significantly inhibits

proliferation of diffuse large B-cell cell lines ...... 39

2.3.4 KPT-8602 possesses reduced central nervous system penetration ...... 40

2.3.5 KPT-8602 prolongs survival in a mouse model of CLL ...... 41

2.3.6 KPT-8602 significantly inhibits proliferation and induces apoptosis of AML

cell lines and primary AML blasts ...... 43

2.3.7 KPT-8602 prolongs survival in a human leukemia xenograft model of AML 44

2.4 Discussion ...... 45

2.5 References ...... 49

Chapter 3: Dual targeting of exportin 1 and Bruton tyrosine kinase in CLL ...... 79

3.1 Introduction ...... 79

3.2 Materials and Methods ...... 80

3.2.1 Cell isolation and reagents ...... 80

3.2.2 Generation of BTK cell lines ...... 80

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3.2.3 Assessment of cell death ...... 81

3.2.4 Animal studies ...... 81

3.2.5 Statistical Analysis ...... 82

3.3 Results and Discussion ...... 82

3.4 References ...... 87

Chapter 4: Conclusions and Future Directions ...... 100

4.1 Conclusions ...... 100

4.2 Future Perspectives ...... 101

4.2.1 Extending current work ...... 101

4.2.2 Exploring additional XPO1 driven combination therapies...... 104

4.2.3 Mechanisms of acquired resistance to XPO1 blockade ...... 106

4.2.4 Identifying druggable vulnerabilities in cancer ...... 107

4.2.5 Determining the role of XPO1 in the pathogenesis of CLL ...... 108

4.3 References ...... 110

Works Cited ...... 114

xiv

List of Tables

Table 2.1 Structure data collection and refinement statistics ...... 54

Table 2.2 Contacts of <4 Å between KPT-8602 and XPO1 ...... 56

Table 2.3 Brain penetration of KPT-8602 and KPT-330 across species ...... 57

Table 3.1 Cytogenetic and molecular features of patient specimens used for in vitro apoptosis assays ...... 91

xv

List of Figures

Figure 1.1 Transport of macromolecules via XPO1 ...... 25

Figure 1.2 Targeting nuclear export via selective inhibitor of nuclear export (SINE)

compounds ...... 26

Figure 2.1 KPT-8602 binds in the NES-binding groove of XPO1 ...... 58

Figure 2.2 Structure of KPT-8602 bound to XPO1 compared to KPT-185 bound structure

and electron density of KPT-8602 ...... 60

Figure 2.3 KPT-8602 inhibits XPO1-NES interactions...... 62

Figure 2.4 KPT-8602 conjugation to XPO1 is reversible ...... 63

Figure 2.5 Inhibitor-induced degradation of XPO1 ...... 64

Figure 2.6 KPT-8602 induces apoptosis in primary CLL cells ...... 65

Figure 2.7 KPT-8602 induces nuclear retention of IκBα in a manner similar to KPT-330

...... 66

Figure 2.8 KPT-8602 inhibits proliferation of diffuse large B-cell lymphoma cell lines ..67

Figure 2.9 KPT-8602 and KPT-330 show similar survival benefit when given twice weekly ...... 68

xvi

Figure 2.10 KPT-8602 improves survival compared to KPT-330 in a mouse model of

CLL ...... 69

Figure 2.11 KPT-8602 decreases leukemic burden beyond KPT-330 in a mouse model of

CLL ...... 70

Figure 2.12 KPT-8602 and ibrutinib improve survival compared either agent alone in a

mouse model of CLL ...... 72

Figure 2.13 KPT-8602 controls disease in a mouse model of CLL ...... 73

Figure 2.14 KPT-8602 significantly inhibits proliferation and induces apoptosis of AML

cell lines ...... 74

Figure 2.15 KPT-8602 significantly inhibits proliferation primary AML blasts ...... 76

Figure 2.16 KPT-8602 restores nuclear localization of XPO1 cargo proteins in AML cell

lines ...... 77

Figure 2.17 KPT-8602 increases survival in a human leukemia xenograft model of AML

compared to KPT-330 ...... 78

Figure 3.1 Selinexor synergizes in vitro with ibrutinib ...... 92

Figure 3.2 Selinexor and ibrutinib retain efficacy in CpG and anti-IgM stimulated CLL

cells ...... 93

Figure 3.3 Selinexor and ibrutinib are cytotoxic to CLL cells co-cultured with bone marrow stromal cells ...... 94

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Figure 3.4 Selinexor synergizes in vivo with ibrutinib ...... 95

Figure 3.5 Selinexor is more effective in post-ibrutinib CLL samples ...... 96

Figure 3.6 Selinexor induces cytotoxicity in cells expressing C481S BTK ...... 97

Figure 3.7 Selinexor induces cytotoxicity in a mouse model of acquired resistance to ibrutinib ...... 98

Figure 3.8 Selinexor is active in the setting of acquired resistance to ibrutinib ...... 99

xviii

List of Abbreviations

AE Adverse event

AKT Protein kinase B

AML

BCR B-cell receptor

BTK Bruton tyrosine kinase

CLL Chronic lymphocytic leukemia

CML Chronic myeloid leukemia

CR Complete response

DLBCL Diffuse large B-cell lymphoma

EC50 Half maximal effective concentration

Eµ Immunoglobulin heavy chain (IgH) enhancer

FLT3-ITD Fms-related tyrosine kinase 3 internal tandem duplication

GRP Growth regulatory protein

IBR Ibrutinib

IC50 Half maximal inhibitory concentration

IGHV Immunoglobulin heavy chain (IgH) variable region gene

IgM Immunoglobulin M protein

IκB Inhibitor of kappa B

LMB Leptomycin B

MCL Mantle cell lymphoma

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MM

mRNA messenger RNA

NES Nuclear export sequence

NF-κB Nuclear factor-κB

NLS Nuclear localization sequence

NPC complex

OR Objective response

PBMC Peripheral blood mononuclear cells

PLC-γ2 Phospholipase C-gamma-2

PD Progressive disease

PDX Patient-derived xenograft

PI Propidium iodide

PR Partial response

Ran Ras-related nuclear protein rRNA ribosomal RNA

SCID Severely combined immunodeficient

SD Stable disease

SEL Selinexor (KPT-330)

SINE Selective inhibitor of nuclear export

TCL1 T-cell leukemia/lymphoma protein

TSP Tumor suppressor protein

XPO1/CRM1 Exportin 1 / Chromosome region maintenance 1

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Chapter 1: Introduction

1.1 Chronic lymphocytic leukemia

1.1.1 Overview of CLL

Chronic lymphocytic leukemia (CLL) is diagnosed in approximately 4-5 individuals per

100,000 in the United States each year and it remains the most prevalent adult leukemia in the Western world1. The clinical presentation of CLL is varied, with some patients

presenting with painless lymphadenopathy and others presenting asymptomatically with

an elevated lymphocyte count on routine blood work. The former presentation is termed

small lymphocytic lymphoma (SLL), which is considered to be a unique presentation of

CLL. An absolute lymphocyte count of >5000 cells per microliter is required to establish

the diagnosis of CLL. In addition, CLL often presents and is diagnosed in older adults

around 70 years of age on average. While most CLL patients present asymptomatically,

the disease is progressive and results in severe immune impairment as the clonal population

of dysfunctional CLL B lymphocytes accumulates. Despite the presence of multiple

comorbidities at diagnosis, the major cause of death in patients with CLL remains disease

progression or related complications2.

At the cellular level, CLL is characterized by the clonal accumulation of malignant

B lymphocytes in the peripheral blood, bone marrow, and secondary lymphoid organs3. 1

These B cells are nonfunctional and as a result, CLL patients are immunocompromised

with increased susceptibility to infectious agents. Infection is a leading cause of mortality

in CLL patients.

The natural history of CLL is quite varied, with survival ranging in some patients from 2 years to 2 decades. The search for prognostic factors to explain this variability in outcome has led to the identification of two major subtypes of CLL, categorized based on the mutational status of the immunoglobulin heavy chain variable (IGHV) gene. Patients with CLL cells harboring somatic IGHV mutations (defined as >2% of the sequenced region) tend to have longer survival and better response to treatment, compared to patients with an unmutated IGHV gene4,5.

1.1.2 Genomic aberrations in CLL

At the genomic level, CLL exhibits considerable complexity. A few genetic subtypes of

CLL have been described with prognostic significance. The most common genomic

aberrations are trisomy 12 and deletions of 11q, 13q, and 17p6. In the past

decade, our understanding of the genomic basis of CLL has expanded greatly due to next- generation sequencing studies of large CLL cohorts7. The identification of novel recurrent

mutations in CLL such as NOTCH1, MYD88, and mutations in RNA maturation and

splicing (SF3B1), and components of nuclear export machinery (XPO1 and RANBP2) has generated interest in understanding their putative role in CLL pathogenesis 8–10. In the context of developing experimental therapeutics for CLL, understanding the role and

2

potential druggable susceptibilities of these mutations may open the door to precise

therapeutic approaches to individual genetic CLL subtypes.

1.1.3 Therapeutic landscape in CLL

The therapeutic landscape in CLL has changed considerably over the past few years. In

particular, the introduction of novel targeted agents that suppress the B cell receptor (BCR) signaling pathway has resulted in significantly higher response rates in relapsed or refractory CLL11,12. In particular, Bruton tyrosine kinase (BTK), a critical component of

BCR signaling, has been identified as a key molecular target in CLL. The recently

approved BTK inhibitor, ibrutinib, has demonstrated exceptional clinical activity in CLL

and other mature B cell malignancies such as mantle cell lymphoma, and Waldenström’s

macroglobulinemia13,14. However, despite the impressive responses with ibrutinib in CLL

compared to other therapies, complete responses (CR) have generally not been achieved

and a subset of patients develop resistant cancer clones making the development of

combination strategies with ibrutinib a high priority. Even with the development of novel

targeted therapies, CLL remains an incurable disease.

1.2 Nuclear transport in leukemia

1.2.1 Nucleocytoplasmic transport—concepts and mechanisms

The nucleus physically separates transcription from translation and is the hallmark of all

eukaryotic cells. This temporospatial separation of transcription and translation allow for

3

multilayered control of the two processes via regulation of nuclear import and export of

proteins and nucleic acids. Integration of these two fundamental and highly intertwined processes requires rapid and controlled movement of proteins and ribonucleic acids between each environment.

The nucleus is surrounded by a double lipid bilayer, the nuclear envelope, which prevents molecules greater than 40 kDa from passively communicating between the nucleus and cytoplasm. While the transport of small molecules, salts, and nucleotides is governed by passive transport (diffusion), the transport of large molecules, including many

proteins and RNA, is a highly regulated, active transport process that occurs at pores in the

nuclear envelope where the outer and inner nuclear membranes meet15. Located at these

pore sites is a large collection of proteins that form the nuclear pore complex (NPC). With

a mass of ~60 MDa, the NPC is a significant structure consisting of ~30 different types of nucleoporin proteins that help to establish polarity, interact with transporters, and maintain

its gatekeeping function16,17.

Macromolecules that traverse the NPC require a transport receptor or chaperone,

termed or exportins, from β family. To interact with these importins or exportins, a macromolecule must harbor a specified sequence, termed a nuclear localization sequence (NLS) or a nuclear export sequence (NES). The macromolecule may then interact with an or exportin either directly via its NLS/NES or indirectly via an adapter protein18. These transport receptors and their cognate NLS or NES recognition

contribute to the overall selectivity of macromolecular transport through the NPC. The

energy and directionality of karyopherin-mediated import and export is controlled by a

4

gradient of RanGTP (Ras-related nuclear protein) across the nuclear envelope. The Ran

cycle is essential for proper directionality of nuclear import and export1.

The exportin family contains 7 transporters (Exportin-1, 2, 4, 5, 6, 7, t), which interact with different types of cargo molecules. Of these exporters, Exportin-1 (XPO1), also known as Chromosome Region Maintenance 1 (CRM1), is the best characterized and transports the broadest array of cargos. XPO1 recognizes a NES characterized by a leucine- rich 8-15 amino acid sequence19. The XPO1 NES sequences are diverse in structure and

sequence and can be difficult to predict. The consensus sequence consists of the form Φ1-

X(2-3)- Φ2-X(2-3)- Φ3-X- Φ4 where Φ indicates a hydrophobic residue M, L, V, I, F and X

is any amino acid20.

1.2.2 XPO1 Cargos

XPO1 shuttles a diverse array of cargo proteins and a more limited set of RNA species

between the nucleus and the cytoplasm (Figure 1.1). It is the major nuclear export receptor

in humans, with >200 cargo proteins that have been experimentally validated. These cargo

proteins are functionally important in an array of cellular processes including transcription,

translation, growth regulation, DNA replication, stress responses and homeostasis. A

complete repertoire of XPO1 cargos has not been fully described and is an area of active

research. Recent proteomic analysis of nuclear and cytoplasmic fractions identified >700

cargos in S. cerevisiae, >1000 in Xenopus oocytes, and >1000 in human cells21. The array

of cargos that XPO1 interacts with likely depends on species and tissue type.

5

Of particular interest to the field of oncology, are the growth regulatory proteins

(GRPs), tumor suppressor proteins (TSPs), and drug targets that are exported by XPO1.

Some of these important drug targets include MAPK signaling proteins, BCR-ABL, topoisomerase IIα, nucleophosmin, COX-2, and cyclin D122,23. By promoting re- localization of drug targets, XPO1 overexpression may represent an important mode of acquired resistance in patients. XPO1 is also responsible for the subcellular translocation of many TSPs such as , retinoblastoma, APC, FOXO proteins, endogenous inhibitor of

NF-κB (IκB), BRCA1, and cell cycle regulators p21CIP1 and p27KIP1 24–26. The TSP p53 is an important protein that can arrest the cell cycle to initiate DNA damage repair pathways and/or induce apoptosis of severely damaged cells27. However, to exert its function, p53 must be properly localized in the nucleus. As such, an imbalance in the nucleocytoplasmic shuttling of p53 may result in mislocalization and functional impairment of p53 in the cell.

In addition to its ability to shuttle proteins to the cytoplasm, XPO1 also plays a role in the export of certain RNA species, such as the ribosomal subunits 40S and 60S and certain mRNAs. The majority of pre-messenger RNAs (pre-mRNAs) are shuttled to the cytoplasm via the Nxf1(TAP)/Nxt1 complex17, which drives bulk export of mRNAs via a host of export factors termed the transcription-export (TREX) complex28. Yet, some transcripts use the saturatable, receptor-mediated XPO1 export pathway instead of the bulk mRNA export pathway. The reasons for this are presently unclear, but may indicate a need for additional plasticity in regulating expression of certain . For example, the ability to rapidly upregulate specific transcripts in response to external stimuli may be important in certain contexts. A class of mRNAs that contain AU-rich elements (ARE) in their 3’-

6

UTR have been shown to undergo XPO1-dependent nuclear egress via the AU-rich RNA binding protein (AUBP), HuR. Current estimates indicate ARE-genes represent 10-15% of all transcripts in the cell29. Notable ARE-genes include BCL2, c-fos, cyclin D1, and

VEGF30. As a class, the functions of ARE-genes are related to sustained production of pro-

inflammatory cytokines31. In the cancer microenvironment these can lead to the hallmarks

of cancer: tumor growth, resistance to apoptosis, angiogenesis, invasion and metastasis. In

addition, a subset of mRNAs such as c-Myc and cyclin D1 that require translation initiation factor 4E (eIF4E) undergo XPO1-dependent export from the nucleus32,33.

1.2.3 Nuclear transport defects in hematologic malignancies including CLL

One of the surprising recent findings in the field of oncology has been the predilection of

cancer cells to co-opt nuclear export machinery for growth advantage and therapy

resistance. It is now more widely acknowledged that mislocalization of key regulatory

molecules is a common feature of many human cancers. By co-opting XPO1 function,

cancer cells can post-transcriptionally amplify multiple growth regulatory pathways

simultaneously.

Overexpression of nuclear export machinery has been identified in a number of

cancers, including solid tumors and hematologic malignancies. In particular,

overexpression of XPO1 has been described in CLL, acute myeloid leukemia, multiple

myeloma, lymphoma as well as solid tumors such lung, bone, brain, pancreatic,

esophageal, , gastric, renal, ovarian, and cervical cancer34–39. In multiple cancer types,

XPO1 overexpression confers a poor prognosis and is implicated in resistance to therapy34.

7

No direct evidence exists yet to prove that XPO1 overexpression leads to tumorigenesis.

However, another component of the nuclear export machinery, eIF4E was recently shown

indirectly to cause tumorigenesis in vitro and remodel the cytoplasmic face of the NPC40.

In hematologic malignancies, evidence has recently accumulated to establish a role

for XPO1 in the pathophysiology of multiple myeloma. It was recently shown that XPO1

is important for maintaining growth and survival of multiple myeloma cells and for

osteoclastogenesis41.

Similarly, nuclear transport is an emerging theme in myeloid neoplasms. This

became clear when cytoplasmic mislocalization was identified as the critical missing link

in explaining the mechanism behind nucleophosmin (NPM1) in and

less than a decade ago42. Cytoplasmic mislocalization of NPM1 (NPMc+) is the major

genetic mutation in patients with cytogenetically normal AML. XPO1 has been shown to

be a prognostic indicator in AML as well43. Other protein components of the NPC machinery have been identified as contributors to leukemogenic fusion proteins in AML.

Nucleoporin 98 (NUP98) has been observed in chromosomal rearrangements with at least

20 different partners44. Furthermore the clinical course associated with these leukemias is

aggressive and has poor response to available therapies. Additionally, XPO1 has been

shown to be overexpressed in in CD34+ progenitors from chronic myeloid leukemia

(CML) patients in blast crisis and from Philadelphia chromosome positive (Ph+) B-cell

acute lymphocytic leukemia (ALL) patients45.

In addition to overexpression of XPO1 protein, somatic mutations in XPO1 have

been described in multiple hematologic malignancies including CLL10,46, primary

8

mediastinal B cell lymphoma (PMBL)47,48, and Hodgkin lymphoma49. Somatic mutational

analysis of 363 CLL patients, identified the two recurrent XPO1 mutations at the 571 amino

acid (p.E571K and p.E571G), which is located in the highly conserved binding pocket10.

This observation has been confirmed in additional CLL cohorts, which indicate the

E571K/G XPO1 mutation occurs in roughly 2.5-5% of CLL patients and 28% of patients with Richter’s transformation46,50,51. The E571K XPO1 mutation was also identified in

24% (28/117) PMBL cases, 26% (5/19) of Hodgkin lymphoma, and few (3/197) cases of

diffuse large B cell lymphoma47,48.

1.2 Targeting nuclear export – theory to practice

1.3.1 Nuclear export inhibitory drugs

Recent basic and translational work has led to the development of promising therapies that target XPO1. Excitement for XPO1 as a therapeutic target has stemmed from the ability to target nucleocytoplasmic transport of multiple TSPs important in the pathogenesis, maintenance, and resistance to therapies in multiple cancer types.

The first inhibitor of XPO1 to be tested in humans was the natural product leptomycin B (LMB). LMB was originally identified as an antifungal agent, but subsequently shown to have potent anticancer properties. XPO1 was identified as the cellular target of LMB. Specifically, the lactone warhead of LMB covalently binds to

Cys52852, a key residue located in the hydrophobic NES-binding groove of XPO153. After 9

binding to Cys528, the lactone ring of LMB is hydrolyzed and which then allows LMB to

form additional bonds with XPO1. Interestingly, Sun and colleagues, showed that the

LMB-XPO1 covalent bond is slowly reversible; however, after hydrolysis of the lactone

ring in LMB, the bond becomes irreversible54. Thus, LMB is an irreversible inhibitor of

XPO1. LMB was the first XPO1 inhibitor to be tested in humans as a cancer therapy, but

the severity of associated toxicities (nausea, vomiting, anorexia, and malaise) precluded

any further clinical development in human subjects55. The irreversible blockade of XPO1 nuclear export by LMB hydrolysis is likely the cause of the strong toxicities observed. This

is consistent with evidence that the XPO1 nuclear export pathway is essential in normal tissues, in addition to cancer cells56.

In a drug screen for inhibitors of the HIV-1 Rev viral protein, a known cargo of

XPO1, PKF050-638 was identified as an XPO1 inhibitor57. The N-azolylacrylate scaffold of PKF050-638 was shown to disrupt XPO1-cargo interactions. A novel method of in silico was used to improve the N-azolylacrylate class of XPO1 inhibitors58, resulting in

the recent development of Selective Inhibitors of Nuclear Export, or SINEs. This current

generation of XPO1 inhibitors disrupts the XPO1-NES interaction by covalently modifying

the reactive Cys528, similar to the mechanism of LMB. However, in contrast to LMB, the

acrylate warhead of these inhibitors contain Michael acceptors which covalently conjugate

the reactive donor of XPO1 via a slowly reversible Michael-type addition54. Thus,

SINEs induce a temporal blockade of XPO1-mediated nuclear export, compared to the irreversible blockade induced by LMB. The anticancer activity of SINEs is a direct result of its binding to Cys528 and inhibition of XPO1 function, which was recently validated 10

using T-ALL Jurkat cells modified with CRISPR/Cas9-directed gene editing to express a missense serine mutation at the Cys528 locus59.

1.3.2 SINEs: Preclinical, Clinical Development in hematologic malignancies

In CLL, proof of concept studies performed with the early SINE compound, KPT-185,

demonstrated that XPO1 is a promising therapeutic target. In primary CLL cells, XPO1

was shown to be overexpressed at the protein and mRNA level compared to normal B

cells60. XPO1 inhibition by KPT-185 demonstrated cytotoxicity in primary CLL cells, with

an EC50 < 500nM compared to an estimated EC50 > 40 uM in normal B cells and

peripheral blood mononuclear cells (PBMCs) at 72 hours of drug exposure60. The impact

of in vivo XPO1 blockade by SINE compounds was evaluated in an adoptive transfer mouse model of CLL (Eµ-TCL1) using KPT-251, which has more favorable oral bioavailability compared to KPT-185, allowing for oral dosing. The Eµ-TCL1 mouse model is a well-characterized model that spontaneously develops a lethal leukemia similar to the aggressive, IGHV unmutated subtype of human CLL61,62. CD19+ leukemic cells

from the spleen of a Eµ-TCL1 mouse were engrafted into SCID mice and the recipient

mice were enrolled into a therapeutic study with KPT-185. In this study, mice treated with

KPT-185 showed significant improvement in overall survival compared to mice treated

with either vehicle control or fludarabine, a standard chemotherapeutic agent used in the

treatment of CLL. Following these seminal studies showing that XPO1 is expressed in CLL

and is a promising target that restores localization and function of TSPs, a new SINE for 11

clinical development, selinexor (KPT-330), emerged. Selinexor was evaluated in the Eµ-

TCL1-SCID adoptive transfer model and when compared to vehicle-treated controls,

selinexor-treated mice showed significantly improved overall survival, when administered

at schedules of twice per week and three times per week63. Regarding drug toxicity, mild

drug-associated weight loss was observed in mice treated with >10 mg/kg either twice or

thrice weekly.

Based on encouraging pre-clinical data, selinexor is currently in phase I/II clinical

trials in both hematologic malignancies and solid tumors. A multi-center phase I trial of

single-agent selinexor in hematologic malignancies (NCT01607892), conducted from June

2012 to June 2016, was recently reported in the literature. In this study, 79 patients with a

spectrum of hematologic malignancies including mantle cell lymphoma (MCL), follicular

lymphoma, diffuse large B cell lymphoma (DLBCL), Richter’s transformation, and CLL

were enrolled in this phase I dose-escalation study64. Selinexor was administered 2-3 times per week at dosages of 3 to 80 mg/m2. The most common grade 1 or 2 treatment-related adverse events (AEs) observed were nausea (66%), fatigue (61%), anorexia (57%), vomiting (37%) and diarrhea (34%) which were treated with supportive therapy such as

D2 antagonists, 5HT3 antagonists, anti-emetic dosing of , megestrol and/or olanzapine. Of note, 38 patients received one or more doses of dexamethasone as supportive care for decreased appetite and nausea while on study64. The most common

grade 3 or 4 AEs were thrombocytopenia (47%), neutropenia (32%), and anemia (27%).

Overall, 22 (31%) patients had an objective response to selinexor, including 4 complete

responses (CRs), 18 partial responses (PRs), and 21 patients with stable disease (SD). Of 12

6 evaluable CLL patients treated with selinexor, 2 (33%) had a PR, 3 (50%) had SD, and

one patient (17%) had progressive disease (PD). Also enrolled in this trial were 5 evaluable

patients with Richter’s transformation, 2 of which attained CRs and 2 achieved SD.

Correlative studies from this trial determined the plasma concentration half-life of

selinexor to be 5-7 hours across tested doses64.

In AML, a phase I trial of single-agent selinexor was conducted January 2013 to

June 2014, and the results were recently reported65. Patient with relapsed or refractory

AML were enrolled. Patients were administered selinexor 2-3 times per week in doses of

3 to 70 mg/m2 and a recommended Phase 2 was established at 60mg (35 mg/m2) given

twice per week. Drug-related adverse events were observed in 5% of AML patients and included fatigue, anorexia, diarrhea, nausea, weight loss, and vomiting, similar to those reported by Kuruvilla and colleagues64. The majority of observed adverse events were grade 1 or 2. Of 81 patients, 14% had an objective response (OR) with an improvement in median progression-free survival and overall survival compared with patients who did not

respond65. Taken together, these trials indicate that selinexor shows efficacy in some

patients, but this must be measured against the observed toxicities to establish a clinical benefit.

1.3.3 Potential XPO1 dual targeting strategies in CLL

13

Since the advent of novel targeted agents such as BTK inhibitors, the path forward in CLL

is likely to feature combination therapy. In particular, rational combinations that leverage

synergy will be of particular value. XPO1 is an attractive combination therapy target in

oncology due to the ability to post-translationally re-activate TSPs such as IκB, p53, and

FoxO3a. In CLL, engagement of the B cell receptor activates multiple downstream pathways that stimulate and secure CLL cells from apoptosis66. BCR signaling leads to phosphorylation of AKT and ERK and activation of the NF-κB pathway. Importantly, NF-

κB signaling induces BTK transcription and protein expression67. XPO1 blockade disrupts

this positive feedback cycle in CLL cells by trapping IκB in the nucleus and inhibiting NF-

κB-driven BTK expression63. Consistent with this model, XPO1 blockade with selinexor prevents phosphorylation of AKT and ERK in primary CLL cells stimulated with anti-

IgM63. Therefore, targeting downstream components of the BCR pathway using multiple modalities such as tyrosine kinase inhibitors and nuclear export blockade may be beneficial to increase the depth and durability of clinical responses and possibly suppressing acquired resistance to any single agent.

1.4 Conclusions and hypothesis

Although initial attempts to establish nuclear export inhibition as an important treatment strategy in cancer were hindered by the toxicity of LMB, it is now evident that aberrant nuclear export plays an important role in human neoplasms. The development of newer targeted inhibitors is a culmination of an emerging field with the potential to substantially 14

impact human health. Due to their unique ability to target multiple deregulated signaling pathways, SINEs show promise in treating acquired resistance to other anticancer agents and in combination with other targeted therapies. Our objective is to provide a basis for the clinical development of SINEs by demonstrating that nuclear transport is a promising point of therapeutic intervention in hematologic malignancies such as CLL.

15

1.5 References

1. Howlader N, Noone A, Krapcho M, et al. SEER Cancer Statistics Review, 1975-

2014. Natl. Cancer Institute. Bethesda, MD. 2017;

2. Strati P, Chaffee K, Achenbach S, et al. Disease Progression and Complications

Are the Main Cause of Death in Patients with Chronic Lymphocytic Leukemia

(CLL) Independent of Age and Comorbidities at Diagnosis. Blood.

2015;126(23):5265 LP-5265.

3. Pekarsky Y, Zanesi N, Croce CM. Molecular basis of CLL. Semin. Cancer Biol.

2010;20(6):370–6.

4. Zenz T, Fröhling S, Mertens D, Döhner H, Stilgenbauer S. Moving from

prognostic to predictive factors in chronic lymphocytic leukaemia (CLL). Best

Pract. Res. Clin. Haematol. 2010;23(1):71–84.

5. Moreno C, Montserrat E. Genetic lesions in chronic lymphocytic leukemia: what’s

ready for prime time use? Haematologica. 2010;95(1):12–5.

6. Zenz T, Mertens D, Döhner H, Stilgenbauer S. Importance of genetics in chronic

lymphocytic leukemia. Blood Rev. 2011;25(3):131–7.

7. Guièze R, Wu CJ. Genomic and epigenomic heterogeneity in chronic lymphocytic

leukemia. Blood. 2015;126(4):445 LP-453.

16

8. Landau D a, Carter SL, Stojanov P, et al. Evolution and impact of subclonal

mutations in chronic lymphocytic leukemia. Cell. 2013;152(4):714–26.

9. Landau DA, Tausch E, Taylor-Weiner AN, et al. Mutations driving CLL and their

evolution in progression and relapse. Nature. 2015;

10. 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. 2011;44(1):47–52.

11. Woyach J a, Johnson AJ, Byrd JC. The B-cell receptor signaling pathway as a

therapeutic target in CLL. Blood. 2012;120(6):1175–1184.

12. Awan FT, Byrd JC. New Strategies in Chronic Lymphocytic Leukemia: Shifting

Treatment Paradigms. Clin. Cancer Res. 2014;

13. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with Ibrutinib in Relapsed

Chronic Lymphocytic Leukemia. N. Engl. J. Med. 2013;369:32–42.

14. Byrd J, Brown J, O’Brien SM, et al. Ibrutinib versus Ofatumumab in Previously

Treated Chronic Lymphoid Leukemia. N. Engl. J. Med. 2014;371:213–223.

15. Bednenko J, Cingolani G, Gerace L. Nucleocytoplasmic Transport: Navigating the

Channel. Traffic. 2003;4(3):127–135.

16. Cronshaw JM, Krutchinsky AN, Zhang W, Chait BT, Matunis MJ. Proteomic

analysis of the mammalian nuclear pore complex. J. Cell Biol. 2002;158(5):915

LP-927.

17. Cautain B, Hill R, Pedro N De, Link W. Components and regulation of nuclear

transport processes. FEBS J. 2014;

17

18. Lange A, Mills RE, Lange CJ, et al. Classical Nuclear Localization Signals:

Definition, Function, and Interaction with Importin α. J. Biol. Chem. .

2007;282(8):5101–5105.

19. la Cour T, Kiemer L, Mølgaard A, et al. Analysis and prediction of leucine-rich

nuclear export signals. Protein Eng. Des. Sel. 2004;17(6):527–536.

20. Chook YM, Fung HYJ. Atomic basis of CRM1-cargo recognition, release and

inhibition. Semin. Cancer Biol. 2014;1–10.

21. Kirli K, Karaca S, Dehne HJ, et al. A deep proteomics perspective on CRM1-

mediated nuclear export and nucleocytoplasmic partitioning. Elife.

2015;4(December):e11466.

22. Ferrigno P, Posas F, Koepp D, Saito H, Silver PA. Regulated nucleo/cytoplasmic

exchange of HOG1 MAPK requires the importin β homologs NMD5 and XPO1.

EMBO J. 1998;17(19):5606 LP-5614.

23. Jang B-C, Muñoz-Najar U, Paik J-H, et al. Leptomycin B, an Inhibitor of the

Nuclear Export Receptor CRM1, Inhibits COX-2 Expression. J. Biol. Chem. .

2003;278(5):2773–2776.

24. Turner JG, Sullivan DM. CRM1-mediated nuclear export of proteins and drug

resistance in cancer. Curr. Med. Chem. 2008;15(26):2648–55.

25. Kanai M, Hanashiro K, Kim S-H, et al. Inhibition of Crm1–p53 interaction and

nuclear export of p53 by poly(ADP-ribosyl)ation. Nat. Cell Biol. 2007;9:1175.

26. Shao C, Lu C, Chen L, et al. p53-dependent anticancer effects of leptomycin B on

lung adenocarcinoma. Cancer Chemother. Pharmacol. 2011;67(6):1369–1380.

18

27. Vousden KH, Lane DP. p53 in health and disease. Nat. Rev. Mol. Cell Biol.

2007;8:275.

28. Cullen BR. Nuclear RNA export. J. Cell Sci. 2003;116(4):587–597.

29. Halees AS, El-Badrawi R, Khabar KSA. ARED Organism: expansion of ARED

reveals AU-rich element cluster variations between human and mouse. Nucleic

Acids Res. 2008;36(suppl_1):D137–D140.

30. Bakheet T, Frevel M, Williams BRG, Greer W, Khabar KSA. ARED: human AU-

rich element-containing mRNA database reveals an unexpectedly diverse

functional repertoire of encoded proteins. Nucleic Acids Res. 2001;29(1):246–254.

31. Khabar KS a. Post-transcriptional control during chronic inflammation and cancer:

a focus on AU-rich elements. Cell. Mol. Life Sci. 2010;67(17):2937–55.

32. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E is

a central node of an RNA regulon that governs cellular proliferation. J. Cell Biol.

2006;175(3):415–26.

33. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E

promotes nuclear export of cyclin D1 mRNAs via an element in the 3’UTR. J. Cell

Biol. 2005;169(2):245–56.

34. Sun Q, Chen X, Zhou Q, et al. Inhibiting cancer cell hallmark features through

nuclear export inhibition. Signal Transduct. Target. Ther. Publ. online 1 July

2016; | doi10.1038/sigtrans.2016.10. 2016;1(April):1041–1050.

35. Noske A, Weichert W, Niesporek S, et al. Expression of the nuclear export protein

chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human

19

. Cancer. 2008;112(8):1733–1743.

36. Yao Y, Dong Y, Lin F, et al. The expression of CRM1 is associated with prognosis

in human osteosarcoma. Oncol. Rep. 2009;21(1):229–235.

37. Huang W, Yue L, Qiu W, et al. Prognostic value of CRM1in pancreas cancer.

Clin. Investig. Med. 2009;32(6):315–321.

38. Shen A, Wang Y, Zhao Y, et al. Expression of CRM1 in human gliomas and its

significance in p27 expression and clinical prognosis. Neurosurgery.

2009;65(1):153–160.

39. Van Der Watt PJ, Maske CP, Hendricks DT, et al. The karyopherin proteins, Crm1

and Karyopherin Beta1, are overexpressed in cervical cancer and are critical for

cancer cell survival and proliferation. Int. J. Cancer. 2009;124(November

2008):1829–1840.

40. Culjkovic-Kraljacic B, Baguet A, Volpon L, Amri A, Borden KLB. The

eIF4E reprograms the nuclear pore complex to promote mRNA export and

oncogenic transformation. Cell Rep. 2012;2(2):207–15.

41. Tai Y-T, Landesman Y, Acharya C, et al. CRM1 inhibition induces tumor cell

cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular

mechanisms and therapeutic implications. Leukemia. 2014;28(1):155–65.

42. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute

myelogenous leukemia with a normal karyotype. N. Engl. J. Med.

2005;352(3):254–66.

43. Kojima K, Kornblau SM, Ruvolo V, et al. Prognostic impact and targeting of

20

CRM1 in acute myeloid leukemia. Blood. 2013;121(20):4166 LP-4174.

44. Gough SM, Slape CI, Aplan PD. NUP98 gene fusions and hematopoietic

malignancies: common themes and new biologic insights. Blood.

2011;118(24):6247–57.

45. Walker CJ, Oaks JJ, Santhanam R, et al. Preclinical and clinical efficacy of

XPO1/CRM1 inhibition by the karyopherin inhibitor KPT-330 in Ph+ leukemias.

2013;122(17):3034–3044.

46. Balatti V, Bottoni A, Palamarchuk A, et al. NOTCH1 mutations in CLL associated

with trisomy 12. Blood. 2012;119(2):329–331.

47. Jardin F, Lehmann-horn K, Sagan S a, et al. Recurrent mutations of the exportin 1

gene (XPO1) and their impact on selective inhibitor of nuclear export compounds

sensitivity in primary mediastinal B-cell lymphoma. Am. J. Hematol. 2016;(1):1–

20.

48. Dubois S, Viailly P-J, Mareschal S, et al. Next Generation Sequencing in Diffuse

Large B Cell Lymphoma Highlights Molecular Divergence and Therapeutic

Opportunities: a LYSA Study. Clin. Cancer Res. 2016;1078-0432.CCR-15-2305-.

49. Janku F, Vergilio JA, Salhia B, et al. Comprehensive genomic profiling reveals

recurrent XPO1 mutations and other alterations in archival samples of patients

with Hodgkin lymphoma. Eur. J. Cancer. 2018;69:S41–S42.

50. Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in

chronic lymphocytic leukemia. N. Engl. J. Med. 2011;365(26):2497–506.

51. Stamatopoulos B, Antoniou P, Vavoulis D, et al. Characterization of Recurrent

21

Mutations in Patient with a Richter Syndrome By Targeted Next Generation

Sequencing. Blood. 2016;128(22):3200 LP-3200.

52. Kudo N, Matsumori N, Taoka H, et al. Leptomycin B inactivates CRM1/exportin 1

by covalent modification at a cysteine residue in the central conserved region.

Proc. Natl. Acad. Sci. U. S. A. 1999;96(August):9112–9117.

53. Dong X, Biswas A, Suel KE, et al. Structural basis for leucine-rich nuclear export

signal recognition by CRM1. Nature. 2009;458(April):1136–1141.

54. Sun Q, Carrasco YP, Hu Y, et al. Nuclear export inhibition through covalent

conjugation and hydrolysis of Leptomycin B by CRM1. Proc. Natl. Acad. Sci. U.

S. A. 2013;110(4):1303–8.

55. Newlands ES, Rustin GJ, Brampton MH. Phase I trial of elactocin. Br. J. Cancer.

1996;74(4):648–649.

56. Crochiere ML, Baloglu E, Klebanov B, et al. A method for quantification of

exportin-1 ( XPO1 ) occupancy by Selective Inhibitor of Nuclear Export ( SINE )

compounds. Oncotarget. 2015;1:.

57. Daelemans D, Afonina E, Nilsson J, et al. A synthetic HIV-1 Rev inhibitor

interfering with the CRM1-mediated nuclear export. Proc. Natl. Acad. Sci. .

2002;99(22):14440–14445.

58. Kalid O, Toledo Warshaviak D, Shechter S, Sherman W, Shacham S. Consensus

Induced Fit Docking (cIFD): methodology, validation, and application to the

discovery of novel Crm1 inhibitors. J. Comput. Aided. Mol. Des.

2012;26(11):1217–28.

22

59. Neggers JE, Vercruysse T, Jacquemyn M, et al. Resource Identifying Drug-Target

Selectivity by CRISPR / Cas9 Genome Editing. Chem. Biol. 2015;1–10.

60. Lapalombella R, Sun Q, Williams K, et al. Selective inhibitors of nuclear export

show that CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood.

2012;120(23):4621–34.

61. Bichi R, Shinton S a, Martin ES, et al. Human chronic lymphocytic leukemia

modeled in mouse by targeted TCL1 expression. Proc. Natl. Acad. Sci. U. S. A.

2002;99(10):6955–60.

62. 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. 2006;108(4):1334–8.

63. Zhong Y, El-Gamal D, Dubovsky J a, et al. Selinexor suppresses downstream

effectors of B-cell activation, proliferation and migration in chronic lymphocytic

leukemia cells. Leukemia. 2014;1–5.

64. Kuruvilla J, Savona M, Baz R, et al. Selective inhibition of nuclear export with

selinexor in patients with non-Hodgkin lymphoma. Blood. 2017;129(24):3175 LP-

3183.

65. Garzon R, Savona M, Baz R, et al. A phase 1 of single-agent selinexor

in acute myeloid leukemia. 2017;129(24):18–21.

66. Longo PG, Laurenti L, Gobessi S, et al. 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. 2008;111(2):846–55.

23

67. Yu L, Mohamed AJ, Simonson OE, et al. -dependent autoregulation of

Bruton tyrosine kinase (Btk) promoter via NF-κB. Blood. 2008;111(9):4617 LP-

4626.

24

1.6 Figures

Figure 1.1. Transport of macromolecules via Exportin 1 (XPO1). A subset of proteins containing nuclear export sequences (NESs) and RNAs are exported from the nucleus to the cytoplasm via the XPO1 complex. More than 200 proteins have been experimentally validated as cargos of XPO1.

25

Figure 1.2 Targeting nuclear export via selective inhibitor of nuclear export (SINE) compounds.

26

Chapter 2: Novel therapeutics targeting exportin 1 in hematologic malignancies

2.1 Introduction

Nuclear-cytoplasmic transport of proteins is a highly regulated and energy- dependent process that is mostly mediated by proteins in the karyopherin family of nuclear

transport receptors (also called importins and exportins). Exportin 1 (XPO1; also called

chromosome region maintenance 1 or CRM1) is the best characterized exportin with a

broad spectrum of over 200 cargo proteins, including key tumor suppressor proteins (TSPs)

such as p53, IκB, and FOXO3a, as well as various RNAs 1,2. Protein cargoes that are

transported by XPO1 contain 10-15 residue nuclear export signals (NESs) that bind to a

hydrophobic groove on the surface of XPO1 3-6. The XPO1-NES interaction is enhanced

by RanGTP to form an XPO1-Ran-cargo complex that is exported from the nucleus

through the nuclear pore complex and then dissociated in the cytoplasm following

hydrolysis of RanGTP to RanGDP 7.

Overexpression of XPO1 has been detected in a variety of hematologic 8 and solid

tumors 9, and has been correlated with poor clinical outcomes related to prognosis and resistance to anticancer therapies. Increased levels of XPO1 in a cancer cell promote the

egress of multiple tumor suppressor proteins (TSPs) from the nucleus to the cytoplasm

where they can no longer regulate cell cycle, proliferation, and apoptosis. Subcellular

27

mislocalization of TSPs such as p53, nucleophosmin, BRCA1, APC, and retinoblastoma

has been observed in cancer and has been linked to cancer progression and maintenance 10.

The importance of the XPO1-dependent nuclear export pathway is highlighted in a subset of adult AML patients with normal cytogenetics (CN-AML) where mutations in the nucleophosmin (NPM1) gene create a novel NES that results in hyperactive XPO1- dependent export of NPM1 into the cytoplasm 11. Mutations in NPM1 are the most common genetic lesion in CN-AML (50-60% of cases) and NPM1 relocation to the nucleus

(thereby restoring tumor suppressor function) by SINE compounds represents a potential therapeutic strategy for this frequent subtype of AML. Additionally, overexpression of

XPO1 in AML correlates with poor clinical outcome 12. In CLL recurrent mutations in

XPO1 have also been described although the impact of these mutations remain uncertain

13.

Selective Inhibitor of Nuclear Export (SINE) compounds developed by

Karyopharm Therapeutics Inc. (Newton, MA), are orally bioavailable small molecules that

bind to and inhibit XPO1, thereby restoring nuclear localization of dysregulated molecules

that control cell proliferation and survival 14. SINE compounds, like all other known small

molecule inhibitors of XPO1, achieve nuclear export blockade by specifically and

covalently binding to a reactive cysteine residue in the NES-binding groove of XPO1

(Cys528 of human XPO1) to prevent NES/cargo binding and export 14.

Our previous published work showed that XPO1 is a validated therapeutic target

for chronic lymphocytic leukeimia (CLL) 14 and acute myeloid leukemia (AML) 15,16, and has facilitated the translation of a SINE compound named KPT-330 (selinexor) to a Phase

28

1 clinical trial in advanced hematologic tumors (NCT01607892) and in multiple solid

tumors (NCT01607905). Anti-tumor activity of selinexor has been observed in patients

with lymphoma 17, CLL 17, multiple myeloma 18, and AML 19. To date >1000 patients have

been treated with selinexor in Phase 1 and Phase 2 clinical trials. While constitutional

symptoms (weight loss, fatigue, etc) were initially therapy-limiting, selinexor tolerability has been improved with supportive care, consisting of appetite stimulants (megesterol plus olanzapine) and anti-nausea agents (odansetron). Despite these improvements, the admistration of selinexor is limited to twice per week.

The other clinical stage XPO1 inhibitor is the widely used natural product

Leptomycin B (LMB), which showed long lasting toxicity in a single phase 1 clinical trial

20 which prevented its clinical development. In contrast, selinexor is much better tolerated in animals and in human patients in presence of supportive care. The difference in tolerance of the two inhibitors, which both bind covalently to the same XPO1 site, may be explained by differences in their chemical mechanisms of XPO1 inhibition 21. LMB is hydrolyzed after forming a covalent bond with the XPO1 NES groove cysteine. This hydrolysis stabilizes the covalent bond between XPO1 and LMB, thus rendering the inhibition irreversible. In contrast, SINE compounds are slowly-reversible inhibitors, a feature that may contribute to their improved tolerance over LMB as sufficient inhibitor release from

XPO1 can allow essential nuclear export to resume in normal cells.

The idea of improved tolerance of reversible XPO1 inhibitors drove us to design a new generation of SINE compounds that bind and inhibit XPO1 with greater reversibility.

29

In this report we describe a new generation SINE compound, KPT-8602, which

shows reduced brain penetration and two-fold increased reversibility compared to first

generation SINE compounds (KPT-185 and KPT-330). The improved pharmacologic properties of KPT-8602 along with its preserved target specificity suggest that it may have

promising clinical efficacy in B cell malignancies, AML, and a variety of other cancers

where upregulation of XPO1 is seen.

2.2 Materials and Methods

2.2.1 Cloning, expression, and protein purification

ScXPO1 (residues 1-1058 with residues 377-413 removed and Thr539 mutated to Cys) was cloned into the previously described pGEX-Tev vector(1, 2). Thr539 of ScXPO1 was

mutated to cysteine (ScXPO1 does not contain a reactive cysteine) to allow covalent

conjugation of KPT-8602. Residues 11-201 ScRanBP1 (Yrb1p) was also cloned into pGEX-TEV and full length human Ran was cloned into the pET-15b vector. All three

proteins were expressed separately in E. coli BL-21(DE3) with induction by 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 10 hrs at 25°C. GST-ScXPO1 and GST-Yrb1

cells were lysed in buffer containing 40 mM Tris (pH 7.5), 2 mM MgOAc, 200 mM

NaCl, 10 mM DTT and protease inhibitors. Both proteins were purified by affinity

chromatography using glutathione Sepharose (GE Healthcare Life Sciences), followed by

cleavage with TEV protease to remove GST and then further purified by size-exclusion

chromatography in buffer containing 20 mM Tris (pH 7.5), 100 mM NaCl, 5 mM 30

MgOAc, 2 mM DTT. His-Ran cells were lysed in buffer containing 50 mM Tris (pH 8.0),

5 mM MgOAc, 400 mM NaCl, 2.5% (v/v) glycerol, 20 mM imidazole (pH 7.8), 0.2 mM

GTP, 2 mM beta-mercaptoethanol and protease inhibitors, purified by affinity

chromatography (HisTrap HP; GE Healthcare Life Sciences) and size exclusion

chromatography into buffer containing 20 mM Tris (pH 8), 110 mM KOAc, 2 mM

MgOAc, 10% (v/v) glycerol and 2 mM DTT.

2.2.2 Assembly of the XPO1-Ran-Yrb1 complex, crystallization and X-ray data collection

Ran was loaded with GTP analog GppNHp on alkaline-acrylic phosphatase beads

(Sigma-Aldrich) for 12 hours. The Ran-Yrb1 heterodimer was first formed and purified over a size-exclusion column and then added in a 2:1 molar ratio to XPO1. The ScXPO1-

Ran-Yrb1 ternary complex was purified over size-exclusion in 20 mM Tris (pH 7.5), 100 mM NaCl and 5 mM MgOAc. 10 molar excess of KPT-8602 (dissolved in DMSO to 10 mM) was added to the purified protein complex, incubated for 1 hr and concentrated to

7.5 mg/mL of protein. KPT-8602-ScXPO1-Ran-Yrb1 complex was crystallized in conditions similar to those used by Koyama and Matsuura (17% PEG3350, 100 mM Bis-

Tris (pH 6.6) and 200 mM ammonium nitrate). Crystals were cryoprotected with the crystallization solution supplemented with up to 23% PEG3350 and 12% (v/v) glycerol and flash cooled in liquid nitrogen. X-ray diffraction data was collected at Advanced

Photon Source (APS) – Structural Biology Center (SBC) 19ID beamline. Data was

indexed, integrated and scaled using HKL3000.

31

2.2.3 X-ray structure determination and refinement

Since crystals of KPT-8602-ScXPO1-Ran-Yrb1 are isomorphous to previously solved

SINE-bound and unbound ScXPO1-Ran-Yrb1, structure was determined by multiple

rounds of refinement of unliganded complex (4HB2) against collected data using

PHENIX(3-5) and manual modeling in Coot(6). Structure figures were generated with

PyMOL(7).

2.2.4 In vitro inhibition assays

NESs were cloned into pGEX-TEV and expressed in E. coli BL-21 (DE3) with induction

by 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 4 hrs at 37°C. GST-NES proteins

were purified and immobilized on glutathione Sepharose beads (GE Healthcare Life

Sciences). For inhibition assays, 2 μM purified XPO1 was incubated with the indicated

inhibitor (0, 5, 10, 20 μM) and 10 μM RanGTP for 90 mins at 4°C in buffer containing

50 mM Tris (pH 7.5), 110 mM KOAc, 2 mM MgOAc, 1 mM EGTA, 20% (v/v) glycerol,

0.005% IGEPAL and 2 mM DTT in total volumes of 200 μL. The mixtures were then added to approximately 10 μg of immobilized NESs and rotated for 2 hrs at 4°C.

Unbound proteins were washed extensively with buffer and remaining bound proteins were separated by SDS/PAGE and visualized by Coomassie Blue staining. DMSO

(instead of inhibitors in DMSO) was used as control. For inhibition time-course experiments, incubation times of XPO1, RanGTP and inhibitors were varied and mixtures were rotated with immobilized GST-PKINES for 1 hr before extensive washing.

Reversibility assays were performed as previously described (8). 2 uM of XPO1 was 32

incubated with 10 uM of inhibitors in total volumes of 200 μL as described above.

Triplicate samples were subjected to (i) binding assay with GST-MVMNES, (ii) dialysis

against 10 mM Tris (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 2 mM DTT for 24 hrs at

room temperature, or (iii) treated with 20 mM DTT for 24 hrs at room temperature. The

latter two samples were subjected to the same inhibition assays as described above. To

compare the relative intensities of XPO1 band to yield an estimate of the degree of

deconjugation of SINE compounds from XPO1, SDS/PAGE gels were scanned with a

desktop scanner (Epson V300) and processed with ImageJ software. Analysis was

performed as previously described. Corrected relative XPO1 band intensities normalized

to the DMSO treated XPO1 controls and their standard errors are plotted as histograms in

Figure 2.5 with GraphPad Prism.

2.2.5 In vivo XPO1 degradation by SINE treatment

Human fibrosarcoma HT1080 cells were cultured (in D-MEM, 10% fetal bovine serum

and penicillin/streptomycin) and grown to 80% confluence. KPT-185, KPT-330, or KPT-

8602 were added to final concentrations of 25–1000 nM and cells were grown for another

18 hr. Treated and control cells were collected by centrifugation and lysed on ice for 30

min (RIPA buffer: 50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1.0 mM EDTA, 0.1% SDS,

1.0% Triton X-100, 1.0% sodium deoxycholate and protease inhibitors). ~7 μg portion of total protein was separated by SDS–PAGE, transferred onto nitrocellulose membranes, which were blocked and probed overnight with XPO1 antibody (H-300; sc-5595, Santa

33

Cruz). Immunodetection was performed with the beta- (MA5-15739, Thermo

Scientific) to confirm equal protein loading.

2.2.6 Cell isolation and reagents

Human CLL and normal B cells were isolated and cultured as previously described(9).

Murine splenic lymphocytes were isolated from spleen by Ficoll-Hypaque density gradient centrifugation. Blood and frozen bone marrow aspirates from newly diagnosed untreated

CLL and AML patients were obtained from the Ohio State University Leukemia Tissue

Bank after getting informed consent approved by the cancer institution review board according to the Declaration of Helsinki. DLBCL cell lines including OCI-Ly1, OCI-Ly2,

OCI-Ly3, OCI-Ly6, and OCI-Ly7 were purchased from the Ontario Cancer Institute and were cultured in IMDM supplemented with 20% FBS and 100U/mL penicillin and

100ug/mL streptomycin. AML cell lines, MV4-11 and Kasumi-1 were purchased from

American Type Culture Collection. OCI-AML3 was purchased from DSMZ, Germany.

AML cell lines were cultured in RPMI supplemented with 10% FBS and 100U/mL penicillin and 100ug/mL streptomycin. Authentication was conducted by deep sequencing on Illumina MiSeq to exclude cross-contamination and last confirmed in February 2015.

2.2.7 Cell lysis and immunoblot

Cells were lysed in RIPA buffer. Nuclear extracts were prepared with NE-PER (Pierce).

Proteins were separated by SDS-PAGE and blots probed with commercially available

34 antibodies and detected by addition of chemiluminescent substrate (Pierce) followed by quantification by Chemi-Doc system with Quantity One software (Bio-Rad Laboratories).

2.2.8 Immunofluorescent Staining

Cells were first counted and diluted to 1x106/ml. Approximately 200,000 cells were cytospinned to slide. The cells were then fixed in 3.7% formaldehyde for 15 min followed by permeabilization with 0.25% Triton. The slides were blocked by 1% goat serum for 30 min. The cells were incubated with primary antibody for 1 hour at room temperature. After washing, the sample was incubated with fluorochrome conjugated secondary antibody for 1 hr at room temperature. Nucleus was stained by DAPI. Samples were analyzed by confocal microscope (OSU Image facility).

2.2.9 Animal studies

All experiments were carried out under protocols approved by The Ohio State University

Institutional Animal Care and Use Committee.

2.2.10 TCL1 transplant mouse model

CD19+CD5+ cells (1 x 107) from the spleen of a TCL1 transgenic mouse with active

CLL-like leukemia and palpable splenomegaly were engrafted by tail vein into a

C57BL/6 mouse. Criteria for active CLL-like leukemia include white blood cell count >

60 and CD45+CD5+CD19+ > 80%. Leukemia onset was defined as >10%

CD45+CD5+CD19+ B cells in peripheral blood by flow cytometry. At leukemia onset,

35

engrafted mice were randomly assigned to treatment with KPT-330 at 15 mg/kg, KPT-

8602 at 15 mg/kg, or 0.5% methylcellulose/1% Tween80 (vehicle control). All treatments

were administered by oral gavage. Disease progression was monitored by weekly by

CD45/CD5/CD19 flow cytometry and smears of peripheral blood, spleen size, health, and

weight loss. Mice were sacrificed on development of splenomegaly and presence of other

disease criteria causing discomfort. Overall survival was the primary endpoint.

2.2.11 MV4-11 xenograft mouse model

Spleen cells (0.5 x 104) from MV4-11 transplanted NSG mice were intravenously

injected into NSG mice vial tail vein as previously described(10). One week after tumor inoculation, mice were given vehicle control five times a week, KPT-8602 at 15 mg/kg

five times a week (daily from Monday to Friday with a break Saturday-Sunday), KPT-

8602 at 15 mg/kg twice per week, or KPT-330 at 20 mg/kg twice per week. All

treatments were administered via oral gavage. Mice were monitored daily for clinical

signs of leukemia, such as weight loss, hind-limb weakness, and hind-limb paralysis.

Overall survival was the primary endpoint.

2.2.12 Statistical analysis

All analyses were performed by the OSU Center for Biostatistics; using SAS/STAT

software, version 9.3 (SAS Institute, Inc., Cary, NC) and previously described

models[14]. For survival experiments, survival curve estimates were calculated using the

Kaplan-Meier method and differences in curves were assessed using the log-rank test.

36

Spleen volume was compared between groups using ANOVA, and mixed effects models

were used to assess changes in %CD19+/CD5+ cells over time. Where applicable, data

were log-transformed to reduce skewness. P-values were adjusted for multiple

comparisons using Holm’s stepdown procedure or Dunnett’s test, were appropriate.

2.3 Results

2.3.1 KPT-8602 binds in the NES-binding groove of XPO1

The novel SINE, KPT-8602, forms a covalent bond with the same reactive cysteine

that KPT-330 binds through a warhead with an activated Michael acceptor in the C that

is similar to KPT-330’s (Figure 2.1a). We have solved the 2.5 Å resolution X-ray structure of KPT-8602 bound to the Saccharomyces cerevisiae XPO1 (ScXPO1) in complex with

human RanGppNHp and S. cerevisiae RanBP1 (Yrb1) (Figure 2.1b, Table 2.1). As

previously reported, Thr539 of ScXPO1 was mutated to cysteine (ScXPO1 does not contain

a reactive cysteine) to allow covalent conjugation of KPT-860215, 22-24. Structure of the

KPT-8602-bound ScXPO1-Ran-Yrb1 complex is very similar to other inhibitor-bound

ScXPO1-Ran-Yrb1 complexes (Cα rmsd of 0.2-0.3 Å). KPT-8602 binds in the NES- binding groove of XPO1, forms a covalent bond with the reactive Cys539 (equivalent to

Cys528 of human XPO1) and buries a surface area of 318.1 Å2.

KPT-8602 is oriented in the groove similar to other SINE compounds15, 23. The NES

groove of XPO1 remains largely the same when bound to KPT-8602, KPT-185 or

Leptomycin B (Cα rmsds of 0.2-0.3 Å)15, 22. KPT-185 is a first generation SINE analog of

37

KPT-330 that shares almost identical in vitro activities and the structure of its complex with XPO1 was previously reported. While both KPT-185 and KPT-8602 contain a similar substitution (trifluoromethyl phenyl triazole) in the Cβ, the Michael acceptor of KPT-8602 is activated with a pyrimidyl group at the C . The trifluoromethyl phenyl core of KPT-

185 and KPT-8602 interact with the XPO1 groove in a similar fashion (Figure 2.1b and

Table 2.2), but the triazole moiety in KPT-8602 is rotated 180° relative to that of KPT-

185, thus positioning the Cβ 2.4 Å away from the Cβ of KPT-185 and towards the solvent.

Repositioning of the Cβ shifts the entire Michael acceptor arm such that its amide and pyrimidine form hydrogen bonds with Lys548 and Lys579 of XPO1 and make van der

Waals contact with Val 540 and Phe583 of XPO1 (Figure 2.1c and Figure 2.2a-d).

Accordingly, the Cys539 sidechain of XPO1 that forms a covalent bond with KPT-8602 adopts an alternate rotamer conformation (Figure 2.1c). By comparison, while the Michael acceptor arm of KPT-8602 point out of the XPO1 groove toward solvent, the equivalent portion of KPT-185 binds deep into the XPO1 NES groove (Figure 2.1d).

2.3.2 KPT-8602 inhibits XPO1-cargo interactions

We confirmed the ability of KPT-8602 to inhibit XPO1-NES interactions in a similar manner to other SINE compounds. We evaluated the ability of KPT-8602 to prevent pull-down of purified recombinant XPO1 with immobilized NESs. KPT-8602 successfully inhibited three different classical NESs/cargos (PKINES, MVM-NS2NES and full-length

Snurportin) efficiently (Figure 2.3a) although higher concentrations are needed to

38

completely inhibit XPO1 (10-20μM; compared to 5μM for KPT-185 or KPT-330). Unlike

KPT-185, both KPT-8602 and KPT-330 required longer incubation times with XPO1 to completely inhibit XPO1 binding to PKINES (60min; compared to 5min for KPT-185;

Figure 2.3b).

To confirm that the novel structure of KPT-8602 did not result in altered binding

kinetics to XPO1 we assessed the reversibility of KPT-8602 conjugation to XPO1 as

previously described22. Treatment with either dialysis or DTT to remove

unconjugated/deconjugated inhibitors was able to remove similar amounts of KPT-8602

and KPT-185 from XPO1 after 24 hours, suggesting that KPT-8602 binds XPO1 in a

slowly reversible manner similar to first-generation SINEs (Figure 2.4).

Finally, we examined inhibitor-induced degradation of XPO1 by KPT-8602. KPT-

8602 induces XPO1 degradation in HT1080 cells in a dose-dependent manner similar to other SINE compounds although higher concentrations of KPT-8602 are required to yield degradation responses similar to those with KPT-185 and KPT-330 (Figure 2.5a). This phenomenon was also evaluated in human primary CLL cells. In contrast to HT1080 cell lines, KPT-8602 and KPT-330 decreased XPO1 total protein as early as 4 hours post- treatment (Figure 2.5b) with similar efficiency.

2.3.3 KPT-8602 induces apoptosis of primary CLL cells and significantly inhibits proliferation of diffuse large B-cell lymphoma cell lines

The ability of KPT-8602 to induce cell death in primary CLL cells was evaluated.

As shown in Figure 2.6a, treatment with KPT-8602 induced dose-dependent killing of 39

primary CLL cells when compared to vehicle as measured by MTS analysis. We then tested

the ability of selected concentrations of KPT-8602 to induce apoptosis of primary CLL

cells in the presence of fetal bovine serum (FBS) or human serum (HS). As shown in

Figure 2.6b, KPT-8602 was effective in inducing apoptosis of CLL cells in both FBS and

HS.

We confirmed the effects of KPT-8602 on XPO1-dependent nuclear export by

examining the cellular localization of IkB, a well-known cargo protein of XPO1, in human

CLL cells after KPT-8602 treatment. Nuclear retention of IkB was observed within 8 hours

of treatment of KPT-330 and KPT-8602 (Figure 2.7). Next, we assessed the ability of

KPT-8602 to inhibit the proliferation of a panel of cell lines representative of diffuse large

B-cell lymphoma (DLBCL) both activated B-cell like (ABC-DLBCL) or germinal center subtypes (GC-DLBCL). As shown in Figure 2.8a KPT-8602 inhibited proliferation in

ABC-DLBCL cell lines and in GC-DLBCL cell lines (Figure 2.8b).

2.3.4 KPT-8602 possesses reduced central nervous system penetration

We assessed the capacity of KPT-8602 to cross the blood brain barrier in three mammalian species (mouse, rat, and monkey) compared to that of KPT-330 (selinexor), which is currently in clinical trials. As shown in Table 2.3, KPT-8602 possesses a reduced central nervous system (CNS) penetration in all species compared to KPT-330.

Additionally, KPT-8602 does not accumulate in plasma after repetitive dosing (data not shown).

40

2.3.5 KPT-8602 prolongs survival in a mouse model of CLL

The Eµ-TCL1 transgenic mouse develops a disease very similar to that observed in

human CLL patients and this model has been used extensively to evaluate experimental

therapeutics in CLL25, 26. We and others have shown27 that the B-cell malignancy occurring

in the Eµ-TCL1 mice is amenable to adoptive cell transfer by engrafting splenic white

blood cells from Eµ-TCL1 mice with active CLL-like leukemia (>10%

CD45+CD5+CD19+ B-cells in peripheral blood; WBC>60) and palpable splenomegaly

into background strain WT C57BL/6 mice. This produces a homogeneous population with

similar pathologic findings to the Eµ-TCL1 transgenic mice and a more rapid disease

acquisition (weeks instead of months), although time-to-disease and disease progression can be quite variable depending on the donor used for the engraftment.

We therefore employed the Eµ-TCL1-C57BL/6 transplant model described above to evaluate the potential of KPT-8602 to abrogate CLL progression in vivo. We hypothesized that increased reversibility combined with the lower brain penetration of

KPT-8602 could reduce the development of constitutional symptoms (weight loss) and allow more frequent dosing, which would in turn impact its efficacy. C57BL/6 mice were engrafted with CD5+CD19+ splenocytes derived from a Eµ-TCL1 donor mouse with active disease. At the time of leukemia onset (defined as ≥10% CD45+CD5+CD19+ cells),

mice were randomized to receive KPT-8602, KPT-330, or vehicle control. Our group

previously showed that KPT-330 dosed 3x/week showed worse overall survival compared

to KPT-330 dosed 2x/week in this model26. We first compared KPT-8602 to KPT-330 by

employing the clinically-used schedule of administration via oral gavage 2x/week as we

41 previously described26, 28. As expected, mice treated with either compound showed similar overall survival which was significantly improved over vehicle (p=0.001 and 0.028, respectively) (Figure 2.9). KPT-8602 shows lower central nervous system penetration and mildly increased reversibility in XPO1 binding qualities that suggest an enhanced tolerability and wider therapeutic index compared to KPT-330. We therefore evaluated the therapeutic benefit of KPT-8602 dosed continuously vs 2x/week in additional C57BL/6 mice engrafted with leukemic cells from a different Eµ-TCL1 donor. At leukemia onset, mice were randomized to receive vehicle or KPT-8602 daily or 2x/week by oral gavage.

Mice treated daily with KPT-8602 had significantly improved survival compared to those treated only 2x/week (p=0.001) (Figure 2.10). Both daily and 2x/week KPT-8602 provided significant survival benefit compared to vehicle treatment (p<0.001). Peripheral blood disease was evaluated weekly by CD45/CD5/CD19 flow cytometry. The percentage of leukemic cells in the blood was similar among groups at enrollment, but the percentage of

CD5+ B-cells in mice treated with daily KPT-8602 was significantly lower than either vehicle control or mice treated with KPT-8602 2x/week at four weeks post-enrollment and at the last available measurement (p<0.001, all comparisons) (Figure 2.11a). Spleen dimensions were taken when study removal criteria were met. Spleens derived from mice treated daily with KPT-8602 were significantly smaller compared to all the other groups

(p<0.001, all comparisons) (Figure 2.11b). Daily KPT-8602 treatment did not cause a significant change in body weight after 28 days of treatment (Figure 2.11c).

We have recently shown that the combination of KPT-330 and ibrutinib (bruton tyrosine kinase inhibitor) elicits a synergistic cytotoxic effect in primary CLL cells and

42 increases overall survival compared to ibrutinib alone in a mouse model of CLL28. We therefore tested the effect of KPT-8602+ibrutinib in an additional cohort of C57BL/6 mice with engrafted leukemic cells from another Eµ-TCL1 donor. At leukemia onset, mice were randomized to receive vehicle, KPT-8602 (daily), ibrutinib (daily), KPT-330 (2x/week), or the combinations of KPT-8602+ibrutinib or KPT-330+ibrutinib. As shown in Figure 12 the combination of KPT-8602+ibrutinib was able to further improve the survival induced by KPT-330+ibrutinib. Additionally, the percentage of leukemic cells in peripheral blood was similar for all groups at week 1 of treatment, but was significantly lower in mice treated with the combination compared to mice treated with either agent alone or vehicle at week

3 (p<0.004, all comparisons) (Figure 2.13a). Similarly, at the last available flow cytometry analysis prior to sacrifice due to disease, peripheral blood disease was lower in mice treated with the combination compared to mice treated with ibrutinib alone or vehicle alone

(p<0.001) (Figure 2.13a). Spleens derived from mice treated daily with KPT-8602 or the combination were significantly smaller compared to vehicle or ibrutinib alone (p<0.001 for all comparisons) (Figure 2.13b).

2.3.6 KPT-8602 significantly inhibits proliferation and induces apoptosis of AML cell lines and primary AML blasts

In order to assess the biological activity of KPT-8602 in AML, we treated a panel of AML cell lines (MV4-11, Kasumi-1, OCI/AML3) with KPT-8602 and KPT-330 and measured cell proliferation using MTS assays (Figure 2.14).

43

Treatment with KPT-8602 at the predetermined IC50 values induced apoptosis in

AML cell lines similar to KPT-330 when compared to DMSO treated controls at 48 hours

(Figure 2.14). We then performed proliferation assays (MTS) using primary AML samples

(n=3) using both KPT-8602 and KPT-330 and found comparable IC50 for all patients,

except one in which the IC50 for KPT-8602 was higher (300nM vs. 100nM) (Figure 2.15).

The effect of KPT-8602 on the expression levels of two known XPO1 cargo proteins; p53 and NPM1 was assessed in AML cell lines after KPT-8602 treatment using confocal microscopy as described in methods. A significant accumulation of p53 and NPM1 in the nucleus of MV4-11 and OCI-AML3 respectively were observed after treatment with KPT-

8602 (Figure 2.16).

2.3.7 KPT-8602 prolongs survival in a human leukemia xenograft model of AML

To compare the activity of KPT-8602 and KPT-330 in AML in vivo, we used a xenograft human AML murine model (MV4-11). This model consists of NOD/SCIDγ mice that were intravenously inoculated (tail vein) with MV4-11 cells obtained from spleens of primary MV4-11 xenografts. One week post-inoculation, mice were treated with KPT-

8602 at 15 mg/kg via oral gavage (5x/week or 2x/week), KPT-330 at 20 mg/kg via oral gavage (2x/week), or vehicle control (5x/week) and monitored for survival. Mice treated with KPT-330 (20mg/kg) or KPT-8602 (15mg/kg) 2x/week showed similar outcomes with improved survival when compared to vehicle-treated mice, p<0.0001.

More importantly, mice treated with KPT-8602 at 15mg/kg but given 5x/week showed strikingly better outcomes compared to KPT-8602 2x/week (15mg/kg, p<0.0001)

44

and KPT-330 (20mg/kg, 2x/week, p<0.0001) (Figure 2.17a). Peripheral blood was collected from mice in each treatment group at day 21 to assess the effect of KPT-8602 on circulating leukemia burden by measuring white blood cell count (WBC). Treatment with

KPT-8602 (daily and x2/week) and KPT-330 significantly reduced WBC in mice compared to those treated with vehicle (Figure 2.17b).

2.4 Discussion

Leptomycin B (LMB) was the first clinically developed XPO1 inhibitor 20, but

suspected off-target effects on molecules such as cysteine proteases and long lasting

toxicity in the clinic led to abandonment of its clinical development. We now believe that

the long lasting toxicity of LMB is due to enhanced irreversibility of its conjugation to

XPO1 as a result of an XPO1-driven hydrolysis/modification of the drug 21. Additionally, dose escalation studies of LMB and other synthetic nuclear export inhibitors in C57BL/6 mice suggest the low maximum tolerated dose (MTD) of LMB limits its efficacy in vivo

34.

Although SINE compounds also bind covalently to XPO1, they are considered

slowly-reversible inhibitors as compared to LMB 21. The slowly reversible XPO1-SINE

compounds interaction is believed to contribute to their improved tolerance over LMB as

sufficient inhibitor release from XPO1 can allow essential nuclear export to resume in

normal cells. This property has allowed the rapid clinical development of selinexor, which

45

has been tested in > 1000 patients demonstrating anti-tumor activity in patients with lymphoma 17, CLL 17, multiple myeloma 18, and acute myeloid leukemia (AML). Despite

notable clinical activity, constitutional symptoms (weight loss, fatigue, anorexia) have

been observed most likely due to the ability of selinexor to cross the brain barrier. Although

these side effects have been mostly manageable with supportive care, consisting of appetite

stimulants (megesterol plus olanzapine) and anti-nausea agents (odansetron), they still

represent a major limitation for a daily administration of KPT-330, which could be

beneficial for sustained inhibition of XPO1 in cancer cells.

KPT-8602 was designed to enhance reversibility of the covalent binding to XPO1

by the addition of a Michael acceptor that is activated by an electron withdrawing

pyrimidyl moiety. Additionally, a second electron withdrawing amide group was

introduced in the Cα to increase the acidity of the Cα proton and potentially make it more

susceptible to abstraction by a base to enhance the reverse Michael reaction and

deconjugation from XPO1 35,36. These modifications appear to enhance reversibility of

XPO1-KPT-8602 binding compared to KPT-185 and KPT-330, although the ~2-fold

higher reversibility was smaller than expected. The attenuated XPO1-KPT-8602

reversibility may be explained in two ways. The extensive van der Waals and polar

interactions between KPT-8602 and XPO1 groove may stabilize their interactions and cancel out a portion of the predicted enhanced intrinsic reversibility afforded by electron withdrawing substituents of KPT-8602. Second, although α-proton acidity is likely increased and may be optimized for intrinsic Michael reaction reversibility, it may not be rate-limiting in the reverse Michael reaction with the protein. Our crystal structure shows

46

the KPT-8602 α-proton pointing into the hydrophobic XPO1 groove instead of being

solvent exposed, limiting access for a base to approach and deprotonate the Cα for the reverse Michael reaction. In both scenarios, the intimate placement of KPT-8602 into the

XPO1 groove and its interactions with XPO1 sidechains likely tempered reversibility of

XPO1-KPT-8602 covalent binding resulting in only a small two-fold enhancement. It has been reported that a similar effect of enhanced reversibility is also substantially diminished when bound to a protein target in the case of newly designed reversible covalent kinase inhibitors 36. Although protein-inhibitor complexes are only slightly more reversible, enhanced intrinsic reversibility of the covalent inhibitors may represent an important therapeutic strategy for improving specificity and cellular stability as off-target conjugations reverse easily due to the inhibitors’ high intrinsic reversibility, while interactions with the protein target is mostly preserved 36.

Our data provide evidence that KPT-8602 inhibits XPO1 and induces similar

nuclear retention of XPO1 targets similar to other SINE compounds, as expected 14,15.

Additionally KPT-8602 induces comparable level of cytotoxicity as well as inhibition of cell proliferation compared to KPT-330 in primary CLL and AML tumors as well as a panel of cell lines representative of AML and both molecular subtypes of DLBCL. In contrast to KPT-330, KPT-8602 possesses a reduced blood brain barrier penetration in all the analyzed species (mouse, rat, and monkey), which combined with the slightly increased reversibility, may contribute to improvement in tolerability.

KPT-8602 was tested in the Eu-TCL1 transplant model of CLL in which splenocytes from an Eµ-TCL1 transgenic donor with active disease were engrafted into

47

C57BL/6 mice. The resulting CLL-like leukemia is uniformly fatal to all recipient mice

that successfully engraft; however, the median survival for vehicle-treated mice in each

experiment depends on donor characteristics such as cell viability and disease burden (% leukemic cells by flow). Additionally, the frequency of oral gavage may impact overall survival as demonstrated in median survival of vehicle-treated mice in Figures 5A and 5D where mice received daily and twice daily oral gavage, respectively.

Our data indicate that KPT-8602 allows a prolonged and frequent dosing schedule compared to KPT-330, which leads to an excellent therapeutic benefit and less toxicity in

two mouse models of hematological malignancies. These results are consistent with the in

vitro finding of a two-fold enhanced reversibility of XPO1-KPT-8602 binding and the lack

of blood brain barrier penetration. Additionally, our data suggest that the wider therapeutic

window of KPT-8602 may allow increased on-target efficacy leading to even more

efficacious combinations with other targeted anticancer therapies 37 as exemplified by the

combination of KPT-8602 with ibrutinib in a mouse model of CLL.

In conclusion, we report here the preclinical therapeutic activity of the novel SINE

compound, KPT-8602, an XPO1 antagonist, in CLL and AML murine models. Our

preclinical results support further development of KPT-8602 as a novel therapeutic strategy

for CLL and AML human patients.

48

2.5 References

1. Xu D, Grishin NV, Chook YM. NESdb: a database of NES-containing CRM1

cargoes. Molecular Biology of the Cell. 2012;23(18):3673-3676.

2. Brennan CM, Gallouzi I-E, Steitz JA. Protein ligands to HuR modulate its

interaction with target mRNAs in vivo. J Cell Biol. 2000;151(1):1-14.

3. Wen W, Meinkoth JL, Tsien RY, Taylor SS. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995;82:463-473.

4. Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 1997;90:1051-1060.

5. Dong X, Biswas A, Suel KE, et al. Structural basis for leucine-rich recognition by CRM1. Nature. 2009;458(7242):1136-1141.

6. Monecke T, Guttler T, Neumann P, Dickmanns A, Gorlich D, Ficner R. Crystal structure of the nuclear export receptor CRM1 in complex with Snurportin1 and RanGTP.

Science. 2009;324(5930):1087-1091.

7. Xu D, Farmer A, Chook YM. Recognition of nuclear targeting signals by

Karyopherin-β proteins. Current opinion in structural biology. 2010;20:782-790.

8. Tai Y-T, Landesman Y, Acharya C, et al. CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications. Leukemia. 2014;28:155-165.

49

9. Van Der Watt PJ, Maske CP, Hendricks DT, et al. The karyopherin proteins, Crm1

and Karyopherin Beta1, are overexpressed in cervical cancer and are critical for cancer cell

survival and proliferation. International Journal of Cancer. 2009;124:1829-1840.

10. Tan DSP, Bedard PL, Kuruvilla J, Siu LL, Razak ARA. Promising SINEs for

Embargoing Nuclear-Cytoplasmic Export as an Anticancer Strategy. Cancer discovery.

2014.

11. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute

myelogenous leukemia with a normal karyotype. The New England journal of medicine.

2005;352:254-266.

12. Yoshimura M, Ishizawa J, Ruvolo V, et al. Induction of p53-mediated transcription and apoptosis by exportin-1 (XPO1) inhibition in mantle cell lymphoma. Cancer Sci.

2014;105(7):795-801.

13. Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature. 2011;475(7354):101-105.

14. Lapalombella R, Sun Q, Williams K, et al. Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood.

2012;120(23):4621-4634.

15. Ranganathan P, Yu X, Na C, et al. Preclinical activity of a novel CRM1 inhibitor

in acute myeloid leukemia. Blood. 2012;120:1765-1773.

16. Ranganathan P, Yu X, Santhanam R, et al. Decitabine priming enhances the anti-

leukemic effects of exportin 1 (XPO1) selective inhibitor selinexor in acute myeloid

leukemia. Blood. 2015.

50

17. Kuruvilla J, Gutierrez M, Shah BD, et al. Preliminary Evidence Of Anti Tumor

Activity Of Selinexor (KPT-330) In a Phase I Trial Ofa First-In-Class Oral Selective

Inhibitor Of Nuclear Export (SINE) In Patients (pts) With Relapsed / Refractory Non

Hodgkin’s Lymphoma (NHL) and Chronic Lymphocytic L…. Vol. 122; 2013.

18. Chen CI, Gutierrez M, de Nully Brown P, et al. Anti Tumor Activity Of Selinexor

(KPT-330), A First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE)

XPO1/CRM1 Antagonist In Patients (pts) With Relapsed/Refractory Multiple Myeloma

(MM) Or Waldenstrom’s Macroglobulinemia (WM). Blood. 2013;122(21):1942-1942.\

19. Savona M, Garzon R, de Nully Brown P, et al. Phase I trial of selinexor (KPT-330), a first-in-class oral selective inhibitor of nuclear export (SINE) in patients (pts) with advanced acute myelogenous leukemia (AML). Blood. 2013;122(21):1440-1440.

20. Newlands ES, Rustin GJ, Brampton MH. Phase I trial of elactocin. British Journal of Cancer. 1996;74(4):648-649.

21. Sun Q, Carrasco YP, Hu Y, et al. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proceedings of the National

Academy of Sciences of the United States of America. 2013;110:1303-1308.

22. Sun Q, Carrasco YP, Hu Y, et al. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proc Natl Acad Sci U S A.

2013;110(4):1303-1308.

23. Chook YM, Blobel G. Structure of the nuclear transport complex karyopherin- beta2-Ran x GppNHp. Nature. 1999;399(6733):230-237.

51

24. Moriarty NW, Grosse-Kunstleve RW, Adams PD. electronic Ligand Builder and

Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation.

Acta Crystallogr D Biol Crystallogr. 2009;65(Pt 10):1074-1080.

25. Adams PD, Afonine PV, Bunkoczi G, et al. PHENIX: a comprehensive Python-

based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr.

2010;66(Pt 2):213-221.

26. Afonine PV, Grosse-Kunstleve RW, Echols N, et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol

Crystallogr. 2012;68(Pt 4):352-367.

27. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot.

Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486-501.

28. Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.7.0.1.;

2010.

29. Etchin J, Sun Q, Kentsis A, et al. Antileukemic activity of nuclear export inhibitors

that spare normal hematopoietic cells. Leukemia. 2013;27(1):66-74.

30. Haines JD, Herbin O, de la Hera B, et al. Nuclear export inhibitors avert progression

in preclinical models of inflammatory demyelination. Nat Neurosci. 2015.

31. 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. 2006;108(4):1334-1338.

52

32. Zhong Y, El-Gamal D, Dubovsky JA, et al. Selinexor suppresses downstream

effectors of B-cell activation, proliferation and migration in chronic lymphocytic

leukemia cells. Leukemia. 2014;28(5):1158-1163.

33. Hing ZA, Mantel R, Beckwith KA, et al. Selinexor is effective in acquired

resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic

leukemia. Blood. 2015.

34. Mutka SC, Yang WQ, Dong SD, et al. Identification of nuclear export inhibitors

with potent anticancer activity in vivo. Cancer Research. 2009;69:510-517.

35. Serafimova IM, Pufall MA, Krishnan S, et al. Reversible targeting of noncatalytic

with chemically tuned electrophiles. Nat Chem Biol. 2012;8(5):471-476.

36. Krishnan S, Miller RM, Tian B, Mullins RD, Jacobson MP, Taunton J. Design of

reversible, cysteine-targeted Michael acceptors guided by kinetic and

computational analysis. J Am Chem Soc. 2014;136(36):12624-12630.

37. Turner JG, Dawson J, Sullivan DM. Nuclear export of proteins and drug resistance

in cancer. Biochemical pharmacology. 2012;83:1021-1032.

53

2.6 Tables

Table 2.1. Structure data collection and refinement statistics. Values in parentheses are for

a the highest-resolution shell. Rmerge = 100 ΣhΣi|Ih,i— Ih|/ΣhΣi Ih,i, where the outer sum

(h) is over the unique reflections and the inner sum (i) is over the set of independent

b 1/2 observations of each unique reflection. Rpim = 100 ΣhΣi [1/(nh - 1)] |Ih,i— Ih|/ΣhΣi Ih,i,

c where nh is the number of observations of reflections h. As defined by the validation suite

MolProbity (Chen, V.B., Arendall, W.B.A., Headd, J.J., Keedy, D.A., Immormino, R.M.,

Kapral, G.J., Murray, L.W., Richardson, J.S., Richardson, D.C. (2010) MolProbity: all-

atom structure validation for macromolecular crystallography. Acta Cryst. D66, 12-21.).

54

(Table 2.1 continued)

KPT8602-ScXPO1-RanGppNHp-Yrb1 Data Collection: Space Group P43212 Unit cell dimentions a, b, c (Å) 105.93, 105.93, 305.14 α, β, γ (degree) 90, 90, 90 Wavelength (Å) 0.9795 Resolution Range (Å) 50.04 – 2.50 (2.54 – 2.50) Unique reflections 60851 (2980) Multiplicity 6.4 (6.5) Completeness (%) 99.9 (100.0) a Rmerge (%) 8.5 (87.7) b Rpim (%) 3.6 (36.5) Mean I/σ 20.5 (1.65) Wilson B-value (Å2) 48.63 Refinement: Number of reflections Rwork/Rfree 57196 (2001) Rwork (%) 19.46 (26.90) Rfree (%) 23.12 (34.50) R.m.s.d. bond lengths (Å) 0.008 R.m.s.d. bond angles (°) 0.86 Average B factors (Å2) Protein 46.1 Ligand/Ion 48.1 Water 35.9 KPT8602 63.0 Ramachandran plot (%)c (Favored/Allowed/Outliers) 97/3/0

55

KPT-8602 Atom XPO1 residue (atom) F1 L580 (CD2,CG), F583 (CE2) F2 V576 (CG1) F3 I555 (CG2, C, O), M556 (CA, CG, N) C12 − F4 T575 (CG2) F5 V576 (CA, CG2), F572 (CE1), T575 (CG2) F6 F572 (CE1), L536 (CD1, CD2) C13 − C6 K579 (CG) C7 L536 (CD2), K579 (CG) C8 K579 (CG) C9 K579 (CG) C10 I555 (CG2), K579(CG) C11 I555 (CG2), K579 (CG), F583 (CE2) C4 C539 (SG, CB), I555 (CD1) C5 L536 (CD2), I555 (CD1) N2 C539 (SG, CB) N3 F583 (CZ), I555 (CD1) N4 L536 (CD2), C539 (SG) C1 C539 (SG) C2 C539 (SG, CB) C3 C539 (SG, CB) O1 K548 (NZ) N1 C539 (SG), V540 (CG2) C1’ − C2’ − C3’ K579 (CD, CE, NZ), F583 (CE1) C4’ −

Table 2.2. Contacts of <4 Å between KPT-8602 and XPO1.

56

Mean Concentration at

Dose 2h post dose Brain/Plasma Compound Species (po; mg/kg) Plasma Brain Ratio

(h.ng/mL) (h.ng/g)

Mouse 5 550 105 0.19

KPT-8602 Rat 5 320 70 0.22

Monkey 10 1310 <32 <0.02

Mouse 10 1760 1295 0.71 KPT-330 Rat 10 1670 1207 0.72 (selinexor) Monkey 10 2545 1565 0.6

Table 2.3. Brain penetration of KPT-8602 and KPT-330 across species.

57

2.7 Figures

Figure 2.1. KPT-8602 binds in the NES-binding groove of XPO1.

(A) Chemical structure of KPT-8602 and KPT-330 (selinexor). (B) KPT-8602 (green) binds to the NES binding groove of XPO1 (yellow). Select inhibitor-XPO1 interactions

(<4Å) are shown as dotted lines. (C) Zoomed-in view of the KPT-8602 (green) Michael acceptor sidechain. Interactions with XPO1 (yellow) are show with dotted lines. (D)

Zoomed-in view of the KPT-185 (PDB ID 4GMX; grey) Michael acceptor sidechain.

Interactions with XPO1 (yellow) are show with dotted lines for comparison with (C).

58

(Figure 2.1 continued)

59

Figure 2.2. Structure of KPT-8602 bound to XPO1 compared to KPT-185 bound structure

and electron density of KPT-8602. (A) Overall structure of the ScXPO1-Ran-Yrb1 complex

bound to KPT-8602 (space-filling model). XPO1 is in yellow, Ran orange and Yrb1 purple.

(B) Surface representation of the NES-binding groove of XPO1 (grey) with KPT-8602

and KPT 185 overlaid for comparison. Both inhibitors are shown as sticks, with KPT-

8602 in green and KPT-185 in grey. (C) Stereo view of electron density of KPT-8602

(2Fo-Fc map contoured at 1.0 σ). (D) Kick omit mFo-DFc electron density of KPT-8602 contoured at 3.0 σ calculated by omitting the ligand in PHENIX (Averaged kick maps: less noise, more signal... and probably less bias. J. Pražnikar, P.V. Afonine, G. Guncar,

P.D. Adams, and D. Turk. Acta Crystallogr D Biol Crystallogr 65, 921-31 (2009).)

60

(Figure 2.2 continued)

61

Figure 2.3. KPT-8602 inhibits XPO1-NES interactions. (A) KPT-8602 inhibits XPO1- cargo interactions. Pull-down binding assays using immobilized GST-MVM-NS2NES or

GST-PKINES or GST-Snurportin with purified recombinant XPO1 (pre-incubated with indicated inhibitors in 1:0, 1:2, 1:4, 1:8 molar ratio) and RanGTP. Bound proteins were resolved with SDS-PAGE and Coomassie Blue staining. (B) KPT-8602 requires slightly longer incubation time than KPT-185 to achieve complete XPO1 inhibition. Pull-down binding assays of immobilized GST-PKINES with XPO1 (pre-incubated with 10uM of indicated inhibitors for different times) and RanGTP were performed as in (A).

62

Figure 2.4. KPT-8602 conjugation to XPO1 is reversible. Triplicate sets of XPO1 was incubated with 10uM of respective inhibitors and subjected to dialysis or DTT treatment before binding assay with RanGTP and immobilized GST-MVMNES. XPO1 band intensities were quantified and normalized to DMSO treated controls and plotted as percentages on the right panel.

63

Figure 2.5. Inhibitor-induced degradation of XPO1. (A) HT1080 cells treated with 25-1000 nM KPT-185, KPT-330 or KPT-8602 for 18 hours were harvested and cell lysates were probed with specific antibodies to observe degradation of XPO1. (B) Freshly isolated CLL cells from a representative patient were treated with vehicle (DMSO), 0.5 μM KPT-330, or 0.5 μM KPT-8602. Total protein was extracted at 2h, 4h, 8h, 18h after treatment initiation. N=3.

64

Figure 2.6. KPT-8602 induces apoptosis in primary CLL cells. (A) KPT-8602 induces a dose-dependent cytotoxicity of CD19+ cells from CLL patients (n=7) as measured by MTS at 24h and 48h. 2faraA indicates fludarabine treatment. Red bars indicate averages. (B)

KPT-8602 induces a dose-dependent cytotoxicity of CD19+ cells from CLL patients (n=3) in the presence of 10% fetal bovine serum (FBS) or human serum. Cytotoxicity after 24h was measured by annexin-V/PI flow cytometry. Viable populations were calculated as a percent of viability of vehicle control (DMSO). Black bars indicate averages.

65

Figure 2.7. KPT-8602 induces nuclear retention of IκBα in a manner similar to KPT-330.

CD19+ cells from CLL patients were isolated from peripheral blood and incubated with

vehicle, 0.5 μM KPT-330, or 0.5 μM KPT-8602. Nuclear and cytosolic fractions were

isolated at 8h and analyzed by immunoblot for BRG-1 and IkBa. Results are from 1

representative patient sample.

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Figure 2.8. KPT-8602 inhibits proliferation of diffuse large B-cell lymphoma cell lines.

(A) KPT-8602 induces a dose-dependent cytotoxicity of activated B-cell (ABC) subtype of diffuse large B-cell lymphoma cell lines as measured by MTS. (B) KPT-8602 induces a dose-dependent cytotoxicity of germinal center (GC) subtype of diffuse large B-cell lymphoma cell lines as measured by MTS.

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Figure 2.9. KPT-8602 and KPT-330 show similar survival benefit when given twice weekly.

At the time of leukemia onset (defined as ≥10% CD5+/CD19+ cells in the leukocyte population), mice were treated with KPT-8602 15 mg/kg 2x/week (n = 8), KPT-330 15 mg/kg 2x/week (n = 12), or vehicle control 2x/week (n = 12). Median OS: 134 days (KPT-

330), 135 (KPT-8602), and 57 days (vehicle). Both KPT-330 and KPT-8602 increased survival compared to vehicle (p=0.001 and 0.028, respectively); survival with both treatments were similar. Survival comparisons were made with the log-rank test; p-values were adjusted using Holm’s method.

68

Figure 2.10. KPT-8602 improves survival compared to KPT-330 in a mouse model of CLL.

Overall survival (OS) curve for C57BL/6 mice engrafted with Eµ-TCL1 leukemic splenocytes treated with KPT-8602 15 mg/kg daily (n = 14), KPT-8602 15 mg/kg 2x/week on Monday and Tuesday (KPT-8602 M,T; n = 12), or vehicle control daily (n = 12). Median

OS: 70 days (KPT-8602 Daily), 50 days (KPT-8602 M,T), and 33 days (vehicle daily).

Survival of mice treated daily with KPT-8602 was significantly increased compared those treated with KPT-8602 M,T (p=0.001) or vehicle daily (p<0.001). KPT-8602 M,T significantly increased survival compared to vehicle daily (p<0.001). Survival comparisons were made with the log-rank test.

69

Figure 2.11. KPT-8602 decreases leukemic burden beyond KPT-330 in a mouse model of

CLL. (A) CD45/CD5/CD19 flow cytometry of peripheral blood was used to assess leukemic burden weekly for C57BL/6 mice engrafted with Eµ-TCL1 leukemic splenocytes treated with KPT-8602 15 mg/kg daily (n = 14), KPT-8602 15 mg/kg 2x/week on Monday and Tuesday (KPT-8602 M,T; n = 12), or vehicle control daily (n = 12). Median OS: 70 days (KPT-8602 Daily), 50 days (KPT-8602 M,T), and 33 days (vehicle daily). Percentage of leukemic cell in peripheral blood was significantly lower in mice treated with KPT-8602 daily compared to all other groups at week 4 and at the last available measurement

(p<0.001 for all comparisons). A mixed effects model was used to assess changes in

%CD19+/CD5+ cells over time. (B) Splenic volume (mm3) was measured at the time of euthanasia for all mice meeting disease progression criteria. Black bars indicate averages of each cohort. Spleen volume in the KPT-8602 daily group was significantly lower compared to all other groups (p<0.001 for all comparisons). ANOVA was used to compare spleen volume (log-transformed) in KPT-8602 vs. all other groups; p-values were adjusted using Dunnett’s method. (C) Body weights (g) of C57BL/6 mice engrafted with Eµ-TCL1 leukemic splenocytes treated with KPT-8602. Body weights of mice were measured at 7 and 28 days post enrollment into treatment arms.

70

(Figure 2.11 continued)

71

Figure 2.12. KPT-8602 and ibrutinib improve survival compared either agent alone in a mouse model of CLL. Overall survival for C57BL/6 mice engrafted with Eµ-TCL1 leukemic splenocytes treated with ibrutinib 30 mg/kg daily (n = 5), ibrutinib plus KPT-

8602 15 mg/kg daily (n = 6), KPT-8602 15 mg/kg daily (n = 7), or vehicle control daily (n

= 6). Median OS: 64 days (KPT-8602 plus ibrutinib), 26 (ibrutinib), and 20 days (vehicle).

KPT-8602 plus ibrutinib increased survival compared to ibrutinib alone (p<0.001).

Survival comparisons were made with the log-rank test and adjusted for multiple comparisons.

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Figure 2.13. KPT-8602 controls disease in a mouse model of CLL. (A) CD45/CD5/CD19 flow cytometry of peripheral blood was used to assess leukemic burden weekly in C57BL/6 mice engrafted with Eµ-TCL1 leukemic splenocytes treated with ibrutinib 30 mg/kg daily

(n = 5), ibrutinib plus KPT-8602 15 mg/kg daily (n = 6), KPT-8602 15 mg/kg daily (n =

7), or vehicle control daily (n = 6). Percentage of leukemic cell in peripheral blood was significantly lower in mice treated with KPT-8602 plus ibrutinib compared to all other groups at week 3 (p<0.004 for all comparisons). A mixed effects model was used to assess changes in %CD19+/CD5+ cells over time. (B) Splenic volume (mm3) was measured at the time of euthanasia for all mice meeting disease progression criteria. Spleens in the combination group and KPT-8602 daily group were significantly smaller compared ibrutinib alone or vehicle (p<0.001 for all comparisons).

73

Figure 2.14. KPT-8602 significantly inhibits proliferation and induces apoptosis of AML cell lines. (A) Apoptosis as measured by AnnexinV/PI staining using FACS at 48 hours.

(B) WST-1 assays in 3 untreated primary AML samples.

74

(Figure 2.14 continued)

75

Figure 2.15. KPT-8602 significantly inhibits proliferation primary AML blasts. WST-1 assays were performed in 3 untreated primary AML samples.

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Figure 2.16. KPT-8602 restores nuclear localization of XPO1 cargo proteins in AML cell lines. Representative confocal microscopy images of p53 and NPM1 in MV4-11 and OCI-

AML3 cells. The left panel shows the DAPI staining (). The middle panel is p53 and NPM1 staining and the right panel is the merger of p53 and NPM1 and DAPI staining.

77

Figure 2.17. KPT-8602 increases survival in a human leukemia xenograft model of AML compared to KPT-330. (A) Survival of MV4-11 xenograft mice after treatment with KPT-

8602 15 mg/kg 5x/week (n = 15), KPT-8602 15mg/kg 2x/week (n = 10), KPT-330 20mg/kg

2x/week (n = 10), or vehicle control 5x/week (n = 15). Median OS: 58 days (KPT-8602

Daily), 35 days (KPT-8602 and KPT-330 M,Th), and 31 days (vehicle). Survival comparisons were made with the log-rank test. (B) White blood cell count in KPT-8602,

KPT-330 treated mice vs. vehicle control at 21 days. Comparisons were made using two- sample t-tests.

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Chapter 3: Dual targeting of exportin 1 and Bruton tyrosine kinase in CLL

3.1 Introduction

Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy of clonal B-cells that exhibit aberrant activation of the B-cell receptor (BCR) signaling pathway. A critical component of this pathway is Bruton agammaglobulinemia tyrosine kinase (BTK), a non- receptor tyrosine kinase expressed predominantly in B-lymphocytes3. Ibrutinib, which irreversibly binds and inhibits BTK activity, has shown promising results in CLL, MCL, and a subset of DLBCL driven by BCR signaling4-6. Despite encouraging results, complete responses are infrequent7. Additionally, acquired resistance to ibrutinib represents an

important clinical challenge wherein no standard treatment approach currently exists.

Mechanisms of ibrutinib resistance were elucidated by our group and others and involve

mutations at the C481S site of BTK or in the immediate downstream target, PLCγ21,8.

Exportin-1 (CRM1/XPO1) is the sole nuclear exporter of tumor suppressor proteins

such as p53, IκB, and FOXO3a9,10. Selective inhibitors of nuclear export (SINEs) inhibit

XPO1 and restore subcellular localization of dysregulated molecules. Our previous

published work showed XPO1 is a therapeutic target for CLL11, and has facilitated

translation of selinexor, a SINE, to a Phase I clinical trial (NCT01607892) where anti- tumor activity has been observed in lymphoma12, CLL12, multiple myeloma13, and acute

myeloid leukemia14. We recently showed that selinexor inhibits activation of downstream

BCR targets such as ERK and AKT and suppresses BTK gene expression15. Based on these

observations, we hypothesized that i) targeting XPO1 via selinexor might be effective in 79

patients with acquired resistance to ibrutinib, and ii) dual targeting of XPO1 alongside BTK

function might produce synergistic activity in CLL and prevent onset of ibrutinib-resistant

clones.

3.2 Materials and Methods

3.2.1 Cell isolation and reagents

Human CLL and normal B cells were isolated and cultured as previously described1. Blood was obtained from patients with CLL under an Ohio State University Institutional Review

Board–approved protocol with informed consent according to the Declaration of Helsinki.

HS5 stromal cells were obtained from ATCC.

3.2.2 Generation of BTK cell lines

The chicken DT40 BTK null cell lines (RCB1468) were obtained from the Riken

bioresource, Japan. The lentiviral constructs pReceiver-LV125 and A0534-Lv125 were

obtained from Genecopoeia and were used to stably transfect DT40 BTK null cells with

empty vector and BTK. The mutation was made using QuikChange site directed

mutagenesis (Stratagene) in the kinase domain at cysteine 481 to serine (primer sequence:

5’- GAG TAC ATG GCC AAT GGC TCC CTC CTG AAC TAC CTG AGG CCT CAG

GTA GTT CAG GAG GGA GCC ATT GGC CAT GTA CTC-3’). Viral particles were

80 produced by co-transfecting the plasmid DNA of interest, packaging and envelope plasmids (psPAX2 and pMD2.G) into the 293T cell line using calcium phosphate precipitation. Confirmation of the DNA sequence and infection of the DT40 cell lines was performed as previously described2. Cells were selected with puromycin.

3.2.3 Assessment of cell death

Cell death was assessed using either Annexin-V/PI staining or MTS assay as previously described1.

3.2.4 Animal studies

All experiments were carried out under protocols approved by The Ohio State University

Institutional Animal Care and Use Committee. CD19+CD5+ cells (1 x 107) from the spleen of a TCL1 transgenic mouse with active CLL-like leukemia and palpable splenomegaly were engrafted by tail vein into C56BL/6 mice. Leukemia onset was defined as >10%

CD45+CD5+CD19+ B cells in peripheral blood by flow cytometry. At leukemia onset, engrafted mice were randomly assigned to one of four groups: selinexor (15mg/kg twice per week [BIW] via oral gavage), ibrutinib (30mg/kg/day via drinking water) or selinexor

+ ibrutinib. Disease progression was monitored by weekly by CD45/CD5/CD19 flow cytometry and smears of peripheral blood, spleen size, health, and weight loss. Mice were sacrificed on development of strict early removal criteria including weight loss (>20%), splenomegaly, impaired motility, and presence of other disease criteria causing discomfort.

Overall survival was the primary endpoint.

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An in vivo model of ibrutinib resistance was developed using C57BL/6 mice engrafted

with splenocytes derived from ibrutinib-refractory Eµ-TCL1 mice that were passaged

through two Eµ-TCL1 animals. Ibrutinib-refractory Eµ-TCL1 mice were generated by

continuous dosing of animals with ibrutinib in drinking water from the time of weaning.

At the time of leukemia onset (see above), mice were randomized to receive vehicle,

ibrutinib alone (~30 mg/kg/day via drinking water) or selinexor alone (15 mg/kg BIW via

oral gavage). Ibrutinib-resistant Eμ-TCL1 mice with active leukemia were injected

intraperitoneally with 100 μg EdU (5-ethynyl-2’-deoxyuridine), and spleens and bone

marrow were collected upon sacrifice. Single-cell suspensions were prepared from spleen and bone marrow and EdU incorporation was detected by flow cytometry according to manufacturer protocol (Life Technologies). Leukemic cells were identified by anti-mouse

CD45, CD19, and CD5 antibodies (BD Biosciences).

3.2.5 Statistical Analysis

Analyses included linear mixed effects models to formally test for synergy for the cell data,

Kaplan-Meier estimates and log rank test for survival data, as previously described1.

3.3 Results and Discussion

We have previously shown that selinexor exhibits pro-apoptotic activity against CLL cells via inhibition of nuclear export of tumor suppressor proteins11. Additionally we have

82

shown that selinexor counteracts BCR signaling partially through the down-modulation of

BTK protein expression15. We therefore hypothesized selinexor would synergize with

ibrutinib as it targets BTK through a completely different mechanism. We examined this

hypothesis in primary CLL patient samples and found that ibrutinib and selinexor in

combination exhibit significant synergistic cytotoxicity (Figure 3.1). We repeated this assay in patient samples stimulated via TLR9 using synthetic CpG oligodeoxynucleotides.

CpG oligodeoxynucleotides mimic bacterial DNA motifs and stimulate TLR9 on CLL cells to induce ex vivo survival and cell cycle entry and progression. CpG stimulation was used

in this study to measure the ability of selinexor and ibrutinib to block the activation and

survival capacity of CLL cells ex vivo. It is important to note that primary CLL cells tend to undergo apoptosis when cultured in vitro unless they are activated. We observed that synergistic cytotoxicity of ibrutinib and selinexor was maintained with CpG stimulation

(Figure 3.2a).

To investigate the efficacy of combined BTK and nuclear export inhibition in the presence of BCR stimulation, CD19+ cells from CLL patients (n=6) were unstimulated or IgM- stimulated in the presence of vehicle, 0.5 μM SEL (24h continuous exposure), 1 μM Ibr

(1h pulse exposure) or SEL + Ibr. Immobilized anti-IgM was used to perform BCR crosslinking. In each condition (with or without anti-IgM) viable cells were normalized to the vehicle control (DMSO). In both the presence and absence of anti-IgM stimulation,

there was a synergistic effect of Ibr and SEL on decreasing viability (p=0.039 and p<0.001,

respectively). That is, the observed drop in viability with the combination was greater than

83

the sum of the individual effects of each drug alone. The synergistic effect was larger with

anti-IgM stimulation compared to the unstimulated condition (p=0.002). When we

considered superiority of SEL + Ibr, viability was also significantly decreased with the

combination compared to each agent alone (p<0.001 for all comparisons). A linear mixed

effects model was used to formally test for synergy.

In the human body, CLL cells derive proliferative and anti-apoptotic signaling via direct

interaction with resident cells of the bone marrow and lymph node microenvironment.

Therefore, we sought to investigate the ability of SEL + Ibr to induce apoptosis in patient-

derived CLL cells in the presence of co-cultured human bone marrow-derived fibroblast

cell line HS-5 that induces survival of normal B-cells and CLL cells ex vivo16,17. The

combination showed a significant increase in cytotoxicity compared to each agent alone

during stromal cell co-culture (Figure 3.3). It is well established that CLL cells rely on

pro-survival signals from the microenvironment to resist cytotoxic agents. This suggests

dual inhibition of BTK kinase function by ibrutinib and BTK protein expression by

selinexor may be an effective strategy to target CLL cells localized to many different

compartments including peripheral blood, bone marrow, and other secondary lymphoid

tissues.

Our prior studies with ibrutinib18 and selinexor15 in the Eµ-TCL1 engraftment mouse

model of CLL showed that each of drug alone can inhibit the expansion phase of CLL in this model. To see if selinexor has potential to improve upon ibrutinib therapy in vivo,

84

since this agent is a current standard of care, we monitored overall survival in a cohort of

engrafted mice randomized to receive either ibrutinib alone or selinexor and ibrutinib. As

shown in Figure 3.4, mice treated with the combination had significantly better survival

compared to mice given ibrutinib alone. Similar to reports of other active agents in the Eµ-

TCL1 model, disease eradication was not achieved for any treatment group due to the

aggressive nature of this model. We next examined the efficacy of selinexor in the common clinical scenario of prolonged lymphocytosis following ibrutinib treatment in patients with

CLL. Our previous data indicate that while BTK is inhibited, downstream mediators of

BCR signaling are activated in the persistent lymphocytes19, and treatment with targeted

kinase inhibitors shows that these cells are not addicted to a single survival pathway19.

Lymphocytes collected at baseline and 9 months after beginning of ibrutinib therapy from the same patients were treated with targeted kinase inhibitors19 or selinexor. While all the

other inhibitors remain equally active at both time points19, selinexor was significantly

more effective in persistent (post-ibrutinib) samples (Figure 3.5), providing additional

evidence for therapeutic combination of these two agents.

Selinexor targets multiple BCR signaling nodes, including BTK, in a manner independent

of BTK tyrosine kinase activity, suggesting that selinexor may possess the ability to

overcome or prevent ibrutinib-mediated resistance in CLL by blocking adaptive signaling

responses in resistant subclones. Our group previously identified a major mechanism of

acquired ibrutinib resistance in CLL patients involving mutation of the BTK cysteine

residue where ibrutinib binding occurs (C481S), changing the binding of ibrutinib from

85

irreversible to reversible1,2. To focus on this important resistance mechanism, we cloned

human wild-type or C481S BTK into DT40 cells lacking endogenous BTK (Supplementary

Materials). Viability was assessed after treating DT40 cells with selinexor for 24 hours.

Selinexor remains active in the presence of the BTK C481S mutation (Figure 3.6). To test our hypothesis in vivo, C57BL/6 mice were engrafted with CD19+CD5+ leukemia derived from ibrutinib-refractory Eµ-TCL1 mice. While these mice are not known to possess the

C481S mutation, they maintain functionally resistant disease as a result of selective pressure from ibrutinib exposure, mimicking acquired resistance in patients. At leukemia onset, mice were randomized to receive vehicle, ibrutinib alone or selinexor alone. As expected, mice retained their resistance to ibrutinib. However, treatment with selinexor induced a significant improvement in survival (Figure 3.7a). We further demonstrated that selinexor effectively inhibited the fraction of proliferating leukemic cells, based on a significant decrease in the percentage of 5-ethynyl-2’-deoxyuridine-(EdU)-positive leukemic cells of ibrutinib-resistant mice treated with selinexor (Figure 3.7b). The ability of selinexor to overcome acquired resistance to ibrutinib was confirmed in vitro in primary

CLL cells derived from patients on ibrutinib that have relapsed with BTK C481S mutations

(n=3), as confirmed by Ion Torrent deep sequencing performed at the time of ibrutinib relapse (Figure 3.8). These data show that selinexor has single-agent activity in ibrutinib- resistant CLL in vitro, suggesting it may be effective in ibrutinib-resistant CLL patients, and may have the potential to prevent expansion of ibrutinib-resistant subclones when used in combination with ibrutinib.

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Together our data suggest the combination of selinexor and ibrutinib as a promising new

therapeutic paradigm in CLL that may elicit more robust initial responses and provide

activity in the setting of acquired resistance to ibrutinib.

3.4 References

1. Woyach JA, Furman RR, Liu TM, et al. Resistance mechanisms for the Bruton's

tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370(24):2286-2294.

2. Jennifer Ann Woyach ASR, Gerard Lozanski, Arletta Lozanski, Nyla A.

Heerema, Weiqiang Zhao, Lynne Abruzzo, Amber Gordon, Jeffrey Alan Jones, Joseph

M. Flynn, Samantha Mary Jaglowski, Leslie A. Andritsos, Farrukh Awan, Kristie A.

Blum, Michael R. Grever, Amy J. Johnson, John C. Byrd, Kami J. Maddocks.

Association of disease progression on ibrutinib therapy with the acquisition of resistance

mutations: A single-center experience of 267 patients. J Clin Oncol. 2014;32(5s):(suppl;

abstr 7010).

3. Mohamed AJ, Yu L, Bäckesjö C-M, et al. Bruton’s tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain.

Immunological Reviews. 2009;228(1):58-73.

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4. Cinar M, Hamedani F, Mo Z, Cinar B, Amin HM, Alkan S. Bruton tyrosine

kinase is commonly overexpressed in mantle cell lymphoma and its attenuation by

Ibrutinib induces apoptosis. Leuk Res. 2013;37(10):1271-1277.

5. Wilson WH, Gerecitano JF, Goy A, et al. The Bruton's Tyrosine Kinase (BTK)

Inhibitor, Ibrutinib (PCI-32765), Has Preferential Activity in the ABC Subtype of

Relapsed/Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): Interim

Results of a Multicenter, Open-Label, Phase 2 Study. ASH Annual Meeting Abstracts.

2012;120(21):686-.

6. Yang Y, Shaffer AL, 3rd, Emre NC, et al. Exploiting synthetic lethality for the

therapy of ABC diffuse large B cell lymphoma. Cancer Cell. 2012;21(6):723-737.

7. Byrd JC, Brown JR, O’Brien S, et al. Ibrutinib versus Ofatumumab in Previously

Treated Chronic Lymphoid Leukemia. N Engl J Med. 2014;In press.

8. Furman RR, Cheng S, Lu P, et al. Ibrutinib Resistance in Chronic Lymphocytic

Leukemia. New England Journal of Medicine. 2014;370(24):2352-2354.

9. Xu D, Grishin NV, Chook YM. NESdb: a database of NES-containing CRM1 cargoes. Molecular Biology of the Cell. 2012;23(18):3673-3676.

10. Brennan CM, Gallouzi I-E, Steitz JA. Protein ligands to HuR modulate its

interaction with target mRNAs in vivo. J Cell Biol. 2000;151(1):1-14.

11. Lapalombella R, Sun Q, Williams K, et al. Selective inhibitors of nuclear export

show that CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood.

2012;120(23):4621-4634.

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12. Kuruvilla J, Gutierrez M, Shah BD, et al. Preliminary Evidence Of Anti Tumor

Activity Of Selinexor (KPT-330) In a Phase I Trial Ofa First-In-Class Oral Selective

Inhibitor Of Nuclear Export (SINE) In Patients (pts) With Relapsed / Refractory Non

Hodgkin’s Lymphoma (NHL) and Chronic Lymphocytic L…. Vol. 122; 2013.

13. Chen CI, Gutierrez M, de Nully Brown P, et al. Anti Tumor Activity Of Selinexor

(KPT-330), A First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE)

XPO1/CRM1 Antagonist In Patients (pts) With Relapsed/Refractory Multiple Myeloma

(MM) Or Waldenstrom’s Macroglobulinemia (WM). Blood. 2013;122(21):1942-1942.

14. Savona M, Garzon R, de Nully Brown P, et al. Phase I trial of selinexor (KPT-

330), a first-in-class oral selective inhibitor of nuclear export (SINE) in patients (pts) with

advanced acute myelogenous leukemia (AML). Blood. 2013;122(21):1440-1440.

15. Zhong Y, El-Gamal D, Dubovsky JA, et al. Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells. Leukemia. 2014.

16. Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes. Vol. 85;

1995.

17. Kay NE, Shanafelt TD, Strege AK, Lee YK, Bone ND, Raza A. Bone biopsy derived marrow stromal elements rescue chronic lymphocytic leukemia B-cells from spontaneous and drug induced cell death and facilitates an “angiogenic switch”.

Leukemia research. 2007;31(7):899-906.

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18. 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. 2014;123(8):1207-1213.

19. 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. 2014;123(12):1810-1817.

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3.5 Tables

Co-culture CpG & IgM

U M U M

IgVH 10 9 3 2

del(17p) 1 0 0 0

del(13q) 3 6 1 2

del(11q) 3 0 2 0

Table 3.1. Cytogenetic and molecular features of patient specimens used for in vitro

apoptosis assays. The apoptosis experiments with and without CpG in Figures 3.2a and

3.2b were performed using cells isolated from 6 CLL patients. Of the 6 CLL patients, 3 were IgVH unmutated, 2 were IgVH mutated, and 1 patient’s IgVH status was unknown.

The apoptosis experiment in the presence and absence of HS5 co-culture in Figure 3.3

were performed using cells isolated from 19 CLL patients. Of these, 10 were IgVH

unmutated and 9 were IgVH mutated. U = unmutated and M = mutated. Patients for

which cytogenetic and IgVH data were not available have not been listed.

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3.6 Figures

Figure 3.1. Selinexor synergizes in vitro with ibrutinib. CD19+ cells from CLL patients

(n=6) were isolated from peripheral blood and incubated with vehicle, 0.5 μM selinexor

(SEL), 1 μM Ibrutinib (Ibr) or selinexor + Ibrutinib. Ibr was given as a 1h pulse exposure followed by washout and SEL was given continuously for 24h. Viability was determined by annexin-V/propidium iodide (PI) flow cytometry, and is shown relative to time-matched

DMSO controls for each group. Horizontal bars represent averages. Each agent alone

(selinexor or ibrutinib) significantly decreased cell viability compared to vehicle (P<0.03).

The combination produced a synergistic effect on viability (P=0.041).

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Figure 3.2. Selinexor and ibrutinib retain efficacy in CpG and anti-IgM stimulated CLL cells. (A) CD19+ cells from CLL patients (n=6) were unstimulated or 3.2µM CpG-

stimulated in the presence of vehicle, 0.5 μM SEL (24h continuous exposure), 1 μM Ibr

(1h pulse exposure with washout) or SEL + Ibr. Viability was determined by annexin-

V/propidium iodide (PI) flow cytometry, and is shown relative to time-matched DMSO controls for each group. Horizontal bars represent averages. Each agent alone (selinexor or ibrutinib) significantly decreased cell viability compared to vehicle (P=0.001). The combination produced a synergistic decrease in viability (P=0.005). (B) CD19+ cells from

CLL patients (n=6) were unstimulated or IgM-stimulated in the presence of vehicle, 0.5

μM SEL (24h continuous exposure), 1 μM Ibr (1h pulse exposure) or SEL + Ibr.

Immobilized anti-IgM was used to perform BCR crosslinking. Viability was determined by annexin-V/propidium iodide (PI) flow cytometry. Horizontal bars represent averages.

In each condition (with or without anti-IgM) viable cells were normalized to the vehicle

93 control (DMSO). Selinexor and ibrutinib together resulted in significantly more cytotoxicity than either agent alone (P<0.001).

Figure 3.3. Selinexor and ibrutinib are cytotoxic to CLL cells co-cultured with bone

marrow stromal cells. CD19+ cells from CLL patients were incubated with 0.5 μM SEL

(24h continuous exposure), 1 μM Ibr (1h pulse exposure with washout) or SEL + Ibr on an

HS5 human bone marrow stromal cell layer for 24 hours. Viability was determined by

annexin-V/propidium iodide (PI) flow cytometry, and is shown relative to the vehicle

(DMSO) control without stromal support. Horizontal bars represent averages. Selinexor

and ibrutinib together resulted in significantly more cytotoxicity than either agent alone

(P<0.001).

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Figure 3.4. Selinexor synergizes in vivo with ibrutinib. Overall survival (OS) curves for

C57BL/6 mice engrafted with spleen lymphocytes derived from the Eμ-TCL1 transgenic

mouse. Mice with active leukemia (defined as ≥10% CD5+/CD19+ cells in the leukocyte

population) were randomized to treatment with ibrutinib (~30 mg/kg/day via drinking

water) or ibrutinib + selinexor (15 mg/kg on two consecutive days each week via oral gavage) (n=6 per group).

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Figure 3.5. Selinexor is more effective in post-ibrutinib CLL samples. Persistent lymphocytes collected at baseline and 9 months after beginning of ibrutinib from the same patients were treated in vitro with selinexor at 0.5 μM (n=13). Cytotoxicity was measured by annexin-V/propidium iodide (PI) flow cytometry after 72 hours.

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Figure 3.6 Selinexor induces cytotoxicity in cells expressing C481S BTK. DT40 BTK null cells with WT or C481S BTK were exposed to 1 μM ibrutinib for 1 hour, 0.5 μM selinexor for 24 hours, or DMSO (vehicle) for 24 hours. Cytotoxicity after 24 hours was measured by AnnexinV/PI flow cytometry. Viable populations were calculated as a percent of viability of vehicle control. Three biological replicates were performed. Selinexor induced significantly more cell death compared to vehicle in cells expressing C481S (P=0.042),

WT (P=0.027), or empty vector (P=0.011).

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Figure 3.7. Selinexor induces cytotoxicity in a mouse model of acquired resistance to ibrutinib. (A) C57BL/6 mice were engrafted with spleen lymphocytes derived from an Eμ-

TCL1 transgenic mouse with acquired resistance to ibrutinib. Mice were followed for leukemia development (defined as ≥10% CD5+/CD19+ cells in the leukocyte population), and once leukemic, randomized to treatment with ibrutinib alone (~30 mg/kg/day via drinking water), selinexor alone (15 mg/kg on two consecutive days each week via oral gavage) or vehicle. As expected, mice treated with ibrutinib did not show any survival advantage compared to vehicle control, while mice treated with selinexor showed improved survival (n=12-14 per group). (B) In vivo EdU labeling was performed in a cohort of mice engrafted as described in (A). Mice were treated for 2 days with vehicle, SEL or

Ibr (n=5 for each group). EdU was injected on day 3. Spleens were analyzed by flow cytometry for percentage of Edu-positive cells within the leukemic population

(CD45+/CD19+/CD5+ cells).

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Figure 3.8. Selinexor is active in the setting of acquired resistance to ibrutinib. CLL cells derived from ibrutinib resistant patients (n=3) were treated in vitro with selinexor at 0.5

μM. Cytotoxicity was measured by annexin/PI after 48 hours.

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Chapter 4: Conclusions and Future Directions

4.1 Conclusions

In this dissertation, we have presented three main conclusions concerning targeted nuclear export therapeutics in CLL. In Chapter 2, using a combination of cell-free techniques, cell line models, and animal models, we have examined the therapeutic potential of a novel exportin 1 inhibitor, KPT-8602. Previously, XPO1 was shown to be overexpressed in CLL and was validated as a target in preclinical models. In addition, an

XPO1 inhibitor, selinexor (KPT-330), has entered clinical trials. Responses have been observed in patients with lymphoma, CLL, multiple myeloma, and AML. However, therapy-limiting constitutional symptoms such as weight loss and fatigue have been observed, limiting selinexor to a twice weekly schedule. An abbreviated dosing schedule results in sub-optimal inhibition of the target, which may result in decreased efficacy.

Our biochemical and in vivo characterization of the novel XPO1 inhibitor, KPT-8602, provides evidence that KPT-8602 possess improved tolerability allowing daily dosing and full target inhibition.

Secondly, we evaluated the efficacy of a novel combination therapy in CLL. In

Chapter 3, in a series of proof-of-principle studies, we demonstrated that dual targeting of XPO1 and BTK elicits a synergistic cytotoxic response in CLL cells. This synergistic 100

response was maintained in the presence of microenvironment stimuli such as stromal

support cells.

Finally, we found that inhibition of XPO1 is a promising therapeutic strategy in

the setting of acquired ibrutinib resistance (Chapter 3). Despite the remarkable responses to the BTK inhibitor, ibrutinib, complete responses are not frequent in CLL patients.

Acquired BTK mutations at the ibrutinib binding site and in the downstream target phospholipase C-γ2 (PLC- γ2) have been identified in CLL patients who relapse on

ibrutinib. Thus, acquired ibrutinib resistance represents a growing challenge in clinical

management. Herein, we have shown that XPO1 inhibition retains its potent cytotoxic effect in the setting of acquired ibrutinib resistance using a mouse model of ibrutinib resistance and cell lines expressing wild-type or mutant C481S BTK. These results were validated in leukemic cells isolated from CLL patients with acquired ibrutinib resistance.

4.2 Future Perspectives

4.2.1 Extending current work

In the current work, we have used a combination of cell lines, mouse models, and primary patient samples to describe the efficacy of XPO1 inhibitors in the setting of acquired resistance to ibrutinib. An important extension of this work is to identify the molecular pathways or target(s) responsible for XPO1 inhibitor efficacy in the setting of ibrutinib resistance. While we have shown that XPO1 targets the B cell receptor pathway,

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in particular BTK, it is likely that XPO1 inhibition has pleiotropic effects on CLL cells.

In mantle cell lymphoma (MCL), BCR signaling is critical to pathogenesis and treatment with ibrutinib has resulted in an overall response rate of 68% in a phase II trial1. Wang

and colleagues assessed the transcriptomic response of six MCL cell lines to selinexor

and ibrutinib via RNA-seq2. Cytotoxicity of selinexor was associated with downregulated

of NF-κB and ibrutinib resistance was associated with unchanged NF-κB expression. These results may suggest the importance of NF-κB as a key point of nuclear export inhibition in leukemia.

There are important questions in the field still unresolved. Namely, what are the key proteins and transcripts transported by XPO1 that mediate its oncogenic effects? The entire scope of proteins and transcripts whose localization is controlled by XPO1 has yet to be defined. This a fundamental question in the field of both cancer biology and nuclear transport and has clinical implications now that nuclear transport therapies are in various stages of clinical development, with promising initial results. As discussed, XPO1- mediated nuclear export is a critical pathway in normal tissues as well as cancer cells. We hypothesize that in cancer, overexpression of XPO1 is advantageous due to the mislocalization of specific XPO1 cargos such as p53 and IκB. Conversely, normal tissues rely on XPO1-mediated nuclear transport to maintain cellular homeostasis. Systemically identifying the oncogenic cargos of XPO1 may help expand the therapeutic window of future XPO1-directed therapies. The answer to this question will likely depend on the context, therefore necessitating rigorous, unbiased studies of XPO1 in various cellular models.

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Up to this point, most studies of XPO1 in cancer have lent support to a paradigm

in which tumor cells co-opt nucleocytoplasmic transport of proteins such as TSPs or

GRPs to obtain fitness or survival advantages3. This paradigm highlights the importance

of post-translational regulation that occurs in normal cells. Another hypothesis focuses on

the ability of XPO1 to transport certain RNA species such as mRNA and rRNA to

cytoplasm, where they undergo translation or ribosome biogenesis, respectively.

Although some RNA cargos of XPO1 are known, the importance of XPO1-mediated

RNA export in cancer has yet to be established. Indeed, little has been done to

characterize the full breadth of RNAs that use the XPO1 pathway in normal cells. It is

known that XPO1 mediates export of certain oncogenic mRNAs that are subsequently

translated by eIF4E4,5. It would be interesting to identify mislocalization of RNAs, if any,

in hematologic malignancies and solid tumors. In this way, XPO1 inhibition may act to

repair regulatory defects at both a post-transcriptional and post-translational level in some

contexts.

Our preclinical studies of KPT-8602 in AML, as well as previous preclinical

experiments performed with selinexor, make use of xenograft mouse models, which have

limitations as a drug development tool6. Xenograft mouse models of leukemia are often

used to study cancer biology and response to investigational therapeutic agents. Mouse-

adapted human cell lines are adoptively transferred into severely immunocompromised

animals, which avoids potential recognition and rejection of human cells7. The limitations

of this approach including a lack of immune microenvironment and an inability to study the interplay between the tumor, drug, and immune system. In addition, cell lines often

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do not adequately represent the complexity of human disease. Patient-derived xenograft

(PDX) models address some of these limitations by making use of patient samples. In

theory, PDX studies that use multiple patient samples may more accurately predict the

eventual response of a candidate drug in the clinic8. Recently, Etchin and colleagues have

shown that KPT-8602 is active against leukemic blasts and leukemia-initiating cells in

PDX experiments derived from high-risk AML groups such as complex karyotype,

cytogenetically normal, and FLT3-ITD9.

4.2.2 Exploring additional XPO1 driven combination therapies

It is increasingly recognized that dysregulated nuclear export, associated with XPO1 overexpression, is a common pathway for cancer cells to obtain proliferative and anti- apoptotic advantages. Indeed, XPO1 directed therapies have shown promising efficacy in

a variety of solid tumors and hematologic malignancies. The synergy observed between

BTK inhibition and nuclear export blockade raises the possibility of additional

combination therapies in CLL, AML and other malignancies.

In some cases, the mechanism of synergy underpinning XPO1 combinations have

been clearly defined. In the setting of drug-resistant multiple myeloma (MM), XPO1

blockade restores nuclear localization of topoisomerase IIα, allowing topo II inhibitors

like doxorubicin to effectively initiate DNA double-strand breaks in the leukemic cell10.

The combination of doxorubicin and XPO1 inhibition displays synergy in vitro11. In

chronic myeloid leukemia (CML), the hallmark oncogenic fusion protein BCR-ABL is

predominately cytoplasmic where it activates AKT; however, BCR-ABL contains an

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XPO1 NES and when it is sequestered in the nucleus, the ABL partner acts as a homolog

of p53 to induce apoptosis12. Thus, XPO1 inhibition in CML results in inactivation of the

BCR-ABL oncogenic properties and reactivation tumor suppressor properties. This

mechanism and the resulting in vivo synergy between LMB and the BCR-ABL tyrosine

kinase inhibitor imatinib were described in a series of experiments by Vigneri and

colleagues12.

In other cases, a molecular explanation for the efficacy of XPO1 driven

combination therapy is an area of active investigation. Mantle cell lymphoma is B cell

neoplasm characterized by cyclin D1 overexpression mediated by a t(11;14)(q13;32)

reciprocal translocation placing cyclin D1 (11q13) under regulation of the

immunoglobulin heavy-chain promoter/enhancer (14q32). Ibrutinib has shown

impressive ORs in MCL, however ibrutinib-resistance is an emerging clinical issue,

which is more generally an issue with single-agent regimens. Thus,

developing an approach that combines targeted therapies may hold promise and is

currently under investigation in MCL. Tabe and colleagues recently showed that

combined ibrutinib and selinexor treatment results in increased suppression of p-4EBP1

and cyclin D1 and downmodulation of p-Rb, c-Myc, and Mcl-1, compared to either agent

alone13. In addition, selinexor and ibrutinib displayed a synergistic decrease in cell

proliferation in 3 of the 4 tested MCL cell lines. Proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) of Mino cells treated with the combination of selinexor + ibrutinib identified upregulation of cytochrome C, HSP10, and histone H1,

which mediate the intrinsic apoptosis pathway. Selinexor has also been investigated in

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combination with proteasome inhibitors (, ) in MM and has been shown to be active in the setting of proteasome-inhibitor resistance and re-sensitize MM

cells to proteasome inhibitors via IκB-mediated downmodulation of NF-κB14. Overall,

these reports point to the promise of nuclear export as a therapeutic target in multidrug

regimens.

4.2.3 Mechanisms of acquired resistance to XPO1 blockade

As nuclear export matures into a promising target in clinical oncology, it will become

important to understand the pathways that cancer cells may use to develop resistance to

inhibition of this pathway. Crochiere and colleagues established a SINE-“resistant”

HT1080 fibrosarcoma cell line by gradual dose escalation to 600 nM of KPT-185 over a

10 month time period15. No XPO1 mutations or activation of multi-drug resistance

transporters were identified in the SINE-resistant cells compared to the parental SINE-

sensitive cell line. Despite a 130-fold decrease in sensitivity to KPT-185, nuclear

retention of cargo proteins such as p53, p21, p27, PP2A, and IκB was still observed in the

SINE-resistant cell line indicating that this cell line may not be fully resistant, but rather,

less sensitive to SINE compounds. Gene expression analysis 8 hours after SINE

treatment demonstrated that the majority of genes responded in the same direction in the

parental and “resistant” cell line. Pro-inflammatory and anti-apoptotic pathways were

more highly upregulated in the SINE-resistant cell line compared to the parental cell line.

Interestingly, Early growth response-1 (EGR1) was solely induced in the SINE-resistant

cell line. EGR1 is a transcription factor that may play a role in the differentiation of B

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cells into plasma cells16. It will be interesting to characterize the mechanisms of

resistance that develop in patients who relapse on selinexor. Deep sequencing of patient samples at relapse to identify the presence of acquired mutations, if any, may shed light on the pathways that tumor cells use to evade or compensate for inactivation of XPO1.

4.2.4 Identifying druggable vulnerabilities in cancer

As we enter the era of precision medicine, it will be important to identify specific tumor subtypes based on genomic or metabolomic profiling that display a strong response or no response to nuclear export blockade. Identifying these druggable vulnerabilities will allow XPO1 inhibitors to be used in a precise and meaningful manner and in this way, the clinical value of XPO1 inhibitors may be maximized. While most CLL patients overexpress XPO1, studies that correlate mutational or epigenetic status with SINE response will be particularly valuable. It will be interesting to understand how CLL patients with genetic aberrations in nuclear export machinery such a XPO1 and RANBP2 will respond to XPO1 inhibition. One such example comes from lung cancer, where genetic dependencies of a panel of non-small-cell lung cancer (NSCLC) cell lines were explored using pools of short interfering RNAs by Kim and colleagues17. They observed

that KRAS-mutant NSCLC cell lines were exclusively dependent on the nuclear export

pathway and chemical inhibition of XPO1 demonstrated synthetic lethality, primarily due

to nuclear retention of IκB. Interestingly, two KRAS-mutant NSCLC cell lines (H2291

and H1573) were insensitive to XPO1 inhibition and were subsequently found to contain co-occurring Follistatin-like 5 (FSTL5) mutations. Kim and colleagues showed that

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FSTL5 loss-of-function mutations activate the Yes-associated protein 1 (YAP1) pathway

which was sufficient to generate resistance to XPO1 inhibition. Furthermore, inhibition

of YAP1 via verteporfin or siRNAs was sufficient to restore XPO1 inhibitor sensitivity.

In AML, patients with double-hit Fms-like tyrosine kinase 3 / internal tandem

duplications (FLT3/ITD mutations) and co-occurring FLT3 D835 mutations are markedly

sensitive to nuclear p53 and suppression of c-Myc, IκB, and HIF1α 18. These

observations and those that follow will help establish a basis for using targeted therapies

in a mechanistically sound fashion.

4.2.5 Determining the role of XPO1 in the pathogenesis of CLL

The role of XPO1 in CLL pathogenesis has yet to be fully established. Interestingly,

recurrent somatic mutations at the highly conserved 571 amino acid residue of XPO1

have been identified in 5% of CLL cases and approximately a quarter (~25%) of patients

with primary mediastinal B cell lymphoma19–21. In CLL, the E571 mutation represents a

significant mutational burden and is predominately clonal prior to therapy with a variant

allele frequency (VAF) consistently at ~0.5, indicating its early role in CLL

pathogenesis22,23.

The functional significance of mutated XPO1 is currently unknown although there

is some published data to suggest that this mutation may change the specificity of XPO1

for certain cargo proteins. García-Santisteban and colleagues developed a cellular reporter system to study the E571K mutation24. In their system, survivin, a known XPO1

cargo protein, was modified and is efficiently exported to the cytoplasm by wild-type

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XPO1 in 293T cells. Structurally, the E571 residue of XPO1 is most proximal to third and fourth hydrophobic NES residues (Φ3 and Φ4, of the consensus sequence Φ1-X(2-3)-

Φ2-X(2-3)- Φ3-X- Φ4 where Φ indicates a hydrophobic residue M, L, V, I, F and X is any

amino acid). They show that the E571K mutant is bound more strongly to the survivin

NES when negative charges were introduced adjacent to Φ3 and Φ4. Conversely, an NES containing positive charges in the vicinity of Φ3 and Φ4 displayed weaker binding to

E571K compared to wild-type XPO124. Additional functional studies are needed to

characterize the role of XPO1 mutations in leukemia, which may help guide therapies

targeting the nuclear export pathway.

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4.3 References

1. Wang ML, Rule S, Martin P, et al. Targeting BTK with Ibrutinib in Relapsed or

Refractory Mantle-Cell Lymphoma. N. Engl. J. Med. 2013;369(6):507–516.

2. Wang YL, Ming M, Xie B, et al. XPO1 Inhibitor Selinexor Overcomes Ibrutinib

Resistance in Mantle Cell Lymphoma Via Nuclear Retention of IκB. Blood.

2017;130(Suppl 1):3837 LP-3837.

3. Tan DSP, Bedard PL, Kuruvilla J, Siu LL, Razak ARA. Promising SINEs for

Embargoing Nuclear-Cytoplasmic Export as an Anticancer Strategy. Cancer

Discov. 2014;

4. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E is

a central node of an RNA regulon that governs cellular proliferation. J. Cell Biol.

2006;175(3):415–26.

5. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E

promotes nuclear export of cyclin D1 mRNAs via an element in the 3’UTR. J. Cell

Biol. 2005;169(2):245–56.

6. Ranganathan P, Yu X, Na C, et al. Preclinical activity of a novel CRM1 inhibitor

in acute myeloid leukemia. Blood. 2012;120(9):1765–73.

110

7. Kohnken R, Porcu P, Mishra A. Overview of the Use of Murine Models in

Leukemia and Lymphoma Research . Front. Oncol. . 2017;7:22.

8. Gao H, Korn JM, Ferretti S, et al. High-throughput screening using patient-derived

tumor xenografts to predict clinical trial drug response. Nat. Med. 2015;21:1318.

9. Etchin J, Berezovskaya A, Conway AS, et al. KPT-8602, a second-generation

inhibitor of XPO1-mediated nuclear export, is well tolerated and highly active

against AML blasts and leukemia-initiating cells. Leukemia. 2016;31:143.

10. Turner JG, Engel R, Derderian JA, Jove R, Sullivan DM. Human topoisomerase

IIα nuclear export is mediated by two CRM-1-dependent nuclear export signals. J.

Cell Sci. 2004;117(14):3061 LP-3071.

11. Turner JG, Marchion DC, Dawson JL, et al. Human Multiple Myeloma Cells Are

Sensitized to Topoisomerase II Inhibitors by CRM1 Inhibition. Cancer Res.

2009;69(17):6899 LP-6905.

12. Vigneri P, Wang JYJ. Induction of apoptosis in chronic myelogenous leukemia

cells through nuclear entrapment of BCR–ABL tyrosine kinase. Nat. Med.

2001;7:228.

13. Tabe Y, Harada M, Miyamae Y, et al. Selective Inhibitor of Nuclear Export

Selinexor (KPT-330) and BCL2 Inhibitor ABT-199 Enhance the Anti-Lymphoma

Effect of BTK Inhibitor Ibrutinib in Mantle Cell Lymphoma. Blood.

2014;124(21):2254 LP-2254.

14. Turner JG, Kashyap T, Dawson JL, et al. XPO1 inhibitor combination therapy

with bortezomib or carfilzomib induces nuclear localization of IκBα and

111

overcomes acquired proteasome inhibitor resistance in human multiple myeloma.

Oncotarget. 2016;7(48):78896.

15. Crochiere M, Kashyap T, Kalid O, et al. Deciphering mechanisms of drug

sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE)

compounds. BMC Cancer. 2015;15(1):910.

16. Oh Y-K, Jang E, Paik D-J, Youn J. Early Growth Response-1 Plays a Non-

redundant Role in the Differentiation of B Cells into Plasma Cells. Immune Netw.

2015;15(3):161–166.

17. Kim J, McMillan E, Kim HS, et al. XPO1-dependent nuclear export is a druggable

vulnerability in KRAS-mutant lung cancer. Nature. 2016;538:114.

18. Yamatani K, Tabe Y, Sekihara K, et al. XPO1 Inhibition Targets Transcriptional

Vulnerability of FLT3-ITD+D835 Double Mutant AML through p53

Accumulation and Inhibition of Oncogenic Transcription Factors: Lesson Learned

from Cage Sequencing of Primary AML Cells. Blood. 2017;130(Suppl 1):1244

LP-1244.

19. Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies

recurrent mutations in chronic lymphocytic leukaemia. Nature.

2011;475(7354):101–5.

20. Jardin F, Lehmann-horn K, Sagan S a, et al. Recurrent mutations of the exportin 1

gene (XPO1) and their impact on selective inhibitor of nuclear export compounds

sensitivity in primary mediastinal B-cell lymphoma. Am. J. Hematol. 2016;(1):1–

20.

112

21. Dubois S, Viailly P-J, Mareschal S, et al. Next Generation Sequencing in Diffuse

Large B Cell Lymphoma Highlights Molecular Divergence and Therapeutic

Opportunities: a LYSA Study. Clin. Cancer Res. 2016;1078-0432.CCR-15-2305-.

22. Lawrence MS, Stojanov P, Mermel CH, et al. Discovery and saturation analysis of

cancer genes across 21 tumour types. Nature. 2014;505:495.

23. Amin NA, Seymour EK, Ulintz P, et al. A Quantitative Analysis of Subclonal and

Clonal Gene Mutations Occurring Pre- and Post-Therapy in 53 Cases of Chronic

Lymphocytic Leukemia. Blood. 2015;126(23):2909 LP-2909.

24. Garcia-Santisteban I, Arregi I, Alonso-Marino M, et al. A cellular reporter to

evaluate CRM1 nuclear export activity : functional analysis of the cancer-related

mutant E571K. Cell. Mol. Life Sci. 2016;

113

Works Cited

1. Howlader N, Noone A, Krapcho M, et al. SEER Cancer Statistics Review, 1975- 2014. Natl. Cancer Institute. Bethesda, MD. 2017; 2. Strati P, Chaffee K, Achenbach S, et al. Disease Progression and Complications Are the Main Cause of Death in Patients with Chronic Lymphocytic Leukemia (CLL) Independent of Age and Comorbidities at Diagnosis. Blood. 2015;126(23):5265 LP-5265. 3. Pekarsky Y, Zanesi N, Croce CM. Molecular basis of CLL. Semin. Cancer Biol. 2010;20(6):370–6. 4. Zenz T, Fröhling S, Mertens D, Döhner H, Stilgenbauer S. Moving from prognostic to predictive factors in chronic lymphocytic leukaemia (CLL). Best Pract. Res. Clin. Haematol. 2010;23(1):71–84. 5. Moreno C, Montserrat E. Genetic lesions in chronic lymphocytic leukemia: what’s ready for prime time use? Haematologica. 2010;95(1):12–5. 6. Zenz T, Mertens D, Döhner H, Stilgenbauer S. Importance of genetics in chronic lymphocytic leukemia. Blood Rev. 2011;25(3):131–7. 7. Guièze R, Wu CJ. Genomic and epigenomic heterogeneity in chronic lymphocytic leukemia. Blood. 2015;126(4):445 LP-453. 8. Landau D a, Carter SL, Stojanov P, et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell. 2013;152(4):714–26. 9. Landau DA, Tausch E, Taylor-Weiner AN, et al. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015; 10. 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. 2011;44(1):47–52. 11. Woyach J a, Johnson AJ, Byrd JC. The B-cell receptor signaling pathway as a therapeutic target in CLL. Blood. 2012;120(6):1175–1184. 12. Awan FT, Byrd JC. New Strategies in Chronic Lymphocytic Leukemia: Shifting Treatment Paradigms. Clin. Cancer Res. 2014; 13. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with Ibrutinib in Relapsed Chronic Lymphocytic Leukemia. N. Engl. J. Med. 2013;369:32–42. 14. Byrd J, Brown J, O’Brien SM, et al. Ibrutinib versus Ofatumumab in Previously Treated Chronic Lymphoid Leukemia. N. Engl. J. Med. 2014;371:213–223. 15. Bednenko J, Cingolani G, Gerace L. Nucleocytoplasmic Transport: Navigating the Channel. Traffic. 2003;4(3):127–135.

114

16. Cronshaw JM, Krutchinsky AN, Zhang W, Chait BT, Matunis MJ. Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 2002;158(5):915 LP-927. 17. Cautain B, Hill R, Pedro N De, Link W. Components and regulation of nuclear transport processes. FEBS J. 2014; 18. Lange A, Mills RE, Lange CJ, et al. Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α. J. Biol. Chem. . 2007;282(8):5101–5105. 19. la Cour T, Kiemer L, Mølgaard A, et al. Analysis and prediction of leucine-rich nuclear export signals. Protein Eng. Des. Sel. 2004;17(6):527–536. 20. Chook YM, Fung HYJ. Atomic basis of CRM1-cargo recognition, release and inhibition. Semin. Cancer Biol. 2014;1–10. 21. Kirli K, Karaca S, Dehne HJ, et al. A deep proteomics perspective on CRM1- mediated nuclear export and nucleocytoplasmic partitioning. Elife. 2015;4(December):e11466. 22. Ferrigno P, Posas F, Koepp D, Saito H, Silver PA. Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin β homologs NMD5 and XPO1. EMBO J. 1998;17(19):5606 LP-5614. 23. Jang B-C, Muñoz-Najar U, Paik J-H, et al. Leptomycin B, an Inhibitor of the Nuclear Export Receptor CRM1, Inhibits COX-2 Expression. J. Biol. Chem. . 2003;278(5):2773–2776. 24. Turner JG, Sullivan DM. CRM1-mediated nuclear export of proteins and drug resistance in cancer. Curr. Med. Chem. 2008;15(26):2648–55. 25. Kanai M, Hanashiro K, Kim S-H, et al. Inhibition of Crm1–p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation. Nat. Cell Biol. 2007;9:1175. 26. Shao C, Lu C, Chen L, et al. p53-dependent anticancer effects of leptomycin B on lung adenocarcinoma. Cancer Chemother. Pharmacol. 2011;67(6):1369–1380. 27. Vousden KH, Lane DP. p53 in health and disease. Nat. Rev. Mol. Cell Biol. 2007;8:275. 28. Cullen BR. Nuclear RNA export. J. Cell Sci. 2003;116(4):587–597. 29. Halees AS, El-Badrawi R, Khabar KSA. ARED Organism: expansion of ARED reveals AU-rich element cluster variations between human and mouse. Nucleic Acids Res. 2008;36(suppl_1):D137–D140. 30. Bakheet T, Frevel M, Williams BRG, Greer W, Khabar KSA. ARED: human AU- rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Res. 2001;29(1):246– 254. 31. Khabar KS a. Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements. Cell. Mol. Life Sci. 2010;67(17):2937–55. 32. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E is a central node of an RNA regulon that governs cellular proliferation. J. Cell Biol. 2006;175(3):415–26.

115

33. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E promotes nuclear export of cyclin D1 mRNAs via an element in the 3’UTR. J. Cell Biol. 2005;169(2):245–56. 34. Sun Q, Chen X, Zhou Q, et al. Inhibiting cancer cell hallmark features through nuclear export inhibition. Signal Transduct. Target. Ther. Publ. online 1 July 2016; | doi10.1038/sigtrans.2016.10. 2016;1(April):1041–1050. 35. Noske A, Weichert W, Niesporek S, et al. Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancer. Cancer. 2008;112(8):1733–1743. 36. Yao Y, Dong Y, Lin F, et al. The expression of CRM1 is associated with prognosis in human osteosarcoma. Oncol. Rep. 2009;21(1):229–235. 37. Huang W, Yue L, Qiu W, et al. Prognostic value of CRM1in pancreas cancer. Clin. Investig. Med. 2009;32(6):315–321. 38. Shen A, Wang Y, Zhao Y, et al. Expression of CRM1 in human gliomas and its significance in p27 expression and clinical prognosis. Neurosurgery. 2009;65(1):153–160. 39. Van Der Watt PJ, Maske CP, Hendricks DT, et al. The karyopherin proteins, Crm1 and Karyopherin Beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation. Int. J. Cancer. 2009;124(November 2008):1829–1840. 40. Culjkovic-Kraljacic B, Baguet A, Volpon L, Amri A, Borden KLB. The oncogene eIF4E reprograms the nuclear pore complex to promote mRNA export and oncogenic transformation. Cell Rep. 2012;2(2):207–15. 41. Tai Y-T, Landesman Y, Acharya C, et al. CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications. Leukemia. 2014;28(1):155–65. 42. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N. Engl. J. Med. 2005;352(3):254–66. 43. Kojima K, Kornblau SM, Ruvolo V, et al. Prognostic impact and targeting of CRM1 in acute myeloid leukemia. Blood. 2013;121(20):4166 LP-4174. 44. Gough SM, Slape CI, Aplan PD. NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights. Blood. 2011;118(24):6247–57. 45. Walker CJ, Oaks JJ, Santhanam R, et al. Preclinical and clinical efficacy of XPO1/CRM1 inhibition by the karyopherin inhibitor KPT-330 in Ph+ leukemias. 2013;122(17):3034–3044. 46. Balatti V, Bottoni A, Palamarchuk A, et al. NOTCH1 mutations in CLL associated with trisomy 12. Blood. 2012;119(2):329–331. 47. Jardin F, Lehmann-horn K, Sagan S a, et al. Recurrent mutations of the exportin 1 gene (XPO1) and their impact on selective inhibitor of nuclear export compounds sensitivity in primary mediastinal B-cell lymphoma. Am. J. Hematol. 2016;(1):1– 20.

116

48. Dubois S, Viailly P-J, Mareschal S, et al. Next Generation Sequencing in Diffuse Large B Cell Lymphoma Highlights Molecular Divergence and Therapeutic Opportunities: a LYSA Study. Clin. Cancer Res. 2016;1078-0432.CCR-15-2305-. 49. Janku F, Vergilio JA, Salhia B, et al. Comprehensive genomic profiling reveals recurrent XPO1 mutations and other alterations in archival samples of patients with Hodgkin lymphoma. Eur. J. Cancer. 2018;69:S41–S42. 50. Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N. Engl. J. Med. 2011;365(26):2497–506. 51. Stamatopoulos B, Antoniou P, Vavoulis D, et al. Characterization of Recurrent Mutations in Patient with a Richter Syndrome By Targeted Next Generation Sequencing. Blood. 2016;128(22):3200 LP-3200. 52. Kudo N, Matsumori N, Taoka H, et al. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. U. S. A. 1999;96(August):9112–9117. 53. Dong X, Biswas A, Suel KE, et al. Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature. 2009;458(April):1136–1141. 54. Sun Q, Carrasco YP, Hu Y, et al. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proc. Natl. Acad. Sci. U. S. A. 2013;110(4):1303–8. 55. Newlands ES, Rustin GJ, Brampton MH. Phase I trial of elactocin. Br. J. Cancer. 1996;74(4):648–649. 56. Crochiere ML, Baloglu E, Klebanov B, et al. A method for quantification of exportin-1 ( XPO1 ) occupancy by Selective Inhibitor of Nuclear Export ( SINE ) compounds. Oncotarget. 2015;1:. 57. Daelemans D, Afonina E, Nilsson J, et al. A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export. Proc. Natl. Acad. Sci. . 2002;99(22):14440–14445. 58. Kalid O, Toledo Warshaviak D, Shechter S, Sherman W, Shacham S. Consensus Induced Fit Docking (cIFD): methodology, validation, and application to the discovery of novel Crm1 inhibitors. J. Comput. Aided. Mol. Des. 2012;26(11):1217–28. 59. Neggers JE, Vercruysse T, Jacquemyn M, et al. Resource Identifying Drug-Target Selectivity by CRISPR / Cas9 Genome Editing. Chem. Biol. 2015;1–10. 60. Lapalombella R, Sun Q, Williams K, et al. Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood. 2012;120(23):4621–34. 61. Bichi R, Shinton S a, Martin ES, et al. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc. Natl. Acad. Sci. U. S. A. 2002;99(10):6955–60. 62. 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. 2006;108(4):1334–8.

117

63. Zhong Y, El-Gamal D, Dubovsky J a, et al. Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells. Leukemia. 2014;1–5. 64. Kuruvilla J, Savona M, Baz R, et al. Selective inhibition of nuclear export with selinexor in patients with non-Hodgkin lymphoma. Blood. 2017;129(24):3175 LP-3183. 65. Garzon R, Savona M, Baz R, et al. A phase 1 clinical trial of single-agent selinexor in acute myeloid leukemia. 2017;129(24):18–21. 66. Longo PG, Laurenti L, Gobessi S, et al. 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. 2008;111(2):846–55. 67. Yu L, Mohamed AJ, Simonson OE, et al. Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-κB. Blood. 2008;111(9):4617 LP- 4626.

68. Xu D, Grishin NV, Chook YM. NESdb: a database of NES-containing CRM1 cargoes. Molecular Biology of the Cell. 2012;23(18):3673-3676. 69. Brennan CM, Gallouzi I-E, Steitz JA. Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol. 2000;151(1):1-14. 70. Wen W, Meinkoth JL, Tsien RY, Taylor SS. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995;82:463-473. 71. Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 1997;90:1051-1060. 72. Dong X, Biswas A, Suel KE, et al. Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature. 2009;458(7242):1136-1141. 73. Monecke T, Guttler T, Neumann P, Dickmanns A, Gorlich D, Ficner R. Crystal structure of the nuclear export receptor CRM1 in complex with Snurportin1 and RanGTP. Science. 2009;324(5930):1087-1091. 74. Xu D, Farmer A, Chook YM. Recognition of nuclear targeting signals by Karyopherin-β proteins. Current opinion in structural biology. 2010;20:782-790. 75. Tai Y-T, Landesman Y, Acharya C, et al. CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications. Leukemia. 2014;28:155-165. 76. Van Der Watt PJ, Maske CP, Hendricks DT, et al. The karyopherin proteins, Crm1 and Karyopherin Beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation. International Journal of Cancer. 2009;124:1829-1840. 77. Tan DSP, Bedard PL, Kuruvilla J, Siu LL, Razak ARA. Promising SINEs for Embargoing Nuclear-Cytoplasmic Export as an Anticancer Strategy. Cancer discovery. 2014. 78. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. The New England journal of medicine. 2005;352:254-266.

118

79. Yoshimura M, Ishizawa J, Ruvolo V, et al. Induction of p53-mediated transcription and apoptosis by exportin-1 (XPO1) inhibition in mantle cell lymphoma. Cancer Sci. 2014;105(7):795-801. 80. Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature. 2011;475(7354):101-105. 81. Lapalombella R, Sun Q, Williams K, et al. Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood. 2012;120(23):4621-4634. 82. Ranganathan P, Yu X, Na C, et al. Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia. Blood. 2012;120:1765-1773. 83. Ranganathan P, Yu X, Santhanam R, et al. Decitabine priming enhances the anti- leukemic effects of exportin 1 (XPO1) selective inhibitor selinexor in acute myeloid leukemia. Blood. 2015. 84. Kuruvilla J, Gutierrez M, Shah BD, et al. Preliminary Evidence Of Anti Tumor Activity Of Selinexor (KPT-330) In a Phase I Trial Ofa First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE) In Patients (pts) With Relapsed / Refractory Non Hodgkin’s Lymphoma (NHL) and Chronic Lymphocytic L…. Vol. 122; 2013. 85. Chen CI, Gutierrez M, de Nully Brown P, et al. Anti Tumor Activity Of Selinexor (KPT-330), A First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE) XPO1/CRM1 Antagonist In Patients (pts) With Relapsed/Refractory Multiple Myeloma (MM) Or Waldenstrom’s Macroglobulinemia (WM). Blood. 2013;122(21):1942-1942.\ 86. Savona M, Garzon R, de Nully Brown P, et al. Phase I trial of selinexor (KPT-330), a first-in-class oral selective inhibitor of nuclear export (SINE) in patients (pts) with advanced acute myelogenous leukemia (AML). Blood. 2013;122(21):1440-1440. 87. Newlands ES, Rustin GJ, Brampton MH. Phase I trial of elactocin. British Journal of Cancer. 1996;74(4):648-649. 88. Sun Q, Carrasco YP, Hu Y, et al. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:1303- 1308. 89. Sun Q, Carrasco YP, Hu Y, et al. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proc Natl Acad Sci U S A. 2013;110(4):1303-1308. 90. Chook YM, Blobel G. Structure of the nuclear transport complex karyopherin- beta2-Ran x GppNHp. Nature. 1999;399(6733):230-237. 91. Moriarty NW, Grosse-Kunstleve RW, Adams PD. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr D Biol Crystallogr. 2009;65(Pt 10):1074-1080. 92. Adams PD, Afonine PV, Bunkoczi G, et al. PHENIX: a comprehensive Python- based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 2):213-221. 119

93. Afonine PV, Grosse-Kunstleve RW, Echols N, et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012;68(Pt 4):352-367. 94. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486-501. 95. Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.7.0.1.; 2010. 96. Etchin J, Sun Q, Kentsis A, et al. Antileukemic activity of nuclear export inhibitors that spare normal hematopoietic cells. Leukemia. 2013;27(1):66-74. 97. Haines JD, Herbin O, de la Hera B, et al. Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination. Nat Neurosci. 2015. 98. 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. 2006;108(4):1334-1338. 99. Zhong Y, El-Gamal D, Dubovsky JA, et al. Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells. Leukemia. 2014;28(5):1158-1163. 100. Hing ZA, Mantel R, Beckwith KA, et al. Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia. Blood. 2015. 101. Mutka SC, Yang WQ, Dong SD, et al. Identification of nuclear export inhibitors with potent anticancer activity in vivo. Cancer Research. 2009;69:510-517. 102. Serafimova IM, Pufall MA, Krishnan S, et al. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat Chem Biol. 2012;8(5):471-476. 103. Krishnan S, Miller RM, Tian B, Mullins RD, Jacobson MP, Taunton J. Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis. J Am Chem Soc. 2014;136(36):12624-12630. 104. Turner JG, Dawson J, Sullivan DM. Nuclear export of proteins and drug resistance in cancer. Biochemical pharmacology. 2012;83:1021-1032.

105. Woyach JA, Furman RR, Liu TM, et al. Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370(24):2286-2294. 106. Jennifer Ann Woyach ASR, Gerard Lozanski, Arletta Lozanski, Nyla A. Heerema, Weiqiang Zhao, Lynne Abruzzo, Amber Gordon, Jeffrey Alan Jones, Joseph M. Flynn, Samantha Mary Jaglowski, Leslie A. Andritsos, Farrukh Awan, Kristie A. Blum, Michael R. Grever, Amy J. Johnson, John C. Byrd, Kami J. Maddocks. Association of disease progression on ibrutinib therapy with the acquisition of resistance mutations: A single-center experience of 267 patients. J Clin Oncol. 2014;32(5s):(suppl; abstr 7010). 107. Mohamed AJ, Yu L, Bäckesjö C-M, et al. Bruton’s tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain. Immunological Reviews. 2009;228(1):58-73.

120

108. Cinar M, Hamedani F, Mo Z, Cinar B, Amin HM, Alkan S. Bruton tyrosine kinase is commonly overexpressed in mantle cell lymphoma and its attenuation by Ibrutinib induces apoptosis. Leuk Res. 2013;37(10):1271-1277. 109. Wilson WH, Gerecitano JF, Goy A, et al. The Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), Has Preferential Activity in the ABC Subtype of Relapsed/Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): Interim Results of a Multicenter, Open-Label, Phase 2 Study. ASH Annual Meeting Abstracts. 2012;120(21):686-. 110. Yang Y, Shaffer AL, 3rd, Emre NC, et al. Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma. Cancer Cell. 2012;21(6):723-737. 111. Byrd JC, Brown JR, O’Brien S, et al. Ibrutinib versus Ofatumumab in Previously Treated Chronic Lymphoid Leukemia. N Engl J Med. 2014;In press. 112. Furman RR, Cheng S, Lu P, et al. Ibrutinib Resistance in Chronic Lymphocytic Leukemia. New England Journal of Medicine. 2014;370(24):2352-2354. 113. Xu D, Grishin NV, Chook YM. NESdb: a database of NES-containing CRM1 cargoes. Molecular Biology of the Cell. 2012;23(18):3673-3676. 114. Brennan CM, Gallouzi I-E, Steitz JA. Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol. 2000;151(1):1-14. 115. Lapalombella R, Sun Q, Williams K, et al. Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood. 2012;120(23):4621-4634.

116. Kuruvilla J, Gutierrez M, Shah BD, et al. Preliminary Evidence Of Anti Tumor Activity Of Selinexor (KPT-330) In a Phase I Trial Ofa First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE) In Patients (pts) With Relapsed / Refractory Non Hodgkin’s Lymphoma (NHL) and Chronic Lymphocytic L…. Vol. 122; 2013. 117. Chen CI, Gutierrez M, de Nully Brown P, et al. Anti Tumor Activity Of Selinexor (KPT-330), A First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE) XPO1/CRM1 Antagonist In Patients (pts) With Relapsed/Refractory Multiple Myeloma (MM) Or Waldenstrom’s Macroglobulinemia (WM). Blood. 2013;122(21):1942-1942. 118. Savona M, Garzon R, de Nully Brown P, et al. Phase I trial of selinexor (KPT- 330), a first-in-class oral selective inhibitor of nuclear export (SINE) in patients (pts) with advanced acute myelogenous leukemia (AML). Blood. 2013;122(21):1440-1440. 119. Zhong Y, El-Gamal D, Dubovsky JA, et al. Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells. Leukemia. 2014. 120. Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes. Vol. 85; 1995. 121. Kay NE, Shanafelt TD, Strege AK, Lee YK, Bone ND, Raza A. Bone biopsy derived marrow stromal elements rescue chronic lymphocytic leukemia B-cells 121

from spontaneous and drug induced cell death and facilitates an “angiogenic switch”. Leukemia research. 2007;31(7):899-906. 122. 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. 2014;123(8):1207-1213. 123. 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. 2014;123(12):1810-1817.

124. Wang ML, Rule S, Martin P, et al. Targeting BTK with Ibrutinib in Relapsed or Refractory Mantle-Cell Lymphoma. N. Engl. J. Med. 2013;369(6):507–516. 125. Wang YL, Ming M, Xie B, et al. XPO1 Inhibitor Selinexor Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma Via Nuclear Retention of IκB. Blood. 2017;130(Suppl 1):3837 LP-3837. 126. Tan DSP, Bedard PL, Kuruvilla J, Siu LL, Razak ARA. Promising SINEs for Embargoing Nuclear-Cytoplasmic Export as an Anticancer Strategy. Cancer Discov. 2014; 127. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E is a central node of an RNA regulon that governs cellular proliferation. J. Cell Biol. 2006;175(3):415–26. 128. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB. eIF4E promotes nuclear export of cyclin D1 mRNAs via an element in the 3’UTR. J. Cell Biol. 2005;169(2):245–56. 129. Ranganathan P, Yu X, Na C, et al. Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia. Blood. 2012;120(9):1765–73.

130. Kohnken R, Porcu P, Mishra A. Overview of the Use of Murine Models in Leukemia and Lymphoma Research . Front. Oncol. . 2017;7:22. 131. Gao H, Korn JM, Ferretti S, et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat. Med. 2015;21:1318. 132. Etchin J, Berezovskaya A, Conway AS, et al. KPT-8602, a second-generation inhibitor of XPO1-mediated nuclear export, is well tolerated and highly active against AML blasts and leukemia-initiating cells. Leukemia. 2016;31:143. 133. Turner JG, Engel R, Derderian JA, Jove R, Sullivan DM. Human topoisomerase IIα nuclear export is mediated by two CRM-1-dependent nuclear export signals. J. Cell Sci. 2004;117(14):3061 LP-3071. 134. Turner JG, Marchion DC, Dawson JL, et al. Human Multiple Myeloma Cells Are Sensitized to Topoisomerase II Inhibitors by CRM1 Inhibition. Cancer Res. 2009;69(17):6899 LP-6905. 135. Vigneri P, Wang JYJ. Induction of apoptosis in chronic myelogenous leukemia cells through nuclear entrapment of BCR–ABL tyrosine kinase. Nat. Med. 2001;7:228. 136. Tabe Y, Harada M, Miyamae Y, et al. Selective Inhibitor of Nuclear Export Selinexor (KPT-330) and BCL2 Inhibitor ABT-199 Enhance the Anti-Lymphoma 122

Effect of BTK Inhibitor Ibrutinib in Mantle Cell Lymphoma. Blood. 2014;124(21):2254 LP-2254. 137. Turner JG, Kashyap T, Dawson JL, et al. XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma. Oncotarget. 2016;7(48):78896. 138. Crochiere M, Kashyap T, Kalid O, et al. Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds. BMC Cancer. 2015;15(1):910. 139. Oh Y-K, Jang E, Paik D-J, Youn J. Early Growth Response-1 Plays a Non- redundant Role in the Differentiation of B Cells into Plasma Cells. Immune Netw. 2015;15(3):161–166. 140. Kim J, McMillan E, Kim HS, et al. XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer. Nature. 2016;538:114. 141. Yamatani K, Tabe Y, Sekihara K, et al. XPO1 Inhibition Targets Transcriptional Vulnerability of FLT3-ITD+D835 Double Mutant AML through p53 Accumulation and Inhibition of Oncogenic Transcription Factors: Lesson Learned from Cage Sequencing of Primary AML Cells. Blood. 2017;130(Suppl 1):1244 LP-1244. 142. Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature. 2011;475(7354):101–5. 143. Jardin F, Lehmann-horn K, Sagan S a, et al. Recurrent mutations of the exportin 1 gene (XPO1) and their impact on selective inhibitor of nuclear export compounds sensitivity in primary mediastinal B-cell lymphoma. Am. J. Hematol. 2016;(1):1– 20. 144. Dubois S, Viailly P-J, Mareschal S, et al. Next Generation Sequencing in Diffuse Large B Cell Lymphoma Highlights Molecular Divergence and Therapeutic Opportunities: a LYSA Study. Clin. Cancer Res. 2016;1078-0432.CCR-15-2305-. 145. Lawrence MS, Stojanov P, Mermel CH, et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature. 2014;505:495. 146. Amin NA, Seymour EK, Ulintz P, et al. A Quantitative Analysis of Subclonal and Clonal Gene Mutations Occurring Pre- and Post-Therapy in 53 Cases of Chronic Lymphocytic Leukemia. Blood. 2015;126(23):2909 LP-2909. 147. Garcia-Santisteban I, Arregi I, Alonso-Marino M, et al. A cellular reporter to evaluate CRM1 nuclear export activity : functional analysis of the cancer-related mutant E571K. Cell. Mol. Life Sci. 2016;

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