Dysregulation of Mitogen-Activated Protein Kinases Signaling and Immune Suppression

in B-cell Leukemia

DISSERTATION

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

Yo-Ting Tsai, M.S.

Graduate Program in Pharmaceutical Sciences

The Ohio State University

2017

Dissertation Committee:

John C. Byrd, M.D., Advisor David M. Lucas, Ph.D., Co-Advisor Michael R. Grever, M.D. Natarajan Muthusamy, DVM, Ph.D. Ching-Shih Chen, Ph.D. A. Mitchell Phelps, Ph.D.

Yo-Ting Tsai

2017

Abstract

Mutant mitogen-activated protein kinases (MAPK) components-mediated dysregulation of the pathway is commonly exhibited in cancers and can drive malignancies by promoting tumor survival and proliferation. Tumor-mediated immune suppression in both solid tumors and hematologic malignancies is significant and the activated MAPK signaling pathway is evident in solid tumors to enhance immune suppression. The improvement of current therapies in chronic lymphocytic leukemia (CLL) controls disease development to a greater extent, but the leukemia-induced immune suppression remains one of the leading causes of death. The refractory/relapse cases and the severe immune- deficiency provide a strong justification to identify the effect(s) of genetic lesions and to develop strategies to reverse it in CLL. Activating mutation of MAPK components including BRAF occurs in approximately 10% of CLL and nearly 100% of hairy cell leukemia (HCL), and a BRAF pseudogene-mediated MAPK activation presents in up to

30% of B-cell lymphomas. However, the contribution of the mutant BRAF-activated

MAPK signaling in B-cell malignancies remains unclear in respects of disease development and tumor-induced immune suppression, and few models of mutant BRAF- mediated MAPK activation are available to address these. We therefore generate both cellular and mouse models of BRAFV600E mutant B-cell leukemia to study these aspects.

The work presented here focuses on the evaluation of the pathomechanism of

BRAFV600E in B-cell leukemias. Chapter one introduces the background of B-cell development, B-cell receptor (BCR) and MAPK signaling in B-cell malignancies, and

MAPK signaling-mediated immune suppression in cancers. In addition, animal models of leukemia with MAPK activation and/or immune suppression are referred. Chapters two evaluates the pathologic effects of BRAFV600E in a CLL cell line, OSUCLL, using a doxycycline transduction manner. We found that transduction of BRAFV600E increases growth rate and enhances activation of OSUCLL cells. Moreover, it leads to changes in transcripts in OSUCLL cells including ABCB1 (aka p-glycoprotein), an ATP-dependent efflux pump and often mediated anti-cancer drug resistance. The mechanism of the ABCB1 induction is evidenced by inhibition of BRAFV600E and MEK kinases using pharmacological agents, and the key mediators are the activated MAPK pathway-induced

AP-1 transcription factors. Chapter three introduces a novel mouse model of B-cell leukemia harboring the B-cell restricted BRAFV600E mutation. This new mouse model with

B-cell specific BRAFV600E expression was characterized to shorten the leukemia onset and overall survival. The early B-cell development and the proliferation of leukemia B cells were not affected by this mutation. The reduction of B-cell apoptosis and mutant BRAF leukemia-enhanced immune suppression in the microenvironment contributed to the more aggressive disease seen in this B-cell leukemia mouse model. Chapter four concludes the findings, addresses the unanswered questions in this work, and proposes future perspectives, especially the applications of the new mouse model in future preclinical studies.

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Together, this work, for the first time, employs a transgenic mouse model of B-cell leukemia with B-cell lineage-restricted BRAFV600E expression and demonstrates the immune-suppressive impact of BRAFV600E in B-cell leukemias. Importantly, this mouse model can be utilized to develop rational combination strategies to both directly target the tumor cells and overcome tumor-mediated immune evasion.

Dedication

This document is dedicated to my family.

Acknowledgments

I would like to thank my mentors Drs. John Byrd and David Lucas for their mentorship, guidance and continued support through my PhD education. I joined the lab with little background about hematologic malignancies, and I am grateful for this opportunity to gain knowledge and start my PhD research in the lab with abundant resources. Your patience, intelligence and optimistic attitude always encourage me along the path. I am always inspired and learn how to deal with the hard time while getting challenges or not exciting results from research whenever Dr. Byrd says “Let’s make lemonade from lemons” and “data are data.” Dr. Lucas is always patient and discreet on research to avoid any misleading conclusion, and now I also can find myself an inner peace while editing slides. I am so blessed to have both mentors providing their insight from MD and PhD aspects to my research projects. I would also like to thank my committee members

Drs. Michael Grever, Natarajan (Raj) Muthusamy, Ching-Shih Chen and Mitch Phelps for providing your expertise and advice and taking time out of your busy schedules to meet with me for my projects. It is really my honor to have many exceptional researchers served on my committee.

I would like to extend my thanks to all the past and present Byrd lab members for their support and advice on both my research and daily life. Especially thank Bonnie

Harrington for performing necropsy, histology and characterizing disease for my mouse model. Thank Minh Tran, Ellen Sass and Aparna Lakshmanan for helping maintain my mouse colonies, monitoring ERC, processing mouse samples, and performing related experiments. Thank Carolyn Cheney and Jennifer Mele for properly maintaining flow cytometers and providing training and consultation. Thank Sue Scott and Laura Hanley for keeping weekly meetings organized and hardly working to find out dates from my mentors’ busy schedules for my committee meetings, candidate exam, and final oral exam. I would also like to thank my colleagues and peers Yuh-Ying Yeh, Ta-Ming Liu, Meixiao Long,

Karilyn Larkin, Rajeswaran Mani, Fabienne Lucas, Shuai Dong, Chi-Ling Chiang,

Zachary Hing, Shaneice Mitchell, Eileen Hu, Joseph Greene, Priscilla Do, Emily

McWilliams, Sean Reiff, and Timothy Chen for their friendship, support, and help on my research and daily life.

Finally, I want to express my sincere appreciation to my family for unconditional love and support throughout my life. Thank my dear parents for sharing experience, caring everything about my life and always being there for me. I become the person I am because of you. Thank my husband, Yu-Chou, for being my friend and partner, taking care our little family, and always encouraging me. I am proud of everything you did for our family. Thank my little boy, Ryan, for being the best baby that a graduate student Mom can ask for. Thank you for coming into my life and bringing us endless joy and love.

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Vita

July 1985 ...... Born in Taipei, Taiwan

2007 ...... B.S. Life Science

National Chung Hsing University, Taiwan

2009...... M.S. Pharmacology

National Cheng Kung University, Taiwan

2012 to Present ...... Graduate Research Associate

Medicinal Chemistry and Pharmacognosy

Program, The Ohio State University

Publications

Tsai YT, Lakshmanan A, Lehman A, Harrington BK, McClanahan F, Tran M, Sass EJ, Long M, Flechtner AD, Jaynes F, La Perle K, Coppola V, Lozanski G, Muthusamy N, Byrd

JC, Grever MR, Lucas DM. (2017). BRAFV600E accelerates disease progression and enhances immune suppression in a mouse model of B-cell leukemia. (Manuscript in revision)

Tsai YT, Lozanski G, Lehman A, Sass EJ, Salunke SB, Chen CS, Grever MR, Byrd JC, Lucas DM. (2015). BRAFV600E induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via AP-1 activation. Leuk Res. 2015 Sep 5 [Epub ahead of print]

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Tang YA, Lin RK, Tsai YT, Hsu HS, Yang YC, Chen CY, Wang YC. (2012). MDM2 overexpression deregulates the transcriptional control of RB/E2F leading to DNA methyltransferase 3A overexpression in lung cancer. Clin Cancer Res. 18(16), 4325-33.

Wu MH, Tsai YT, Hua KT, Chang KC, Kuo ML, Lin MT. (2012). Eicosapentaenoic acid and docosahexaenoic acid inhibit macrophage-induced gastric cancer cell migration by attenuating the expression of matrix metalloproteinase 10. J Nutr Biochem. 23(11), 1434- 9.

Hsu HS, Lin JH, Huang WC, Hsu TW, Su K, Chiou SH, Tsai YT, Hung SC. (2011). Chemoresistance of lung cancer stemlike cells depends on activation of Hsp27. Cancer. 117(7), 1516-28.

Ho TF, Ma CJ, Lu CH, Tsai YT, Wei YH, Chang JS, Lai JK, Cheuh PJ, Yeh CT, Tang PC, Tsai Chang J, Ko JL, Liu FS, Yen HE, Chang CC. (2007). Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53. Toxicol Appl Pharmacol. 225(3), 318-28.

Fields of Study

Major Field: Pharmaceutical Sciences

Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments ...... vi

Vita ...... viii

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations ...... xiv

Chapter 1 ...... 1

Introduction

1.1 B-cell development, B-cell Receptor (BCR) Signaling, and Leukemia ...... 1

1.2 Activated MAPK Signaling in B-cell Malignancies ...... 6

1.3 MAPK Signaling and Immune Suppression ...... 12

1.4 Animal Models of B-cell Leukemia with MAPK Activation and Immune

Suppression ...... 17

1.5 Conclusion and Hypothesis ...... 22

Chapter 2 ...... 29

BRAFV600E induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via

AP-1 activation

2.1 Introduction ...... 29

2.2 Materials and Methods ...... 31

2.3 Results ...... 35

2.4 Discussion ...... 40

Chapter 3 ...... 55

BRAFV600E accelerates disease progression and enhances immune suppression in a

mouse model of B-cell leukemia

3.1 Introduction ...... 55

3.2 Materials and Methods ...... 57

3.3 Results ...... 61

3.4 Discussion ...... 70

Chapter 4 ...... 98

Discussion and Future Perspectives

4.1 Overview ...... 98

4.2 Future Perspectives ...... 101

References ...... 109

List of Tables

Table 1. 1 Rai and Binet staging systems for chronic lymphocytic leukemia ...... 25 Table 2. 1 Microarray results showing top 30 BRAFV600E up- or down-regulated genes 45

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List of Figures

Figure 1. 1 Stages of B-cell development...... 26 Figure 1. 2 B-cell receptor (BCR) signaling...... 27 Figure 1. 3 Overview of immune checkpoint molecules associated with T-cell exhaustion...... 28 Figure 2. 1 Effects of transfected BRAFV600E on the MAPK pathway and cell growth. .. 46 Figure 2. 2 BRAFV600E induces the expression of ABCB1 and functional P-gp in OSUCLL cells...... 47 Figure 2. 3 Constitutive BRAFV600E expression drives P-gp expression in OSUCLL cells...... 49 Figure 2. 4 BRAFV600E activates MAPK signaling and P-gp expression in a subset of CLL patient samples...... 50 Figure 2. 5 BRAFV600E and MEK inhibition block ABCB1/P-gp expression in OSUCLL cells...... 51

Figure 2. 6 BRAFV600E enhances ABCB1 promoter activity via MAPK and AP-1...... 52 Figure 2. 7 P-gp induction is not via CREB or NF-kB pathway ...... 54 Figure 3. 1 BRAFV600E protein is expressed in the B cells of BRAFVExTCL1 mice…….75 Figure 3. 2 BRAFV600E produces more aggressive disease in the Eµ-TCL1 mouse model...... 76 Figure 3. 3 Phenotypes of leukemic BRAFWT and BRAFVE TCL1 transgenic mice...... 77 Figure 3. 4 Phenotypes of leukemic BRAFWT and BRAFVE TCL1 transgenic mice...... 78 Figure 3. 5 Cell populations in transgenic mice prior to leukemia onset...... 81 Figure 3. 6 Spleens of transgenic mice at two months of age...... 82 Figure 3. 7 Histology of mice engrafted with leukemia cells from BRAFVE TCL1 mice. 83 Figure 3. 8 Effect of BRAFV600E on cell proliferation and apoptosis in vivo...... 84 Figure 3. 9 Immunosuppressive effect of BRAFV600E in B cells in vitro...... 86 Figure 3. 10 Cytokine analysis in vitro...... 93 Figure 3. 11 Immunosuppressive effect of BRAFV600E in vivo...... 94 Figure 3. 12 Mice received BRAFWT or BRAFVE leukemia cells by adoptive transfer (single donor)...... 97

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List of Abbreviations

ALL acute lymphoblastic leukemia

AML acute myeloid leukemia

APC antigen-presenting cells

AT adoptive transfer

BAD Bcl-2-associated death promoter

BAFF B-cell activating factor

BCL-2 anti-apoptotic B-cell lymphoma 2

BCL-XL B-cell lymphoma-extra large

BCR B-cell Receptor

BTK Bruton's tyrosine kinase

BTLA B- and T-lymphocyte attenuator

CBA cytometric bead array

CBC complete blood count

CBF core-binding factor

CFSE carboxyfluorescein succinimidyl ester

CLL chronic lymphocytic leukemia

CSR class switch recombination

CTLA4 cytotoxic T-lymphocyte-associated protein 4

DLBCL diffuse large B-cell lymphoma xiv

DUSP6 dual-specificity phosphatase 6

EGFR epidermal growth factor receptor

ELK1 ETS domain-containing protein

ERK extracellular signal-regulated kinases

ETS1 ERK1/2-responsive E-twenty-six 1

FBS fetal bovine serum

FGFR1 fibroblast growth factor receptor 1

FRS2 fibroblast growth factor receptor substrate 2

GRB2 growth factor receptor-bound protein 2

GSK-3 glycogen synthase kinase 3

H&E hematoxylin and eosin

HCL hairy cell leukemia

IκB phosphorylation of inhibitor of κB

Ig immunoglobulin

ITAM immunoreceptor tyrosine-based activation motif

LPS lipopolysaccharide

LYN Lck/Yes novel tyrosine kinase

MAPK mitogen-activated protein kinase

MCL mantle cell lymphoma

MCL-1 myeloid cell leukemia 1

MDA melanoma differentiation antigens

MDR multi-drug resistance

MDSC myeloid-derived suppressor cells

MKP MAPK phosphatases

MZ marginal zone

NFκB nuclear factor kappa-light-chain-enhancer of activated B

cells

NLC monocyte-derived nurse-like cells

P-gp P-glycoprotein

PD-L1 programmed death-ligand 1

PD-1 programmed cell death protein 1

PLCγ2 phosphorylates phospholipase C γ 2

PROTAC proteolysis targeting chimera

RAG recombination activating gene

RSSs recombination signal sequences

SHM somatic hypermutation

SOS Son of Sevenless

SPRY sprouty proteins

SYK spleen tyrosine kinase

TAM tumor-associate macrophages

TCR T cell receptor

TLRs Toll-like receptors

TIM-3 T-cell immunoglobulin and mucin-domain containing-3

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Treg regulatory T cell

VEGF vascular endothelial growth factor

WBC white blood cell

XID X-linked immunodeficiency

XLA X-linked agammaglobulinemia

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Chapter 1

Introduction

1.1 B-cell development, B-cell Receptor (BCR) Signaling, and Leukemia

Hematopoietic stem cells residing in the bone marrow have self-renewal capability and can branch to the common lymphoid progenitors and the common myeloid progenitors.

These progenitors then commit to lymphoid lineage and myeloid lineages, respectively.

Lymphocytes include B cells, T cells (small lymphocytes), and natural killer (NK) cells

(large lymphocytes). NK cells and leukocytes from myeloid lineage including monocytes

(can further differentiate to macrophages and dendritic cells) and granulocytes (neutrophil, eosinophils, basophils, and mast cells) are the main cell types in the innate immune system, which fight against infection in a generic manner while B cells and T cells function in the adaptive immune system, which specifically responds to pathogens and can create memory cells for long-term protection.

B cells develop structurally along with serial immunoglobulin (Ig) rearrangements in the bone marrow and then mature in the spleen (Figure 1.1). The first rearrangement of

D (diversity) and J (joining) gene segments of the heavy chain occurs in the Pro-B cells, followed by a second recombination of a V (variable) gene segment, upstream of D and J segments, with the newly rearranged DJ segment to form a VDJ gene segment. VDJ 1 recombinases including recombination activating genes 1 and 2 (RAG1 and RAG2), artemis nuclease and terminal deoxynucleotidyl transferase (TdT). The RAG1/2 complex recognizes the recombination signal sequences (RSSs), a spacer composed of 12 base pairs or 23 base pairs, a conserved heptamer (CACAGTG), and a conserved nonamer

(ACAAAAACC), flanking the V, D, and J gene segments and follows the 12/23 rule to maintain the specificity of this process1,2. At this stage, B cell marker CD19 and surface antigen CD43 are expressed on Pro-B cells. More surface antigen information of B-cell subsets is shown in Figure 1.1. The assembly of the rearranged µ heavy chain and surrogate light chains (Vpre-B and lambda 5) in the Pre-B cell receptor (Pre-BCR) on the membrane differentiates Pre-B cells. Undergoing proliferation and rearrangement of V and J gene segments of the light chain through Pre-BCR signaling, Pre-B cells combine the heavy chain with the k or l light chain to form membrane-bound IgM and become immature B cells. These immature B cells go through a negative selection in the bone marrow, where

B cells with the strong binding affinity of B cell receptor (BCR) to self-antigens will undergo anergy, receptor editing or programmed cell death (clonal deletion), whereas B cells that do not bind self-antigens express IgD and migrate from the bone marrow to the spleen to finalize maturation into either marginal zone (MZ) B cells or follicular (FO) B cells depending on signals received through BCR, B-cell activating factor (BAFF) receptor, and cytokine receptors. Once B cells encounter antigen stimulation from T helper cells, which usually occurs in germinal centers of secondary lymphoid organs, they undergo class switch recombination (CSR) and somatic hypermutation (SHM) and further differentiate to plasma cells or memory B cells in order to release different antibodies against antigens.

The BCR is a transmembrane multiprotein complex that consists of a membrane immunoglobulin for antigen binding and a signal transducing complex, the Iga and Igb heterodimer. The disulfide-bound Iga and Igb contains an immunoreceptor tyrosine-based activation motif (ITAM) in their cytoplasmic tails to transduce signals upon BCR engagement. BCR signaling (Figure 1.2), including the upstream factor Bruton's tyrosine kinase (BTK), plays a pivotal role in regulating B cell development, survival and proliferation. BTK, in addition to TEC, ITK, TXK/RLK and BMX/ETK, is a non-receptor tyrosine kinase belonging to the TEC family kinases, the second largest family of non- receptor tyrosine kinases. BTK is predominantly expressed in B cells but not in plasma cells, and also is expressed in other hematopoietic cells except T lymphocytes3-7. Several reports have shown that expression of BTK is required for B-cell proliferation, development, survival, and apoptosis8-10. A loss-of-function BTK mutation even causes disease in humans. This disease, X-linked agammaglobulinemia (XLA), is a primary immunodeficiency disease characterized by block of B cell development at pre-B stage, with a resulting failure to generate immunoglobulins of all classes. Therefore, XLA patients are unable to develop humoral immune responses11,12. Btk-deficient mice, including the X-linked immunodeficiency (XID) strain and mice with a targeted disruption of the Btk gene, show mild phenotypes relative to the human XLA condition. However, impaired differentiation and survival of mature B cells and reduced B cell numbers in spleen and lymph nodes are observed in these Btk-deficient mice13. In B cells, BTK functions are based on the interactions between five domains (PH, TH, SH3, SH2 and

3 kinase domain) of BTK and a variety of factors that are critical for diverse intracellular signaling.

The BCR/BTK signaling starts following engagement of antigen and BCR, and Src family kinases, mostly Lck/Yes novel tyrosine kinase (LYN), are consequently recruited to phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) of Igα and

Igβ. The activated ITAMs serve a docking site for spleen tyrosine kinase (SYK) binding, and LYN and SYK subsequently phosphorylate BTK at residue Y551 in the kinase domain after BTK translocates to the cell membrane through the interaction of PIP3 and its PH domain. Then residue Y223 in the SH3 domain is autophosphorylated, and activated BTK further phosphorylates phospholipase C γ 2 (PLCγ2) and transactivates the mitogen- activated protein kinase (MAPK), nuclear factor kappa-light-chain-enhancer of activated

B cells (NFκB), and AKT signaling pathways. BTK activity can be negatively regulated by phosphatases PTEN and SHIP1, which dephosphorylate PIP3 to thereby inhibit BTK membrane association13. These downstream effects of BCR/BTK usually modulate transcription factors that regulate cell cycle, cell proliferation, differentiation, and apoptosis. For example, extracellular signal-regulated kinases (ERK) activation can phosphorylate myeloid cell leukemia 1 (MCL-1) in the cytosol to prevent cell apoptosis, and also phosphorylate transcription factors ETS domain-containing protein (ELK1) and c-MYC in the nucleus to regulate gene expression involved in cell proliferation.

Phosphorylation of inhibitor of κB (IκB) allows NFκB to translocate into the nucleus as a transcription factor to modulate gene expression for cell proliferation and cell survival.

Phosphorylation by AKT will sequester Bcl-2-associated death promoter (BAD) from B-

4 cell lymphoma-extra large (BCL-XL), an anti-apoptotic B-cell lymphoma 2 (BCL-2) family protein, thereby inhibiting cell apoptosis. Additionally, AKT activation inhibits glycogen synthase kinase 3 (GSK-3) activity, which results in cell cycle dysregulation.

In addition to normal B cell development, BCR signaling is also believed to be crucial for the initiation and maintenance of certain B cell malignancies such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). Thus, targeting components of the BCR signaling pathway has provided a new therapeutic strategy in these B-cell malignancies. Several agents have been developed to target BCR pathway components, including inhibitors of SYK, LYN, BTK,

PI3K, AKT, PKCβ, and MEK14,15. As a key component in the BCR pathway, BTK is an attractive target for drug design. This is evidenced by the success to date of ibrutinib, an irreversible inhibitor that covalent binds to residue C481 to inhibit BTK kinase activity.

Besides CLL and MCL for which it is approved, ibrutinib also displays promising activity and tolerability in hairy cell leukemia (HCL), some subtypes of DLBCL, and multiple myeloma in clinical phase 1 and 2 studies16-22.

CLL, the most prevalent adult leukemia in the Western world and mainly seen in older people, results from the uncontrolled accumulation of B cells in the bone marrow, secondary lymphoid organs, and peripheral blood to impact healthy blood cells, but the exact causes of CLL is still uncertain. Symptoms of patients with advanced CLL include enlarged liver and spleen, high white blood count (WBC, >5000/µl)23, and impaired immunity. The malignant B cells are dysfunctional and also impact normal blood production, leading to increased risk for infection. Due to the compromised immunity,

5 infection becomes one of the major causes of death in leukemia. The immunophenotype of

CLL cells includes expression of CD5, CD19, CD20, CD23 and CD79b23,24. The Rai25 and the Binet26 clinical staging systems (Table 1.1) are commonly used in CLL to define disease stages and predict the risk by presence of organomegly and cytopenia. Prediction of disease progression and response to treatment also rely on the mutational status of IgVH, expression of CD38 and ZAP-7027-29 and other genetic abnormalities such as deletion of

13q14, deletion of 11q22, deletion of 17p13, and trisomy 1230-32. CLL with unmutated

IgVH, expression of CD38, expression of ZAP-70, or unfavorable gene aberrations

(deletion of 11q, deletion of 17p) is associated with rapid disease progression and shorter survival, while CLL with mutated IgVH or favorable gene aberration (deletion of 13q) shows slow progression and longer survival28,33-37. Other than ibrutinib, current treatments for this disease include chemotherapy (such as fludarabine and cyclophosphamide), targeted therapy (such as rituximab, idelalisib, venetoclax) and stem cell transplant.

Although these therapies control disease progression, resistance or relapse occurs and immune deficiency remains a serious clinical problem.

1.2 Activated MAPK Signaling in B-cell Malignancies

As stated above, one of the signaling pathways downstream of BCR/BTK that regulates B-cell lymphopoiesis and functions is the MAPK pathway38-40, composed of

RAS, RAF, MEK and ERK as key effectors. In addition to the BCR, growth factor receptors, cytokine receptors, and G-coupled protein receptors mediate signals through

MAPK pathway as well, and the RAF protein kinases function as both an effector and an activator to transduce signaling in this pathway. The RAF kinase family consists of 6 serine/threonine-specific protein kinases and have three members, CRAF (RAF1), ARAF, and BRAF, which were discovered in 1985, 1986 and 1988 respectively. Conserved regions (CR) 1, 2, and 3 are shared in each RAF member. CR1 contains a RAS-binding domain and a cysteine-rich domain. CR2 (hinge region) is a serine/threonine rich domain that can be bound by 14-3-3 to keep RAF in an inhibitory state. CR3 is the protein kinase domain, composed of a small lobe at N terminal and a large lobe at C terminal. The small lobe contains a glycine-rich ATP binding loop (also called P-loop) and the large lobe interacts with MEK, a direct downstream effector of RAF kinases41. The catalytic site of the RAF protein kinase locates in the cleft between the two lobes. Upon activation, BRAF shows the highest kinase activity, and ARAF has the lowest kinase activity42-45. All three

RAF proteins function as homo- or heterodimers, and the BRAF/CRAF heterodimer has higher kinase activity relative to other RAF dimers46,47. Although with different expression levels depending on tissue types, all three RAF proteins are ubiquitously expressed48-50 and generally, RAF monomer exists as an autoinhibitory (closed) state by binding of 14-3-3 dimer to the hinge region.

Upon activation, RAS kinase (RAS-GTP) binds to the RAS-binding domain in the

CR1 region of RAF protein and recruits the RAF protein to the membrane48, the 14-3-3 dimer is dephosphorylated by phosphatases such as PP1 and PP2A, and the closed RAF is now opened but still not activated yet. The opened RAF immediately dimerizes with another RAF monomer at the cell membrane, usually forming BRAF and CRAF heterodimers and resulting in a partially active conformation. In turn, the two linked RAF kinases phosphorylate each other at their catalytic site to become a fully active RAF protein

7 kinase51. The signal cascade continues with the activated RAF kinase phosphorylating its direct effectors, the dual-specificity enzymes MEK1 and MEK2, at serine residues. MEK1 and MEK2 then transactivate ERK1 and ERK2 at both threonine and tyrosine residues (the conserved threonine-glutamate-tyrosine motif) in their activation loop. The activated

ERK1/2 kinases enter into the nucleus to regulate gene expression via modulating the activities of transcription factors such as c-MYC, ELK1, and AP1, but also keep phosphorylating additional downstream substrates in the cytoplasm that control cell proliferation, survival, differentiation, and protein translation.

On the other hand, the activated ERK protein kinases act as feedback regulators for the MAPK signaling cascade, which regulate most components in the MAPK pathway through phosphorylation at different residues on the key activators in this signaling pathway. For example, activated ERK kinases further phosphorylate and/or interrupt the dimerized structure of the growth factor receptors such as the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) to reduce the activation level of receptor kinases52-55. The ERK protein kinase-dependent hyperphosphorylation of the Son of Sevenless (SOS), a guanine nucleotide exchange factor, not only disassociates the binding with Growth factor receptor-bound protein 2 (GRB2), an adaptor protein, but also reduces SOS membrane localization56-61, resulting in an inactive state of RAS kinase.

In addition, the activated ERK-mediated feedback loops disrupt the association between

RAS and BRAF or CRAF and delocalize the RAS kinase from the plasma membrane56,62-

64. Also, this negative regulation disrupts the dimerization of BRAF and CRAF or KSR1 kinase, and also reduces kinase activity of MEK protein as well as BRAF protein at the

8 unique C-terminal region of BRAF upon BCR engagement64-68. ERK protein kinases are fully activated upon phosphorylation of threonine and tyrosine residues in the activation loop, thus another negative MAPK regulation mechanism by which the activated ERK protein kinase activity is suppressed is by protein phosphatases69. One of the protein phosphatases that specifically modulate MAPK signal cascade is dual-specificity MAPK phosphatases (MKP), expression of which is transcriptionally increased by MAPK signal activation. These factors in turn dephosphorylate and deactivate ERK kinases70,71. For instance, the expression of fibroblast growth factor-induced dual-specificity phosphatase 6

(DUSP6) is mediated by direct binding of the ERK1/2-responsive E-twenty-six 1 (ETS1) transcription factor on the DUSP6 promoter, leading to feedback deactivation of ERK1/2 and potentially delaying ERK1/2 reactivation via anchoring them in the nucleus or cytoplasm72-75. In addition, the sprouty proteins (SPRY) are transcriptionally induced by

MAPK signaling76-78. Phosphorylated SPRY1 and SPRY2 generate a SH2 domain binding site for GRB2, which disrupts the binding of GRB2 with the adaptor protein Fibroblast growth factor receptor substrate 2 (FRS2), to in turn deactivate RAS. SPRY-mediated

MAPK signaling feedback inhibition also functions via binding with CRAF79-81. Taken together, the MAPK signaling pathway plays an important role in regulating many diverse factors of cell biology with appropriately complex layers of feedback regulation.

Mutations that activate the MAPK pathway have been identified in many types of solid tumors. Activating mutations of RAS kinases typically occur at amino acid 12, 13,

6182 with the prevalence of K-RASG12 in 69-95% of pancreatic cancer, 40% of all colorectal carcinoma83-85, N-RASQ61 in 20-30% malignant melanoma86,87 and H-RASG12 in 12% of

9 bladder cancer88,89. In hematologic malignancies, N-RASQ61 is present in approximately

12% of patients with acute myeloid leukemia (AML), and both K-RAS and N-RAS mutations have been detected in multiple myeloma90. Another frequently mutated MAPK component is BRAF protein kinase. Following the first documentation of mutant BRAF in

2002, it was subsequently shown that many types of solid tumors carry a somatic BRAF mutation that results in constitutive activation of MAPK signaling and cell transformation; most are activating mutations and result in stimulation of the MAPK pathway91. Among the distinct point mutations of the BRAF kinase domain that have been identified, a mutation altering amino acid 600 from valine to glutamate (V600E) in the activation segment of the kinase domain (resulting from a transversion of thymidine to adenosine at the nucleotide 1799, T1799A) shows the highest incidence. BRAFV600E is present in 40-

70% of malignant melanomas, 45% of papillary thyroid cancer, 10% of colorectal cancer, as well as lower percentages in a variety of other cancers91-95. In addition, recent reports show that almost 10% of patients with CLL, the most common type of adult leukemia, carry an activating mutation in a MAPK pathway component including RAS, BRAF, and

MEK96-98. BRAF mutations were also identified as one of the acquired initiating mutations in early hematopoietic cells of CLL patients, leading to deregulation of BCR signaling99.

Importantly, nearly 100% of classic HCL cases show the BRAFV600E mutation100, suggesting a key function for the mutated protein in the development of disease. Multiple reports now demonstrate that HCL can be successfully treated with vemurafenib, a selective BRAFV600E inhibitor, further supporting this hypothesis101. Furthermore, a BRAF pseudogene transcript is aberrantly expressed in primary human diffuse large B-cell

10 lymphoma and is positively correlated with BRAF expression, resulting in MAPK signaling activation. Global expression of this pseudogene in mice results in aggressive B- cell lymphoma102. Together, these findings indicate a clear role for activated MAPK in a significant subset of B-cell malignancies.

To block this constitutive activation, the selective BRAFV600E inhibitors vemurafenib and dabrafenib were developed. These two drugs, approved by the FDA in

2011 and 2013 respectively, show excellent clinical responses in many cases of malignant melanoma. However, responses are short-lived (progression-free survival, PFS, is approximately seven months) with relapse occurring in nearly every melanoma case, suggesting the existence of intrinsic BRAF inhibitor resistance103-105. Also, only 5% response rate was seen in colorectal cancer patients with BRAFV600E 106, suggesting there exists different intrinsic resistance in cancers. In addition, emerging data also indicate that

BRAFV600E HCL patients relapse following vemurafenib treatment that was initially effective107. Interestingly, it was recently reported that a patient with BRAFV600E-driven melanoma who responded to vemurafenib developed CLL-like disease, possibly due to paradoxical BRAF inhibitor-associated ERK activation in B-cells via the

BCR/SYK/RAS/RAF axis108. ERK is also a key downstream effector of the BCR pathway, and inhibition of this pathway by ibrutinib leads to loss of ERK phosphorylation both in vitro109 and in samples from ibrutinib-treated patients110. Thus, in B-cells, ERK is a point of convergence of the MAPK pathway and the critical BCR pathway, further supporting the relevance of MAPK signaling in B-cell malignancies. Despite the prevalence of

BRAFV600E in HCL and the importance of mutant BRAF in leukemia development, and potentially its treatment, the functional role of BRAF in mature B-cells remains unclear.

1.3 MAPK Signaling and Immune Suppression

Malignant B cells not only have impaired B-cell function but also impact on the tumor microenvironment, where tumor cells interact with non-malignant cells such as stromal cells, immune cells, endothelial cells, and monocyte-derived nurse-like cells

(NLC), through secretion of cytokines, chemokines, growth factors, and cell-contact by surface antigens expression111. B-cell tumors reprogram the cells in tumor environment to evade immune surveillance, where these dysfunctional B cells dampen cytotoxic T cell activation, increase regulatory T cell (Treg) population, repress NK cell and dendritic cell function, induce tumor-associate macrophages (TAM) and myeloid-derived suppressor cells (MDSC), and receive growth/survival signals from stromal cells, endothelial cells,

NLCs112.

In healthy human subjects, upon engaging with ligands, the immune checkpoint molecules transduce signals that negatively regulate immune and inflammatory reactions and prevent autoimmune responses; however, the expression of these molecules are elevated on tumor cells and/or their surrounding cells in order to escape from immune surveillance. Therefore, blockade of immune checkpoints becomes attractive targeted therapies (Figure 1.3). Emerging evidence demonstrates the clinical benefits of immune checkpoint blockade in variety of cancers based on the fact that these immune checkpoint inhibitors restore immune functions against tumor cells. Such inhibitors include the anti- cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies ipilimumab and 12 tremelimumab, anti-programmed cell death protein 1 (PD-1) antibodies nivolumab, pidilizumab, and pembrolizumab, and anti-programmed death-ligand 1 (PD-L1) antibodies atezolizumab and avelumab113-118. CTLA4, also known as CD152, is mainly expressed on

T cells and functions as a feedback regulation of T cell responses. When the T cell receptor

(TCR) and the co-stimulator CD28 are activated, CTLA4 is then transported to the membrane to compete for the shared ligands CD80 (B7.1) and CD86 (B7.2) with CD28119-

121. This engagement induces an inhibitory signal to down-regulate T cell activation by recruiting phosphatase SHP2 to inhibit the activation of TCR and CD28 signaling.

Additionally, this interaction reduces CD80/86 expression on the antigen-presenting cells

(APC) by removal of CD80/CD86 and trans-endocytosis of the CTLA4-CD80/CD86 complex that further reduce the CD28 co-stimulatory signal122. Oppositely, the expression of CTLA4 enhances Treg immune suppressive function123. The hematologic clinical relevance of CTLA4 is that both membrane and intracellular CTLA4 expression of T cells in CLL patients are higher compared to healthy human subjects, and its expression is associated with disease stage and genetic alteration124. Another immune checkpoint expressed on activated T cells is PD-1 (CD279), transcriptionally increased by TCR signaling activation. Similar to CTLA4, PD-1 inhibits T cell activation after binding of its ligands PD-L1 or PD-L2 by inducing an inhibitory signal with recruitment of phosphatases

SHP2 and PP2A to TCR signaling but not interfering the CD28 co-stimulating signaling125.

In addition to activated T cells, PD-1 is also expressed on B cells and NK cells, where the engagement of PD-1 and its ligands induces inhibitory signals to these cells as well. In contrast, this engagement activates Treg and MDSC function126-128. PD-L1 (CD274, B7-

H1) and PD-L2 (CD273, B7-DC) are expressed on tumor cells and can be induced on the surrounding cells by IFN-g secreted from activated T cells and NK cells129. Moreover, PD-

L1 and CD80 can be binding partners to inhibit T cell proliferation and cytokine production130. Together the activation of the PD-1 and PD-L1/PD-L2 axis affects the function of a wide range of immune cells, further compromising patients’ immunity.

CD200, also known as OX2, is another potential immune checkpoint target in leukemia. It is normally expressed on B cells, T cells, neurons, some dendritic cells and endothelial cells but also commonly expressed on CLL cells131,132 and a subset of AML cells associated with core-binding factor (CBF) abnormalities. CD200 induces an inhibitory signal upon binding to the CD200 receptor (CD200R), expression of which is limited to myeloid cells, mainly macrophages and dendritic cells, and a subset of T cells.

In leukemia, the expression of CD200 is associated with worse survival133 and has been demonstrated to induce Treg numbers and attenuate T cell and NK cell function. These immunosuppressive effects can be partially reversed by an anti-CD200 neutralizing antibody133-137. A more recent study shows that the expression of the immunosuppressive ligand PD-L1 and CD200 are linked in AML138, identifying a new therapeutic target for

CD200 blockade. The expression of immune checkpoint molecules and immunosuppressive cytokines in the tumor microenvironment contributes to severe immune suppression seen in patients with leukemia. Aside from leukemia-induced immune suppression, the further enhanced MAPK activation in malignant B cells may suppress patients’ immunity to a greater extent.

The MAPK pathway regulates cell proliferation, differentiation, survival, and senescence following external signals and is commonly aberrant in tumor cells139. Because mutated MAPK pathway components can be active in the absence of external stimuli, they can constitutively signal to activate transcription and translation factors that subsequently drive tumor survival and proliferation. Inhibitors of MAPK factors are being aggressively pursued to combat these effects, such as the BRAFV600E inhibitors vemurafenib and dabrafenib and the MEK inhibitor trametinib103,104,140-142. In addition, MAPK signaling can induce immune-suppressive behavior in tumor cells via several mechanisms such as cytokine production, cytoskeletal remodeling, and induction of immune checkpoint molecules.

For example, Toll-like receptors (TLRs) function via MAPK to regulate production of immune suppressive cytokines such as IL-10 and MCP-1 (CCL2) via AP-1 and IRF transcription factors143-145. MAPK pathway activation in melanoma cells without MAPK activating mutations, such as through TLR stimulation or TGF-β signaling, can promote tumor infiltration and immune suppression at least in part via CD200 and PD-L1 induction, and these effects can be reversed by MEK inhibition146-149. Similar findings are reported for BRAFV600E mutant melanoma, where inhibition of BRAF and MEK increases dendritic cell function and T-cell recognition150,151. In vitro studies based on co-culture of monocyte- derived dendritic cells induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid with polylysine and carboxymethylcellulose (poly-ICLC) with cell-cell contact or with supernatant from melanoma cells carrying the BRAFV600E mutation reveal that inhibition of MAPK activation using inhibitors or RNAi against BRAFV600E restores IL-12

15 and TNFa secreted by dendritic cells and/or abrogates the secretion of activated MAPK pathway-induced immunosuppressive cytokines IL-6, IL-10, and vascular endothelial growth factor (VEGF) from melanoma cells 150,152. This resulted in reduced recruitment of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) in the tumor microenvironment. The same group also found that the BRAFV600E melanoma co-cultured dendritic cells decrease CD80, CD83, and CD86 expression, which can be partially reversed by BRAF inhibition150. The restoration of CD80 and CD86 presented on dendritic cells may contribute to T cell activation. Moreover, Boni et al. demonstrated that BRAF and MEK inhibition increases the expression of melanoma differentiation antigens (MDA), including gp100 and MART-1, on melanoma cells, to be better recognized by TCR- transgenic cytotoxic T cells specific for gp100 and MART-1 and subsequently induce IFN- g secretion151. Another group showed that melanoma cells increase IL-1α and IL-1β secretion upon transduction of BRAFV600E. These cytokines suppress melanoma-specific cytotoxic T cells through the upregulation of COX2, PD-L1, PD-L2 on fibroblasts153. In addition, knock-down of CD200, induced by BRAF/MAPK cascade, with shRNA in melanoma cells enhances the dendritic cells-induced T cell activation via reducing the

CD200 receptor signals received in dendritic cells 147. Moreover, T cell function is improved as evidenced by increased of IFN-g and intra-tumor lysis upon MAPK inhibition in adoptive transfer animal models of pmel-1 and OVA TCR-transgenic T cells with

BRAFV600E SM1 melanoma154,155. Vemurafenib treatment in patients with advanced melanoma increases CD8 T cell infiltration and decreases immunosuppressive cytokines

IL-6 and IL-8. However, inhibition of BRAFV600E elevates the expression of T-cell

16 exhaustion markers T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and

PD-1 as well as the immunosuppressive ligand PD-L1, suggesting that combined therapies of BRAF and immune checkpoint molecules inhibition are needed for optimal efficacy156,157.

The above evidence strongly supports that MAPK signaling leads to immunosuppression via cytokine secretion and immune checkpoint molecule expression, and that targeting this pathway could reverse tumor-induced immune suppression and lead to longer disease control as well as improved resistance to opportunistic infections in cancer patients. However, despite the prevalence of mutation mediated activation of BRAF in some B-cell malignancies, its function with regard to immune suppression in this context is unclear.

1.4 Animal Models of B-cell Leukemia with MAPK Activation and Immune Suppression

Animal models are essential to elucidate a pathological role of a candidate gene in disease progression and assess a drug’s efficacy and toxicity before human clinical trials, and also to provide an opportunity to investigate pharmacokinetics – not only of each agent individually, but whether pharmacokinetic properties of one agent in a combination are affected by the other. Furthermore, animal models allow the hypothesized mechanism of action, and potentially of synergy, to be validated and correlated with efficacy.

A well-established mouse model of human CLL, Eµ-TCL1, was described by Bichi et al. in 2002 and has been widely utilized in CLL research158. TCL1, a proto-oncoprotein, is not typically found in mature B cells but is highly expressed in human-derived B cell lines and primary malignant B cells. Based on this, a transgenic mouse model carrying the 17 human TCL1 gene under control of a VH promoter and IgH-Eµ enhancer, driving the expression of TCL1 in immature and mature B cells, was generated to study its pathologic role in B-cell malignancy. The Eµ-TCL1 mouse model is well-described to spontaneously develop a CLL-like disease at 8 to 12 months of age, characterized by clonal expansion of

IgM+/CD5+ cells in peripheral blood, spleen, bone marrow, and lymphoid tissues, organomegaly associated with high white blood cell (WBC) count and tumor-mediated immune suppression158-161, demonstrating the importance of TCL1 pathway in CLL development. Importantly, this is also an established model for drug development as well as biological and immune suppression studies related to human CLL159,162. John Gribben’s group identified T-cell defects with gene and protein alterations in Eµ-TCL1 transgenic mice, resembling what is seen in human CLL. These changes include increased expression of CTLA4 and decreased expression of CD28 and B- and T-lymphocyte attenuator

(BTLA). As disease develops, Eµ-TCL1 mice show diminished mitogen-induced T cell proliferation, antigen-specific T-cell activation, and T-cell cytotoxicity toward CLL cells, as well as increases in a subset of Treg with CTLA4 expression. In addition, increased T- cell cytokines IL-1, IL-4, IL-6 and decreased IL-2, IL-12b, and IFN-g are seen in the mice with leukemia (data not shown in the publication). Moreover, impaired F-actin polymerization is seen at the synapse between T cells and CLL cells in the Eµ-TCL1 mice.

These T-cell defects can be at least partially repaired by the immunomodulatory agent lenalidomide and/or blockade of PD-1/PD-L1 activation159,163. In addition to T-cell defects, skewing of myeloid population toward to immunosuppressive phenotype is seen in the Eµ-

TCL1 transgenic mice164. Hanna et al. show that leukemic Eµ-TCL1 mice accumulate 18 monocytes and CD11bintF4/80int macrophages with M2-like phenotypes in the peritoneal cavity, compared to CD11bhiF4/80hi resident macrophages in the wild-type control mice.

Other studies describe that the CD11bintF4/80int macrophages are recruited to the peritoneal cavity in response to inflammation and are known to increase MHC-II expression in Eµ-

TCL1 mice165,166. The M2-macrophage skewing phenotypes are confirmed by elevated M2 markers arginase 1, CD206 (mannose receptor) and CD124 (IL-4Rα), reduced expression of CD86, higher expression of PD-L2, and stronger response to IL-10 and IL-4 stimulation, resulting in higher STAT3 and STAT6 phosphorylation. While inflammatory monocytes

(Ly6ChiCD43low) are the major subset in control wild-type mice, the leukemic cells lead to the accumulation of patrolling monocytes (Ly6ClowCD43hi) and reduction of macrophage numbers and MHC-IIhi dendritic cells in the spleen of Eµ-TCL1 mice. In addition, PD-L1 expression is increased on the dendritic cells as well as both inflammatory and patrolling monocytes in healthy mice that received TCL1 leukemia cells via adoptive transfer (AT).

The TREM-1 (triggering receptor expressed on myeloid cells 1)-associated genes (Itgb1,

Il-10, Stat3, Nfkb2, Tlr2 and Tnf) and cytokines (Il-10, Cxcl9, Il-1b, Cxcl10, Tnf, Il-1rn and Cxcl16) are on the top upregulated genes in the TCL1 AT monocytes, indicating that

TREM-1 regulates inflammation in this model164. Taken together, besides T-cell defects, the immunosuppressive myeloid cells with expression of PD-L1 and inflammatory cytokines further contribute to leukemia-induced immune suppression in the Eµ-TCL1 transgenic mice.

Since the establishment of the Eµ-TCL1 human CLL model, many transgenic mouse models have become available to study other crucial players in CLL development

19 by crossing the Eµ-TCL1 transgenic mouse with other transgenic mice carrying genes involved in B-cell development, proliferation, survival, and apoptosis. Such transgenes include APRIL, BCL-2, TP53, ROR1, CD44, XID, miR-15a, miR-16, miR-29. However, none of the resulting strains harbors a B-cell specific MAPK activating mutation in order to address the role of enhanced MAPK activation in B-cell malignancy development. Due to the high frequency of BRAFV600E mutation in cancers, conditional allele BRAF transgenic mice (BRAFCA) were generated to allow tissue-specific expression, at the normal physiologic level of BRAFV600E. A target vector carrying the loxP-flanked mouse exons 15-18 and followed by a modified exon 15 encoding V600E was used to generate this strain by homology recombination in the embryonic stem cells. These mice carry a silent mutated BRAF sequence that becomes expressed under mouse naïve promoter following cre recombinase-mediated cleavage of the loxP-flanked normal Braf sequence and the physiological expression level of BRAFV600E recapitulates the situation seen in human cells, where most patients have one copy of normal BRAF converted to V600E167.

This strain has been used in many tumors with the prevalence of BRAFV600E mutation to study the impact of this mutation in different contexts, such as in the lung by intranasal instillation of adenoviral Cre167, in melanocytes by inducible Tyr:Cre-ERT2 mice168-170, in hematopoietic cells by inducible Mx1-Cre strain171,172, in epithelia of the small and large intestine by Villin-Cre mice173.

We employed the BRAFCA strain to study the contribution of mutant BRAF to B- cell cancer. As will be described in Chapter 3, transgenic mice with B-cell restricted

BRAFV600E expression, resulting from the cross of BRAFCA mice with CD19-cre transgenic

20 mice, do not exhibit signs of disease by the age of one year, suggesting that a second hit is needed to induce disease development. In contrast to this B-lineage specific expression model, Chung et al. explored a mouse model carrying postnatal hematopoietic stem cell- specific BRAFV600E expression172, based on the hypothesis that the HCL cells with

BRAFV600E mutation originate from hematopoietic stem cells. To generate these mice, the conditional BRAF transgenic mouse and the Mx1-cre transgenic mouse were crossed to express BRAFV600E mutation in pan-hematopoietic stem cells. Hematopoietic cell-specific

BRAFV600E expression results in splenomegaly and hepatomegaly due to extramedullary hematopoiesis, anemia due to impaired erythroid differentiation, and thrombocytopenia by three weeks of age, and the sub-lethally irradiated NSG mice with the whole bone marrow transplant from Mx-1 cre mice show increase of spleen weight, liver weight, and a HCL marker soluble CD25172. However, no specific B-cell malignancies were described in the

Mx-1 cre BRAFV600E transgenic mice. Additionally, the Mx1-cre induced BRAFV600E expression leads to death within four weeks of age with proliferative disorder characterized by amplification of circulating histiocytes/macrophages171. However, mice with ubiquitously expressed and prenatal hematopoietic stem cell-expressed BRAFV600E by

CMV-cre and Vav-cre induce embryonic lethality before embryonic day 7.5 and beyond day 12.5, respectively171,172. Interestingly, BRAF deficiency also leads to embryonic death174, suggesting that BRAF is not redundant and the balance of MAPK activation is important in gestation. The Mx1-cre BRAFV600E bone marrow adoptive transferred mice show some phenotypic characteristics of HCL; however, neither B-cell disease is described nor B-cell morphology changes are seen in these mice. This indicates that Mx-1 cre

BRAFV600E is not an ideal model to study B-cell malignancies and suggests that BRAFV600E needs to cooperate with other genetic alterations to produce HCL. Therefore, there is currently no appropriate mouse model available to address the pathologic role of

BRAFV600E specifically in B-cell malignancy.

1.5 Conclusion and Hypothesis

Tumor-mediated immune suppression still remains a serious clinical problem in both solid tumors and hematological malignancies. More and more studies focus on the mechanisms of tumor-induce immune suppression and development of combination therapeutic strategies that decrease tumor burden and simultaneously enhance immunity in patients. The mechanisms of immune dysfunction in cancers are believed to occur via the increased immune suppressive cytokines secretion, immune checkpoints expression, cytoskeletal remodeling. In addition, the activated MAPK signaling pathway is evident to drive immune suppression in solid tumors, but whether the enhanced MAPK signaling contributes to further immune suppression and/or disease progression remains largely unknown in hematologic malignancies.

BRAF mutations occurs in approximately seven percent of cancers91, and these activating mutations constitutively activate the MAPK pathway to enhance tumorigenesis.

Activating mutations of MAPK components are not as common in hematologic malignancies as in solid tumors, but involve 10% or more of patients with B-cell malignancies including CLL, and nearly 100% of classic HCL. In addition, next-generation sequencing identifies BRAF mutations in the hematopoietic stem cells from CLL patients99, suggesting that BRAF may be involved in the development of B-cell cancers. 22

Moreover, the MAPK pathway is one of the BCR/BTK downstream signals to regulate B- cell development, survival and tumor growth. However, the role of mutated BRAF still remains uncertain in B-cell malignancies.

Current treatments for B-cell malignancies control tumor growth to some extent, but the immune deficiency leads to one of the main causes of death. It has been demonstrated that the enhanced MAPK activation in melanoma induces immunosuppressive cytokines such as IL-6, IL-8, IL-10, VEGF and immune checkpoint molecules such as PD-L1, CD200 in order to skew the tumor microenvironment toward immunosuppressive phenotypes. These observations bring up the question of whether the constitutive MAPK activation in B-cell tumors further enhances the leukemia-driven immune suppression in the tumor microenvironment. We hypothesized that B-cell leukemia-mediated immune suppression will be further dampened by the presence of

BRAF activating mutation. Given that vemurafenib and dabrafenib combined with trametinib have been FDA-approved for metastatic melanoma carrying the BRAFV600E mutation, and vemurafenib shows promising effects in clinical trials of HCL, these

BRAFV600E targeted therapies have potential in patients with other cancers harboring this mutation. However, emerging evidence indicates that not all BRAFV600E tumors respond to these agents; for example, only approximately 5% of colorectal cancer patients respond, and short response duration and relapse occur in most cases of melanoma. Therefore, examination of drug efficacy in different cancer types with the same gene alteration and investigation of the mechanisms of drug resistance are needed.

Taken together, the pathological effects of BRAFV600E have been examined nearly entirely in solid tumors, and the mechanism by which activated MAPK might drive immune suppression in a disseminated malignancy such as leukemia is unknown.

Moreover, lack of preclinical cell and animal models are the challenges to elucidate the effects of MAPK activation in B-cell malignancy172,175,176. This dissertation will directly address these issues. We hypothesized that BRAF activating mutations in malignant B- cells will produce more aggressive and/or treatment-resistant disease, and enhance immune suppressive effects. The goals of this work are: (1) to generate cellular and mouse models of B-cell leukemia with mutated BRAF; (2) to characterize immune defects in a mouse model of B-cell leukemia with B-cell-restricted expression of BRAFV600E; (3) to define the mechanism of this immune suppression in T-cells and myeloid cells; (4) to utilize this model to determine if BRAFV600E inhibition can reverse tumor-mediated immune suppression in vivo in BRAF-mutant B-cell leukemia.

Kipps et al. Page 52 Table 1. 1 Rai and Binet staging systems for chronic lymphocytic leukemia

Table 1

Author ManuscriptAuthor Rai Manuscript Author staging system Manuscript Author Manuscript Author

Risk group Clinical features Median life expectancy* Low risk Lymphocytosis without cytopenia, 13 years (Rai stage 0/I) lymphadenopathy or splenomegaly Intermediate risk Lymphocytosis, lymphadenopathy and/or 8 years (Rai stage II) splenomegaly, but without cytopenia High risk Lymphocytosis and cytopenia (a haemoglobin 2 years (Rai stage III/IV) level of ≤11 g per dl and/or a platelet count of Kipps et al. ≤100,000 cells per µl) Page 53 * These life-expectancy estimates are increasing with the advent of newer therapies.

Table 2

Author ManuscriptAuthor Binet Manuscript Author staging system Manuscript Author Manuscript Author

Risk group Clinical features Median life expectancy*

Low risk Less than three palpably enlarged sites‡ without 13 years (Binet stage A) cytopenia

Intermediate risk Three or more palpably enlarged sites‡ without 8 years (Binet stage B) cytopenia High risk Cytopenia (a haemoglobin level of ≤10 g per dl 2 years (Binet stage C) and/or a platelet count of ≤100,000 cells per µl)

* These life-expectancy estimates are increasing with the advent of newer therapies. ‡ There are five sites of lymphoid organs: cervical, axillary and inguinal nodes, the spleen and the liver.

Table taken from Kipps et al. 2017177

Nat Rev Dis Primers. Author manuscript; available in PMC 2017 March 04.

Nat Rev Dis Primers. Author manuscript; available in PMC 2017 March 04. Figure 1. 1 Stages of B-cell development. B-cell lineage structurally develops in the bone marrow and the spleen. The loci on immunoglobulin of heavy chain and light chain are rearranged, Igµ and surrogate light chains form pre-B-cell receptor (BCR), and mature BCR are presented during the differentiation from Pro-B cells, Pre-B cells to the immature B cells. The immature B cells successfully completing selections leave the bone marrow and enter into the spleen for further maturation, where these B cells encounter antigen stimulation from T helper cells, and undergo class switch recombination (CSR) and somatic hypermutation (SHM) to further differentiate to plasma cells or memory B cells. Figure taken from Cambier et al. 2007178.

Figure 1. 2 B-cell receptor (BCR) signaling. BCR/BTK activation transduces signaling to three main cascades: MAPK pathway, AKT pathway and calcium influx/NFkB, NFAT pathway. More detailed components of each pathway are shown in the figure. Figure taken from Hendriks et al. 201413.

Figure 1. 3 Overview of immune checkpoint molecules associated with T-cell exhaustion. The engagement of ligands on antigen-presenting cells (APC) and receptors on T cells leads to recruit phosphatases such as SHP1 and SHP2 via inhibitory intracellular motifs to deactivate the TCR-mediated signals, resulting in an exhausted phenotype of T cells. 179 REVIEWSFigure taken from Wherry and Kurachi 2015 .

MHC Antigen-presenting cell CD80 CD86 PDL1 PDL2 class II CEACAM1 HVEM CD48 CD155 or target cell

Galectin 9

Ectodomain competition TCR CTLA4 PD1 LAG3 TIM3 BTLA CD160 2B4 TIGIT

Exhausted Co-stimulatory T cell ITIM KIEELE BAT3 ITIM ITSM ITIM receptor

YVKM YVKM ITSM ITSM ITSM ZAP70 PI3K ITSM ITSM GRB2 PLCγ AKT SHP1 and SHP2 Modulation of intracellular mediators

Cytoplasm

NF-κB NFAT AP-1 Induction of Nucleus inhibitory genes

Figure 3 | Molecular pathways of inhibitory receptors associated with T cell exhaustion. Ligand and receptor pairs for inhibitory pathways are depicted, showing the intracellular domains of receptorsNature that contribute Reviews |to Immunology T cell exhaustion. Many inhibitory receptors have immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and/or immunoreceptor tyrosine-based switch motifs (ITSMs) in their intracellular domains; however, some receptors have specific motifs, such as YVKM for cytotoxic T lymphocyte antigen 4 (CTLA4) and KIEELE for lymphocyte activation gene 3 protein (LAG3). The molecular mechanisms of inhibitory receptor signalling are also illustrated and can be classified as: ectodomain competition (inhibitory receptors sequester target receptors or ligands); modulation of intracellular mediators (local and transient intracellular attenuation of positive signals from activating receptors such as T cell receptors and co-stimulatory receptors); and induction of inhibitory genes. Multiple inhibitory receptors are responsible for these three mechanisms. AP-1, activator protein 1; BAT3, HLA-B-associated transcript 3 (also known as BAG6); BTLA, B and T lymphocyte attenuator; CEACAM1, carcinoembryonic antigen-related cell adhesion molecule 1; GRB2, growth factor receptor-bound protein 2; HVEM, herpes virus entry mediator (also known as TNFRSF14); NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; PD1, programmed cell death protein 1; PDL1, PD1 ligand 1; PI3K, phosphoinositide 3-kinase; PLCγ, phospholipase Cγ; TIGIT, T cell immunoreceptor with immunoglobulin and ITIM domains; TIM3, T cell immunoglobulin and mucin domain-containing protein 3.

expression of effector genes, such as BATF, which encodes In addition to PD1, exhausted T cells express a the activator protein 1 (AP-1) family member basic leu- range of other cell surface inhibitory molecules (FIG. 3). cine zipper transcription factor ATF-like14. Despite this Exhausted T cells can co-express PD1 together with elegant work, it is unclear how these observations relate lymphocyte activation gene 3 protein (LAG3), 2B4 (also to exhausted T cells exposed to chronic infection in vivo. known as CD244), CD160, T cell immunoglobulin PD1 expression is rapidly upregulated upon T cell domain and mucin domain-containing protein 3 (TIM3; activation30, and it may persist at moderate levels in also known as HAVCR2), CTLA4 and many other inhib- healthy humans40,41, indicating that PD1 expression alone itory receptors18. Typically, the higher the number of

is not a unique feature of exhausted T cells. However, inhibitory receptors co-expressed by exhausted T cells, during chronic infections PD1 expression 28 can be sub- the more severe the exhaustion. Indeed, although indi- stantially higher than observed on functional effector vidual expression of PD1 or other inhibitory receptors or memory CD8+ T cells42,43. During chronic infection, is not indicative of exhaustion, co-expression of mul- sustained upregulation of PD1 is usually dependent on tiple inhibitory receptors is a cardinal feature. These continued epitope recognition44, although examples co-expression patterns are mechanistically relevant, as exist of residual PD1 expression even after removal of simultaneous blockade of multiple inhibitory receptors persisting antigen signalling20,45. results in synergistic reversal of T cell exhaustion. This

490 | AUGUST 2015 | VOLUME 15 www.nature.com/reviews/immunol

Chapter 2

BRAFV600E induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via

AP-1 activation

2.1 Introduction

Several types of solid tumors, especially melanoma, papillary thyroid cancer, and colon cancer, have been found to carry a somatic BRAF mutation that results in constitutive activation and cell transformation. Since the initial description of BRAF mutations in

200291, over 40 distinct point mutations affecting the BRAF kinase domain have been identified94. Among these, a mutation altering valine at amino acid 600 (V600) in the activation segment of the kinase domain shows the highest incidence, and most frequently changes this residue to glutamic acid (V600E). While earlier studies found little or no occurrence of BRAF mutations in hematologic diseases, rarer disease types were not studied. More recently, Tiacci et al. found that nearly 100% of classic hairy cell leukemia

(HCL) cells bear the BRAFV600E mutation100, and Dietrich and others now report that HCL can be successfully treated with vemurafenib101. In addition, the BRAFV600E mutation is expressed in hematopoietic stem cells in HCL patients, and a murine model with pan- hematopoietic BRAFV600E expression shows HCL clinical features. However, these features were not seen with B-cell lineage-restricted BRAFV600E expression172, implying that a 29 second hit is needed to develop HCL. A subset of patients with chronic lymphocytic leukemia (CLL) have also been shown to have a BRAF mutation96,98,180, and BRAF mutations were recently identified as one of the acquired initiating mutations in early hematopoietic cells of CLL patients, leading to deregulation of B-cell receptor (BCR) signaling99. Furthermore, a BRAF pseudogene transcript is aberrantly expressed in primary human diffuse large B-cell lymphoma and is positively correlated with BRAF expression, resulting in MAPK signaling activation. Global expression of this pseudogene in a murine model results in aggressive B-cell lymphoma102. Together, these findings clearly implicate

BRAFV600E in the development of a subset of B-cell malignancies.

Although not all BRAF mutations identified to date are V600E, most are activating mutations and result in stimulation of the MAPK pathway. BRAF is upstream of MEK and

ERK, which are involved in regulating cell proliferation, survival, differentiation and senescence following external signals139. Because BRAFV600E is active in the absence of external stimuli, it constitutively activates the MAPK pathway to stimulate cell transformation by driving increased transcription (e.g. via c-Fos, Elk-1) and translation (e.g. via RSK, eIF4E) of factors that subsequently drive survival and proliferation (e.g. cyclin

D1, c-myc). To abolish this constitutive activation, BRAFV600E inhibitors including vemurafenib and dabrafenib have been developed and show clinical responses in many cases of BRAFV600E mutated cancers. However, resistance to these agents commonly develops103,104. Emerging data also indicate that BRAFV600E HCL patients relapse following vemurafenib treatment that was initially effective181. Interestingly, it was recently reported that a patient with BRAFV600E-driven melanoma who responded to vemurafenib developed

CLL-like disease, possibly due to paradoxical BRAF inhibitor-associated ERK activation in B-cells via the BCR/SYK/RAS/RAF axis108. ERK is also a key downstream effector of the BCR pathway, and inhibition of this pathway by ibrutinib leads to loss of ERK phosphorylation both in vitro109 and in samples from ibrutinib-treated patients110. Thus, in

B-cells, ERK is a point of convergence of the MAPK pathway and the critical BCR pathway, further supporting the relevance of MAPK signaling in B-cell malignancies.

Despite the prevalence of BRAFV600E in HCL and the importance of mutant BRAF in leukemia development, and potentially its treatment, downstream targets of this pathway in B-cells remain unclear. Here, we sought to identify transcriptional events resulting from constitutive BRAF activation in transformed B-cells. We demonstrate that BRAFV600E induces the expression of the multi-drug resistance (MDR) gene ABCB1 and its product,

P-glycoprotein (P-gp). Further, we determined that MAPK pathway-mediated induction of

AP-1 could be a potential mechanism for this effect. This finding may have clinical implications for the long-term use of MDR substrate agents in patients with BRAF-mutated cancers.

2.2 Materials and Methods

2.2.1 Cells and cell culture

The cytogenetics and immunophenotype of the OSUCLL cell line are similar to the patient from whom it was derived and have been described previously182. The cell lines used here were generated through retroviral infection of OSUCLL cells using the Tet-On

3G Inducible Expression System (OSUCLL-Tet), then transfecting with pRetroX-tight-pur

31 vectors (Clontech, Mountain View, CA) expressing wild-type BRAF (OSUCLL-BRAF)

V600E or mutant BRAF (OSUCLL-BRAF ). Cells were cultured at 37°C and 5% CO2 in

RPMI 1640 with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (all from Sigma-Aldrich, St. Louis, MO).

2.2.2 Reagents

Vemurafenib (PLX-4032) was purchased from Selleck Chemicals (Houston, TX).

CI-1040 (PD184352) was synthesized in the OSU College of Pharmacy according to a published procedure183. Doxycycline (dox) was purchased from Clontech. Verapamil, vincristine and rhodamine 123 were obtained from Sigma-Aldrich. The BRAFV600E cDNA construct was purchased from Addgene (Cambridge, MA).

2.2.3 Viability and proliferation assays

MTS assays were performed according to the manufacturer’s instructions

(CellTiter 96, Promega, Madison WI). Cells (105 cells/well) were incubated in 96-well plates with or without dox (1 µg/ml) and other agents (as noted) for 48 hrs, and MTS reagent was added for an additional 2 hrs before analysis. To detect differences in growth rate, cells were cultured at 5x105 cells/well in 96-well plate for 20 hrs in the presence or absence of dox, then BrdU was added for an additional 4 hrs. BrdU was detected according to the manufacturer’s instructions (Millipore, Billerica, MA).

2.2.4 Microarray analysis

RNA samples from the OSUCLL-Tet and OSUCLL-BRAFV600E cells incubated with dox for 48 hr were analyzed using U133 plus 2.0 Gene Chips (Affymetrix, Santa Clara,

CA) in collaboration with the OSU Comprehensive Cancer Center Nucleic Acids Shared

Resource. Expression changes in genes of interest were validated by real-time RT-PCR.

2.2.5 Flow cytometry

To assess cell activation and validate microarray results, cells were incubated in the presence or absence of dox for 48 hrs, and 106 cells were then labeled using anti-CD69 or isotype antibodies (BD Biosciences, San Jose, CA). Cells were analyzed on a FC500 flow cytometer and results processed using Kaluza software (both Beckman Coulter, Brea, CA).

To measure P-gp activity, cells were cultured with dox for 48 hrs then incubated 1 hr with rhodamine 123 (2.6 µM). After two washes in media, cells were incubated 90 mins without or with verapamil (10 µg/ml), and rhodamine-positive cells were detected by flow cytometry.

2.2.6 Real-time reverse transcription–polymerase chain reaction (RT-PCR)

Total RNA was extracted by TRIzol (Invitrogen, Carlsbad, CA) and analyzed by real-time RT-PCR performed on the ABI ViiA 7 system (Applied Biosystems, Foster City

CA), using GAPDH as an endogenous control. TaqMan Universal Master Mix, primers, and labeled probes were used according to the manufacturer’s procedure (Applied

Biosystems). Mean threshold cycle (Ct) values were calculated by ABI ViiA 7 software to determine fold differences according to the manufacturer’s instructions (Applied

Biosystems).

2.2.7 Immunoblot analysis

SDS-PAGE and immunoblotting were performed according to standard procedures.

Antibodies against phospho-MEK, MEK, phospho-ERK, ERK, phospho-c-Fos, c-Fos,

FosB/B2, phosphor-Fra-1, Fra-1, phospho-c-Jun, c-Jun, JunB and JunD were obtained from Cell Signaling Technology (Danvers, MA), and BRAF, P-gp, and GAPDH from

Santa Cruz (Santa Cruz, CA). Human specific BRAFV600E antibody was obtained from

Spring Bioscience (Pleasanton, CA). Each immunoblot analysis was repeated a minimum of three times.

2.2.8 ABCB1 promoter activity

The ABCB1 promoter reporter construct pTL-MDR1 was purchased from

Affymetrix, and the pBabepuro-BRAFV600E plasmid was obtained from Addgene

(Cambridge, MA). NEK-293T cells were incubated at 105 cells/well in a 24-well plate and transiently transfected with pTL-MDR1 and pBabepuro plasmids for 8 hrs using FuGENE

6 transfection reagent (Promega). Cells were then incubated 16 hrs with vehicle, vemurafenib (2 µM), CI-1040 (1 µM), or both. Luciferase activity was measured using the

ABCB1 reporter assay system (Promega). Luciferase activities (relative luciferase units,

RLU) were normalized to ug protein, as determined by BCA protein assay.

2.2.9 Electrophoretic mobility shift assay (EMSA)

AP-1 double-stranded probes (5’-CGCTTGATGAGTCAGCCGGAA-3’ for wild- type and 5’-CGCTTGATAGTAGTGCCGGAA-3’ for mutant-type, respectively184,185) were end-labeled with 32P dCTP using Klenow DNA polymerase (Life Technologies,

Grand Island, NY). Nuclear lysates (10 µg) were incubated 1 hr with 2 µl AP-1 antibodies.

Antibodies to c-Jun, c-Fos, JunB, JunD, and Fra-1 were purchased from Santa Cruz

Biotechnology (Dallas, TX). MEK, phospho-c-Jun, and phospho-c-Fos antibodies were obtained from Cell Signaling. Labeled probes were then added and incubations continued at room temperature for another 30 mins. Complexes were separated by electrophoresis on

4% native polyacrylamide gels. Gels were dried and signals detected using a Phosphor

Screen (GE Healthcare, Pittsburgh, PA).

2.2.10 Statistical analysis

Mixed effects models were applied to log2-transformed data. Differences and p- values were then estimated from the model. p-values less than 0.05 were considered significant.

2.3 Results

2.3.1 Generation of B-cells with inducible BRAFV600E expression

BRAF mutations are found in a subset of CLL, and are early acquired events that may contribute to disease development or progression99 and potentially drug resistance. To investigate effects of this mutant protein in malignant B-cells, we stably transfected the

CLL cell line OSUCLL182 with the Tet-On doxycycline (dox)-inducible vector system to generate OSUCLL-Tet cells. This cell line was then infected with a retroviral vector containing either wild-type or mutant BRAF (OSUCLL-BRAF and OSUCLL-BRAFV600E, respectively). The resulting stable clones were incubated 24 hrs without or with dox (1

µg/ml) and examined by immunoblot. OSUCLL-BRAFV600E cells strongly upregulated the mutant protein following dox treatment, as detected using a BRAFV600E-specific antibody.

A moderate but consistent increase in wild-type BRAF was observed in OSUCLL-BRAF compared to non-transfected cells (OSUCLL-Tet) (Figure 2.1A). Under these same conditions, dox-mediated induction of BRAFV600E was accompanied by increased phosphorylation of the BRAF downstream signaling factors MEK and ERK, indicating a functional effect of the transfected protein. Minimal effects on MEK and ERK were detected in OSUCLL-BRAF cells incubated in dox. The effects on cell growth were then investigated. In comparison with OSUCLL-Tet, both OSUCLL-BRAF and OSUCLL-

BRAFV600E cells showed a moderate increase in growth rate in the presence of dox, as determined by BrdU proliferation assays (Figure 2.1B). These results demonstrate that the introduction of BRAFV600E into OSUCLL cells produces the expected biochemical downstream changes in MEK/ERK phosphorylation as well as increased cell growth.

Based on these results, we conducted a microarray analysis to examine transcriptional changes resulting from the presence vs. absence of BRAFV600E expression. The top 30 genes found to be increased or decreased in OSUCLL-BRAFV600E cells relative to

OSUCLL-Tet cells are shown in Table 2.1. Among these, CD69 and multidrug resistance gene ABCB1 were observed to be increased in OSUCLL-BRAFV600E cells compared with

OSUCLL-Tet cells. To validate these results, OSUCLL cells were examined for CD69 expression by flow cytometry following 48-hr incubations with or without dox. In comparison with OSUCLL-Tet and OSUCLL-BRAF cells, OSUCLL-BRAFV600E cells showed a marked increase in CD69 expression, an early activation marker in lymphocytes186,187 (Figure 2.1C), confirming that BRAFV600Eexpression enhances activation markers in the OSUCLL cell line.

2.3.2 BRAFV600E induces ABCB1 mRNA and P-gp protein expression in OSUCLL cells

To investigate the mechanism of BRAFV600E-mediated ABCB1/P-gp induction, transfected OSUCLL cells (Tet, BRAF, and BRAFV600E) were incubated with dox for 48 hrs. As early as 24 hrs, but consistently by 48 hrs, ABCB1 mRNA and P-gp protein were notably up-regulated in dox-treated OSUCLL-BRAFV600E cells (Figures 2.2A, 2.2B,

2.3A). Drug-resistant 697R cells established to have increased P-gp expression188 were included as a positive control. In the absence of dox treatment, a low level of BRAFV600E protein was observed in OSUCLL-BRAFV600E cells with longer exposures (Figure 2.2B), and ABCB1 expression in these cells was slightly higher relative to OSUCLL-Tet cells

(Figures 2.2A, 2.3A), indicating that transcription from this promoter is not completely shut off in the absence of dox. However, increased P-gp expression was not clearly detected in OSUCLL-BRAFV600E cells in the absence of dox, nor was ABCB1 mRNA increased in dox-treated OSUCLL-BRAF cells (Figure 2.2A). As an additional demonstration of this effect, OSUCLL cells were transfected with cDNAs driven by a constitutive promoter.

Cells transfected with BRAFV600E again showed increases in MAPK signaling and in P-gp expression relative to cells transfected with normal BRAF or the empty vector (Figure

2.3).

As P-gp expression may not necessarily correlate with its function189, efflux of the fluorescent P-gp substrate rhodamine 123 was examined by flow cytometry in dox-induced

OSUCLL cells. One and a half hours after staining and returning cells to rhodamine-free media, OSUCLL-BRAFV600E cells were essentially negative for rhodamine compared to

OSUCLL-Tet and OSUCLL-BRAF cells (p = 0.0003) (Figure 2.2C, black bars). This

37 effect was significantly reversed by the P-gp inhibitor verapamil (p = 0.0025) (Figure

2.2C, grey bars). As a further determination of P-gp function, cells were examined for sensitivity to the prototypical P-gp substrate vincristine. Cells were incubated for 24 hrs without or with dox, then treated with vincristine in the presence or absence of verapamil.

After an additional 48 hrs, cell proliferation was evaluated by MTS assay. As shown in

Figure 2.2D, dox-mediated induction of BRAFV600E resulted in a significant increase in resistance to vincristine (p<0.001). Furthermore, the addition of verapamil significantly reduced this vicristine resistance (p<0.001). Together, these data indicate that BRAFV600E induces P-gp expression and function in OSUCLL cells. In addition, MAPK signaling activation and P-gp expression are observed in a subset primary CLL samples with

BRAFV600E mutation (Figure 2.4).

2.3.3 BRAFV600E and MEK inhibition blocks P-gp induction in OSUCLL cells

Previous studies using various tumor cell lines have shown that ABCB1 expression can be regulated through the MAPK pathway190,191, involvement of the transcription factor

AP-1192 or reactive oxygen species193, and/or NF-κB signaling and CRE transcriptional activity194. Given that BRAFV600E causes constitutive MEK-ERK activation, this pathway was first examined to identify the mechanism of BRAFV600E-induced P-gp expression in

OSUCLL cells. Vemurafenib, an ATP-competitive inhibitor of BRAFV600E kinase, shows clinical benefits in several cancers with BRAFV600E expression such as melanoma195 and thyroid cancer196,197, and more recently, the B-cell malignancy HCL101. Vemurafenib therefore was used to identify impacts of BRAFV600E activity in OSUCLL cells.

Vemurafenib by itself did not increase cytotoxicity in OSUCLL cells by 48 hrs, even at 38 concentrations up to 8 µM (data not shown). Following a 24-hr incubation with dox,

OSUCLL cells were treated with vehicle, vemurafenib (2 µM), and/or the MEK inhibitor

CI-1040 (1 µM) for 16 hrs and expression of ABCB1 mRNA and P-gp protein were assessed. As shown in Figures 2.2A and 2.2B and 2.5A and 2.5B, the induction of

BRAFV600E by dox caused a notable increase in P-gp protein as well as ABCB1 mRNA.

This effect could be partially inhibited by vemurafenib or CI-1040, and stronger inhibition was noted with the combination.

2.3.4 BRAFV600E enhances ABCB1 promoter activity

To identify the transcriptional mechanism of BRAFV600E-mediated ABCB1 induction, a construct containing 1 kb of the ABCB1 promoter driving a luciferase reporter

(pTL-ABCB1) was transiently co-transfected with either an empty vector (pBabepuro) or one containing BRAFV600E (pBabepuro-BRAFV600E) into HEK293T cells. Following an 8- hr recovery, cells were treated without or with vemurafenib and/or CI-1040 and cell lysates were measured for luciferase activity (Figure 2.6A). This experiment demonstrated that

ABCB1 promoter activity was significantly increased with BRAFV600E expression, and that inhibition of BRAFV600E and MEK reduced this effect. Based on data from Roy et al.192 demonstrating the role of AP-1 in ABCB1 regulation, a panel of AP-1 proteins were assessed by immunoblot in OSUCLL cells following 24-hr BRAFV600E induction with dox.

As shown in Figure 2.6B, c-Fos, FosB/B2, Fra1, c-Jun, JunB, and JunD all appeared to be upregulated and/or phosphorylated upon BRAFV600E induction, and these effects could be blocked by vemurafenib and/or CI-1040.

As the NF-κB pathway and CRE activity have been implicated in ABCB1 regulation194, these factors were also examined. Phosphorylation of NFκB p65 was observed with BRAFV600E induction; however, this effect was inconsistent despite the consistent increase in ABCB1 mRNA. Also, phosphorylation of CREB was unaltered upon

BRAFV600E induction (Figure 2.7). Together, these results further support that the

BRAFV600E-induced ABCB1 expression occurs via the MEK/ERK/AP-1 pathway. Thus, to identify potential AP-1 factors involved in ABCB1 regulation in OSUCLL cells, EMSAs were performed using AP-1 elements from the ABCB1 promoter as previously identified184,185. Following a 24-hr dox incubation, nuclear extracts were prepared from

OSUCLL cells, incubated with labeled probe in the absence or presence of antibodies to

AP-1 elements, and separated on a non-denaturing acrylamide gel (Figure 2.4C). A mobility shift was identified that was notably increased upon induction of BRAFV600E

(lanes 1 vs. 7). This appeared to be specific for AP-1, as it was reduced following addition of excess cold probe or substitution of a mutated probe (lanes 2 and 3, respectively). While most of the antibodies to AP-1 family members produced no effect, an anti-JunD antibody resulted in a clear supershift in the AP-1 complex. Overall, these results indicate that

BRAFV600E mediates ABCB1/P-gp expression in OSUCLL cells, and that JunD is likely to be an important component of this regulatory mechanism.

2.4 Discussion

A subset of patients with B cell malignancies carry activating BRAF mutations, but the role of activated BRAF in B cell leukemia development is unclear. We investigated the pathological role of BRAFV600E in B cell leukemia using a model system in which we could 40 control BRAFV600E expression to better assess specific signaling events and outcomes.

Following stable transfection and dox treatment to induce functional BRAFV600E protein, we examined transcriptional changes using microarray analysis. While the list of genes potentially affected by increased BRAF signaling includes several interesting candidates, we chose to focus on ABCB1 because many melanoma patients with BRAFV600E are either resistant to therapy or relapse following treatment, hinting at a correlation between

BRAFV600E and drug resistance.

The protein product of ABCB1, P-gp, is a member of a class of glycoproteins that export xenobiotic agents across the cytoplasmic membrane via an ATP-dependent mechanism. Thus, its aberrant expression in tumor cells is responsible for reducing intracellular accumulation of anti-cancer drugs, resulting in relative resistance to those agents. P-gp is normally expressed in the liver, pancreas, kidney, and gut, but its level has been found to be increased in drug-resistant tumors such as breast and colon cancers. Its expression has also been reported in some hematologic malignancies191,198, but is less common compared to solid tumors.

Multiple mechanisms have been put forth for how P-gp and other such proteins are upregulated, and to date, several mechanisms of drug resistance have been reported in cancers bearing the BRAFV600E mutation. These include concurrent activation of the PI3K pathway through PTEN loss170,199-202, amplification of cyclin D1203, and feedback activation of EGFR204. However, the vast majority of these studies have focused on increased gene expression as a consequence of chronic exposure to chemotherapeutic agents, and to date no studies have described the involvement of P-gp in de novo

BRAFV600E-mediated drug resistance. Here, we demonstrate that mutated BRAF contributes to increased P-gp expression through constitutive MAPK pathway activation and AP-1 mediated transcriptional induction.

Importantly, previous reports indicate that the expression levels of ABCB1 mRNA and P-gp protein do not necessarily correlate with P-gp function189. Furthermore, the size and significant glycosylation of P-gp make this protein challenging to detect by immunoblot. Therefore, following real-time RT-PCR validation of increased ABCB1 mRNA expression following BRAFV600E induction, we assessed P-gp function via rhodamine exclusion and drug sensitivity with and without the P-gp inhibitor verapamil.

Both experiments confirmed increased P-gp function in BRAFV600E-expressing cells. To investigate the relationship between BRAF activity and ABCB1 regulation, we treated cells with the BRAFV600E inhibitor vemurafenib. Interestingly, this agent abolished ERK phosphorylation in OSUCLL cells only transiently (data not shown). This transient effect, which is also seen in melanoma and other BRAFV600E-mutant cell lines205,206, could be the result of P-gp-mediated drug elimination, as vemurafenib has been reported to be a substrate of efflux pumps such as P-gp. In support of this, brain distribution of vemurafenib is diminished via P-gp and BCRP/ABCG2 in patients with metastatic melanoma, and can be enhanced by inhibition of these proteins207,208. These observations further support that elevated MDR protein expression might constitute an important mechanism of resistance to vemurafenib. Hence, our data support that the BRAFV600E mutation may in fact contribute to resistance to drugs specifically targeting it. In addition to V600E, other mutations in

BRAF have been identified (e.g. G466, G469, L597, K601) that result in enhanced BRAF

42 kinase activity. While these are rarer and therefore less characterized, we hypothesize that these mutations would increase ABCB1 expression to cause resistance to chemotherapies including vemurafenib. However, more experiments as well as studies using primary samples are needed to address this.

BRAFV600E kinase inhibitor-induced paradoxical MAPK reactivation is known to reactivate receptor tyrosine kinases, Ras, or c-Raf to re-induce MAPK signaling.

Interestingly, it was recently reported that a patient with BRAFV600E-driven melanoma who responded to vemurafenib developed CLL-like disease, possibly due to paradoxical BRAF inhibitor-associated ERK activation in B-cells via the BCR/SYK/RAS/RAF axis108. To avoid this and enhance drug efficacy, combinations of MEK inhibitors with vemurafenib are now being explored, and studies are also emerging with new inhibitors of ERK. In this study, we found that MAPK pathway-induced AP-1 protein expression results in increased

ABCB1/P-gp expression. We observed increased expression and/or phosphorylation of several proteins in the Fos and Jun family including c-Fos, FosB/B2, Fra-1, c-Jun, JunB, and JunD. Although only JunD was identified to interact with the ABCB1 promoter element in EMSAs, other AP-1 components could be also crucial for ABCB1 expression, and combinations of MAPK pathway inhibitors will likely be needed to effectively prevent this effect in patients.

While we demonstrate that increased P-gp expression and function can result from constitutive BRAF activity, the interpretations of these results are limited by the use of cell lines. It will be essential to demonstrate that these effects are noted in tumor cells derived from patients with the BRAFV600E mutation, and such studies are now underway. The

43 strategy utilized here also does not address the role of BRAFV600E in disease development, as transfections were performed in a cell line derived from malignant B-cells. Although

Chung et al. demonstrated that BRAFV600E expression in earlier hematopoietic cells develops a HCL-like disease in a murine model172, a murine model with B-lineage restricted co-expression of BRAFV600E and a second hit will likely provide a more accurate model to investigate the pathological role of BRAFV600E in the development of B cell leukemia.

Table 2. 1 Microarray results showing top 30 BRAFV600E up- or down-regulated genes

Upregulated genes Fold change Gene Symbol Gene Description 1 10.342 NRCAM neuronal cell adhesion molecule 2 8.907 GBP4 guanylate binding protein 4 3 8.137 CLDN1 claudin 1 4 7.943 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 5 7.042 SERPINB2 serpin peptidase inhibitor, clade B (ovalbumin), member 2 6 5.320 DENND1B DENN/MADD domain containing 1B 7 5.294 DOCK4 dedicator of cytokinesis 4 8 5.292 NCKAP1 NCK-associated protein 1 9 5.185 HERC5 HECT and RLD domain containing E3 ubiquitin protein ligase 5 10 4.971 LOC284215 uncharacterized LOC284215 11 4.869 GPR84 G protein-coupled receptor 84 12 4.395 MIR548T microRNA 548t 13 4.268 KIR3DX1 killer cell immunoglobulin-like receptor, three domains, X1 14 4.266 EGR1 early growth response 1 15 4.229 IFI6 interferon, alpha-inducible protein 6 16 4.115 SLC9A2 solute carrier family 9, subfamily A (NHE2, cation proton antiporter 2), member 2 17 3.963 ETV4 ets variant 4 18 3.932 RHOBTB3 Rho-related BTB domain containing 3 19 3.851 IFI44L interferon-induced protein 44-like 20 3.784 CLLU1OS chronic lymphocytic leukemia up-regulated 1 opposite strand 21 3.715 IFIT1 interferon-induced protein with tetratricopeptide repeats 1 22 3.692 SPRY4 sprouty homolog 4 (Drosophila) 23 3.625 IFI44 interferon-induced protein 44 24 3.460 ITGA5 integrin, alpha 5 (fibronectin receptor, alpha polypeptide) 25 3.451 TMEM154 transmembrane protein 154 26 3.446 ZC3H12C zinc finger CCCH-type containing 12C 27 3.442 IRAK3 interleukin-1 receptor-associated kinase 3 28 3.368 TDO2 tryptophan 2,3-dioxygenase 29 3.363 IFI27 interferon, alpha-inducible protein 27 30 3.350 ABCB1 ATP-binding cassette, sub-family B (MDR/TAP), member 1

Down-regulated genes Fold change Gene Symbol Gene Description 1 0.127 GPR52 G protein-coupled receptor 52 2 0.134 AICDA activation-induced cytidine deaminase 3 0.137 CLEC4C C-type lectin domain family 4, member C 4 0.183 C1orf186 chromosome 1 open reading frame 186 5 0.206 CD180 CD180 molecule 6 0.218 MIR4524A microRNA 4524a 7 0.226 CNR1 cannabinoid receptor 1 (brain) 8 0.227 SPARC secreted protein, acidic, cysteine-rich (osteonectin) 9 0.230 OR2G6 olfactory receptor, family 2, subfamily G, member 6 10 0.237 GLYATL2 glycine-N-acyltransferase-like 2 11 0.251 KIAA0226L KIAA0226-like 12 0.260 SLC1A1 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 13 0.264 SNORD56B small nucleolar RNA, C/D box 56B 14 0.266 TNFRSF17 tumor necrosis factor receptor superfamily, member 17 15 0.296 ABCA6 ATP-binding cassette, sub-family A (ABC1), member 6 16 0.303 LRMP lymphoid-restricted membrane protein 17 0.311 CD27 CD27 molecule 18 0.312 FAM169A family with sequence similarity 169, member A 19 0.313 KMO kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) 20 0.319 EDA2R ectodysplasin A2 receptor 21 0.322 PECAM1 platelet/endothelial cell adhesion molecule 1 22 0.323 MGST1 microsomal glutathione S-transferase 1 23 0.323 CNR1 cannabinoid receptor 1 (brain) 24 0.325 EMR4P egf-like module containing, mucin-like, hormone receptor-like 4 pseudogene 25 0.326 CNR1 cannabinoid receptor 1 (brain) 26 0.326 RN5S46 RNA, 5S ribosomal 46 27 0.332 TAS2R14 taste receptor, type 2, member 14 28 0.334 FGF9 fibroblast growth factor 9 (glia-activating factor) 29 0.336 ANTXR2 anthrax toxin receptor 2 30 0.342 OR2T5 olfactory receptor, family 2, subfamily T, member 5

Figure 2. 1 Effects of transfected BRAFV600E on the MAPK pathway and cell growth. (A) Immunoblot analysis of wild-type and mutant BRAF expression and MAPK signaling in transfected OSUCLL cells following 24 h incubation without or with dox (1 µg/mL). (B) Proliferation in OSUCLL-Tet, OSUCLL-BRAF, and OSUCLL-BRAFV600E cells without or with dox treatment (1 µg/mL) for 24 h. BrdU was added for the last 4 h of the incubations. Data are shown relative to the OSUCLL-Tet control cell line without dox treatment (N = 4, *p < 0.05; **p < 0.005). (C) OSUCLL-Tet, OSUCLL-BRAF, and OSUCLL-BRAFV600E cells were incubated 48 h with or without dox, then stained with anti-CD69 or isotype antibodies and assessed by flow cytometry. Data are representative

of three independent experiment

1272 Y.-T. Tsai et al. / Leukemia Research 39 (2015) 1270–1277

V600E

Fig. 1. Effects of transfected BRAF on the MAPK pathway and cell growth. (A). Immunoblot analysis of wild-type and mutant BRAF expression and MAPK signaling in

V600E

transfected OSUCLL cells following 24 h incubation without or with dox (1 ␮g/mL). (B). Proliferation in OSUCLL-Tet, OSUCLL-BRAF, and OSUCLL-BRAF cells without or

with dox treatment (1 ␮g/mL) for 24 h. BrdU was added for the last 4 h of the incubations. Data are shown relative to the OSUCLL-Tet control cell line without dox treatment

V600E

(N = 4, *p < 0.05; **p < 0.005). (C). OSUCLL-Tet, OSUCLL-BRAF, and OSUCLL-BRAF cells were incubated 48 h with or without dox, then stained with anti-CD69 or isotype

antibodies and assessed by ﬂow cytometry. Data are representative of three independent 46 experiments.

analyses were performed using SAS/STAT software, version 9.3 (SAS could be detected in these cells with prolonged exposures (Fig. 2B),

Institute, Inc., Cary, NC). indicating transcription from this promoter is not completely

absent in the absence of dox. In comparison with OSUCLL-Tet,

V600E

3. Results both OSUCLL-BRAF and OSUCLL-BRAF cells showed a signif-

icant increase in growth rate as determined by BrdU proliferation

V600E

3.1. Generation of B-cells with inducible BRAF expression assays (Fig. 1B). OSUCLL-BRAF cells appeared to grow slightly

faster than OSUCLL-BRAF cells, but this difference was not signif-

BRAF mutations are found in a subset of CLL, and are early icant. Interestingly, the addition of dox did not further increase

acquired events that may contribute to disease development or proliferation, suggesting that whatever effects transfected BRAF or

V600E

progression [8] and potentially drug resistance. To investigate BRAF have on growth rate occur at very low expression levels.

V600E

effects of this mutant protein in malignant B-cells, we stably trans- These results demonstrate that transfected BRAF produces the

fected OSUCLL cells with a dox-inducible vector system to generate expected downstream biochemical changes in OSUCLL cells, but its

OSUCLL-Tet. This cell line was infected with retroviral vectors impact on proliferation relative to normal BRAF is limited.

V600E

containing wild-type or mutant BRAF to produce OSUCLL-BRAF Based on the increased signaling in BRAF transfected cells,

V600E V600E

and OSUCLL-BRAF . OSUCLL-BRAF cells strongly upregu- we examined transcriptional changes resulting from the presence

V600E

lated mutant BRAF following 24 h dox treatment, as detected by versus absence of BRAF expression using microarray. The top

V600E

immunoblot using a BRAF -speciﬁc antibody (Fig. 1A). A mod- 30 genes increased or decreased in dox-treated OSUCLL-BRAF

erate but consistent increase in wild-type BRAF was observed in cells relative to dox-treated OSUCLL-Tet cells are shown in Supple-

dox-treated OSUCLL-BRAF versus OSUCLL-Tet cells. Under these mentary Table S1. Among these, the lymphocyte activation marker

V600E

conditions, dox-mediated BRAF induction was accompanied CD69 and the multidrug resistance gene ABCB1 were observed to

V600E

by increased MEK and ERK phosphorylation, indicating functional be increased in OSUCLL-BRAF cells versus the control. In vali-

transfected protein. Minimal effects on MEK and ERK phosphory- dation of these results, CD69 expression was found to be markedly

V600E

lation were detected in dox-treated OSUCLL-BRAF cells. However, increased in dox-treated OSUCLL-BRAF compared to OSUCLL-

a slight increase in ERK phosphorylation was detected in OSUCLL- Tet and OSUCLL-BRAF cells (Fig. 1C).

V600E V600E

BRAF cells in the absence of dox, and a low level of BRAF Figure 2. 2 BRAFV600E induces the expression of ABCB1 and functional P-gp in OSUCLL cells. (A) ABCB1 mRNA levels were assessed in OSUCLL cells using real-time RT-PCR (N = 3, **p < 0.001). (B) Immunoblot of normal BRAF, mutant BRAF, and P-gp in OSUCLL cells incubated 48 h without or with dox. (C) OSUCLL cells cultured 48 h with dox were incubated 1 h with the fluorescent P-gp substrate rhodamine 123, then transferred to rhodamine-free media with or without 10 µM verapamil. Retained rhodamine was assessed by flow cytometry after 90 min. Parental 697 cells and drug-resisant 697-R cells were included as controls. Results were averaged from three identical experiments (**p < 0.005) and are shown as percent rhodamine-positive cells relative to vehicle-treated OSUCLL-Tet (far left). (D) OSUCLL-BRAFV600E cells were incubated 48 h with or without dox, vincristine, and/or verapamil, and mitochondrial activity was evaluated by MTS assay. Results shown are averaged from three identical experiments. At each concentration of vincristine, comparisons were made between the dox and no dox conditions, as well as between the verapamil and control conditions in the presence of dox (**p < 0.001).

Y.-T. Tsai et al. / Leukemia Research 39 (2015) 1270–1277 1273

Continued 47

V600E

Fig. 2. BRAF induces the expression of ABCB1 and functional P-gp in OSUCLL cells. (A). ABCB1 mRNA levels were assessed in OSUCLL cells using real-time RT-PCR (N = 3,

**p < 0.001). (B). Immunoblot of normal BRAF, mutant BRAF, and P-gp in OSUCLL cells incubated 48 h without or with dox. (C). OSUCLL cells cultured 48 h with dox were

incubated 1 h with the ﬂuorescent P-gp substrate rhodamine 123, then transferred to rhodamine-free media with or without 10 ␮M verapamil. Retained rhodamine was

assessed by ﬂow cytometry after 90 min. Parental 697 cells and drug-resisant 697-R cells were included as controls. Results were averaged from three identical experiments

V600E

(**p < 0.005) and are shown as percent rhodamine-positive cells relative to vehicle-treated OSUCLL-Tet (far left). (D). OSUCLL-BRAF cells were incubated 48 h with or

without dox, vincristine, and/or verapamil, and mitochondrial activity was evaluated by MTS assay. Results shown are averaged from three identical experiments. At each

concentration of vincristine, comparisons were made between the dox and no dox conditions, as well as between the verapamil and control conditions in the presence of

dox (**p < 0.001).

V600E

3.2. BRAF induces ABCB1 mRNA and P-gp protein expression OSUCLL-Tet cells (Figs. 2 and 3A). However, increased P-gp expres-

V600E

in OSUCLL cells sion was not clearly detected in OSUCLL-BRAF cells in the

absence of dox, nor was ABCB1 mRNA increased in dox-treated

V600E

To conﬁrm BRAF -mediated ABCB1/P-gp induction, OSUCLL- OSUCLL-BRAF cells (Fig. 2A). As an additional validation, OSUCLL

V600E

Tet, -BRAF, and -BRAF cells were incubated with or without cells were transfected with cDNAs driven by a constitutive pro-

V600E

dox and examined by real-time RT-PCR and immunoblot. As early as moter. Cells transfected with BRAF again showed increases in

24 h, but consistently by 48 h, ABCB1 mRNA and P-gp protein were MAPK signaling and in P-gp expression relative to cells transfected

V600E

clearly up-regulated in dox-treated OSUCLL-BRAF cells (Figs. with normal BRAF or the empty vector (Supplementary Fig. S1).

2A and B and 3A). Drug-resistant 697R cells that have increased As P-gp expression may not necessarily correlate with its

P-gp expression [21] were included as a positive control. As previ- function [22], efﬂux of the ﬂuorescent P-gp substrate rhodamine

V600E

ously noted, a low level of BRAF protein could be detected in 123 was examined by ﬂow cytometry in dox-induced OSUCLL

V600E

OSUCLL-BRAF cells in the absence of dox treatment (Fig. 2B), cells. Within 90 min of removal from rhodamine-containing media,

V600E

and ABCB1 expression in these cells was slightly higher relative to OSUCLL-BRAF cells were essentially negative for rhodamine

Y.-T. Tsai et al. / Leukemia Research 39 (2015) 1270–1277 1273

Figure 2.2 Continued

V600E

Fig. 2. BRAF induces the expression of ABCB1 and functional P-gp in OSUCLL cells. (A). ABCB1 mRNA levels were assessed in OSUCLL cells using real-time RT-PCR (N = 3,

**p < 0.001). (B). Immunoblot of normal BRAF, mutant BRAF, and P-gp in OSUCLL cells incubated 48 h without or with dox. (C). OSUCLL cells cultured 48 h with dox were

incubated 1 h with the ﬂuorescent P-gp substrate rhodamine 123, then transferred to rhodamine-free media with or without 10 ␮M verapamil. Retained rhodamine was

assessed by ﬂow cytometry after 90 min. Parental 697 cells and drug-resisant 697-R cells were included as controls. Results were averaged from three identical experiments

V600E

(**p < 0.005) and are shown as percent rhodamine-positive cells relative to vehicle-treated OSUCLL-Tet (far left). (D). OSUCLL-BRAF cells were incubated 48 h with or

without dox, vincristine, and/or verapamil, and mitochondrial activity was evaluated by MTS assay. Results shown are averaged from three identical experiments. At each

concentration of vincristine, comparisons were made between the dox and no dox conditions, as well as between the verapamil and control conditions in the presence of

dox (**p < 0.001).

V600E

3.2. BRAF induces ABCB1 mRNA and P-gp protein expression OSUCLL-Tet cells (Figs. 2 and 3A). However, increased P-gp expres-

48 V600E

in OSUCLL cells sion was not clearly detected in OSUCLL-BRAF cells in the

absence of dox, nor was ABCB1 mRNA increased in dox-treated

V600E

To conﬁrm BRAF -mediated ABCB1/P-gp induction, OSUCLL- OSUCLL-BRAF cells (Fig. 2A). As an additional validation, OSUCLL

V600E

Tet, -BRAF, and -BRAF cells were incubated with or without cells were transfected with cDNAs driven by a constitutive pro-

V600E

dox and examined by real-time RT-PCR and immunoblot. As early as moter. Cells transfected with BRAF again showed increases in

24 h, but consistently by 48 h, ABCB1 mRNA and P-gp protein were MAPK signaling and in P-gp expression relative to cells transfected

V600E

clearly up-regulated in dox-treated OSUCLL-BRAF cells (Figs. with normal BRAF or the empty vector (Supplementary Fig. S1).

2A and B and 3A). Drug-resistant 697R cells that have increased As P-gp expression may not necessarily correlate with its

P-gp expression [21] were included as a positive control. As previ- function [22], efﬂux of the ﬂuorescent P-gp substrate rhodamine

V600E

ously noted, a low level of BRAF protein could be detected in 123 was examined by ﬂow cytometry in dox-induced OSUCLL

V600E

OSUCLL-BRAF cells in the absence of dox treatment (Fig. 2B), cells. Within 90 min of removal from rhodamine-containing media,

V600E

and ABCB1 expression in these cells was slightly higher relative to OSUCLL-BRAF cells were essentially negative for rhodamine Figure 2. 3 Constitutive BRAFV600E expression drives P-gp expression in OSUCLL cells. V600E Figure S1. Constitutive BRAF expression Figure S2. P-gp induction is not drives P-gp expression in OSUCLL cells via CREB or NF-kB pathways

OSUCLL V600E -Tet -BRAF -BRAFV600E BRAF BRAF - - dox: - + - + - + 697R 697

V600E

pBabepuro pBabe pBabe BRAF - - -

BRAF OSUCLL parental OSUCLL OSUCLL OSUCLL OSUCLL P-gp p-c-Fos

BRAFV600E c-Fos

Total BRAF p-ERK

p-ERK ERK

ERK GAPDH

GAPDH p-p65 p-MEK p65 MEK p-CREB GAPDH CREB

GAPDH

Figure 2. 4 BRAFV600E activates MAPK signaling and P-gp expression in a subset of CLL patient samples.

mut BRAF +dox V600E wt BRAF BRAF - 250063 251253 251491 251577 251614 250575 D594N 250733 G466A 250883 V600E 250937 L597Q OSUCLLtet+d OSUCLL P-gp

BRAFV600E

Total BRAF

p-ERK

p-ERK (darker)

ERK GAPDH

p-MEK

MEK

GAPDH

Figure 2. 5 BRAFV600E and MEK inhibition block ABCB1/P-gp expression in OSUCLL cells. (A) Real-time RT-PCR analysis of ABCB1 expression in OSUCLL cells in the presence or absence of BRAFV600E or MEK inhibitors. Cells were incubated without or with dox 24 h, then inhibitors (2 µM vemurafenib and/or 1 µM CI-1040) were added for an additional 16 h. Inhibitor comparisons in the presence of dox were performed versus BRAFV600E + dox (N = 5, *p < 0.05; **p < 0.001). (B) Immunoblot analysis of P-gp, normal BRAF, and BRAFV600E expression in OSUCLL cells treated as in A. Results shown are representative of three individual experiments.

Figure 2. 6 BRAFV600E enhances ABCB1 promoter activity via MAPK and AP-1. (A) HEK293T cells were transiently co-transfected with 1 µg ABCB1 reporter construct (pTL-MDR1) and 1 µg empty vector (pBabepuro) or mutant BRAF plasmid (pBabepuro- BRAFV600E ). After 8 h, inhibitors are added as in Figure 2.5A. After an additional 16 h, luciferase activity was assessed in total cell lysates. Results are shown normalized to the amount of lysate; each inhibitor was compared to control, and in addition, the control was compared to empty vector (N = 3, *p < 0.05; **p < 0.001). (B) The immunoblot from Figure 2.5B was additionally analyzed for c-Fos and c-Jun proteins. (C) Nuclear extracts were prepared from OSUCLL cells incubated 24 h with or without dox, then mixed with 32P-labeled wild-type (wt) or mutant (mut) AP-1 probes, 100× cold probe (*), and antibodies as indicated: A. c-Fos; B. c-Jun; C. JunB; D. JunD; E. MEK (irrelevant control). The final lane is free probe without nuclear extract. Mixtures were separated by native acrylamide electrophoresis and bands detected by autoradiography. Results shown are representative of three individual experiments.

Y.-T. Tsai et al. / Leukemia Research 39 (2015) 1270–1277 1275

Continued

V600E

Fig. 4. BRAF enhances ABCB1 promoter activity via MAPK and AP-1. (A). HEK293T cells were transiently co-transfected with 1 ␮g ABCB1 reporter construct (pTL-MDR1)

V600E

and 1 ␮g empty vector (pBabepuro) or mutant BRAF plasmid (pBabepuro-BRAF ). After 8 h, inhibitors are added as in Fig. 3A. After an additional 16 h, luciferase activity

was assessed in total cell lysates. Results are shown normalized to the amount of lysate; each inhibitor was compared to control, and in addition, the control was compared

to empty vector (N = 3, *p < 0.05; **p < 0.001). (B). The immunoblot from Fig. 3B was additionally analyzed for c-Fos and c-Jun proteins. (C). Nuclear extracts were prepared

from OSUCLL cells incubated 24 h with or without dox, then mixed with [32] P-labeled wild-type (wt) or mutant (mut) AP-1 probes, 100 cold probe (*), and antibodies as

indicated: A. c-Fos; B. c-Jun; C. JunB; D. JunD; E. MEK (irrelevant control). The ﬁnal lane is free probe without nuclear extract. Mixtures were separated by native acrylamide

electrophoresis and bands detected by autoradiography. Results shown are representative of three individual experiments.

V600E

describe the involvement of P-gp in de novo BRAF -mediated protein challenging to detect by immunoblot. Therefore, following

drug resistance. Here, we report that mutationally activated BRAF real-time RT-PCR validation of increased ABCB1 mRNA expression

V600E

drives ABCB1 transcription via AP-1 activity in a model of B-cell following BRAF induction, we assessed P-gp function via rho-

malignancy, leading to enhanced P-gp expression and function. damine exclusion as well as drug sensitivity in the presence and

Previous reports indicate that the expression levels of ABCB1 absence of the P-gp inhibitor verapamil. These experiments con-

V600E

mRNA and P-gp protein do not necessarily correlate with P-gp func- ﬁrmed increased P-gp function in BRAF -expressing cells. To

tion [22]. Furthermore, the size and glycosylation of P-gp make this investigate the relationship between BRAF activity and ABCB1 reg-

Y.-T. Tsai et al. / Leukemia Research 39 (2015) 1270–1277 1275

Figure 2.6 Continued

V600E

Fig. 4. BRAF enhances ABCB1 promoter activity via MAPK and AP-1. (A). HEK293T cells were transiently co-transfected with 1 ␮g ABCB1 reporter construct (pTL-MDR1)

V600E

and 1 ␮g empty vector (pBabepuro) or mutant BRAF plasmid (pBabepuro-BRAF ). After 8 h, inhibitors are added as in Fig. 3A. After an additional 16 h, luciferase activity

was assessed in total cell lysates. Results are shown normalized to the amount of lysate; each inhibitor was compared to control, and in addition, the control was compared

to empty vector (N = 3, *p < 0.05; **p < 0.001). (B). The immunoblot from Fig. 3B was additionally analyzed for c-Fos and c-Jun proteins. (C). Nuclear extracts were prepared

from OSUCLL cells incubated 24 h with or without dox, then mixed with [32] P-labeled wild-type (wt) or mutant (mut) AP-1 probes, 100 cold probe (*), and antibodies as

indicated: A. c-Fos; B. c-Jun; C. JunB; D. JunD; E. MEK (irrelevant control). The ﬁnal lane is free probe without nuclear extract. Mixtures were separated by native acrylamide

electrophoresis and bands detected by autoradiography. Results shown are representative of three individual experiments.

V600E

describe the involvement of P-gp in de novo BRAF -mediated protein challenging to detect by immunoblot. Therefore, following

drug resistance. Here, we report that mutationally activated BRAF real-time RT-PCR validation of increased ABCB1 mRNA expression

V600E

drives ABCB1 transcription via AP-1 activity in a model of B-cell following BRAF induction, we assessed P-gp function via rho-

malignancy, leading to enhanced P-gp expression and function. 53damine exclusion as well as drug sensitivity in the presence and

Previous reports indicate that the expression levels of ABCB1 absence of the P-gp inhibitor verapamil. These experiments con-

V600E

mRNA and P-gp protein do not necessarily correlate with P-gp func- ﬁrmed increased P-gp function in BRAF -expressing cells. To

tion [22]. Furthermore, the size and glycosylation of P-gp make this investigate the relationship between BRAF activity and ABCB1 reg- Figure 2. 7 P-gp induction is not via CREB or NF-kB pathway

Figure S1. Constitutive BRAFV600E expression Figure S2. P-gp induction is not drives P-gp expression in OSUCLL cells via CREB or NF-kB pathways

OSUCLL V600E -Tet -BRAF -BRAFV600E BRAF BRAF - - dox: - + - + - + 697R 697

V600E

pBabepuro pBabe pBabe BRAF - - -

BRAF OSUCLL parental OSUCLL OSUCLL OSUCLL OSUCLL P-gp p-c-Fos

BRAFV600E c-Fos

Total BRAF p-ERK p-ERK ERK

ERK GAPDH

GAPDH p-p65 p-MEK p65 MEK p-CREB GAPDH CREB

GAPDH

Chapter 3

BRAFV600E accelerates disease progression and enhances immune suppression in a

mouse model of B-cell leukemia

3.1 Introduction

The mitogen-activated protein kinase (MAPK) pathway regulates diverse cell functions downstream of many cell-surface receptors by controlling transcription, cell cycle and apoptosis, and is commonly aberrant in tumor cells209. Mutated MAPK pathway components drive tumor survival and proliferation in the absence of external stimuli, and therapeutic MAPK inhibitors are being pursued to block these effects103,104,140-142. MAPK signaling also induces immune-suppressive behavior of tumor cells via multiple mechanisms including production and secretion of cytokines, induction of immune checkpoint molecules, and cytoskeletal remodeling143-145. MAPK pathway activation in tumor cells without MAPK mutations, such as through TLR stimulation or TGF-β signaling, can promote tumor infiltration and immune suppression at least in part via

CD200 and PD-L1 induction, and these effects can be reversed by MEK inhibition146-149.

Similar findings are reported in BRAFV600E mutant melanoma, where inhibition of BRAF and MEK increases T-cell recognition and dendritic cell function150,151. This evidence strongly supports that targeting MAPK signaling in cancer patients could reverse tumor- 55 induced immune suppression, leading to longer disease control, improved resistance to opportunistic infections, and enhanced efficacy of immune-based therapies.

The MAPK pathway is mutationally activated in hematologic malignancies, although generally less frequently than in solid tumors. Most notably, nearly 100% of classic hairy cell leukemia (HCL) cases carry the BRAFV600E mutation100, suggesting a key function for the mutated protein in disease development. Multiple reports demonstrate

HCL can be successfully treated with vemurafenib, further supporting this hypothesis101.

Approximately 10% of chronic lymphocytic leukemia (CLL) patients carry an activating mutation in a MAPK pathway component including BRAF96-98, and BRAF mutations were identified as one of the acquired initiating mutations in early hematopoietic cells of CLL patients99. A recent case report describes the clinical efficacy of MAPK inhibition in a patient with BRAFV600E mutated multiple myeloma210. Moreover, a BRAF pseudogene transcript is aberrantly expressed in primary human diffuse large B-cell lymphoma and is positively correlated with BRAF expression, resulting in MAPK signaling activation.

Global expression of this pseudogene in mice results in aggressive B-cell lymphoma102.

Together, these findings indicate a role for activated MAPK in a meaningful subset of B- cell malignancies. However, its function with regard to immune suppression in these diseases is unclear, and no suitable mouse model with a B-cell specific MAPK activating mutation has been available. Here, we describe a new model of BRAF-mutated B-cell leukemia to investigate this critical aspect.

In this study, we generated a murine model of BRAFV600E-mutated B-cell leukemia based on the well-established Eµ-TCL1 strain158,211-214 and documented the phenotype of

56 the resulting disease. Eµ-TCL1 mice with BRAFV600E mutated B-cells develop a B-cell leukemia significantly earlier than standard Eµ-TCL1 mice, and have a shorter lifespan.

Additionally, BRAFV600E-mutated leukemic B cells exert a greater immunosuppressive effect on T cells, and this can be reversed by pharmacologic inhibition of BRAFV600E. This study provides a new model of MAPK-driven immune defects in B-cell leukemia and supports the introduction of combination strategies using MAPK pathway inhibitors together with BTK and/or immune checkpoint inhibitors in patients with BRAFV600E mutated B-cell malignancies.

3.2 Materials and Methods

3.2.1 Cell lines and reagents

OSUCLL cells with doxycycline-inducible expression of normal or mutated BRAF were previously described215. Vemurafenib and dabrafenib were obtained from

SelleckChem (Houston TX) and doxycycline from Clontech (Mountain View CA).

3.2.2 Transgenic mice

BRafCA (conditional allele) mice (B6.129P2(Cg)-Braftm1Mmcm/J) with loxP-flanked wild-type BRaf followed by human BRafV600E sequence167 and CD19Cre mice

(B6.129P2(C)-Cd19tm1(cre)Cgn/J) with the Cre recombinase gene driven by the CD19 promoter216 were purchased from Jackson Labs (Bar Harbor, ME). Eµ-TCL1 mice were previously described158. All mice are on the C57BL/6 background. Homozygous BRafCA mice were crossed with homozygous CD19cre mice, and after genotypic confirmation via

PCR on tail snip DNA, offspring were crossed with homozygous Eµ-TCL1 mice to

57 generate BRAFVExCD19crexTCL1 (referred to in the text as “BRAFVExTCL1”).

CD19crexTCL1 (referred to in the text as “BRAFWTxTCL1”), BRAFCAxTCL1, and

BRAFVExCD19cre mice were used as controls (CA = conditional allele, no CD19-Cre).

The presence of BRAFV600E, Cre, and human TCL1 was again confirmed via PCR on genomic DNA. Mice were monitored biweekly by flow cytometry and spleen palpation.

Leukemia was defined as enlarged (palpable) spleen and ≥10% CD5+CD19+ cells in the peripheral blood CD45+ population. For adoptive transfer experiments, spleen cells were isolated from transgenic mice with leukemia and assessed by flow cytometry. Live

CD5+CD19+ cells (107) were injected intravenously into 8-10 weeks old wild-type female

C57BL/6 mice217. Mice were sacrificed upon reaching pre-determined euthanasia criteria

(≥20% weight loss, hind limb paralysis, respiratory distress, rough coat, or ≥10% weight loss with other signs). All mouse experiments were performed under a protocol approved by the Ohio State University Institutional Animal Care and Use Committee.

3.2.3 Cell isolation

Mouse CD19 MicroBeads (Miltenyi Biotec, San Diego CA), EasySep™ mouse

Pan-B cell isolation kit, and EasySep™ mouse T-cell isolation kit (StemCell Technologies,

Vancouver BC) were used to isolate B and T cells from mouse spleens. Purity was confirmed by flow cytometry and was at least 90% for all experiments.

3.2.4 Immunoblotting

Lysates from B- and T-cells from spleens were immunoblotted with antibodies for human BRAFV600E (Spring Bioscience, Pleasanton CA), total BRAF (Santa Cruz

Biotechnology, Santa Cruz CA), phosphorylated and total MEK and ERK (Cell Signaling

Technology, Danvers MA) and actin (Santa Cruz) using standard procedures.

3.2.5 Flow cytometry

Flow cytometry was performed using either a Gallios (Beckman Coulter, Brea CA) or a Fortessa (BD Biosciences, San Jose, CA) cytometer, and results were processed using

Kaluza software (Beckman Coulter). Antibodies to CD5, CD19, and CD45 (BD

Biosciences, San Jose, CA) were used to monitor leukemia in blood. Blood was stained with anti-CD160 (eBioscience, San Diego, CA), anti-CD244.2 (BD Biosciences), and anti-

PD-1 (eBioscience) to investigate the T cell exhaustion phenotype. Mouse spleen and marrow cells were stained with anti-B220, anti-IgD (eBioscience), anti-CD43, and anti-

IgM (BioLegend, San Diego, CA) to evaluate B cell development. Leukemia cells were stained with anti-PD-L1 (BioLegend) and anti-CD200 (eBioscience) to investigate immune suppressive phenotype. LIVE/DEAD fixable Near-IR stain (ThermoFisher,

Waltham MA) was used to exclude dead cells. To investigate myeloid phenotype, cells were stained with anti-CD19, anti-CD5, anti-Gr1 Ly6G, anti-PD-L1, viability dye

FVDefl780 (eBioscience), anti-CD45, anti-CD11b (BD Biosciences), anti-F4/80 and anti-

Ly6C (BioLegend).

3.2.6 Cell proliferation assay

Mice were intraperitoneally injected with 50 µg 5-ethynyl-2'-deoxyuridine (EdU;

Santa Cruz) per gram body weight. After 24 hours, spleen cells were collected and stained

59 with anti-CD5 FITC, anti-CD19 PE, and anti-CD45 Alexa700, followed by Click-iT EdU labeling (ThermoFisher). Cells were analyzed immediately by flow cytometry.

3.2.7 Histology

Organs (spleen, liver, marrow, thymus, gastrointestinal tract, kidneys, lungs, heart and lymph nodes) were harvested and fixed in 10% neutral formalin. Tissues were paraffin embedded, sectioned onto slides, and stained with hematoxylin and eosin (H&E). Slides were reviewed by a board-certified veterinary pathologist. Gross and histologic evaluation of lymph nodes, thymus, bone marrow, liver and spleen was also performed on syngeneic mice (n=11) engrafted with cells from a leukemic BRAFVExTCL1 mouse. These evaluations were conducted 8 weeks following engraftment, early in disease process as determined by peripheral leukocyte counts and absence of palpable spleens.

Immunohistochemistry (IHC) with antibodies to CD3 (Dako, Santa Clara CA), B220 (BD

Pharmingen, San Jose CA) and F4/80 (BioRad, Hercules CA) was performed as previously described218 on tissues from 5 BRAFWTxTCL1 mice and 6 BRAFVExTCL1 mice that reached euthanasia criteria.

3.2.8 Functional assays

T cells from peripheral blood of healthy adults, or from spleens of wild type

C57BL/6 mice, were negatively selected and stained with carboxyfluorescein succinimidyl ester (CFSE; ThermoFisher), and stimulated with plate-bound anti-CD3 and soluble anti-

CD28 antibodies (BD Biosciences). Human T cells were co-cultured with OSUCLL cells expressing normal BRAF or BRAFV600E. All conditions, including T cells alone, also

60 received 1 µg/mL doxycycline (dox). For cytokine analyses, supernatants from co-cultures of stimulated healthy T-cells with or without leukemic B cells were analyzed by Cytokine

Bead Array (CBA; BD Biosciences) per manufacturer’s protocol. Cytokines in mouse plasma were also assessed using MILLIPLEX® Multiplex Assays (EMD Millipore,

Billerica MA) according to the manufacturer’s instructions.

3.2.9 Statistics

Kaplan-Meier estimates and the log-rank test were used to compare both time from birth to development of leukemia as well as overall survival from birth between the

BRAFWTxTCL1, BRAFVExTCL1 and BRAFCAxTCL1 groups. Organ weight comparisons were made using two-sample t-tests assuming unequal variances; for the spleen data, the nonparametric Wilcoxon rank sum test was used instead. Similarly, differences in EdU incorporation and annexin-V binding were assessed using two-sample t-tests or Wilcoxon rank sum tests. For the mRNA data, mixed effects models were applied to the ΔCT values and estimated differences were transformed back into fold changes. Changes in F4/80+ macrophages, myeloid-derived suppressor cells (MDSCs), and patrolling monocytes were also estimated using mixed effects models to allow for correlations among observations from the same donors. All analyses were performed using SAS/STAT software Version

9.4 (SAS Institute Inc., Cary NC).

3.3 Results

3.3.1 B-cell selective expression of BRAFV600E in Eµ-TCL1 mice accelerates disease and shortens survival

Mouse strains carrying CD19-driven Cre recombinase and Ig Eµ-driven human

TCL1, without or with a conditional Cre-driven BRAFV600E allele (BRAFWTxTCL1 and

BRAFVExTCL1, respectively) were generated and genotypically confirmed. Immunoblot analysis showed that BRAFV600E protein is expressed in B-cells but not T-cells from the spleens of BRAFVExTCL1 mice, and this expression is associated with increased phosphorylation of downstream kinases MEK and ERK (Figure 3.1). The onset of leukemia, as defined by at least 10% CD19+CD5+ double-positive cells among the CD45+ population in the peripheral blood, was evaluated biweekly by flow cytometry starting at

6 weeks. BRAFVExTCL1 mice (n=34) developed leukemia at a median of 4.9 months (95%

CI: 4.5-5.6 months), significantly earlier than BRAFWTxTCL1 (median 8.1 months; 95%

CI: 7.6-9.2 months; n=46) or BRAFCAxTCL1 (median 8.1 months; n=22) (p<0.001 for both comparisons; Figure 3.2A). Leukemia onset in the control groups was comparable to standard Eµ-TCL1 mice158,219. There was no evidence of leukemia during this period in

BRAFVExCD19cre mice as expected, as BRAFV600E by itself is not sufficient to induce malignancy167,172. This group was therefore not included in the remaining experiments.

BRAFVExTCL1 mice exhibited significantly shorter survival (median 7.3 months; 95% CI:

6.9-8.0 months; n=34) compared to BRAFWTxTCL1 mice (median 12.1 months; 95% CI:

11.5-12.7 months; n=46) or BRAFCAxTCL1 mice (median 11.3 months; 95% CI: 9.9-11.9 months; n=22) (Figure 3.2B; p<0.001 for both comparisons).

3.3.2 Phenotype of disease in BRAFVExTCL1 mice

Detailed necropsies were performed on mice meeting euthanasia criteria due to advanced disease. Organ-to-body weight ratios for liver and spleen were higher in BRAFVE 62 vs. BRAFWT TCL1 mice (n=19 per group for spleen, p=0.043; n=18 per group for liver, p=0.004) (Figure 3.3A). Gross appearances of the spleen and liver are shown in Figure

3.3B. BRAFVE animals showed thymic enlargement and reduced hematocrit relative to

BRAFWT mice, although differences did not reach statistical significance (n=15 per group for thymus, p=0.058; n=16 and 13 respectively for hematocrit, p=0.079), as well as larger submandibular, mediastinal and intra-abdominal lymph nodes. Blood smears from

BRAFVExTCL1 mice with advanced disease showed atypical lymphocytes with decreased numbers of mature monocytes, whereas smears of BRAFWTxTCL1 mice with similar disease load as determined by circulating tumor cells revealed small lymphocytes with condensed chromatin and mature monocytes as seen in Eµ-TCL1 mice (Figure 3.3C)158.

Tissues from 20 BRAFWTxTCL1 and 22 BRAFVExTCL1 mice that reached euthanasia criteria were evaluated histologically. Lymph nodes, spleen, liver, lung and marrow from

19 of the 22 BRAFVExTCL1 mice were markedly infiltrated by a pleomorphic population of neoplastic cells, consistent with a diagnosis of histiocyte-associated B-cell leukemia/lymphoma (Figure 3.4A-B). Infiltrates were composed of macrophages, multinucleated giant cells of macrophage lineage, B-lymphocytes and plasma cells. B-cell and macrophage lineages were confirmed by immunohistochemical staining for B220 and

F4/80, respectively (Figure 3.4C). Scattered CD3+ cells consistent with tumor infiltrating

T-lymphocytes were also observed. In contrast, 18 of 20 BRAFWTxTCL1 mice with late- stage disease developed lymphocytic tissue infiltrates as described for Eµ-TCL1 transgenic mice158 (Figure 3.4D). Neoplasms in these mice were mostly B220+, but also had scattered

CD3+ and F4/80+ cells, interpreted as tumor infiltrating T-cells and macrophages,

63 respectively (Figure 3.4C). Of the 3 remaining BRAFVExTCL1 mice, 1 developed purely

B-cell infiltrates, 1 developed purely histiocytic infiltrates, and the third had bacterial bronchopneumonia without evidence of neoplasia. Two BRAFWTxTCL1 mice had infiltrates consistent with BRAFVE mice, suggesting these phenotypes may not be qualitatively distinct but degrees of the same disease. Both groups showed striking changes in complete blood count (CBC), including mild to moderate leukocytosis composed mostly of circulating neoplastic lymphocytes. Mice also showed mild to severe regenerative anemia and thrombocytopenia, likely due to myelophthisis.

To investigate B-cell development prior to leukemia onset, cells from spleen and marrow from 2-month-old BRAFVExTCL1 and BRAFWTxTCL1 mice (n=7 each) were assessed by flow cytometry. Figure 3.5A shows that pro (B220+CD43+IgM-), pre

(B220+CD43-IgM-), immature (B220+CD43-IgM+), and mature (splenic IgMlowIgDhi) B- cells220 developed similarly in the two groups. However, BRAFVExTCL1 mice showed a slightly higher percentage of myeloid cells (p=0.042) and a trend toward higher B cell and lower T cell numbers in spleens compared to BRAFWTxTCL1 mice (Figure 3.5B).

Complete necropsies were also performed on BRAFWTxTCL1 and BRAFVExTCL1 mice

(n=5 and 6, respectively) at 2 months of age, prior to leukemia onset. Histologically, spleens from all BRAFVExTCL1 animals showed increased numbers of atypical lymphocytes throughout the red pulp, which was not observed in the BRAFWTxTCL1 mice

(Figure 3.6). No gross lesions were observed in either group at this stage. To determine if this model is recapitulated by adoptive transfer of neoplastic cells to allow larger, more uniform studies, leukemia cells from BRAFVExTCL1 mice were intravenously injected

64 into 7-week-old syngeneic wild type recipients (n=11). By 2 weeks, CD5+CD19+ leukemia cells were detected in the peripheral blood of all mice. Complete necropsies with histologic evaluation of spleen, bone marrow, lymph nodes, thymus and liver was performed at 3 weeks. All mice demonstrated histiocyte-associated B cell leukemia similar to their transgenic counterparts (Figure 3.7).

Together, these data demonstrate that expression of BRAFV600E in the B-cell compartment of Eµ-TCL1 mice results in earlier onset, more aggressive disease. Unlike

Eµ-TCL1 mice with unmutated BRAF, the disease in BRAFVExTCL1 mice later progresses to a tissue-phase cancer consistent with histiocyte-associated B-cell lymphoma, as occasionally observed in Richter’s transformation221,222.

3.3.3 BRAFV600E expression does not increase cell proliferation but reduces cell apoptosis

The expression of mutated BRAF in tumor cells alters their proliferative and apoptotic properties, in part via cyclin D1 and BCL2 family proteins, respectively223. To examine the effect of BRAFV600E expression on these parameters in pre-leukemic B-cells in vivo, EdU was injected into BRAFVExTCL1 (n=6) or BRAFWTxTCL1 (n=5) transgenic mice at approximately 2 months of age, prior to onset of leukemia). After 24 hr, spleen cells were collected and EdU incorporation and annexin-V binding were assessed.

Proliferation of CD19+CD5- B cells, as measured by EdU incorporation, was not different between the groups (p=0.319). However, B cells from BRAFVE mice showed significantly lower apoptosis as assessed by annexin-V binding (p<0.001) (Figure 3.8A.).

These effects were further investigated in vivo using the adoptive transfer model.

In the first experiment, pooled leukemic B cells from BRAFVExTCL1 or BRAFWTxTCL1 65 mice were engrafted into wild-type syngeneic mice (n=14 for BRAFVExTCL1; n=24 for

BRAFWTxTCL1). Following leukemia onset, EdU was injected and 24 hr later, spleen cells were isolated for flow cytometric analysis as for Figure 3.8A. Figure 3.8B shows that

BRAFV600E did not increase B-cell proliferation in vivo and in fact mildly decreased it

(p=0.047). Apoptosis was slightly but not significantly reduced (p=0.105). A second adoptive transfer experiment using a single leukemic B cell donor for each genotype confirmed that cell proliferation was not significantly increased in the presence of

BRAFV600E, but cell apoptosis was significantly reduced (p=0.756 and p<0.001, respectively) (Figure 3.8C). These experiments support that the introduction of mutated

BRAF in B-cell leukemia does not impact proliferation and only moderately reduces spontaneous apoptosis.

3.3.4 BRAFV600E B-cells suppress T cell proliferation and function

The above results, together with the immunophenotypic characterization, suggest that the BRAFV600E mutation might accelerate disease development in B-cell leukemia by promoting immune evasion or suppression as observed in some solid tumors146-148,150,151,224, rather than simply by affecting proliferation or apoptosis. We therefore examined the effects of B cells expressing BRAFV600E on other immune compartments. We first tested this mechanistically in vitro, using OSUCLL cells with doxycycline (dox)-inducible expression of normal or mutated BRAF215. These cells were co-cultured with T cells from normal donors that were labeled with CFSE and stimulated with anti-CD3/anti-CD28 antibodies. While OSUCLL cells in general impaired proliferation of stimulated T cells, this was markedly enhanced in BRAFV600E-expressing cells compared to those transfected 66 with an empty vector (OSUCLL-tet) or wild-type BRAF (Figure 3.9A). Proliferation was rescued by the BRAFV600E inhibitor dabrafenib (Figure 3.9B), further supporting the contribution of mutant BRAF to this effect. Dabrafenib had no direct effect on T cell proliferation. Interestingly, vemurafenib at the same dose led to a notable impairment of T cell proliferation, in contrast to what has been reported in vitro225 and in support of clinical data226, and was therefore not used.

These experiments were repeated using transwell plates in which OSUCLL cells and normal T cells were separated by a 0.4 µm filter. As shown in Figure 3.9C, OSUCLL cells still caused an impairment of T cell proliferation, but to a lesser extent, indicating that soluble factors as well as cell-cell contact contribute to T cell inhibition. To further interrogate candidate factors, OSUCLL cells were analyzed by flow cytometry for differences in expression of known immune suppressive molecules including CD200 and

PD-L1. In contrast to what has been reported in melanoma and AML cells147,149, expression of BRAFV600E did not affect the expression of either of these on OSUCLL cells (data not shown). Next, levels of immunomodulatory cytokines produced by anti-CD3/CD28- stimulated T cells (IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ) were measured by Cytometric

Bead Array (CBA). Each of these was strongly induced by anti-CD3/CD28 stimulation, and all except IL-2 were reduced in the presence of OSUCLL cells. However, these effects were not different in BRAFV600E cells compared to cells transfected with wild-type BRAF or an empty vector, suggesting these factors are not responsible for the increased inhibitory effect of OSUCLL-BRAFV600E cells on T cell proliferation (Figure 3.10). Additionally, blocking antibodies against IL-10 or TNF did not rescue T-cell proliferation in co-cultures

67 with OSUCLL-BRAFV600E cells, despite effective blocking as assessed by CBA (data not shown).

To identify candidate immune suppressive factors in vivo, normal mice were engrafted with spleen cells from leukemic BRAFVE or BRAFWT TCL1 mice. After mice achieved a substantial tumor burden (all mice exceeded 35% CD19+/CD5+ cells in the periphery), plasma from these animals was analyzed by a multiplex cytokine assay (n=4 and 5 for BRAFWT and BRAFVE mice, respectively; Figure 3.11A). While most differences did not reach statistical significance, trends toward elevated IL-5, IL-6 and MIP-2 (p=096,

0.095, and 0.108, respectively), and decreased IL-10, IL-12 and IFN-γ (p=0.100, 0.444 and

0.024, respectively) were observed in BRAFVE-transplanted mice. Elevated TNF mRNA expression (2.7-fold change; p=0.001) and decreased IL-10 mRNA expression (0.6-fold change; p=0.027) were observed in BRAFVE-mutated leukemic B cells compared with wild-type BRAF leukemic B cells, consistent with the plasma results.

To investigate the effect of BRAFVE leukemic B cells on T cells in vivo, we analyzed T cell subsets in the adoptive transfer model. Mice were engrafted with leukemia cells from each genotype and monitored until the onset of leukemia. After 3 weeks, peripheral blood T cells were examined by flow cytometry. Relative to BRAFWT leukemia cells, BRAFVE leukemic B cells induced the T-cell activation/exhaustion markers PD-1,

CD244, and CD160 (n=25 and 11 for BRAFWT and BRAFVE, respectively; p<0.01 for each) (Figure 3.11B). Additionally, BRAFVE leukemic B cells did not alter the CD4:CD8 ratio, but significantly increased the population of CD44-expressing (antigen-experienced)

T cells161. These results demonstrate that BRAF-mutated leukemia cells generally produce

68 a more immune-suppressive T-cell phenotype in vivo compared to wild-type BRAF leukemia cells.

3.3.5 BRAFVE B cell leukemia impacts myeloid cells

As previously reported, CLL-associated myeloid cells are aberrant in the Eµ-TCL1 model and show skewing toward a patrolling phenotype, with enhanced PD-L1 expression and secretion of inflammatory and immunosuppressive cytokines164. The impact of the

BRAFVE mutation on the myeloid compartment was therefore evaluated, again using the adoptive transfer model. Mice were engrafted with either BRAFWT or BRAFVE leukemia cells, and upon achieving disease, spleens were collected and cells were evaluated by flow cytometry. Interestingly, the presence of BRAFVE leukemia cells resulted in significant increases in PD-L1 expression on peripheral myeloid (CD11b+) cells (p<0.001; Figure

3.11C). Additionally, there was an increase in F4/80+ macrophages (19.8%; p=0.002; n=15 each; Figure 3.11D). However, in another trial of a smaller cohort (n=8 BRAFWT; 11

BRAFVE), changes were observed only in a subset and overall differences were not significant (Figure 3.12). There was an increasing trend towards CD11b+Ly6CintLy6Ghi

MDSCs and decreasing trend towards CD11b+Ly6CloLy6Glo patrolling monocytes in spleens of mice with BRAFVE leukemia in both studies (Figure 3.11E and Figure 3.12).

These results indicate that in addition to T-cells, myeloid cells are also affected toward to a more immune suppressive phenotype by BRAFVE mutated cells to result in additional remodeling of the leukemia microenvironment.

3.4 Discussion

BRAF activating mutations, particularly BRAFV600E, have been most commonly described in solid tumors including melanoma. Such mutations are less frequent across hematologic diseases, with the notable exceptions of HCL100, Langerhans cell histiocytosis227, and Erdheim-Chester disease228. However, the numbers of BRAF-mutant leukemias, lymphomas and myelomas identified are increasing due to broader and deeper sequencing efforts96,98,180,229. Besides increased growth and decreased apoptosis, an established effect of aberrantly activated MAPK in tumor cells is enhanced immune suppression, mediated by multiple factors including immune checkpoint molecules and cytokines153,155. In melanoma models in vitro, selective BRAFV600E inhibition improves T- cell recognition of tumors151. In both a mouse xenograft melanoma model155 and patients with metastatic melanoma156,157, selective BRAF inhibitor therapy leads to improved CD8+

T cell tumor infiltration, decreases in immunosuppressive cytokines such as IL-6, IL-8 and

VEGF, and increased levels of activation/exhaustion markers such as PD-1 and TIM-3157.

However, the role of BRAF activating mutations in hematologic malignancies and how these impact immune function are unclear, and few mouse models are available to evaluate this.

Here, we generated a novel mouse strain in which BRAFV600E is selectively expressed in B-cells of the established Eµ-TCL1 B-leukemia mouse model, and investigated the pathological function of this mutation in B-cell leukemia development, behavior and immune evasion/suppression in vivo. The Eµ-TCL1 model was selected due to its extensive prior characterization, including studies that demonstrate its utility in

70 investigating tumor-induced immune defects159,217,230. Our results demonstrate that

BRAFV600E accelerates Eµ-TCL1 B-cell leukemia development, likely due to a combination of decreased spontaneous apoptosis and enhanced immune suppression rather than increased proliferation. Additionally, BRAFVE leukemia cells produced greater T-cell effects than BRAFWT leukemia cells, including an enhanced activated/exhausted phenotype that was previously described in CLL patients231. Myeloid subsets were affected as well, with increased expression of PD-L1 on circulating monocytes and elevated percentage of F4/80+ macrophages in spleens. Alterations in monocytic subsets were also observed, with a shift from a patrolling to an MDSC immunophenotype in contrast to what has been reported in Eµ-TCL1 mice164. Depletion of myeloid cells including patrolling monocytes in the Eµ-TCL1 adoptive transfer model controls CLL-like disease development164, but the patrolling monocytes also have anti-tumor function to suppress tumor metastasis to lung by increasing NK cells-mediated killing of metastatic tumor cells in multiple metastatic mouse models232. Although the function of patrolling cells in the cancer context is controversial, the observation of the affected myeloid cells in our mouse model indicates that BRAFV600E leukemia cells skew these cells to an immune suppressive phenotype. Regardless, functional analyses will be required to fully define these populations and these changes suggest a tumor-mediated shift of innate and adaptive immunity in BRAFVE mice toward a more tumor-tolerant environment.

Selective inhibitors of BRAFV600E have clear clinical benefit in BRAF-mutated cancers, but duration of response is typically short. Thus, efforts are now directed toward combination strategies with additional kinase inhibitors (e.g. trametinib) as well as immune

71 checkpoint inhibitors and other immune-based therapies, based on preclinical studies demonstrating activity233,234. Numerous clinical trials of such combinations are underway, but to evaluate these systematically in hematologic malignancies where BRAF mutations are less common will be a lengthy process. A relevant model of BRAF-mutated leukemia will be a valuable tool not just to understand the mechanism of immune modulation but to quickly evaluate various combinations to support clinical trial design.

We investigated several factors that might mediate the enhanced immune suppressive effects of BRAFVE-mutated leukemia cells. In agreement with previous reports in solid tumors235, PD-L1 and CD200 are not further induced in BRAFVE cells compared to BRAFWT cells, despite reports these markers are increased in BRAF-inhibitor resistant cells148,235. However, we detected substantial increases in IL-10 and TNF in a CLL cell line with mutated BRAF, which is in line with the TGF-β induced IL-10 in the melanoma line

A375 with endogenous expression of BRAFV600E 146. Although blocking antibodies to these cytokines failed to reverse T-cell inhibitory effects of BRAFVE-mutant cells in vitro and

IL-10 was not changed in vivo, the potential contributions of TNF and other candidate cytokines in vivo remain to be evaluated. TNF transcript is increased in mouse BRAFVE leukemia cells, and adoptively transferred BRAFVE leukemia cells increase TNF in recipient mice. TNF exerts both pro-tumor and anti-tumor activities in different cancers. In

CLL, TNF supports proliferation of CLL cells and induces IL-6 expression to protect from spontaneous apoptosis236,237. Furthermore, we found that IFN-γ levels in recipient mice are reduced in the presence of BRAFVE leukemia, which may be due to BRAFVE leukemia- induced T cell suppression. IFN-γ, primarily secreted by T cells and NK cells, is one

72 indicator of T-cell activation and can induce PD-L1 expression on tumor cells. Thus, experiments addressing the role of IFN-γ in vivo are also required to determine its role. It has been demonstrated the leukemia B cells induce immune suppression in the Eµ-TCL1 mice as evidenced by changes in expression of cytokines and immune checkpoints and remodeling of cytoskeleton. For example, increase of IL-1, IL-4, IL-6, and CTLA4+ Treg cells, and decrease of IL-2, IFN-g, IL-12b, BTLA are seen in the Eµ-TCL1 mice compared with wild-type control mice159. In addition, PD-L1 is known to be overexpressed on CLL cells and MDSCs from periphery blood of CLL patients132,238, and blockade of PD-1/PD-

L1 axis prevents immune dysfunction in the Eµ-TCL1 adoptive transfer model163.

Moreover, IL-10high and IL-12low phenotypes are often see in most TAMs239-241. Our data suggested that the decrease of IL-12b combined with the decrease of IFN-g and increase of IL-5 may polarize the Th2 CD4 T cell response in the BRAFV600E mutant Eµ-TCL1 mice. Additionally, the further up-regulation of IL-6 and down-regulation of IFN-g and IL-

12b in the BRAFV600E mutant Eµ-TCL1 mouse model compared with the wild-type BRAF

Eµ-TCL1 model may contribute to the more aggressive disease and immune evasion.

Regardless, our experiments demonstrate BRAFVE leukemia cells likely mediate suppression simultaneously through several factors, both soluble and contact-dependent, suggesting therapeutic combinations of more than two agents may be needed.

The phenotype of the disease at earlier stages is similar in BRAFVE and BRAFWT

TCL1 mice, with progressive accumulation of abnormal B-lymphocytes in the organs and periphery. However, the leukemia in BRAFVE mice (19 of 22 examined for histology; 86%) transforms to a histiocyte-associated B-cell lymphoma, which was observed in just 2 of the 73

20 BRAFWT leukemic mice examined for histology. This disease transformation has characteristics of a variant of Richter’s syndrome, a disease transformation to aggressive lymphoma arising from CLL. It was reproduced in normal mice receiving leukemia cells by adoptive transfer, indicating these effects are due to an intrinsic property of the tumor cells. This progression to a lymphoma-type disease may in part explain the interesting finding that, despite the relative frequency of MAPK activating mutations in CLL (~9%),

BRAFV600E mutations are quite rare242. It is possible that the greater activity of BRAFV600E vs. other pathway mutations (even within BRAF) produces a disease phenotype inconsistent with CLL, and such cases are thus not included in the study cohort.

In conclusion, the introduction of BRAFV600E into B-cell leukemia cells in vivo provides a novel and valuable tool to investigate the impact of this mutation on immune function in disseminated disease, building on the extensive work conducted in melanoma models. Current studies are investigating the impact of BRAF inhibition in these mice on the immune microenvironment. Our goal is to use this model to test novel combination strategies that will simultaneously block tumor growth and restore immune function for long-term disease control.

Figure 3. 1 BRAFV600E protein is expressed in the B cells of BRAFVExTCL1 mice. Lysates from purified B or T cells from spleens of BRAFWTxTCL1 or BRAFVExTCL1 transgenic mice were analyzed by immunoblot for mutant and normal BRAF as well as total and phosphorylated MEK and ERK. Lysate from OSUCLL-BRAFV600E+dox was included as a positive control (+).

BRAFWTxTCL1 BRAFVExTCL1 Tg mouse: #1 #2 #3 #4

T B T B T B T B + BRAFV600E BRAF p-MEK

MEK

p-ERK

ERK actin

Figure 1 BRAFWTxTCL1 BRAFVExTCL1 Tg mouse: #1 #2 #3 #4

TBB TBTBT + BRAFV600E BRAF p-MEK

MEK

p-ERK

ERK actin Figure 3. 2 BRAFV600E produces more aggressive disease in the Eµ-TCL1 mouse Figuremodel. 1. BRAFV600E protein is expressed in the B cells of BRAFVExTCL1 mice. Lysates from V600E B-cell specific expression of BRAF significantlyWT shortensVE (A) leukemia onset and (B) purified B or T cells from spleens of BRAF VExTCL1 or BRAFWT xTCL1 transgenicCA mice were analyzedoverall survival by immunoblot (p<0.001 forcomparing mutant BRAFand normal vs. BRAFBRAF as well and asBRAF total and for phosphorylated both onset MEKand OS).and ERK. Lysate from OSUCLL-BRAFV600E+dox was included as a positive control (+).

Figure 2 A. Leukemia Onset BRAFVExTCL1 (n=34) WT 1.0 BRAF xTCL1 (n=46) BRAFCA x TCL1 (n=22) 0.8 p<0.001 0.6

0.4

Survival Probability Survival 0.2

0 0246810121416 Months from Birth B. Overall survival 1.0

0.8 p<0.001 0.6

0.4

Survival Probability Survival 0.2

0 0246810121416 Months from Birth

Figure 2. BRAFV600E produces more aggressive disease in the Eμ-TCL1 mouse model. B-cell specific expression of BRAFV600E significantly shortens (A) leukemia onset and (B) overall survival (p<0.001 comparing BRAFVE vs. BRAF 76 WT and BRAFCA for both onset and OS).

Figure 3. 3 Phenotypes of leukemic BRAFWT and BRAFVE TCL1 transgenic mice. (A) Weight ratios of spleens (n=19 for each genotype, p=0.043) and livers (n=18 for each genotype, p=0.004) in BRAFWT and BRAFVE TCL1 mice. (B) Gross appearances of spleen (left panel) and liver (right panel) of a normal adult mouse (left) and leukemic BRAFVE (middle) and BRAFWT (right) transgenic mice. Pictures are representative of 20 BRAFWT and 22 BRAFVE mice examined. (C) Peripheral blood smears (100x magnification) from (left) BRAFWTxTCL1 mice and (right) BRAFVExTCL1 mice with advanced disease show Figurecirculating 3 leukemia cells. Decreased red blood cell density and numerous polychromatophils in BRAFVExTCL1 mice indicate a regenerative anemia.

A A p=0.004 p=0.043 Figure 3 20 30

15 20 A 10 10 p=0.004 5 p=0.043 20 30 SupplementalSupplemental Fig. 1. Gross Fig. 1.appearances Gross appearances of A spleen of A andspleen B liver and ofB a livernormal of a normal 0 0 adult mouse (left) and leuemicVE BRAFVE (middle) andWT BRAFWT (right) transgenic adult mouse15 (left)BRAF andWT leuemicBRAF BRAFVE (middle) 100 x weight liver/body and BRAFBRAF(right)WT transgenicBRAFVE mice. Pictures 100 x weight spleen/body mice. Picturesare representative are representative of 20 BRAF of 20WT BRAFand20 22WT BRAFand 22VE miceBRAF examined.VE mice examined. 10 B 10 B5 B ABBRAFABWTxTCL1 BRAFVExTCL1 0 0

BRAFWT BRAFVE 100 x weight liver/body BRAFWT BRAFVE spleen/body weight x 100 x weight spleen/body

B B WT VE C BRAF xTCL1 BRAF xTCL1

SupplementalSupplemental Fig. 2. henotypes Fig. 2. henotypes of leukemic of leukemic BRAFWT BRAFand BRAFWT andVE TCL1BRAF VE TCL1 transgenictransgenic mice. A mice. Spleen Ai Spleen and mandibulari and mandibular lymph node lymph ii node(10x magnifications)ii (10x magnifications) in BRAFVEinxTCL1 BRAF VEmicexTCL1 showing mice showingeffacement effacement of organ ofarchitecture organ architecture by neoplas bytic neoplas cells. tic cells. iii igheriii magnification igher magnification (60x) of lymph(60x) ofnode lymph shows node that shows cells that inclu cellsde macrophages include macrophages Figure 3. Phenotypes of leukemic BRAFWT and BRAFVE TCL1 transgenic mice. (A) Weight and multinucleatedand multinucleated giant cells giant (arrow). cells iv(arrow). Numerous iv Numerous scattered scattered foci of large foci atypicalof large atypical ratios of spleens (n=19 for each genotype, p=0.043) and livers (n=18 for each genotype, lymphocytes many of which demonstrate plasmacytoid differentiation (arrow). lymphocytesp=0.004) manyin BRAF ofWT whichand BRAF demonstrateVE TCL1 mice. plasmacytoid(B) Peripheral blood differentiat smears (100xion (arrow). magnification) from (left) BRAFWTxTCL1 mice and (right) BRAFVExTCL1 mice with advanced disease show circulating leukemia cells. Decreased red blood cell density and numerous polychromatophils in BRAFVExTCL1 mice indicate a regenerative anemia.

Figure 3. Phenotypesi ofi leukemic BRAFWT and BRAFii VE TCL1ii transgenic mice. (A) Weight 77 ratios of spleens (n=19 for each genotype, p=0.043) and livers (n=18 for each genotype, p=0.004) in BRAFWT and BRAFVE TCL1 mice. (B) Peripheral blood smears (100x magnification) from (left) BRAFWTxTCL1 mice and (right) BRAFVExTCL1 mice with advanced disease show circulating leukemia cells. Decreased red blood cell density and numerous polychromatophils in BRAFVExTCL1 mice indicate a regenerative anemia.

iii iii iv iv Supplemental Fig. 1. Gross appearances of A spleen and B liver of a normal adult mouse (left) and leuemic BRAFVE (middle) and BRAFWT (right) transgenic mice. Pictures are representative of 20 BRAFWT and 22 BRAFVE mice examined.

Figure 3. 4 Phenotypes of leukemic BRAFWT and BRAFVE TCL1 transgenic mice. (A) Spleen (i) and mandibular lymph node (ii) (10x magnifications) in BRAFVExTCL1 mice showing effacement of organ architecture by neoplastic cells. (iii) higher magnification (60x) of lymph node shows that cells include macrophages and multinucleated giant cells (arrow). (iv) Numerous scattered foci of large atypical lymphocytes many of which demonstrate plasmacytoid differentiation (arrow). (B) Extended histology of organs from BRAFVExTCL1 mice with advanced disease. Infiltration of neoplastic cells is shown in (i) liver (60x), (ii) kidney (60x), (iii) lung (40x), and (iv) bone marrow (40x). Representative of 22 mice examined. (C) Lymph nodes of BRAFSupplementalVExTCL1 show Fig. 2. increased henotypes numbers of ofleukemic cells of BRAF B-cellWT (B220and+ )BRAF and macVE TCL1rophage (F4/80transgenic+) lineages. mice. There A Spleen were alsoi smalland mandibular CD3+ lymphocytes lymph node throughout ii (10x the magnifications) neoplastic populationin BRAFVE interpretedxTCL1 mice as tumor showing infiltrating effacement T- cells of organ(magnification architecture 60x). by(D) neoplas Spleenstic (i) cells. andiii lymph igher nodes magnification (ii) (10x magnifications) (60x) of lymph from node BRAF showsWT xTCL1that cells mice inclu arede effac macrophagesed by a neoplasticand multinucleated population. (giantiii) h ighercells magnification(arrow). iv Numerous(60x) of the scatteredlymph node foci reveals of large sheets atypical of neoplasticlymphocytes lymphocytes many of which consistent demonstrate with the plasmacytoid previously described differentiat Eµion-TCL1 (arrow). mouse phenotype.

A i ii

iii iv Supplemental Fig. 2. B Extended histology of organs from BRAFVExTCL1 mice with advanced disease. Infiltration of neoplastic cells is shown in i liver (60x) ii idney (60x) iii lung (40x) and iv bone marrow (40x). Representative of 22 mice examined.

B iii

Continued

iii iv Supplemental Fig. 1. Gross appearances of A spleen and B liver of a normal adult mouse (left) and leuemic BRAFVE (middle) and BRAFWT (right) transgenic mice. Pictures are representative of 20 BRAFWT and 22 BRAFVE mice examined.

Supplemental Fig. 2. henotypes of leukemic BRAFWT and BRAFVE TCL1 transgenic mice. A Spleen i and mandibular lymph node ii (10x magnifications) in BRAFVExTCL1 mice showing effacement of organ architecture by neoplastic cells. iii igher magnification (60x) of lymph node shows that cells include macrophages and multinucleated giant cells (arrow). iv Numerous scattered foci of large atypical lymphocytes many of which demonstrate plasmacytoid differentiation (arrow).

A i ii

B iii

iii iv

Continued

Supplemental Fig. 2 C Lymph nodes of BRAFVExTCL1 show increased numbers of cells of B-cell (B220+) and macrophage (F4/80+) lineages. There were also small CD3+ lymphocytes throughout the neoplastic population interpreted as tumor infiltrating T- cells (magnification 60x). D Spleens i and lymph nodes ii (10x magnifications) from BRAFWTxTCL1 mice are effaced by a neoplastic population. iii igher magnification (60x) of the lymph node reveals sheets of neoplastic lymphocytes consistent with the previouslyFigure 3.4 described Continued E-TCL1 mouse phenotype.

C BRAFWT BRAFVE B220 F4/80 CD3

i ii iii

Figure 3. 5 Cell populations in transgenic mice prior to leukemia onset. (A) Spleen and bone marrow cells from 2-month-old transgenic mice (n=6-7 for each genotype) were analyzed by flow cytometry for pro-B cells (B220+CD43+IgM-), pre-B cells (B220+CD43-IgM-), immature B cells (B220+CD43-IgM+), and mature B cells (splenic IgMlowIgDhi). Differences between groups were not significant. (B) Spleen cells from 2-month-old BRAFWT or BRAFVE TCL1 transgenic mice (n=6 for each genotype) were analyzed by flow cytometry for B, T and myeloid cell populations. BRAFVExTCL1 + Figuremice 4 showed a minor but significant increase in myeloid (CD11b ) cells prior to leukemia onset and trends toward increased B (B220+) and decreased T (CD3+) cells.

A Bone marrow Spleen 60 50

40 40 30

20 20 10

0 0 % of B220+ population % of B220+ % of CD45+ population of CD45+ %

pro B pre B immature B IgM IgMloIgDhi

B Spleen 80

40 p=0.014

% of CD45+ population 0

B220+ CD3+ CD11b+

Figure 4. Cell populations in transgenic mice 81 prior to leukemia onset. (A) Spleen and bone marrow cells from 2 month old transgenic mice (n=6-7 for each genotype) were analyzed by flow cytometry for pro-B cells (B220+CD43+IgM-), pre-B cells (B220+CD43-IgM-), immature B cells (B220+CD43-IgM+), and mature B cells (splenic IgMlowIgDhi). Differences between groups were not significant. (B) Spleen cells from 2 month old BRAFWT or BRAFVE TCL1 transgenic mice (n=6 for each genotype) were analyzed by flow cytometry for B, T and myeloid cell populations. BRAFVExTCL1 mice showed a minor but significant increase in myeloid (CD11b+) cells prior to leukemia onset and trends toward increased B (B220+) and decreased T (CD3+) cells. Figure 3. 6 Spleens of transgenic mice at two months of age. Spleens of transgenic mice at 2 months of age, prior to the onset of leukemia were examined histologically (n= 5 and 6 for BRAFWT and BRAFVE respectively. (A) VE WT SupplementalBRAF Fig.xTCL1 3. Spleens are effaced of bytransgenic neoplastic cellsmice while at 2 (monthsB) BRAF of xTCL1age prior mice to have the onset normal splenic architecture with well-defined red (R) and whiteWT (W) pulp (10xVE of leuemiamagnifications). were examined Higher histologically magnification (60x) (n= of 5 spleensand 6 from for BRAF(C) BRAFVEandxTCL1 BRAF and (D) VE WT respectively.BRAF AWTBRAFxTCL1 mice.xTCL1 are effaced by neoplastic cells while B BRAF xTCL1 mice have normal splenic architecture with well-defined red (R) and white (W) pulp (10x magnifications). igher magnification (60x) of spleens from C BRAFVExTCL1 and D WT BRAF xTCL1 mice.

AB W W

Supplemental Fig. 4. Mice engrafted with leuemia cells from BRAFVE mice demonstrate neoplastic infiltrates composed of lymphocytes and macrophages in A spleen (60x) B lymph node (60x) C bone marrow (40x) and D liver (40x) among other organs.

AB 82

CD Supplemental Fig. 3. Spleens of transgenic mice at 2 months of age prior to the onset of leuemia were examined histologically (n= 5 and 6 for BRAFWT and BRAFVE respectively. A BRAFVExTCL1 are effaced by neoplastic cells while B BRAFWTxTCL1 mice have normal splenic architecture with well-defined red (R) and white (W) pulp (10x magnifications). igher magnification (60x) of spleens from C BRAFVExTCL1 and D BRAFWTxTCL1 mice.

AB W W

Figure 3. 7 Histology of mice engrafted with leukemia cells from BRAFVE TCL1 mice. Mice engrafted with leukemia cells from BRAFVE mice demonstrate neoplastic infiltrates composed of lymphocytes and macrophages in (A) spleen (60x), (B) lymphVE node (60x), Supplemental(C) bone Fig. marrow 4. Mice (40x), engrafted and (D) liver with (40x) leuemia among other cells organs. from BRAF mice demonstrate neoplastic infiltrates composed of lymphocytes and macrophages in A spleen (60x) B lymph node (60x) C bone marrow (40x) and D liver (40x) among other organs.

Figure 3. 8 Effect of BRAFV600E on cell proliferation and apoptosis in vivo. (A) Proliferation (EdU incorporation) and apoptosis (Annexin-V binding) was analyzed by flow cytometry in normal B cells (CD19+CD5-) from the spleens of pre-leukemic transgenic mice (~two months; n=6 for both BRAFWTxTCL1 and BRAFVExTCL1). (B) The same experiment was conducted using healthy mice engrafted with leukemia cells pooled from three leukemic donor animals, following the onset of leukemia (>10% CD19+CD5+ cells in peripheral blood CD45+ population) (n=18 and 24 BRAFWTxTCL1 for EdU and Annexin-V, respectively; n=14 BRAFVExTCL1 for both EdU and Annexin- V). (C) The experiment in (B) was repeated using single donors for each genotype (EdU: n=8 and n=9 for BRAFWTxTCL1 and BRAFVExTCL1, respectively; Annexin-V: n=8 and n=19 for BRAFWTxTCL1 and BRAFVExTCL1, respectively).

Figure 5

A 10 20 p<0.001 8 15 6 p=0.319 10 4 5 2 CD19+CD5-EdU+ % CD19+CD5-EdU+ 0 % CD19+CD5-AnnV+ 0 BRAFWT BRAFVE BRAFWT BRAFVE

B 50 20 p=0.047

40 p=0.105 15 30 10 20 5 10

% CD19+CD5+EdU+ CD19+CD5+AnnV+ % CD19+CD5+AnnV+ 0 0 BRAFWT BRAFVE BRAFWT BRAFVE

C 50 20

40 p=0.756 p<0.001 15

30 10 Continued 20 84 5 10 CD19+CD5+EdU+ % CD19+CD5+EdU+ CD19+CD5+AnnV+ % CD19+CD5+AnnV+ 0 0 BRAFWT BRAFVE BRAFWT BRAFVE

Figure 5. Effect of BRAFV600E on cell proliferation and apoptosis in vivo. (A) Proliferation (EdU incorporation) and apoptosis (annexin-V binding) was analyzed by flow cytometry in normal B cells (CD5-CD19+) from the spleens of pre-leukemic transgenic mice (~2 months; n=6 for both BRAFWTxTCL1 and BRAFVExTCL1). (B) The same experiment was conducted using healthy mice engrafted with leukemia cells pooled from three leukemic donor animals, following the onset of leukemia (>10% CD19+CD5+ cells in peripheral blood CD45+ population) (n=18 and 24 BRAFWTxTCL1 for EdU and annexin, respectively; n=14 BRAFVExTCL1 for both EdU and annexin). (C) The experiment in (B) was repeated using single donors for each genotype (EdU: n=8 and n=9 for BRAFWTxTCL1 and BRAFVExTCL1, respectively; Annexin-V: n=8 and n=19 for BRAFWTxTCL1 and BRAFVExTCL1, respectively). Figure 5

A 10 20 p<0.001 8 15 Figure 3.8 Continued 6 p=0.319 10 4 5 2 CD19+CD5-EdU+ % CD19+CD5-EdU+ 0 % CD19+CD5-AnnV+ 0 BRAFWT BRAFVE BRAFWT BRAFVE

B 50 20 p=0.047 40 p=0.105 15 30 10 20 5 10 CD19+CD5+EdU+ % CD19+CD5+EdU+ CD19+CD5+AnnV+ % CD19+CD5+AnnV+ 0 0 BRAFWT BRAFVE BRAFWT BRAFVE

C 50 20

40 p=0.756 p<0.001 15 30 10 20 5 10 CD19+CD5+EdU+ % CD19+CD5+EdU+ CD19+CD5+AnnV+ % CD19+CD5+AnnV+ 0 0 BRAFWT BRAFVE BRAFWT BRAFVE

Figure 3. 9 Immunosuppressive effect of BRAFV600E in B cells in vitro. (A) OSUCLL cells with Dox-induced BRAFV600E expression were incubated 1:1 with normal human T cells labeled with CFSE and stimulated with antibodies to CD3 and CD28. After a 4-day incubation, proliferation of CD4+ and CD8+ T cells was determined by CFSE flow cytometry. Proliferation causes dilution of the CFSE and is evidenced by the appearance of lower- intensity peaks. Data are representative of at least 3 separate experiments using T cells from different donors. (B) The experiment from (A) was repeated -/+ dabrafenib (2 µM). Data are representative of at least 3 similar experiments using T cells from different donors. (C) The experiment in (A) was repeated with the different cell types in the same wells or in opposite chambers of 0.4 µm transwell plates.

Continued 86

Figure 6

Figure 3.9 Continued

A CD4 CD8

Unstim. T cells

Stim. T cells

Stim. T cells + OSUCLL-tet

Stim. T cells + OSUCLL-BRAF

Stim. T cells + OSUCLL- Count BRAFV600E

CFSE

V600E Figure 6. Immunosuppressive effect of BRAF in B cells in vitro. (A)Continued OSUCLL cells with Dox-induced BRAFV600E expression were incubated 1:1 with normal human T cells labeled with CFSE and stimulated with antibodies to CD3 and CD28. After a 4-day incubation, proliferation of CD4+ and CD8+ T 87cells was determined by CFSE flow cytometry. Proliferation causes dilution of the CFSE and is evidenced by the appearance of lower- intensity peaks. Data are representative of at least 4 separate experiments using T cells from different donors. Figure 3.9 Continued

CD4 2.0 CD8 p<0.001

1.5 p<0.001

1.0 p<0.001

p<0.001 0.5 division index (DI) index division

0.0 T T T+tet T+BWT T+BVE anti-CD3/CD28

CD4 0.8 CD8 p=0.002

0.6 p=0.001 p<0.001 0.4 p<0.001 0.2

precursor frequency (PF) frequency precursor 0.0 T T T+tet T+BWT T+BVE anti-CD3/CD28

Continued

Figure 6

Figure 3.9 Continued

B CD4 CD8

Unstim. T cells +DMSO

Stim. T cells +DMSO

Stim. T cells + OSUCLL-BRAFV600E +DMSO

Stim. T cells +dabrafenib

Stim. T cells + Count OSUCLL-BRAFV600E +dabrafenib

CFSE

V600E Figure 6. Immunosuppressive effect of BRAF in B cells in vitro. (B) The experiment from (A) was repeated -/+ dabrafenib (2 μM). Data are representative ofC atontinued least 4 similar experiments using T cells from different donors.

Figure 3.9 Continued

CD4 p=0.006 0.8 CD8 p=0.003

0.6

0.4

0.2 division index (DI) index division

0.0 T T T+BVE T T+BVE anti-CD3/CD28 DMSO dabrafenib

CD4 p<0.001 0.5 CD8 p=0.003 0.4

0.3

0.2

0.1

precursor frequency (PF) frequency precursor 0.0 T T T+BVE T T+BVE anti-CD3/CD28 DMSO dabrafenib

Continued 90

Figure 3.9 Continued

T cells + T cells + T cells + OSUCLL- OSUCLL- T cells OSUCLL- tet BRAFWT BRAFVE

only Co - culture Transwell CD4 Count Co - culture CD8 Transwell

CFSE

Continue 91

Figure 3.9 Continued

CD4 2.0 CD8 p=0.013

1.5

1.0

0.5 division index (DI) index division

0.0 VE VE WT WT . T . . T . T+tet T+tet T+B T+B T+B T+B stim

unstim transwell coculture

CD4 0.8 CD8

0.6

0.4

0.2

precursor frequency (PF) frequency precursor 0.0 VE VE WT WT . T . . T . T+tet T+tet T+B T+B T+B T+B stim

unstim transwell coculture

FigureSupplemental 3. 10 Cytokine Figure analysis5. Supernatants in vitro .from the experiment in Figure 6A (n=3 each) Supernatantswere analyed from for thecytoines experiment by Cytometric in Figure Bead 3.9A Array.(n=3 each) T = norma were lanaly T cellszed tet for = cytokSUCLL-ines WT byTet Cytometric B WT = SUCLL-BRAF Bead Array. BTVE = =normal SUCLL-BRAF T cells; tetV600E = .O p<0.05SUCLL - Tet p<0.001.; B = OSUCLL- BRAF; BVE = SUCLL-BRAFV600E. p<0.05, p<0.001.

IL-2 IL-6 10000 100 8000 80 6000 60 pg/ml 4000 pg/ml 40 2000 20 0 0 T TT+tet T+BWT T+BVE T TT+tet T+BWT T+BVE + anti-CD3/CD28 + anti-CD3/CD28

IL-4 IL-10 1600 4000 1200 400 3000

2000

pg/ml 200 pg/ml 1000

0 0 T TT+tet T+BWT T+BVE T TT+tet T+BWT T+BVE + anti-CD3/CD28 + anti-CD3/CD28

TF IFNγ 15000 40000

30000 10000 20000

pg/ml pg/ml 5000 10000

0 0 T TT+tet T+BWT T+BVE T TT+tet T+BWT T+BVE + anti-CD3/CD28 + anti-CD3/CD28

Figure 3. 11 Immunosuppressive effect of BRAFV600E in vivo. (A) Plasma from leukemic mice was collected at the point when each mouse exceeded 35% tumor cells in the peripheral blood CD45+ population, and was evaluated using a Milliplex cytokine assay (n= 4 and 5 for BRAFWT and BRAFVE, respectively). (B) T cell immune parameters in AT mice were assessed by flow cytometry (n=25 and 11 for BRAFWT and BRAFVE, respectively; p<0.01 for each). (C) PD-L1 expression on CD11b+ cells (monocytes/macrophages) was assessed in peripheral blood from AT mice by flow cytometry (n=25 and 11 for BRAFWT and BRAFVE, respectively; p<0.001). (D) The percentage of F4/80+ cells (macrophages) was measured in the non-lymphocyte (CD5- /CD19-) population (n=15 for each; p=0.002). (E) CD11b+ cells were further analyzed for expression of Ly6C and Ly6G, markers differentiating patrolling monocytes and myeloid derived suppressor cells (MDSCs) (n=15 each).

Figure 7 A 2000 Eotaxin150 TNF

1500 100 1000 pg/mL pg/mL 50 500