Utilizing Reversible Bruton’s Tyrosine Kinase Inhibitors to Circumvent Acquired Resistance to Ibrutinib
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Sean David Reiff, B.S.
Biomedical Sciences Graduate Program
The Ohio State University
2018
Dissertation Committee:
Jennifer Woyach, MD, Advisor
John Byrd, MD, Advisor
Robert Baiocchi, MD, PhD
William Carson III, MD
i
Copyright by
Sean David Reiff
2018
ii
Abstract
Chronic lymphocytic leukemia (CLL) is a cancer of monoclonal B cells caused by dysregulated proliferation within the bone marrow which disrupts normal hematopoiesis leading to anemia, immune deficiencies, and increased rates of morbidity and mortality. With approximately 150,000 affected individuals and an annual incidence exceeding 20,000 in the United States, CLL is the most prevalent leukemia in the western hemisphere. Recent appreciation for the extent to which
B cell receptor (BCR) signaling contributes to the pathogenesis of CLL has spurred the development of small molecule inhibitors which block signaling initiated at the
BCR. One such molecule designed to abrogate BCR signaling is ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase (BTK).
Patients treated with ibrutinib benefit from durable remission and prolonged progression free survival. However, despite ibrutinib’s multiple Breakthrough
Therapy Designation, it is not a panacea and resistance to therapy occurs in many patients. Resistance to ibrutinib is most commonly mediated by mutation of BTK’s
Cys481 amino acid to serine (C481S), which prevents ibrutinib’s covalent binding, reducing its potency.
The kinase inhibitors GDC-0853 and ARQ 531 reversibly and potently inhibit BTK at low nanomolar concentrations. Like ibrutinib, these compounds are ii mildly cytotoxic, reduce chemotaxis, and abrogate NF-kB mediated gene transcription. Because GDC-0853 and ARQ 531 are reversible inhibitors which do not rely upon the Cys481 amino acid of BTK for activity, we hypothesized that these compounds would maintain efficacy in mutated C481S BTK. As expected, both GDC-0853 and ARQ 531 inhibit C481S BTK in biochemical assays, as well as cell lines and patient cells expressing C481S BTK.
While GDC-0853 possesses exquisite specificity for BTK, ARQ 531 is a relatively promiscuous kinase inhibitor which targets multiple SRC and TEC family kinases. Interestingly, their distinct inhibitory profiles bestow unique properties of potential clinical benefit. For example, GDC-0853 lacks the ITK inhibition possessed by ibrutinib. Because ITK is necessary for antibody dependent NK cell mediated cytotoxicity, immunotherapies are much more effectively combined with
GDC-0853 than with ibrutinib. Conversely, our results with ARQ 531 suggest that non-specific kinase inhibition may also provide clinical benefit. In vivo, ARQ 531 improved survival over ibrutinib the Eµ-TCL1 murine model of CLL and improved survival in the Eµ-MYC/TCL1 murine model of Richter’s syndrome (in which ibrutinib was ineffective). Additionally, ARQ 531 inhibits downstream signaling in models with ibrutinib resistance due to PLCγ2 mutations.
As the prevalence and duration of ibrutinib therapy continue to increase, so too will the incidence of ibrutinib resistance in patients. The observation that the majority of patients who develop resistance to ibrutinib do so by mutating components of the BCR pathway rather than by upregulating an accessory survival iii pathway indicates that BCR signaling is critical to CLL progression. Therefore, there is strong rationale to utilize reversible BTK inhibitors like GDC-0853 and ARQ
531 in order to maintain inhibitory pressure on BTK in the setting of ibrutinib resistance. Based upon the findings contained herein I propose that reversible BTK inhibition may be a viable therapeutic option for patients with acquired ibrutinib resistance.
iv
Dedication
Dedicated to the patients for whom we struggle in the hope that they need not.
v
Acknowledgments
First and foremost, I would like to thank Drs. Jennifer Woyach and John
Byrd for their support of my PhD training both pedagogically and financially. As a
PhD candidate with minimal prior laboratory experience they showed me extreme patience as I strived to accumulate technical proficiency throughout my tenure in the laboratory. More importantly, working closely with them these past years to understand their unique perspectives on cancer biology and observe their rigorous scientific discipline is an opportunity few enjoy and for which I am incredibly appreciative. Their mentorship has been supremely humbling. Additionally, I would like to acknowledge Drs. Robert Baiocchi and William Carson III for serving on my dissertation committee and guiding me towards the completion of my PhD. Their knowledge and academic experience have ensured that the body of work contained herein is of sufficient merit and quality to allow me to advance to the next stage of my academic career.
Finally, I would like to thank everyone in the Experimental Hematology
Laboratory for fostering such a vibrant and unique environment in which to pursue my doctorate. I will look back on this time in my life fondly not because of the techniques I learned, nor the ideas that I fostered, nor the papers that I published, but because of the relationships I made with so many of you. Specifically, I want to acknowledge J.T. Greene, Emily McWilliams, Fabienne Lucas, and Libby
Muhowski for serving as constant sources of support and scientific discourse. I will miss you the most. vi
Vita
June 2, 1989…………………………………..Born – Cincinnati, Ohio
2007……………..……………………………..LaSalle High School
2011………………………………...... B.S. Biology, B.S. Chemistry
Lee University
2011-Present………………………………….Graduate Research Associate
The Ohio State University
Publications
Reiff SD, Mantel R, Smith LL, Greene JT, Muhowski EM, Fabian CA, Goettl V,
Tran M, Harrington BK, Rogers K, Awan F, Maddocks K, Andritsos L, Lehman A,
Sampath D, Lapalombella R, Eathiraj S, Abbadessa G, Schwartz B, Johnson AJ,
Byrd JC, Woyach JA. The BTK Inhibitor ARQ 531 Targets Ibrutinib Resistant CLL and Richter’s Transformation. In review.
Reiff SD, Muhowski EM, Guinn D, Lehman A, Fabian CA, Cheney C, Mantel R,
Smith L, Johnson AJ, Young WB, Johnson AR, Liu L, Byrd JC, Woyach JA. Non-
vii covalent inhibition of C481S Bruton’s tyrosine kinase by GDC-0853: A new treatment strategy for ibrutinib resistant CLL. Blood. In review.
Fabian CA,* Reiff SD,* Guinn D, Lehman A, Neuman L, Fox JA, Wilson W, Byrd
JC, Woyach JA, Johnson AJ. SNS-062 demonstrates efficacy against C481S mutated BTK in vitro and may serve as a targeted therapy for patients with acquired ibrutinib resistance. In preparation.
Lucas F, Reiff SD, Smith E, Smith L, Goettl V, Harrington B, Breitbach J, Byrd
JC, Woyach JA. Targeting the BTK-signalosome in tumor associated neutrophils in CLL. In preparation.
Eathiraj S, Yu Y, Savage RE, Schwartz B, Reiff SD, Woyach JA, Johnson AJ,
Abbadessa G. ARQ 531 a Reversible BTK Inhibitor, Demonstrates Potent
Antitumor Activity in ABC-DLBCL and GCB-DLBCL. In preparation.
Bottoni A, Rizzotto L, Lai TH, Liu C, Smith LL, Mantel R, Reiff S, El-Gamal D,
Larkin K, Johnson AJ, Lapalombella R, Lehman A, Plunkett W, Byrd JC, Blachly
JS, Woyach JA, Sampath D. Targeting BTK through microRNA in chronic lymphocytic leukemia. Blood. 2016;128(26):3101-12.
Stiff A, Trinkha P, Wesolowski R, Kendra K, Hsu V, Uppati S, McMichael E,
Duggan M, Campbell A, Keller K, Landi I, Zhong Y, Dubovsky J, Howard JH, Yu
L, Harrington B, Old M, Reiff S, Mace T, Tridandapani S, Muthusamy N, Caligiuri
MA, Byrd JC, Carson WE 3rd. Myeloid-Derived Suppressor Cells Express
viii
Bruton’s Tyrosine Kinase and Can Be Depleted in Tumor-Bearing Hosts by
Ibrutinib Treatment. Cancer Research. 2016;76(8):2125-36.
Fields of Study
Major Field: Biomedical Sciences
Area of Research Emphasis: Experimental Therapeutics
ix
Table of Contents
Abstract…………………………………………………………..……………...…….…ii
Dedication………………………………………………………………………..………v
Acknowledgments………………………………………………………..……………..vi
Vita……………………………………………………………………………………….vii
List of Tables……………………………………………………………………...……xiv
List of Figures……………………………………………………………….…..………xv
List of Abbreviations…………………………………………………………………..xvii
Chapter 1: Background and Introduction…………………………………………...…1
1.1 B Lymphocytes……………………………………………………………...1
1.2 Chronic Lymphocytic Leukemia…………………….……………………..6
1.3 Bruton’s Tyrosine Kinase……..…………………………………………..12
1.4 Ibrutinib……………………………………………………………………..18
1.5 Ibrutinib Resistance……………………………………………………….24
Chapter 2: Non-Covalent Inhibition of C481S Bruton’s Tyrosine Kinase by GDC-0853: A New Treatment Strategy for Ibrutinib Resistant CLL……...... 28
2.1 Introduction………………………………………………………………...28
2.2 Materials and Methods………………………………………...... 32 x
2.2.1 Subject Population and Lymphocyte Isolation……………...32
2.2.2 Cell Culture and Drug Treatments…………………….……...32
2.2.3 Immunoblotting……………………………………………...... 33
2.2.4 Cytotoxicity Analysis…………………………………….….....34
2.2.5 CpG Induced Activation……………………………………….35
2.2.6 CXCL12 Induced Migration…………………………………...35
2.2.7 Real-Time PCR………………………………………………...35
2.2.8 Kinase Assay………………………………………….………..36
2.2.9 CCL3 ELISA………………………………………...………….36
2.2.10 NK Cell Mediated ADCC……………………………....………36
2.2.11 Statistical Analysis………………………………………..……37
2.3 Results……………………………………...... 38
2.3.1 GDC-0853 Inhibits BCR Signaling……………………...……38
2.3.2 GDC-0853 Induces Modest Cytotoxicity and Overcomes
Stromal Protection………………………………….………….39
2.3.3 GDC-0853 Inhibits NF-kB Dependent Transcription,
Reduces Activation, and Impairs Migration……………....…39
2.3.4 GDC-0853 Does Not Inhibit Cellular EGFR or ITK……....…40
2.3.5 GDC-0853 Preserves NK Mediated ADCC……………..…..41
2.3.6 GDC-0853 Demonstrates Equivalent Inhibition of
Wild-Type and C481S Mutated BTK…………………...……42
2.4 Discussion…………………………………………………………….……43
xi
2.5 Figures……………………………………………………………...………47
Chapter 3: The BTK Inhibitor ARQ 531 Targets Ibrutinib Resistant CLL and
Richter’s Transformation………………………………………………………..…….57
3.1 Introduction…………………………………………………….……….….57
3.2 Materials and Methods……………………………………………………60
3.2.1 Subject Population and Lymphocyte Isolation……..……….60
3.2.2 BTK Biochemical and Kinase Selectivity Assay…………....60
3.2.3 Crystal Structure Determination………………….…………..61
3.2.4 Cell Culture and Drug Treatment……………….……………61
3.2.5 Immunoblotting…………………………………………………62
3.2.6 Viability………………………………………………………….63
3.2.7 Migration……………………………………………….……….63
3.2.8 CpG Induced Activation……………………………………….64
3.2.9 Real-Time PCR………………………………………...………64
3.2.10 Eµ-TCL1Mouse Model……………………………...…………64
3.2.11 Eµ-MYC/TCL1 Mouse Model…………………………………65
3.2.12 Statistics………………………………………………………...65
3.2.13 Study Approval…………………………………………………66
3.3 Results…………………………………………………………...…………67
3.3.1 ARQ 531 Is a Potent Inhibitor of BCR Signaling………...…67
3.3.2 ARQ 531 Is Cytotoxic to CLL Cells In Vitro…………………68
xii
3.3.3 ARQ 531 Abrogates CpG Mediated Activation and
Chemokine Induced Migration of CLL Cells…...……………69
3.3.4 ARQ 531 Is Superior to Ibrutinib in the Eµ-TCL1
Engraftment Mouse Model……………………………………69
3.3.5 ARQ 531 Is Superior to Ibrutinib in a Murine Model of
Richter’s Transformation……………...………………………70
3.3.6 ARQ 531 Maintains Efficacy in Cells with C481S
Mutated BTK……………………………………………………71
3.3.7 ARQ 531 Effectively Inhibits Downstream Signaling
in Ibrutinib Resistant PLCγ2 Mutations…...…………………72
3.4 Discussion...... 73
3.5 Figures…..……….…………………………………………………………77
Chapter 4: Concluding Remarks..……………………………………………………99
Works Cited…………………………………………………………………………...107
xiii
List of Tables
Table 1. ARQ 531 inhibits multiple kinases including members of the TEC and
Src families and RAS/RAF pathway…………………………………………………81
xiv
List of Figures
Figure 1. BCR signaling is inhibited by GDC-0853 in CLL patient cells…………47
Figure 2. GDC-0853 is modestly cytotoxic to CLL B cells and limits stroma induced survival……………………………………………………………………..…49
Figure 3. GDC-0853 represses NF-κB dependent gene transcription………...…50
Figure 4. GDC-0853 abrogates important cellular functions including CLL activation and migration…………………………………………………………..…..51
Figure 5. GDC-0853 lacks inhibition of EGFR and ITK in cells………………..…52
Figure 6. GDC-0853 preserves NK cell mediated ADCC in response to anti-CD20 antibodies………………………………………………………………….53
Figure 7. GDC-0853 inhibits both wild type and C481S mutated
BTK variants...... 54
Figure 8. Inhibition of BCR mediated signaling by ARQ 531……………………..77
Figure 9. Kinome tree illustrating the selectivity profile of ARQ 531……………..82
Figure 10. ARQ 531 is cytotoxic to CLL cells and more potently inhibits proximal BCR pathway kinases than ibrutinib…………………………….83
Figure 11. Changes in NF-κB function, activation, and CLL migration
xv due to ARQ 531……………………………………………………………….……….85
Figure 12. ARQ 531 improves survival in the Eμ-TCL1 engraftment model compared to ibrutinib………………………………………………………….………89
Figure 13. ARQ 531 improves survival in the Eμ-MYC/TCL1 model compared to ibrutinib………………………………………………………….………92
Figure 14. C481S BTK is inhibited by ARQ 531………………………….………..94
Figure 15. ARQ 531 inhibits BCR signaling in cells with ibrutinib-resistant PLCγ2 mutations………………………………………………….97
xvi
List of Abbreviations
7-AAD 7-Aminoactinomycin D
ADCC Antibody-dependent cell-mediated cytotoxicity
ADP Adenosine diphosphate
AKT AKT8 virus oncogene cellular homolog
ANOVA Analysis of variance
ATP Adenosine triphosphate
BCR B cell receptor
BLK B lymphocyte kinase
BMX Bone marrow expressed kinase
BTK Bruton’s tyrosine kinase
CCL3 Chemokine (C-C motif) ligand 3
CD Cluster of differentiation cGVHD Chronic graft versus host disease
CLL Chronic lymphocytic leukemia
CpG Cysteine-phosphate-Glycine
xvii
CXCL C-X-C motif chemokine ligand
CXCR C-X-C motif chemokine receptor
DMSO Dimethylsulfoxide
EGFR Epidermal growth factor receptor
ELISA Enzyme linked immunosorbent assay
ERK Extracellular signal regulated kinase
FITC Fluorescein isothiocyanate
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GFP Green fluorescent protein
HER Human epidermal growth factor receptor
HSC Hematopoietic stem cell
IACUC Intuitional animal care and use committee
IgM Immunoglobulin M
IgVH Immunoglobulin variable-region heavy chain
IκBα Inhibitor of κBα
IRB Institutional review board
ITK Interleukin 2 inducible T cell kinase
xviii
JAK Janus kinase
LYN Lck/Yes novel tyrosine kinase
MCL1 Myeloid cell leukemia 1
MEK MAPK/ERK kinase
MFI Median fluorescence intensity miRNA MicroRNA
MYC Avian myelocytomatosis virus oncogene cellular homolog
MZL Marginal zone lymphoma
NFAT Nuclear factor of activated T cells
NF-κB Nuclear factor κB
NK Natural killer
OCT2 Octamer binding protein 2 p Phosphorylated
PAMP Pathogen associated molecular pattern
PBMC Peripheral blood mononuclear cell
PCR Polymerase chain reaction
xix
PLCγ2 Phospholipase Cγ2
PI Propidium iodide
RNA Ribonucleic acid
RT-PCR Real-time polymerase chain reaction
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SHIP1 Src homology 2 domain-containing inositol-5’-phosphatase 1
SHP-1 Src homology 2 domain phosphatase-1
SLL Small lymphocytic lymphoma
SYK Spleen tyrosine kinase
TBP TATA binding protein
TCL1 T cell leukemia/lymphoma 1
TCR T cell receptor
TEC Tyrosine kinase expressed in hepatocellular carcinoma
TLR9 Toll like receptor 9
WM Waldenström Macroglobulinemia
WT Wild type
xx
CHAPTER 1
Background and Introduction
1.1 B Lymphocytes
Cells of the human immune system provide protection from pathogenic organisms including viruses, bacteria, fungi, and parasites and can be broadly grouped into two categories, those contributing to innate immunity and those responsible for adaptive immunity1. Innate immunity provides the first line of defense against infection via anatomical barriers including the skin, constitutively secreted antimicrobial compounds, and effector cells which non-specifically destroy invading organisms1. Effector cell types of the innate immune system include neutrophils, macrophages, and natural killer (NK) cells1. These cells rapidly respond to pathogens that evade physical and chemical barriers in part by recognizing pathogen associated molecular patterns (PAMPs) on the surface of invaders2. PAMPs are ubiquitously expressed by pathogens but not by human cells, thereby allowing effector cells of the innate immune system to broadly recognize and target entire classes of foreign invaders without damaging host tissues2. Upon recognizing a PAMP, effector cells of the innate immune system attempt to destroy the pathogen by using several mechanisms including the 1 activation of complement (a series of proteins which perforate the outer membrane of the invader), phagocytic engulfment of the pathogen and subsequent lysosome- mediated destruction, the secretion of antimicrobial compounds, and the release of cytokines1,2. Cytokines recruit immune cells of the innate and adaptive systems to sites of infection and integrate the cooperative responses between these two complimentary systems.
While the innate immune system indiscriminately targets broad classes of foreign invaders based on their conserved molecular patterns, the adaptive immune response relies upon cells which specifically recognize a unique antigen derived from a particular invading pathogen. Cells of the adaptive immune system are highly specialized and possess an immunological memory component that facilitates the rapid clearance of previously encountered pathogens from the host1.
The adaptive immune system is composed of T lymphocytes which facilitate cell- mediated responses through the secretion of cytotoxins, and B lymphocytes which facilitate humoral responses through the secretion of antibodies1.
In the bone marrow, early B cell lineages are derived from hematopoietic stem cells (HSCs). HSCs differentiate into multipotent progenitor cells, which are then differentiated into common lymphoid progenitor cells, which in turn give rise to pre-pro-B lymphocytes3. The maturation of B cells depends upon the expression of immunoglobulins on the surface of the developing B cell and the engagement of these immunoglobulins with antigen4. Surface immunoglobulins are derived from immunoglobulin gene segments that are randomly rearranged and will 2 ultimately constitute the mature lymphocyte’s B cell receptor (BCR), which is an important transducer of extracellular signaling through the plasma membrane of B cells1. Through a process known as positive selection, pre-B cells whose surface immunoglobulin molecules sufficiently recognize antigen will proceed through the maturation process while those whose surface immunoglobulins do not recognize antigen undergo apoptosis5-7. Additionally, negative selection removes pre-B cells whose surface immunoglobulins recognize self-derived antigen, thereby promoting immunological tolerance and reducing the likelihood of autoimmunity5-7. Following positive and negative selection, immature B cells expressing BCRs which recognize foreign antigen without recognizing self-derived tissues will migrate from the bone marrow into the peripheral circulation and secondary lymphoid tissues, including the spleen and lymph nodes1,4. It is in these secondary lymphoid tissues that immature B cells may recognize antigen, subsequently allowing immature B cells to proliferate in response to infection1,4. The process of B cell development is highly selective. It has been estimated that only 10% of pre-pro B cells proceed through positive and negative selection and that the majority of B cells which do reach secondary lymphoid organs will never become mature lymphoctyes8,9.
Through random recombination of V(D)J gene segments, which constitute the antigen binding domain of the BCR, it is estimated that approximately 100 billion unique immunoglobulin genes can be formed1. More incredibly, this repertoire of rearranged immunoglobulins is further refined during B cell maturation to select for B cells with further refined antigenic specificity through a process
3 known as somatic hypermutation10. Somatic hypermutation is an important process during affinity maturation in which cytosine nucleotides in DNA are deaminated to uracil by activation-induced cytidine deaminase (AICDA)10-12.
Cytosine deamination by AICDA produces uracil-guanine mismatches in the V(D)J gene segments that are removed by base excision repair enzymes11. The excised
DNA fragments are then acted upon by low fidelity DNA polymerase enzymes to repair the rearranged V(D)J gene, introducing additional genetic diversity11,12.
Following somatic hypermutation, B cells that effectively recognize antigen will outcompete unfit clones for the limited available resources in secondary lymphoid tissues and proliferate to a greater extent than B cells with sub-optimal BCRs12.
Gene rearrangement and somatic hypermutation during lymphocyte maturation generates incredible diversity which facilitates selective defense against invading pathogens.
The major function of normal B cells in the adaptive immune response is the secretion of antibody in response to antigen. Secreted antibodies are the major effectors of humoral immunity and are critical in the recognition and clearance of immunologic challenges. The main functions of antibodies are neutralization, antibody-mediated activation of complement, and activation of effector immune cells1. Neutralization occurs when antibodies recognize surface antigens thereby blocking their interaction with host tissues and rendering the pathogen less virulent13. The complement cascade can be initiated by immunoglobulin which recruits soluble complement proteins found in the plasma to form a membrane
4 attack complex which kills the pathogen14. In addition to the cell-free mechanisms by which antibodies can facilitate immunity, antibodies can also facilitate cell- mediated immunity. The Fc region of an antibody can trigger several cell-based responses including ingestion by phagocytic cells, degranulation of neutrophils, and the activation of natural killer (NK) cells through a process known as antibody- dependent cell-mediated cytotoxicity (ADCC)15. During ADCC, NK cells recognize and destroy antibody coated pathogens through the release of granzymes and pro- inflammatory cytokines. Importantly, NK cell mediated ADCC contributes to the efficacy of immunotherapies commonly used to treat cancer16.
B lymphocytes are critical mediators of adaptive immunity capable of selectively recognizing a virtually limitless number of potential antigens due to the genetic recombination and selective pressures that occur during their development. When their randomly generated BCRs recognize antigen, B cells can produce corresponding immunoglobulins which mediate immune clearance by directly complexing with antigen and recruiting effector immune cells of the innate and adaptive immune systems. The humoral response generated by B cells serves an indispensable function in the adaptive human immune system which must be precisely controlled. Dysregulated B cell proliferation can have devastating consequences which result in significant morbidity and mortality.
5
1.2 Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in the western hemisphere with approximately 150,000 affected individuals and an annual incidence of 20,000 new cases in the United States17. The median age at diagnosis is 72 years and portends a five-year survival rate of approximately 75% percent following the initial diagnosis17. The median survival for patients under the age of 50 years is 12.3 years post diagnosis compared to 31.2 years in age matched controls indicating that CLL is indeed associated with decreased survival and not merely an innocuous finding associated with advanced age18. CLL is more common in Caucasians than other races and men are affected at nearly twice the rate of women17. Considering that the incidence of CLL is correlated with age, it is likely that demographic changes associated with an aging population will increase the prevalence of CLL in the United States through the foreseeable future.
Despite decades of research, the origin of CLL remains elusive although multipotent, self-renewing HSCs19 and the B1 cell type20 have been posited as putative progenitors. Gene expression studies of CLL B cells have shown that these malignant lymphocytes most closely resemble mature memory B cells21. It is unclear whether there is one common progenitor of CLL or whether multiple subtypes of CLL may arise from distinct precursor cells. Further contributing to the mysterious origin of CLL, having a first-degree relative with CLL increases the risk of developing CLL approximately seven-fold, although the mechanism by which this heritability occurs as yet to be described22. 6
Diagnosis of CLL is most commonly made on the basis of immunophenotyping, blood count, and bone marrow aspirate23. CLL is recognized as a clonal proliferation of lymphocytes expressing the B cell markers CD19,
CD20, and CD23 as well as the T cell marker CD5 in the absence of other T cell antigens23,24. A B cell count ≥5,000 lymphocytes/µL in the peripheral blood for a duration of three months is necessary for diagnosis23,25. On microscopic examination, CLL B cells appear as mature lymphocytes with a narrow border of cytoplasm and demonstrate a pathognomonic smudge like appearance23,26. Bone marrow aspirates indicative of CLL demonstrate that greater than 30% of all nucleated cells in the smear are of lymphoid origin or show infiltrates consistent with CLL B cells23.
CLL is a low grade indolent malignancy typically presenting with slow progression over the course of several years although aggressive cases of CLL also occur infrequently23. Closely related to CLL, small lymphocytic lymphoma
(SLL) is a monoclonal B lymphocyte proliferative disease with bone marrow involvement and an immunophenotype identical to CLL but with lymphoid disease in lieu of a peripheral blood count ≥5,000 lymphocytes/µL23,27. The management of patients with SLL is typically similar to those with CLL23,27. Infrequently, CLL may also transform to a diffuse large B cell lymphoma (DLBCL) in a process termed
Richter’s transformation23,28,29. Patients with DLBCL commonly present with a large mediastinal mass, constitutional symptoms, and/or high serum LDH levels28,29. Patients with Richter’s transformation have abbreviated survival with
7 standard CLL therapies which include chemotherapy often in combination with immunotherapy29. For younger patients with Richter’s transformation bone marrow transplantation can be curative29.
The disease course of CLL is associated with the accumulation and unchecked proliferation of B cells in the bone marrow, lymph nodes, spleen, and peripheral blood resulting in lymphadenopathy, splenomegaly, and the ablation of normal bone marrow architecture23. CLL manifests itself as anemia, fatigue, night sweats, weight loss, and immune dysregulation with an increased susceptibility for infection or autoimmune disorders including hemolytic anemia and immune thrombocytopenia23. Despite being a hyperproliferative disease of B cells, CLL is presents with hypogammaglobulinemia due to the fact that the hyperproliferative
B lymphocytes are functionally abnormal23. Frequently, patients with CLL will ultimately succumb to infection as a result of the immune deficiencies associated with bone marrow failure23,27.
Approximately 80% of patients with CLL have acquired chromosomal aberrations30. Several genetic abnormalities are recurrently associated with CLL and are accepted as important biomarkers indicating relative prognosis in patients31. Deletion of the long arm of 13q (del(13q14)) is the most common genomic abnormality in CLL occurring in approximately half of all patients32. 13q14 deletion as the only genetic aberration indicates that the patient is likely to experience an indolent disease course33. The miRNAs miR-15a and miR-16-1 have been mapped to the 13q14 region of chromosome 13 and are deleted in 8 nearly all cases of 13q14 deletion regardless of the size of the deletion in an individual patient34. These miRNAs recognize and target the anti-apoptotic protein
B-cell lymphoma 2 (BCL2) transcript for degradation. Their deletion therefore predisposes patients with 13q14 deletion to increases in BCL2 protein expression and subsequent dysregulation of cell death35. Trisomy of chromosome 12 is the second most common recurrent chromosomal aberration in CLL with approximately 20% of patients being affected, however there is no consensus as to the underlying molecular mechanism responsible for the prevalence of this mutation in CLL30. These patients nearly always respond to traditional chemoimmunotherapy and demonstrate rates of remission above the median30.
Deletions in the long arm of chromosome 11 are found in approximately 10% of patients30. The deleted region here frequently occurs at del(11q23) and correlates with the ataxia-telangiectasia mutated (ATM) gene, a protein responsible for DNA damage repair in response to double stranded DNA breaks after exposure to ionizing radiation or DNA damaging chemicals36,37. Patients with 11q23 deletions typically have bulky lymphadenopathy with reduced overall survival due to rapid disease progression38. Deletions in the short arm of chromosome 17
(del(17p13.1)) are found infrequently at the time of CLL diagnosis with an approximate prevalence of 5-10%30. Deletions in 17p13.1 include the p53 gene and are associated with very poor outcomes and poor response to DNA damaging therapies such as alkylating agents and purine analogues owing to the fact that these therapies rely upon p53 for their induction of apoptosis39,40. Because p53
9 function is associated with preservation of DNA integrity, this mutation is rarely found as the only chromosomal abnormality and is frequently associated with complex karyotype (≥3 chromosomal aberrations)41. p53 mutation may provide a clonal background upon which genomic changes may accumulate and persist thereby engendering the production of aggressive CLL subclones.
In addition to the recurrent mutations described above, the extent to which the IgVH gene of the predominant CLL clone has been rearranged by somatic hypermutation also informs a patient’s prognosis42,43. Somatic hypermutation of the IgVH gene is a normal physiological process during the course of B cell development which increases the diversity of antigens that B cells can potentially
1 recognize . CLL patients whose B cells possess IgVH genes with greater than 2% variation from germline DNA are said to have a mutated phenotype while patients whose B cells possess less than 2% variation from germline DNA are said to possess an unmutated phenotype42,43. The presence of an unmutated BCR is associated with more aggressive disease and is correlated with inferior outcomes in CLL44,45. Patients with mutated phenotypes tend to respond more favorably to standard chemotherapeutic agents and display longer progression free survival
(PFS) following treatment46. Considering that somatic hypermutation occurs fairly late in the process of B cell development, the positive benefit observed in IgVH mutated patients may reflect a relative increase in differentiation.
CLL is considered incurable outside of stem cell transplant, a therapeutic option reserved for younger, healthy patients47. Due to the generally indolent 10 course of CLL, immediate treatment is not necessary nor recommended and has not been shown to improve patient outcomes48,49. Instead, treatment begins when there is evidence of progressive disease such as organomegaly, anemia, repeated infections, or constitutional B symptoms including weakness, weight loss, and night sweats49. Patients lacking these indications to initiate treatment are monitored with regular blood tests and physical examination49. In recent years CLL has been treated with combination chemoimmunotherapy utilizing nucleoside analogous like fludarabine, alkylating agents such as chlorambucil, and immunotherapies including rituximab50. For young patients with symptomatic disease, the combination of fludarabine, cyclophosphamide, and rituximab (FCR) is indicated as first-line therapy51. Although effective, FCR regimens are associated with persistent cytopenias and recurrent infections due to immune suppression.
Additionally, fludarabine regimens are not well tolerated in older patients, in whom bendamustine may substitute as an appropriate alkylating agent23. Treatment related toxicities associated with chemotherapy limit the clinical applicability of these therapeutics for many patients.
More recently, orally bioavailable small molecule inhibitors designed to target aberrantly expressed signaling pathways are becoming more utilized52. The most successful of these small molecule inhibitors abrogate the effects of Bruton’s tyrosine kinase (BTK)53, phosphoinositol-3-kinase (PI3K)54, and BCL255.
11
1.3 Bruton’s Tyrosine Kinase
BTK belongs to the tyrosine kinase expressed in hepatocellular carcinoma
(TEC) family of kinases, a group of five non-receptor tyrosine kinases which propagate extracellular receptor stimulation56. Aside from bone marrow expressed kinase (BMX), which is primarily restricted to endothelial cells, TEC family kinases are almost exclusively found in lymphocytes56,57. The TEC family of kinases also includes interleukin-2-inducible T cell kinase (ITK) and resting lymphocyte kinase
(RLK) in addition to BTK, TEC, and BMX58,59. Expression of TEC family kinases is unique to individual cell types, typically with redundant expression of multiple TEC family kinases in any given cell. T cells for instance, express ITK, RLK, and TEC, although B cells are unique in that they exclusively express BTK and no other TEC family kinase58,60. TEC family kinases possess a pleckstrin homology (PH) domain which allows these kinases to transiently associate with the plasma membrane by binding to phosphatidylinositol-3,4,5-trisphosphate (PIP3), the product of PI3K action on phosphatidylinositol-4,5-bisphosphate (PIP2)61. BTK and the other TEC family kinases also possess Src homology 2 (SH2) and Src homology 3 (SH3) domains which regulate signal transduction pathways by controlling protein-protein interactions via binding of phosphorylated tyrosine residues on associated proteins62,63.
BTK is the only member of the TEC kinase family whose isolated loss of expression is associated with significant human disease. Loss of function mutations in BTK manifest as X-linked agammaglobulinemia (XLA), an immune 12 disorder which almost exclusively affects males due to BTK’s expression on the X chromosome64,65. Mutation or deletion of BTK essentially eliminates B lymphocytes and plasma cells from the circulation resulting in hypogammaglobulinemia which predisposes individuals with XLA to higher rates of infection and increased mortality65. Intravenous infusion of immunoglobulin is palliative for these patients, further establishing that XLA arises from a B cell deficiency23. The murine Xid model, which possesses Btk with a mutated PH domain, demonstrates a milder disease phenotype than that found in human
XLA66. The milder phenotype observed in Xid mice compared to patients with XLA is potentially attributable to the observation that Tec expression can partially compensate for the loss of Btk in mice67. Due to the lack of redundant TEC family kinase expression in human B cells, the loss of BTK in humans causes significant
B cell dysfunction, emphasizing the important role that BTK serves in B lymphocyte function, survival, and proliferation.
The most important BTK mediated pathway contributing to the pathogenesis of CLL is the BCR pathway. The BCR is composed of membrane bound immunoglobulin noncovalently associated with the signal transduction molecules CD79a and CD79b, both of which possess immunoreceptor tyrosine- based activation motifs (ITAMs)61. Antigenic ligation of the BCR results in ITAM phosphorylation by Lck/Yes novel tyrosine kinase (LYN) and/or Spleen tyrosine kinase (SYK) thereby generating docking sites on CD79a/b that allow the SH2 domains of LYN and SYK to bind to the ITAM61,68-71. After LYN and SYK are bound
13 to the ITAMs of CD79a/b, LYN subsequently phosphorylates and activates both
SYK and the cytoplasmic tail of the BCR coreceptor CD1972-74. CD19 phosphorylation activates PI3K which in turn phosphorylates PIP2 leading to the generation of PIP361. PIP3 facilitates the recruitment of PH domain containing proteins, including BTK, to the plasma membrane. The recruitment of the signalosome proteins LYN, SYK, BTK, Vav, growth factor receptor-bound protein
2 (GRB2), and phospholipase C-gamma 2 (PLCγ2) is enhanced by B-cell linker
(BLNK) which serves as a molecular scaffold by binding the non-ITAM region of
CD79a/b and the SH2 domains of signalosome constituent proteins75. Following
BCR signalosome assembly, SYK transphosphorylates BTK’s Y551 amino acid which then enables BTK to autophosphorylate its own Y223 residue leading to
BTK activation63. One of BTK’s direct targets is PLCγ2, the activated form of which cleaves PIP2 to produce diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3) which activate PKCβ and stimulate calcium release, respectively76. PKCβ activation leads to increases nuclear factor κB (NF-κB) mediated transcription and activation of the mitogen-activated protein kinase (MAPK) family including extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) which cumulatively increases transcription, cytokine production, and cell survival77,78. Activation of
ERK1/ERK2 signaling has also been observed following the initiation of BCR signaling through the action of the RAS/RAF pathway79. Calcium influx by IP3 promotes the action of several transcription factors including NF-κB, nuclear factor of activated T cells (NFAT), and Jun leading to changes in B cell activity and
14 function61. Additionally, the generation of PIP3 by PI3K recruits AKT to the plasma membrane leading to AKT phosphorylation and activation80. Activated AKT then returns to the cytoplasm where it promotes survival signaling by activating the pro- survival transcription factors NF-κB and NFAT, while inhibiting the pro-apoptotic forkhead transcription factor (FOXO) proteins80. AKT is also able to interact with
Bad, another pro-apoptotic protein, and inhibit its activity81.
Regulation of BCR signaling is complex with evidence suggesting that some kinases can serve both activating and inhibitory roles. For example, LYN can activate negative regulators of BCR signaling including Src homology region 2 domain-containing phosphatase-1 (SHP-1) and Src homology 2 domain- containing inositol phosphatase 1 (SHIP1)82. SHP-1 cleaves activating phosphate groups found on the SH2 domains of BCR signalosome proteins leading to their dissociation while SHIP1 dephosphorylates PIP3 thereby reducing the recruitment of PH domain containing proteins to the plasma membrane. Additionally, activated
AKT has been shown to inhibit BCR signaling through direct phosphorylation of
BTK’s S51 amino acid leading to ubiquitination and degradation of BTK by 14-3-
3ζ83. Cumulatively, however, BCR stimulation is a BTK dependent process that increases B cell proliferation, survival, and differentiation by promoting changes in gene transcription and suppressing apoptotic signals.
In addition to propagating transduction cascades originating from the BCR,
BTK also transduces signaling in several other pathways necessary for normal B cell function. Among these pathways are chemokine mediated signaling and toll- 15 like receptor (TLR) signaling84. The role of BTK in these pathways is important for
B cell migration, activation, survival, and proliferation.
Biochemical analyses and migration assays have established that BTK propagates the effects of CXCL12 and CXCL13 which initiate signaling through the B cell chemokine receptors CXCR4 and CXCR5, respectively85. CXCL12 and
CXCL13 are secreted by stromal cells in the bone marrow and lymph node germinal centers, attracting B lymphocytes to secondary lymphoid tissues which serve as protective microenvironments86. BTK propagates chemokine signaling through direct interaction of its PH domain with the Gβγ subunits of CXCR4 and
CXCR587,88. The downstream effect of BTK recruitment in chemokine signaling centers on actin cytoskeletal rearrangement and B cell expression of integrin very late antigen-4 (VLA-4) which binds vascular cell adhesion protein 1 (VCAM-1) and fibronectin, integrins expressed on the surface of endothelial cells89. Further proof that BTK regulates chemokine mediated migration comes from adoptive transfer experiments in which BTK deficient B cells demonstrate impaired migration to secondary lymphoid tissues85. By promoting B cell migration through chemokine receptors, BTK facilitates recruitment to protective microenvironments and improves cell survival.
TLRs expressed on the surface of B lymphocytes recognize PAMPs thereby facilitating B cell activation in response to infection. Among the TLRs for which
BTK has been implicated in their signaling are TLR2, TLR3, TLR4, TLR7, and
TLR990. Following ligation with their respective PAMPs, TLRs recruits myeloid 16 differentiation primary response 88 (MYD88) which activates interleukin-1 receptor associated kinase (IRAK1)84,91. BTK interacts with both MYD88 and IRAK1 to induce NF-κB mediated gene transcription and the production of inflammatory cytokines in response to TLR signaling92. The expression of TLRs allows B cells to respond to innate immune signals with the help of BTK, demonstrating the important role of BTK in integrating adaptive and innate immune responses.
As a kinase upon which several proliferation, activation, and survival pathways proximately converge, BTK represents an ideal drug target for hyperproliferative B cell disorders including CLL. CLL cells overexpress BTK transcript and protein, suggesting that BTK contributes to the pathogenesis of this malignancy93. Because BTK is the only TEC family kinase member expressed in
B cells but is redundantly expressed in other immune cell types, its selective inhibition would be expected to abrogate B cell survival with little to no toxicity in non-B cell lineages. Indeed, Xid mice exhibit slower rates of CLL development and have significantly improved survival when crossed with the Eμ-T cell leukemia/lymphoma protein-1 (TCL1) murine CLL model compared to the parental
TCL1 strain94. This suggests that loss of BTK is sufficient to extend survival and reduce morbidity in CLL, at least in a murine model. Further implicating BTK in B cell hyperactivation is the observation that its overexpression in mice generates an autoimmune phenotype driven solely by B lymphocytes95. More interesting than the observed autoimmunity in these BTK overexpressing mice is the finding that their disease phenotype could be reversed with the BTK inhibitor PCI-32765. PCI-
17
32765, now known as ibrutinib, has unequivocally validated BTK inhibition as a powerful treatment strategy for the treatment of CLL and other hyperproliferative
B cell malignancies.
1.4 Ibrutinib
Ibrutinib is a first in class, orally bioavailable, small molecule inhibitor of BTK that irreversibly binds the C481 amino acid of BTK’s ATP binding site to prevent its kinase activity. Ibrutinib was first approved for mantle cell lymphoma (MCL) in
2013 after achieving a Breakthrough Therapy Designation from the US FDA due to its efficacy in human clinical trials. Shortly thereafter, ibrutinib was approved for
CLL/SLL. Since then, ibrutinib has received approval for Waldenstrom macroglobulinemia (WM) as front-line therapy, marginal zone lymphoma (MZL) as second-line therapy in patients who have received at least one prior anti-CD20- based regimen, and chronic graft versus host disease (cGVHD) after failure of one or more lines of systemic therapy.
One of the first attempts to characterize the effects BTK inhibition by ibrutinib in vitro found that ibrutinib diminishes pathogenic features of patient derived CLL cells including BCR signaling and activation-induced proliferation93.
Ibrutinib treatment also modestly increased apoptosis of CLL B cells without affecting T cell viability, although the expression of the T cell cytokines IL-6, IL-10, and TNF-α were all shown to decrease in response to ibrutinib93. Another in vitro
18 study showed that ibrutinib decreases the response of CLL cells to chemotactic signaling, an effect which was later shown to be partially attributable to decreased
VLA-4 expression on the surface of CLL cells96,97. In vivo analysis of ibrutinib in murine models demonstrated that ibrutinib delayed disease progression and improved survival in a TCL1 adoptive transfer model96 and in the TCL1 model itself94. These preclinical studies strongly suggested the potential efficacy of ibrutinib in CLL and justified subsequent clinical trials.
The first clinical trial of ibrutinib investigated its efficacy in patients with relapsed/refractory B cell lymphoma or CLL and determined its pharmacodynamic properties98. 54% of the patients in this study achieved complete or partial response with the greatest benefit from therapy observed in patients with MCL
(78% response) or CLL (69% response). Greater than 95% BTK occupancy by ibrutinib was achieved 4 hours post dosing and was sustained out to 24 hours post dosing at drug concentrations ≥ 2.5 mg/kg/day. Patients in this study demonstrated reduced lymphadenopathy with transient increases in peripheral blood lymphocyte count which decreased to baseline levels approximately four months after the initiation of therapy. Treatment related adverse events in this study were typically low grade in severity and included diarrhea, nausea, respiratory infection, and fatigue.
Based on the promising results of the previous clinical, in which patients had received a median of three prior therapies, a phase Ib/II clinical trial was conducted in patients with relapsed or refractory CLL or SLL in which patients had 19 a median of four prior therapies99. The majority of patients in this study were considered to have high-risk features including advanced-stage disease (65%),
17p13.1 deletion (33%), 11q23 deletion (36%), and/or unmutated IgVH (81%).
Overall response rate was 71% after a median follow-up of 20.9 months in both the 420 mg/day and 840 mg/day treatment cohorts with an additional 18% of patients having partial response with lymphocytosis. Surprisingly, rates of overall response were independent of the previously mentioned high risk features, although progressive disease tended to occur in patients with either 17p13.1 or
11q23 deletion. The 26-month estimated rate of PFS in this study was found to be
75% with an overall survival of 83%. The most common adverse events associated with treatment were manageable and included diarrhea, upper respiratory tract infection, and fatigue.
Following these previous clinical trials, which enrolled patients with heavily pre-treated disease, the efficacy of ibrutinib was compared against the alkylating agent chlorambucil in 269 patients with treatment naïve CLL who were 65 years of age or older100. In this phase III trial, median PFS was significantly longer with ibrutinib (not reached) than with chlorambucil (18.9 months). Ibrutinib also significantly prolonged overall survival with an estimated survival at two years of
98% as compared to 85% in the chlorambucil cohort. Additionally, hemoglobin and platelet counts were improved to a greater extent with ibrutinib than with chlorambucil. In this study ibrutinib was found to be superior to traditional alkylating
20 chemotherapy in terms of disease progression, survival, and other hematologic variables.
The clinical efficacy of ibrutinib has also been compared to immunotherapy with positive results. In a multicenter phase III trial of 391 patients with relapsed or refractory CLL or SLL, patients were randomized to treatment with ibrutinib or the anti-CD20-antibody ofatumumab101. Median PFS was significantly improved by ibrutinib (not reached, 88% at 6 months) compared to ofatumumab (8.1 months).
Additionally, at one year the overall survival of the ibrutinib cohort (90%) was statistically greater than in the ofatumumab cohort (81%). The overall response rate was also found to be significantly higher in the ibrutinib group (42.6%) than in the immunotherapy group (4.1%). Taken together these data demonstrate the superiority of ibrutinib therapy over ofatumumab.
The use of ibrutinib in combination with other therapeutics has also been explored in clinical trials with variable results. One such phase III trial compared the efficacy of bendamustine plus rituximab (BR) with or without ibrutinib in previously treated CLL/SLL102. At a median follow up of 17 months, PFS was increased in the BR plus ibrutinib cohort (not reached) compared to the BR plus placebo cohort (13.3 months). Another such combination study investigated the efficacy of ibrutinib plus rituximab compared to treatment with ibrutinib alone103. In this study of 206 CLL patients, the combination of ibrutinib with immunotherapy was not shown to improve PFS when compared to ibrutinib alone. Based upon the
21 results of these two studies, it can be inferred that chemotherapy is a better option for combinatorial therapy with ibrutinib than immunotherapy.
The observation that the combination of chemoimmunotherapy with ibrutinib improves PFS102 while the combination of immunotherapy with ibrutinib does not improve PFS103 suggests that immunotherapy is ineffectively combined with ibrutinib. The reason for this may be attributable to the inhibitory profile of ibrutinib and the mechanism by which immunotherapies exert their effect. Although ibrutinib inhibits BTK at low nanomolar concentrations, there are other targets of ibrutinib, which when inhibited, may have deleterious consequences. For example, inhibition of ITK has been shown to inhibit NK cell mediated antibody dependent cellular cytotoxicity (ADCC), an important mechanism of immunotherapeutic efficacy104. Because antibody therapies like rituximab are a modern mainstay of
CLL therapy and have been shown to improve survival in CLL and other hematologic malignancies105,106, it is disappointing that ibrutinib has not yet been shown to be effectively combined with immunotherapy. A more selective BTK inhibitor than ibrutinib, which lacks ITK inhibition, might enable more complete responses when combined with immunotherapies.
Side effects of ibrutinib are consistent among clinical trials and are generally mild in severity. One observation which is frequently noted in clinical trials of ibrutinib is transient lymphocytosis immediately following initial dosing. This increase in absolute lymphocyte count has been explained by in vitro studies which
22 demonstrate decreased integrin expression and impaired chemotaxis by ibrutinib96,97. Importantly, the lymphocytosis observed with ibrutinib is not associated with poor response to therapy and typically decreases with increased duration of therapy107. Diarrhea and rash are a common observation in patients treated with ibrutinib and is likely due to off-target inhibition of epidermal growth factor receptor (EGFR)108. The most common grade 3 and 4 adverse events are usually hematological in nature and include thrombocytopenia and neutropenia109.
Finally, the prevalence of atrial fibrillation has been reported to be as high as 16% in patients receiving ibrutinib, the cumulative incidence of which increases with the duration of therapy with a median time to onset of 7.6 months110.
In the longest follow-up of CLL/SLL patients treated with ibrutinib, the estimated five-year PFS rate was measured to be 92% for treatment naïve patients111. For patients with relapsed/refractory disease, the five-year PFS was
44% with median PFS being reached at 51 months. Additionally, rates of disease progression were found to vary significantly based upon traditional risk factors.
Median PFS in patients with del(17p13.1), unmutated IgVH, and del(11q23) was found to be 26 months, 43 months, and 51 months, respectively. Treatment related adverse events were also found to be more common in the first year when compared to later time periods, indicating improved tolerability of ibrutinib with longer follow up.
Ibrutinib demonstrates remarkable efficacy in CLL, especially in patients with high risk cytogenetic abnormalities like del(17p13.1) in whom ibrutinib is 23 indicated as front-line therapy. Randomized phase 3 trials have also shown a survival advantage with ibrutinib treatment over standard therapies for both treatment naïve and relapsed/refractory CLL. The preclinical work and clinical success of ibrutinib validates BTK inhibition as an effective strategy for treating many low grade hematologic malignancies. However, despite the efficacy of ibrutinib in CLL, its curative potential is limited with patients requiring life-long therapy. Additionally, resistance to ibrutinib can develop in the form of acquired mutations in BTK or its downstream partner PLCγ2 or in the form of disease transformation to DLBCL, an aggressive lymphoma which is refractory to ibrutinib therapy.
1.5 Ibrutinib Resistance
Ibrutinib, though clinically effective in the majority of patients with CLL, is often discontinued due to progressive disease, transformation to DLBCL (Richter’s transformation), or unacceptable toxicities112-116. Patients who relapse on ibrutinib due to progressive disease or Richter’s transformation have poor survival and limited therapeutic options. Several studies have found that the time to relapse and survival post relapse differ greatly in these two groups112-116. The study with the longest median follow-up (3.4 years) found that Richter’s transformation tends to occur within the first two years following the initiation of ibrutinib therapy with a median survival following relapse of 3.9 months and a cumulative incidence of
24
9.6% at four years112. Conversely, the rate of progressive CLL escalates gradually over time with a cumulative incidence of 0.7% at one year post therapy compared to 19.1% at four years post therapy and demonstrates a median survival of 22.7 months post relapse112. Complex karyotype was found to be a risk factor for both
Richter’s transformation and progressive CLL in this study112. Additionally, del(17p13.1) and age less than 65 years were found to positively associate with risk for progressive CLL112. In patients under 65 years of age with complex karyotype and del(17p13.1), the risk of progressive CLL 4 years after the initiation of therapy was 44% compared to 1.9% in patients lacking these high risk features112.
Progressive CLL is highly correlated with acquired mutations in BTK or
PLCγ2117-119. Among 46 patients with progressive CLL, targeted deep sequencing revealed that 37 patients (80.4%) possessed the C481S BTK mutation, 9 patients
(19.6%) possessed mutated PLCγ2, and 3 patients (6.5%) possessed both of these mutation at the time of relapse112. BTK mutations associated with ibrutinib resistance almost always occur at the C481 amino acid to which ibrutinib irreversibly binds, whereas PLCγ2 mutations occur preferentially in the autoinhibitory domain of PLCγ2117-119. The most common mechanism of acquired resistance to ibrutinib facilitating progressive disease is the highly recurrent C481S
BTK mutation112,117. This point mutation prevents the irreversible action of ibrutinib on BTK, essentially transforming ibrutinib into a reversible inhibitor of BTK and consequently decreasing ibrutinib’s potency an estimated 500-fold117,118. Due to
25 the relatively short half-life and rapid clearance of ibrutinib, disrupting its irreversible action greatly diminishes the activity of ibrutinib. Ibrutinib is unable to inhibit downstream BCR in CLL cells harboring C481S BTK mutations, thereby abrogating its therapeutic efficacy117. Among the characterized PLCγ2 mutations are L845F, R665W, and S707Y. The S707Y mutation is a gain-of-function mutation which disrupts a portion of PLCγ2’s auto-inhibitory domain120 while the R665W mutation allows SYK and LYN to bypass BTK and directly activate PLCγ2119. In patient cells with PLCγ2 mutations, ibrutinib was unable to affect changes in calcium signaling following BCR stimulation, indicating that PLCγ2 mutations are constitutively activating downstream of BTK and exert their effects regardless of the inhibitory pressure applied to PLCγ2’s upstream partner119. Regardless of whether relapsed patients acquire mutation in BTK, PLCγ2, or both of these proteins, resistance to ibrutinib reestablishes BCR signaling thereby facilitating
CLL disease progression.
Resistance to ibrutinib in the form of Richter’s transformation is not as well characterized as resistance in the form of progressive CLL. Although C481S BTK mutations can occur in Richter’s transformation, their rate of occurrence is much lower than in progressive CLL117. DLBCL is commonly divided into activated B cell
(ABC-DLBCL) and germinal center B cell (GCB-DLBCL) subtypes with each subtype demonstrating distinct gene expression profiles and differential responses to therapy121. GCB-DLBCL is inherently resistant to ibrutinib therapy while ABC-
DLBCL subtypes occasionally possess activating mutations in their CD79a/b
26 motifs which facilitate dependence upon BCR signaling and confer sensitivity to ibrutinib122. However, ABC-DLBCL also frequently harbors mutated MYD88, which indirectly activates NF-κB, essentially bypassing BTK123. Therefore, it is likely that
Richter’s transformation in the context of ibrutinib resistance is mediated by pharmacologic pressure which selects for GCB-DLBCL or MYD88 mutated ABC-
DLBCL.
The observation that resistance to ibrutinib is most frequently facilitated by mutation of BTK or its downstream partner PLCγ2 implies that BCR signaling is necessary for the perpetuation of CLL, and suggests that this pathway remains a relevant target in patients who relapse on ibrutinib. Maintaining BTK inhibition in the context of ibrutinib resistance may therefore provide further clinical benefit for patients with progressive CLL. An ongoing need exists to develop novel BTK targeting therapies for the population of CLL patients whose disease progresses on ibrutinib therapy.
27
CHAPTER 2
Non-Covalent Inhibition of C481S Bruton’s Tyrosine Kinase by GDC-0853: A
New Treatment Strategy for Ibrutinib Resistant CLL
2.1 Introduction
Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia with an estimated annual incidence greater than 18,000 in the United States17.
Treatment of CLL has changed dramatically in recent years with the introduction of therapies which target B cell receptor (BCR) signaling, a pathway known to enhance proliferation, immune function, and survival of malignant B lymphocytes20,61,124,125. Among the components of the BCR pathway, Bruton tyrosine kinase (BTK) has emerged as an effective therapeutic target in a number of B cell malignancies including CLL94,126. In addition to its role in the BCR pathway,
BTK propagates signaling cascades originating from toll-like receptors (TLRs), chemokine receptors, and a variety of survival inducing cytokine receptors85,127-129.
Mouse models which overexpress BTK in B cells show increased mortality due to systemic autoimmune disease95. B lymphocytes from these mice demonstrate hyper-responsiveness to BCR stimulation, increased NF-κB activity, and resistance to Fas-mediated apoptosis95. Conversely, XID mice, which lack BTK 28 kinase activity due to a point mutation in the pleckstrin homology domain of BTK, exhibit slower rates of CLL development and have significantly improved survival when crossed with the Eμ-TCL1 (TCL1) murine CLL model compared to the parental TCL1 strain94. Taken together, these observations implicate BTK as an important driver of CLL disease progression.
Ibrutinib is a first in class irreversible BTK inhibitor developed for the treatment of B cell malignancies and is currently approved for the treatment of CLL, relapsed mantle cell lymphoma, marginal zone lymphoma, and Waldenström macroglobulinemia. Ibrutinib irreversibly inhibits BTK kinase activity by covalently reacting with the C481 amino acid residue in the ATP binding site93,109,130,131.
Ibrutinib has been extraordinarily successful in CLL therapy, including in patients with high risk cytogenetic abnormalities including del(17p13.1)99. Randomized phase 3 trials have also shown a survival advantage with ibrutinib treatment over standard therapies for both treatment naïve and relapsed/refractory CLL100,101. The preclinical work and clinical success of ibrutinib validates BTK inhibition as an effective strategy for treating many low grade hematologic malignancies.
Despite ibrutinib’s activity in CLL, acquired resistance to ibrutinib does develop in a subset of heavily pretreated patients and is most commonly mediated by mutation of BTK cysteine-481, the amino acid of BTK to which ibrutinib irreversibly binds, to serine113,117. C481S BTK mutations have been reported to diminish ibrutinib’s potency 500-fold and prevent its covalent binding, rendering it unable to effect irreversible inhibition of BTK117,118. This same mode of resistance 29 has also been seen with acalabrutinib, a second generation irreversible BTK inhibitor,132 although the incidence of resistance mutations associated with this more selective agent requires further investigation. The observation that acquired resistance to irreversible BTK inhibitors is facilitated via a mutation of ibrutinib’s binding site in BTK while other irreversible targets of ibrutinib are not mutated suggests that BTK is extremely important to disease progression. Maintaining BTK inhibition in the context of ibrutinib resistance may therefore provide further clinical benefit.
Ibrutinib’s irreversible inhibition of alternative targets other than BTK (e.g.
EGFR, ITK, TEC, BMX, BLK, HER2, HER4, and JAK3) has been suggested to be associated with adverse treatment related events and immune modulation.
Ibrutinib associated adverse events include rash, diarrhea, atrial fibrillation, and thrombocytopenia99,100. Patients who discontinue ibrutinib as the result of treatment related adverse events have been found to outnumber those who stop treatment due to disease progression112,115. EGFR inhibition has been previously linked to rash and diarrhea108,133 and is proposed to mediate these adverse events in patients who experience them on ibrutinib. Inhibition of ITK by ibrutinib modulates T cell populations134 and has been shown to suppress antibody dependent cell mediated cytotoxicity (ADCC) by NK cells in response to therapeutic anti-CD20 antibodies in vitro104,135. Combinations of ibrutinib with antibody therapies, while clinically effective, have not yet been shown to be more effective than ibrutinib alone136,137. Development of a therapeutic agent which
30 could inhibit multiple BTK variants and retain selectivity against alternative targets such as EGFR and ITK may offer a potential therapeutic advantage.
Herein, we characterize GDC-0853, a reversible small molecule drug that is extremely selective for inhibition of BTK138. GDC-0853 binds to BTK without covalently reacting with C481139-141. Due to the non-covalent mechanism of inhibition and novel BTK binding mode of GDC-0853 and other similar inhibitors,141 we expected that GDC-0853 would maintain efficacy in situations of ibrutinib resistance mediated by C481S BTK mutations. Recent work has shown that non- covalent inhibitors similar in structure to GDC-0853 are able to fully retain potent inhibition of C481S BTK mutant enzyme in biochemical and cellular systems141.
We attempt to extend these findings to patient derived CLL B cells and other models in order to further validate the efficacy of non-covalent inhibition on C481S
BTK. We further hypothesize that due to its high degree of specificity for BTK that
GDC-0853 lacks ITK and EGFR inhibition, thereby diminishing alternative on- target effects of ibrutinib. This work supports the clinical trial design and first-in- human phase 1 study of the BTK inhibitor GDC-0853 in relapsed or refractory B cell NHL and CLL in which patients that had relapsed on ibrutinib and were identified to have the C481S mutation were enrolled142.
31
2.2 Methods
2.2.1 Subject Population and Lymphocyte Isolation
Blood samples were obtained from CLL patients at our institution who consented to an IRB-approved tissue procurement protocol or who were enrolled in IRB approved clinical trials of ibrutinib in CLL. Primary CLL cells were either used fresh or were viably cryopreserved and used at later dates. Human T cells were obtained from healthy volunteers. All patients gave written informed consent in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells
(PBMCs) were isolated from whole blood through ficoll density gradient centrifugation. Specific leukocyte populations were negatively selected using
Rosette-sep isolation from whole blood or Easy-sep negative selection from
PBMCs (STEMCELL Technologies).
2.2.2 Cell Culture and Drug Treatments
RPMI-1640 media supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum was used for all cell cultures. To specifically inhibit the irreversible targets of ibrutinib, treatment with this agent occurred for one hour followed by washout in which cells were pelleted and re- suspended in fresh RPMI-1640 medium. Cells treated with DMSO or GDC-0853
(obtained under a material transfer agreement with Genentech) were similarly pelleted then re-suspended in 10% FBS RPMI-1640 medium containing DMSO or
32
GDC-0853. Experiments which occurred over several days included daily addition of drug and medium replacement.
2.2.3 Immunoblotting
Proteins were detected using the following Cell Signaling antibodies: anti- phospho-BTK (Y223, Cat. #5082), anti-BTK (Cat. #8547), anti-phospho-PLCγ2
(Y1217, Cat. #3871), anti-PLCγ2 (Cat. #3872), anti-phospho-AKT (S473, Cat.
#9271), anti-AKT (Cat. #4685), anti-phospho-ERK (T202/Y204, Cat. #9101), anti-
ERK (Cat. #4695), anti-phospho-IκBα (S32, Cat. #2859), anti- IκBα (Cat. #4812), anti-phospho-EGFR (Y1068, Cat. #2234), anti-EGFR (Cat. #54359), anti-NFAT
(Cat. #4389), anti-GAPDH (Cat. #5179), anti-Actin (Cat. #3700), and anti-Lamin
(Cat. #13435). Blots were probed with primary antibodies and HRP-conjugated secondary antibodies (Santa Cruz Biotechnologies) then visualized with
SuperSignal chemiluminescent substrate (Thermo Fisher Scientific) on X-ray film.
Densitometry analysis was performed using AlphaView software.
CLL cells treated with GDC-0853 or ibrutinib were stimulated through their
BCR by spinning onto a six-well plate coated with 10 µg/mL anti-IgM antibody
(Jackson Laboratories). Following 15 minutes of stimulation, cells lysates were collected and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Healthy human T cells were treated with BTK inhibitor and stimulated for 45 minutes with a combination of 2 µg/mL plate bound anti-CD3 and 10 µg/mL soluble anti-CD28 (eBiosciences). A549 (ATCC CCL-185)
33 lung cancer cells were incubated with BTK inhibitor for one hour prior to lysis and
SDS-PAGE analysis as described above. Nuclear and cytoplasmic lysates were collected using ThermoScientific NE-PER nuclear and cytoplasmic extraction reagents (Cat. # 78833) and analyzed by SDS-PAGE.
2.2.4 Cytotoxicity Analysis
HS5 (ATCC CRL-11882) stromal cells were stably transfected with green fluorescent protein (GFP) allowing us to selectively gate on our GFP-negative CLL population in addition to gates based upon cell size. GFP-HS5 stromal cells were plated and permitted to reach 50-75% confluence before being co-cultured with
1x106 cells/mL primary CLL. Both populations were then treated with drug and viability was measured at 48 hours. Samples with DMSO treated viabilities below
40% at 48 hours were omitted from analysis due to their rapid rate of ex vivo apoptosis and unpredictable behavior in vitro. Viability was defined as the percentage of PBMCs staining negative for 7-AAD (Life Technologies). Viability of
C481S BTK mutated patient CLL cells was determined by annexin V and propidium iodine staining (eBioscience). Viability was defined as the percentage of cells staining dually negative for both annexin V and propidium iodine. All viability measurements were acquired with a Beckman Coulter FC3000 flow cytometer and Kaluza software was used for analysis.
34
2.2.5 CpG Induced Activation
Following BTK inhibitor treatment CLL cells were stimulated with 3.2 µg/mL
Class A oligodeoxynucleotide CpG (Eurofins MWG Operon). Subsequent expression of CD86 was determined by Mean Fluorescence Intensity (MFI) after
48 hours with a CD86-PE conjugated antibody (BD Biosciences). Staining intensity of CD86-PE was measured using a Beckman Coulter FC3000 flow cytometer and
Kaluza software was used for analysis.
2.2.6 CXCL12 Induced Migration
Following 1 µM BTK inhibitor treatment for one hour, CLL cells were placed into an 8.0 micron transwell insert (Sigma-Aldrich) resting in media containing 200 ng/mL CXCL12 (R&D Systems). After incubating for four hours the inserts were removed and the number of cells migrating through the transwell insert towards
CXCL12 were counted by flow cytometry and normalized to input controls. Kaluza software was used for analysis.
2.2.7 Real-Time PCR
CLL cells were treated with 1 µM BTK inhibitor with or without stimulation with 10 µg/mL anti-IgM antibody (Jackson Laboratories) for 72 hours prior to Trizol lysis and RNA purification via an RNeasy isolation kit (Qiagen). Lysates were analyzed for mRNA expression via RT-PCR using the following primers: BCL2
(Cat.# Hs00608023_m1), MCL1 (Cat.# Hs01050896_m1), MYC (Cat.#
Hs00153408_m1), OCT2 (Cat.# Hs00922172_m1), TBP (Cat.# Hs00427620_m1) 35
(Applied Biosystems). RNA expression was measured using a Viia7 Real-Time
PCR system and normalized against TBP expression.
2.2.8 Kinase Assay
160 ng human recombinant wild-type and C481S BTK (SignalChem) were incubated with DMSO or 1 µM GDC-0853 for 30 minutes. Recombinant protein was then combined with 50 µM ATP and 5 µg poly (4:1, Glu:Tyr) peptide for 30 minutes at room temperature in 1x reaction buffer (Promega Cat. # V6930) to allow for phosphorylation of the peptide substrate. ADP-glo kinase reagent and kinase detection reagent (Promega Cat. # V6930) were then used to quench and quantify the reaction, respectively. Luminescence was measured using a DTX880 plate reader.
2.2.9 CCL3 ELISA
1x106 B cells from CLL patients with C481S BTK mutations were treated with 1 µM BTK inhibitor and stimulated with 10 µg/mL soluble anti-IgM (Jackson
Laboratories) for 24 hours. Supernatants were then collected and CCL3 production was measured using a human CCL3/MIP-1 alpha Quantikine ELISA kit (R&D
Systems) according to manufacturer’s instructions.
2.2.10 NK cell mediated ADCC
Effector NK cells were isolated from Leukopaks obtained through the
American Red Cross and incubated with target CLL cells loaded with radioactive
Cr51 at an effector to target ratio of 25:1. Following treatment of purified NK cells 36 with DMSO, 1 µM GDC-0853, or 1 µM ibrutinib for one hour, CLL cells were incubated with trastuzumab, alemtuzumab, rituximab, ofatumumab, or obinutuzumab at a concentration of 10 µg/mL and co-cultured with NK cells to allow for lysis. After four hours of co-culture, supernatant was collected and measured for radiation using a PerkinElmer Wizard2 gamma counter. Beta decay measurements were scaled according to a no NK cell co-culture group with baseline CLL lysis and a detergent treated CLL group with complete lysis.
2.2.11 Statistical Analysis
Mixed effects models were used to assess changes in BTK, PLC2, AKT,
ERK activation as well as cytotoxicity between GDC-0853 vs. DMSO, in order to account for correlations among observations from the same patient. For RT-PCR data, models were fit using the ΔCT values to reduce skewness and stabilize variances, and fold changes were estimated from the models. P-values for the primary comparisons (GDC-0853 vs. untreated) were adjusted for multiple comparisons using Holm’s procedure. Similarly, differences in migration, CD86 expression, and BTK inhibition (via kinase assay), and CCL3 production were assessed using mixed effects models; data were log-transformed to reduce skewness. Finally, differences in cytotoxicity between GDC-0853 and ibrutinib in combination with other antibodies were assessed directly using interaction contrasts. P-values were again adjusted for multiple comparisons using Holm’s method. All analyses were performed using SAS/STAT software, Version 9.4 (SAS
Institute Inc., Cary, NC). 37
2.3 Results
2.3.1 GDC-0853 Inhibits BCR Signaling
Inhibition of BTK and subsequent down-stream BCR signaling is thought to be the most important mechanism by which ibrutinib exerts its therapeutic benefit.
In order to investigate pharmacologically effective concentrations of GDC-0853, we assessed its ability to inhibit BCR signaling over a range of concentrations. We found that CLL cells treated with GDC-0853 in vitro prior to BCR stimulation demonstrate reduced levels of BTK phosphorylation and diminished activation of downstream targets including PLCγ2, AKT and ERK (Figure 1A). In the representative immunoblot, 10 nM GDC-0853 is sufficient to abrogate activation of both BTK (Y223) and AKT (S473) while PLCγ2 (Y1217) and ERK (T202/Y204) demonstrate modest inhibition at concentrations ranging from 10 nM to 1 µM.
Using immunoblot densitometry analysis we quantified BCR pathway activation in five CLL patients treated with GDC-0853 or ibrutinib. As expected, we found significant decreases in the mean phosphorylation of BTK (78.6%, p=0.001),
PLCγ2 (43.7%, p=0.023), AKT (60.1%, p=0.033), and ERK (85.8%, p<0.001) following treatment with 1 µM GDC-0853 (Figure 1B-E). Pharmacokinetic data obtained from a clinical trial of GDC-0853 established a mean maximal plasma concentration ranging from 0.354 to 2.10 µM (100 mg and 400 mg daily dose, respectively) on day 15 of the study, well above the concentrations observed in the present study that were necessary for effective BTK inhibition142.
38
2.3.2 GDC-0853 Induces Modest Cytotoxicity and Overcomes Stromal Protection
CLL cells were treated with GDC-0853 for 48 hours and assessed for viability to determine whether this inhibitor mediates direct cytotoxicity. 1 µM GDC-
0853 induced a modest but statistically significant mean viability reduction of 9.8% normalized to DMSO treatment (p=0.031) (Figure 2), similar to the effect observed with 1 µM ibrutinib. Cytotoxicity was also analyzed following co-culture with the
HS5 stromal cell line, a model used to recapitulate the protective bone marrow microenvironment that is not dependent on BCR signaling. GDC-0853 was found to induce a slight increase in cytotoxicity (15.3%, p<0.001) in the presence of HS5 stroma. Furthermore, the viability following treatment with GDC-0853 was not found to significantly differ in the presence or absence of stromal co-culture
(p=0.234) suggesting that GDC-0853 may abrogate the survival benefit from the bone marrow microenvironment in addition to its direct, albeit modest, cytotoxicity.
2.3.3 GDC-0853 Inhibits NF-κB Dependent Transcription, Reduces Activation, and
Impairs Migration
Multiple B cell pathways signal through BTK leading to subsequent intracellular calcium release, activation of NF-κB, and changes in gene transcription143. To determine the influence of GDC-0853 on NF-κB dependent transcription we treated CLL cells with 1 µM BTK inhibitor for 72 hours and stimulated with anti-IgM to induce BCR signaling. We found that the mean transcript levels of BCL2, MCL1, MYC, and OCT2 significantly decreased in anti-
39
IgM stimulated CLL cells following treatment with 1 µM GDC-0853 (46%, 44%,
62%, and 31% compared to DMSO treated conditions, respectively; p< 0.01 in all comparisons) (Figure 3). The effect of GDC-0853 on gene transcription was found to be comparable to that of ibrutinib in all cases.
To determine whether GDC-0853 could prevent B cell activation through
TLR9 signaling, we treated primary CLL cells with CpG deoxynucleotides, which activate TLR9, and then measured expression of the activation marker CD86. We found a mean 39.4% reduction in CD86 expression among patient samples treated with 1 µM GDC-0853 when compared to those treated with DMSO (p=0.024)
(Figure 4A), similar to what we observed with ibrutinib.
Chemokine mediated migration of malignant CLL lymphocytes to protective lymphoid microenvironments is also a BTK dependent process. Both GDC-0853 and ibrutinib were found to prevent CXCR4 mediated migration of CLL cells toward its ligand CXCL12 (Figure 4B), with GDC-0853 showing a 51% reduction in migration as compared to DMSO (p=0.003).
2.3.4 GDC-0853 Does Not Inhibit Cellular EGFR or ITK
To assess the cellular specificity of GDC-0853 for BTK, two alternative targets of ibrutinib, EGFR and ITK, were tested for inhibition. While ibrutinib was found to decrease the constitutive activation of EGFR in the A549 cell line, GDC-
0853 spared EGFR phosphorylation up to 10 µM (Figure 5A), confirming the results of prior biochemical assays139,140 in a cellular system.
40
ITK propagates T cell receptor stimulation leading to phosphorylation of
IKBα, nuclear translocation of the transcription factor NFAT, and subsequent changes in gene expression143. Due to its inhibition of ITK, ibrutinib was found to inhibit IKBα phosphorylation and NFAT nuclear localization in healthy human T cells. GDC-0853, however, which does not inhibit ITK enzyme139,140, preserved
TCR signaling allowing for the activation of IKBα and NFAT nuclear localization confirming that GDC-0853 lacks ITK inhibition in a cellular system and does not affect T cell receptor activation (Figure 5B).
2.3.5 GDC-0853 Preserves NK Cell-Mediated ADCC
CD20 antibody therapy with rituximab or ofatumumab when combined with chemotherapy has been used to prolong patient survival in previously untreated
CLL144,145. However, in vitro data suggests that ITK inhibition by ibrutinib antagonizes the efficacy of NK cell mediated ADCC104. Knowing that GDC-0853 does not inhibit ITK in biochemical assays or in cells, we hypothesized that GDC-
0853 would be effective in combination with anti-CD20 antibody therapies. Indeed, we found that combinations of GDC-0853 with antibody therapies were significantly more effective at inducing NK cell mediated lysis of target CLL cells than combinations of ibrutinib with antibody (Figure 6). Combining GDC-0853 with obinutuzumab, the most pharmacologically active anti-CD20 antibody in CLL, produced 128% greater cytotoxicity compared to ibrutinib (p=0.01). The differences in cytotoxicity between GDC-0853 and ibrutinib in combination with the
41 less active anti-CD20 antibodies rituximab (31.8% vs 4.0%, p<0.001) and ofatumumab (28.5% vs 0.2%, p<0.001) were even more pronounced.
2.3.6 GDC-0853 Demonstrates Equivalent Inhibition of Wild Type and C481S
Mutated BTK
The most common acquired means of ibrutinib resistance occurs via a cysteine to serine mutation of BTK’s ibrutinib binding site (C481S) which prevents ibrutinib’s irreversible inhibition and results in a reduced apparent potency117.
Based on data141 for similar inhibitors with a common mode of action as GDC-0853 and recently published data140 for GDC-0853 we confirmed that, as a non-covalent inhibitor, GDC-0853 maintains equivalent efficacy against both wild type and
C481S mutant BTK. Using a biochemical BTK kinase assay we sought to determine the efficacy of GDC-0853 against both wild type and C481S BTK. 1 µM of GDC-0853 effectively inhibited wild type BTK and the C481S BTK variant
(Figure 7A). The relative inhibition of wild type and C481S BTK by GDC-0853 were similar at 96.6% and 94.1%, respectively (p<0.001 for both comparisons). These results are in agreement with previous results140 for GDC-0853.
GDC-0853 and ibrutinib were tested in HEK 293T cell lines expressing either wild type or C481S BTK to determine their ability to inhibit BTK activation.
Though effective against wild type BTK, ibrutinib did not reduce the phosphorylation of C481S mutated BTK. GDC-0853, however, was able to inhibit
42 both wild type and C481S mutated BTK (Figure 7B). The results in this cell system are also in agreement with previous results140 for GDC-0853.
We further validated the efficacy of GDC-0853 against C481S mutated BTK by testing its cytotoxicity in CLL patient samples harboring this mutation. We found a 10.3% normalized decrease in mean viability for C481S BTK patient samples treated with GDC-0853 (p=0.030) after 72 hours compared to a 2.9% decrease in viability following treatment with ibrutinib (p=0.661) (Figure 7C). Additionally, the cytokine CCL3, which is produced by B cells following BCR stimulation, was decreased by GDC-0853 in CLL cells from patients with C481S BTK mutations
(Figure 7D). GDC-0853 was found to decrease CCL3 expression by 70% compared to DMSO treated cells (p=0.003) and 59% compared to ibrutinib treated cells (p=0.015) indicating that GDC-0853 more effectively inhibits BCR signaling in CLL cells with C481S BTK than ibrutinib.
2.4 Discussion
Mutation of the C481 amino acid of BTK to which ibrutinib irreversibly covalently and irreversibly reacts is the most common and important mechanism of resistance to ibrutinib in CLL. As the clinical usage of ibrutinib continues to increase in patients with heavily pre-treated CLL, so too does the incidence of acquired resistance to this covalent BTK inhibitor. Follow up on previously untreated cohorts of CLL patients receiving ibrutinib is shorter so the frequency of
43 resistance in this population is less well defined. Non-covalent inhibition of wild- type and C481S BTK by GDC-0853 validates that reversible inhibitors are capable of producing sustained BTK inhibition that can overcome the ibrutinib-resistant mutation. Developing potent BTK inhibitors like GDC-0853 which can circumvent acquired mutations in patients is therefore of high importance. Additionally, the inhibition of targets other than BTK likely causes some of the adverse events seen with ibrutinib, such as the diarrhea and rash which may be associated with EGFR inhibition. As well, in vitro data suggest that inhibition of ITK reduces the efficacy of therapeutic antibodies when given in combination with ibrutinib. Because GDC-
0853 lacks EGFR and ITK inhibition, we expect fewer off-target drug related toxicities and the potential for more effective combination therapies. Here, we have profiled GDC-0853, a non-covalent and highly selective BTK inhibitor with the ability to inhibit ibrutinib resistant patient clones and to enable more effective combinations of BTK inhibitor with anti-CD20 antibody therapies.
Among patients treated with ibrutinib, 19.1% discontinue treatment at or before four years of therapy due to CLL progression and greater than 80% of these patients have been found to possess BTK mutations112. Data also exist which demonstrate that BTK mutations facilitate resistance to ibrutinib in Waldenström
Macroglobulinemia146 and mantle cell lymphoma147. Due to its non-covalent mechanism of action, GDC-0853 was expected retain efficacy against C481S mutant BTK variants. Indeed, GDC-0853 demonstrates preclinical efficacy in biochemical assays, cell lines, and primary CLL cells harboring C481S mutated
44
BTK. Thus, our work demonstrates that BTK inhibition can be maintained in ibrutinib resistant CLL cells by employing a reversible inhibitor that does not rely on reaction with the C481 amino acid of BTK. Given that patients who develop acquired resistance to ibrutinib have few available therapeutic options, the need for effective therapies in this population is great. Our data provide evidence that
BTK inhibition can still be achieved even in the context of ibrutinib resistance.
Though GDC-0853 is currently not being actively pursued for development in CLL, our results are an exciting proof of principle that non-covalent BTK inhibitors like
GDC-0853 can overcome acquired resistance to ibrutinib in patients. Further investigation of reversible BTK inhibitors for use in acquired resistance to ibrutinib is justified.
The addition of anti-CD20 monoclonal antibodies to chemotherapy is known to improve patient survival when compared to chemotherapy alone, making the combination of a BTK inhibitor with an anti-CD20 monoclonal antibody appealing.
Ibrutinib in combination with clinical antibodies used in CLL has been effective, but it is unclear whether the combination is superior to single agent ibrutinib therapy.
By blocking BTK signaling and promoting CLL clearance through immune effector cell activation, combinations of antibody therapy with ITK sparing BTK inhibitors, such as GDC-0853 and other irreversible selective BTK inhibitors (acalabrutinib,
ONO-4059, BGB3111) may be more synergistic than similar combinations with ibrutinib. Determining this will require trials directly comparing these combinations.
45
GDC-0853 and potentially other selective reversible BTK inhibitors currently in development represent a novel treatment strategy for CLL patients who acquire
C481S mutations. This molecule performs comparably to ibrutinib in terms of its ability to mitigate BCR signaling, CLL survival, activation, migration, and its ability to repress the transcription of disease drivers including MYC. As such, these data justify further investigation of GDC-0853 and related agents for the treatment of
C481S mutated CLL.
46
2.5 Figures
Figure 1. BCR signaling is inhibited by GDC-0853 in CLL patient cells. CLL cells were treated with BTK inhibitor for one hour and then stimulated with Anti-
IgM to elicit BCR signaling. (A) Representative immunoblot demonstrating the effect of GDC-0853 on BCR signaling. (B-E) Quantification of BCR signaling from five CLL patients using immunoblot densitometry analysis; *, P≤0.05; ***, P≤0.001.
A A A
(Continued on next page)
47
B C
D E
48
Figure 2. GDC-0853 is modestly cytotoxic to CLL B cells and limits stroma induced survival. Viability of CLL cells with or without stromal support following
48 hours of BTK inhibitor treatment (N=9). Cells were treated with 1 µM BTK inhibitor and measured for viability by flow cytometry. Results were normalized to untreated CLL cells in medium; n.s., not significant; *, P≤0.05; ***, P≤0.001.
49
Figure 3. GDC-0853 represses NF-κB dependent gene transcription. CLL patient cells were treated with 1 µM BTK inhibitor for 72 hours with or without anti-
IgM stimulation and assessed for gene expression changes of NF-κB targets by
RT-PCR (N=8); **, P≤0.01; ***, P≤0.001.
50
Figure 4. GDC-0853 abrogates important cellular functions including CLL activation and migration. (A) Activation of CLL cells 48 hours following 3.2 µM
CpG stimulation as determined by CD86 expression via flow cytometry (N=22).
MFI results were normalized to untreated paired samples. (B) Migration of CLL cells towards the chemokine CXCL12 in the presence of DMSO, 1 µM GDC-0853, or 1 µM ibrutinib (N=11); *, P≤0.05; **, P<0.01.
A B A A A A
51
Figure 5. GDC-0853 lacks inhibition of EGFR and ITK in cells. (A) The A549 lung cancer cell line was treated with varying concentrations of BTK inhibitor to determine the effect on EGFR activation. (B) T cell activation in healthy donor T cells following treatment with 1 µM BTK inhibitor prior to TCR stimulation with anti-
CD3 and anti-CD28.
A A A
B A A
52
Figure 6. GDC-0853 preserves NK cell mediated ADCC in response to anti-
CD20 antibodies. NK cells treated with 1 µM BTK inhibitor were co-cultured with
CLL cells loaded with radioactive chromium in the presence of alemtuzumab, rituximab, ofatumumab, or obinutuzumab at a concentration of 10 µg/mL.
Supernatant fluids were collected after four hours and measured for radioactivity to determine the efficacy of CLL cell lysis by NK cell mediated ADCC (N=4); **,
P≤0.01; ***, P≤0.001.
53
Figure 7. GDC-0853 inhibits both wild type and C481S mutated BTK variants.
(A) Inhibition of wild type and C481S recombinant BTK protein by 1 µM GDC-0853 as measured by a biochemical kinase activity assay (N=3). (B) The HEK 293T cell line was stably transfected with either wild type or C481S mutated BTK. Following
BTK inhibitor treatment these cells were immunoblotted to determine the phosphorylation status of BTK (Y223). (C) Primary CLL cells from patients expressing the C481S BTK mutation were treated with DMSO, 1 µM GDC-0853, or 1 µM ibrutinib for 48 hours and assessed for viability (N=9). (D) CLL cells from patients acquiring C481S BTK mutations during the course of ibrutinib therapy were treated with GDC-0853 to determine its ability to inhibit BCR mediated production of CCL3 (N=8); *, P≤0.05; **, P≤0.01; ***, P≤0.001.
A
A A
(Continued on next page)
54
)
Continued on next page Continued next on (
B
55
D C
56
CHAPTER 3
The BTK Inhibitor ARQ 531 Targets Ibrutinib Resistant CLL and Richter’s
Transformation
3.1 Introduction
Recent appreciation for the extent to which Bruton’s tyrosine kinase (BTK) drives select hematological malignancies such as chronic lymphocytic leukemia
(CLL)61,94,124 has spurred the development of targeted BTK inhibitors, most notably the irreversible inhibitor ibrutinib98,100,101,132,149,150. Several pathways which work to increase the survival and proliferation of CLL B cells converge proximally on BTK; these pathways are initiated at the B cell receptor (BCR)61, Toll-like receptors
(TLRs)127,128, and multiple chemokine receptors85,129, making BTK an attractive therapeutic target. BTK is overexpressed at the transcript and protein level in CLL
B cells compared to healthy donors leading to constitutive BCR activation93. In vitro, BTK inhibition induces CLL cytotoxicity, decreases NF-κB dependent transcription, and abrogates chemokine-mediated migration of CLL cells to protective lymphoid microenvironments93,151. Clinically, ibrutinib produces durable responses with manageable drug related toxicities in the majority of CLL
57 patients111,112. In the longest follow-up of patients treated with ibrutinib, the five- year progression free survival (PFS) rate was estimated to be 92% for treatment naïve patients while the median PFS for patients with relapsed/refractory disease was 52 months111. Ibrutinib has broad regulatory approval for marketing in patients with CLL and is clinically effective regardless of most traditional prognostic factors, although complex karyotype, del(17p13.1), and age less than 65 years are risk factors for late relapse112.
Despite its initial clinical benefit for most patients, resistance to ibrutinib continues to emerge as more patients are being treated and follow-up time extends. Relapse on ibrutinib arises via Richter’s transformation to aggressive lymphoma, which occurs early in treatment, and CLL progression, which typically occurs beyond 1-2 years of therapy112-114. Unfortunately, patients who relapse on ibrutinib have poor survival and limited therapeutic options112-116, particularly those with Richter’s transformation. Among patients who develop progressive CLL on ibrutinib, approximately 86% acquire mutations in BTK and/or PLCγ2, BTK’s immediate downstream partner112,114. BTK mutations associated with ibrutinib resistance occur at the C481 amino acid to which ibrutinib irreversibly binds, whereas PLCγ2 mutations occur preferentially in the autoinhibitory domain of
PLCγ2117,118. The observation that resistance to ibrutinib is most frequently facilitated by mutation of BTK or its downstream partner implies that BTK function is necessary for the perpetuation of CLL, and suggests that this pathway remains a relevant target in patients who relapse on ibrutinib. Therefore, an ongoing need 58 exists to develop novel BTK targeting therapies for the population of CLL patients whose disease progresses on ibrutinib therapy due to Richter’s transformation or ibrutinib resistant CLL.
CLL patients who experience Richter’s transformation while on ibrutinib have dismal survival of less than 6 months112,113. This type of resistance is generally not associated with BTK or PLCγ2 mutations113,114. Recently, our laboratory developed a transgenic mouse model, which spontaneously develops an aggressive B cell lymphoma resembling Richter’s transformation on the background of CLL152. These mice overexpress MYC via an Eµ enhancer sequence in addition to overexpression of TCL1. The transgenic Eµ-TCL1 is a standard CLL mouse model which specifically overexpresses the TCL1 oncogene
153 via a B cell specific IgVH promoter and Eµ enhancer . The spontaneous CLL-like leukemia that develops is dependent on BCR signaling and BTK94,96,129,153. The
Eμ-MYC/TCL1 mouse utilized in this study fills an unmet need for murine models which recapitulate the disease phenotype of Richter’s transformation.
Although selective inhibition of BTK is effective in CLL, the extent to which concurrent inhibition of additional kinases may enhance treatment response is unknown. Here we characterize ARQ 531, a potent, ATP-competitive, reversible inhibitor of BTK and several additional kinases important to the viability, proliferation, activation, and motility of CLL B cells. Among these targets are LYN, the kinase which initiates intracellular BCR signaling, RAF1, and MEK1 which
59 directly activates ERK leading to B cell proliferation. Additionally, the activity of
ARQ 531 is not predicated upon its ability to irreversibly bind the C481 amino acid of BTK which is commonly mutated in patients who develop resistance to ibrutinib.
Therefore, we hypothesized that ARQ 531 would be effective in both BTK inhibitor- naïve CLL and ibrutinib resistant CLL arising from the C481S BTK and/or activating
PLCγ2 mutations. Herein, we demonstrate the preclinical efficacy of this molecule.
3.2 Methods
3.2.1 Subject Population and Lymphocyte Isolation
Blood was obtained from CLL patients at our institution who consented to an IRB-approved tissue procurement protocol or who were enrolled in IRB- approved clinical trials of ibrutinib in CLL. All patients provided written informed consent in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood through ficoll density gradient centrifugation. B cells from CLL patients were negatively selected using
Rosette-sep isolation from whole blood or Easy-sep negative selection from
PBMCs (STEMCELL Technologies, Vancouver, BC).
3.2.2 BTK Biochemical and Kinase Selectivity Assay
Biochemical inhibition of BTK was measured using full length BTK constructs of wild-type and C481S mutant proteins (Reaction Biology, Malvern,
60
PA). ARQ 531 was tested in a 10-point concentration mode with 3-fold serial dilutions starting at 1 µM in the presence of 10 µM ATP and the IC50 was determined. Kinase selectivity was assessed against 236 kinases at 200 nM concentration of ARQ 531, the percent inhibition was determined relative to DMSO control. Off-target kinases demonstrating greater than 50% inhibition at 200 nM
ARQ 531 were subjected to IC50 determination at a physiological 1 mM ATP concentration.
3.2.3 Crystal structure determination
Kinase domain of BTK (387-659) was expressed as a 6xHis-tagged protein in Sf9 insect cells and the protein was purified on a Ni-NTA column. ion exchange and size exclusion chromatography. Crystals were obtained by the hanging drop method. Initially, apo-BTK kinase domain crystals were obtained from drops containing 0.2M imidazole pH 7.0, 16% (w/v) PEG 4000, 12.5% ethylene glycol at
4 oC. Subsequently, BTK-ARQ 531 complex was generated by micro seeding of the apo-BTK crystals. Data to 1.1 Å were collected at 100K at station I03 (λ =
0.9763 Å), Diamond Light Source (Didcot, England) equipped with a Pilatus3 6M detector.
3.2.4 Cell Culture and Drug Treatment
RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum was used for cell cultures. To specifically inhibit the irreversible targets of ibrutinib, cells were treated with
61 ibrutinib for 1 hour followed by washout in which cells were pelleted and resuspended in fresh media. Cells treated with DMSO or ARQ 531 were pelleted then resuspended in 10% FBS RPMI-1640 media containing DMSO or ARQ 531.
Cells used in experiments which occurred over multiple days received fresh media and drug every 24 hours. ARQ 531 ((2-chloro-4-phenoxyphenyl)(4-(((3R,6S)-6-
(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-5- yl)methanone) was supplied by ArQule Inc., (Burlington, MA).
3.2.5 Immunoblotting
Freshly isolated patient CLL cells treated with ARQ 531 or ibrutinib were stimulated by spinning onto a six-well plate coated with anti-IgM antibody (Jackson
Laboratories, Bay Harbor, ME) at a concentration of 10 µg/mL. Following 15 minutes of stimulation, cells lysates were collected and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Blots were probed with primary antibodies and HRP-conjugated secondary antibodies (Santa
Cruz Biotechnologies, Santa Cruz, CA), and then visualized with SuperSignal chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) on X-ray film. The following antibodies obtained from Cell Signaling Technologies (Danvers,
MA) were utilized for immunoblot experiments: anti-phospho-BTK (Y223, Cat.
#5082), anti-BTK (Cat. #8547), anti-phospho-PLCγ2 (Y1217, Cat. #3871), anti-
PLCγ2 (Cat. #3872), anti-phospho-AKT (S473, Cat. #9271), anti-AKT (Cat.
#4685), anti-phospho-ERK (T202/Y204, Cat. #9101), anti-ERK (Cat. #4695), anti-
62 phospho-MEK1/2 (S217/221, Cat. #9121), anti-MEK1/2 (Cat. #8727), anti- phospho-SYK (Y525/526, Cat. #2710), anti-SYK (Cat. #2712), anti-phospho-Src family (Y416, Cat. #2101), anti-LYN (Cat. #2796), anti-phospho-IκBα (S32, Cat.
#2859), anti-IκBα (Cat. #4812), anti-GAPDH (Cat. #5179), anti-Lamin (Cat.
#13435), anti-p65 (8242). Anti-phospho-BTK (Y551, Cat. #MAB7659) was ordered from R&D Systems (Minneapolis, MN)
3.2.6 Viability
Viability of patient CLL cells was determined by annexin V and propidium iodine staining (eBioscience, San Diego, CA) followed by flow cytometry analysis.
Viability was defined as the percentage of cells staining dually negative for both annexin V and propidium iodine. All viability measurements were acquired with a
Beckman Coulter FC500 flow cytometer and Kaluza software (Beckman Coulter,
Brea, CA) was used for data analysis.
3.2.7 Migration
Following treatment with ARQ 531 or ibrutinib CLL cells were placed into an
8.0 micron transwell insert (Sigma-Aldrich, Saint Louis, MO) resting in media containing 200 ng/mL CXCL12 (R&D Systems, Minneapolis, MN) or 1 µg/mL
CXCL13 (R&D Systems, Minneapolis, MN). After incubating for 4 hours, the inserts were removed and the number of cells migrating through the transwell insert towards CXCL12 or CXCL13 were counted by flow cytometry and normalized to input controls. Data analysis was conducted using Kaluza software. 63
3.2.8 CpG Induced Activation
Following treatment with DMSO, ARQ 531, or ibrutinib, CLL cells were stimulated with 3.2 µg/mL oligodeoxynucleotide CpG (Eurofins MWG Operon,
Louisville, KY). Subsequent expression of CD40 and CD86 was determined by
Mean Fluorescence Intensity (MFI) after 48 hours with a CD40-PE or CD86-PE conjugated antibody (BD Biosciences, Franklin Lakes, NJ). Staining intensity of
CD40-PE and CD86-PE was measured using a Beckman Coulter FC3000 flow cytometer and data analysis was conducted using Kaluza software.
3.2.9 Real-Time PCR (RT-PCR)
CLL cells were treated with DMSO, ARQ 531, or ibrutinib for 72 hours prior to Trizol lysis and RNA purification via an RNeasy isolation kit (Qiagen, Hilden,
Germany). Lysates were analyzed for mRNA expression via RT-PCR using the following Applied Biosystems (Waltham, MA) primers and probes: MCL1 (Cat. #
Hs01050896_m1), MYC (Cat. # Hs00153408_m1), CD40 (Cat. #
Hs01002915_g1), and TBP (Cat. # Hs00427620_m1). RNA expression was measured using a Viia7 Real-Time PCR system and normalized against TBP expression.
3.2.10 Eμ-TCL1 Mouse Model
C57BL/6 mice were engrafted via tail vein injection with 1x107 live
CD5+/CD19+ B cells isolated from the spleen of a single Eµ-TCL1 mouse. Once
64 a peripheral population of CD5+/CD19+ cells was established at greater than 10%, the mice were randomly assigned to experimental cohorts. Mice were treated with vehicle consisting of 10% Cremophor EL and 10% ethanol in saline, 75 mg/kg ARQ
531, 50 mg/kg ARQ 531, or 25 mg/kg ibrutinib via daily oral gavage and tracked for overall survival. Two concentrations of ARQ 531 were chosen to assess efficacy and potential drug related toxicity while 25 mg/kg of ibrutinib has been established as an effective concentration for use in this model94,154. Weekly bleeding was performed in order to assess CD5+/CD19+ cell populations and to prepare slides for microscopic examination. Cessation of drug treatment occurred at 74 days.
3.2.11 Eμ-MYC/TCL1 Mouse Model
C57BL/6 mice were engrafted via tail vein injection with 5x106 live
CD5+/CD19+ B cells isolated from the spleen of a single Eµ-Myc/TCL1 donor mouse. 10 days following engraftment, mice were randomly assigned to experimental cohorts. Mice were treated with vehicle, 75 mg/kg ARQ 531, or 25 mg/kg ibrutinib via daily oral gavage and tracked for overall survival. Weekly bleeding was performed in order to assess CD5+/CD19+ cell populations and to prepare slides for microscopic examination.
3.2.12 Statistics
Differences in phosphorylation (Figure 8D), cytotoxicity (Figures 10A, 10B,
14C), transcript expression (ΔCt values; Figure 11A), CD40/CD86 MFI (log- 65 transformed values; Figures 11B, 11C), and migration towards CXCL12/CXCL13
(log-transformed values; Figures 11D, 11E) between groups were estimated using mixed models to account for correlations among observations from the same patient. Survival curves were estimated using the Kaplan-Meier method and curve differences between groups assessed using the log-rank test (Figures 12A, 13A).
Analysis of variance (ANOVA) was used to compare spleen weight (Figure 12C) among groups. Mixed effects models were also used to compare group differences in the percentage of CD19+ cells over time (Figure 13B). Data represent mean ±
SEM. For experiments with multiple endpoints, p-values were adjusted for multiple comparisons using Holm’s procedure. All analyses were performed using
SAS/STAT software, version 9.4 of the SAS system for Windows (SAS Institute,
Inc., Cary, NC).
3.2.13 Study Approval
All studies conducted with the use of human samples were reviewed and approved by The Ohio State University IRBs. Animal experiments were conducted in accordance with the guidelines established by The Ohio State University and approved by the IACUC.
66
3.3 Results
3.3.1 ARQ 531 Is a Potent Inhibitor of BCR Signaling
ARQ 531 was designed as a reversible, orally bioavailable, ATP- competitive inhibitor of BTK (Figure 8A). Crystal structure of BTK in complex with
ARQ 531 at 1.1 Å resolution shows that ARQ 531 inhibits BTK by competing with
ATP (Figure 8B). The core pyrrolopyrimidine moiety forms bidentate hydrogen bonding with the hinge backbone of G475 and Y476 residues and the chlorine atom is positioned between the A428 and K430 side chains. Similar to ibrutinib154, the phenoxyphenyl group occupies the hydrophobic pocket of the ATP binding region. The polar tetrahydropyran methanol side chain is exposed to the solvent area and facilitates water-mediated extensive hydrogen bonding network.
Importantly, ARQ 531 does not interact with C481, suggesting that C481S mutation would not affect binding.
Initial determination of the kinase inhibitory profile of ARQ 531 was performed via a biochemical kinase screen (Table 1 and Figure 9). ARQ 531 possesses nanomolar potency against TEC family kinases including the target kinase BTK as well as several Src family kinases. Additionally, RAF1 and MEK1 were identified as targets of ARQ 531. To confirm inhibition of BCR-associated kinases in a cellular model, primary CLL cells were treated with escalating doses of ARQ 531. As expected, BCR-mediated activation of BTK, AKT, and ERK was inhibited in a dose dependent manner by ARQ 531 (Figure 8C and 8D). Although
67
ARQ 531 and ibrutinib comparably decreased BTK and AKT phosphorylation, ARQ
531 decreased ERK phosphorylation more effectively than ibrutinib, likely due to predicted inhibition of RAF1 and MEK1. We also found that phosphorylation of Src family kinases and SYK was also inhibited by ARQ 531, but not by ibrutinib in patient derived primary CLL cells (Figure 10C). After observing this differential ability to inhibit kinases upstream of BTK in the BCR pathway, we sought to determine if the Y551 amino acid of BTK, which is transphosphorylated by SYK leading to autophosphorylation of Y223 and BTK kinase activity, is affected by
ARQ 531. Indeed, we found that both the Y551 and Y223 activation sites of BTK were dephosphorylated by ARQ 531 whereas only the Y223 autoactivation site of
BTK was dephosphorylated with ibrutinib treatment (Figure 8E).
3.3.2 ARQ 531 Is Cytotoxic to CLL Cells In Vitro
We next sought to determine if ARQ 531 was cytotoxic to CLL cells. At 48 hours, the viability of CLL cells treated with continuous exposure to 0.1 µM, 1.0
µM, or 10.0 µM ARQ 531 was found to decrease in a dose-dependent manner by
19% (p=0.001), 40% (p<0.001), and 59% (p<0.001) respectively (Figure 10A).
Additionally, we evaluated direct cytotoxicity in primary CLL cells treated with ARQ
531 for two hours followed by drug washout and media replacement. Cytotoxicity following washout did not significantly differ from the continuous drug exposure experiment (p=0.859) suggesting that ARQ 531 maintains sufficient residency of its targets to elicit cytotoxicity even with short drug exposure (Figure 10B).
68
3.3.3 ARQ 531 Abrogates CpG Mediated Activation and Chemokine Induced
Migration of CLL Cells
BTK activation has been shown to increase NF-κB activity and thus CLL cell activation155,156, events which can be blocked by ibrutinib157. To determine whether
ARQ 531 similarly affects NF-κB target genes, we performed real-time PCR in primary CLL cells following treatment with ARQ 531 and ibrutinib. ARQ 531 significantly decreased transcription of MYC, MCL1, and CD40 compared to vehicle by 73% (p=0.003), 23% (p=0.003), 40% (p=0.027), respectively (Figure
11A). As well, in vitro treatment of CLL cells with 1 µM ARQ 531 for 48 hours decreased CpG-induced upregulation of CD40 and CD86 by 25% (p=0.006) and
40% (p=0.001), respectively (Figure 11B&C). We next investigated if ARQ 531 inhibits migration of CLL cells toward cytokines (CXCL12 and CXCL13) secreted by the protective marrow microenvironment. Using a transwell assay system we found that ARQ 531 decreased migration of primary CLL cells towards CXCL12 by
51% (p=0.002) and CXCL13 by 66% (p=0.001) (Figure 11D&E).
3.3.4 ARQ 531 Is Superior to Ibrutinib in the Eμ-TCL1 Engraftment Mouse Model
We and others have demonstrated that the Eμ-TCL1 mouse model of CLL displays active BCR signaling and responds to treatment with the BTK inhibitors ibrutinib and acalabrutinib96,97. Following the establishment of leukemia in an Eμ-
TCL1 adaptive transfer model, we randomized mice to treatment with vehicle
(n=14), 25 mg/kg ibrutinib (n=6), 50 mg/kg ARQ 531 (n=14) or 75 mg/kg ARQ 531
69
(n=14) given by daily oral gavage. ARQ 531 given at either 50 mg/kg or 75 mg/kg was found to significantly improve survival over ibrutinib treatment (p=0.044 and p=0.009, respectively). Median survival of mice treated with vehicle, ibrutinib, and
ARQ 531 was 36 days, 53 days, and not reached by 74 days for both dose levels, respectively (Figure 12A). ARQ 531 was discontinued on day 74 (60 total days of administration) to assess the rate of disease progression. Following observation off ARQ 531 therapy, mice rapidly died of progressive disease, with a final median survival of 76 days for the 50 mg/kg ARQ 531 cohort and 78 days for the 75 mg/kg
ARQ 531 cohort. Mice receiving ARQ 531 had lower blood lymphocyte counts on microscopic examination compared to those treated with vehicle or ibrutinib
(Figure 12B). In a cohort of mice sacrificed two weeks after the initiation of therapy, splenic weight was significantly reduced in mice treated with ARQ 531 compared to vehicle (p=0.001 for 50 mg/kg and p<0.001 for 75 mg/kg) (Figure 12C).
Collectively, these studies establish the superior efficacy of ARQ 531 in the Eμ-
TCL1 transgenic mouse model of CLL as compared to ibrutinib.
3.3.5 ARQ 531 Is Superior to Ibrutinib in a Murine Model of Richter’s
Transformation
Given the poor survival for patients with CLL whose disease progresses to
Richter’s transformation, we next investigated if ARQ 531 demonstrates efficacy in a murine model of aggressive lymphoma that we have recently described152 in which ibrutinib is ineffective. B cells isolated from the spleen of an Eµ-MYC/TCL1
70 mouse were engrafted into C57BL/6 mice via tail vein injection followed by treatment 10 days post engraftment. 75 mg/kg ARQ 531 prolonged survival in this model with a median survival of 47 days compared to ibrutinib (median survival 38 days, p=0.036) and vehicle treatment (median survival 35.5 days, p=0.008) (Figure
13A). Additionally, the percentage of CD19+ cells in the peripheral blood of these mice was lower throughout the course of the study in the cohort treated with ARQ
531 compared to mice treated with either ibrutinib or vehicle (Figure 13B).
3.3.6 ARQ 531 Maintains Efficacy in Cells with C481S Mutated BTK
We hypothesized that because ARQ 531 does not require stabilization from the C481 amino acid within BTK’s ATP binding domain that this molecule would possess inhibitory activity against C481S BTK mutations which mediate clinical ibrutinib resistance. To determine the activity of ARQ 531 against C481S BTK, we performed a biochemical kinase assay utilizing recombinant BTK protein and found that the IC50 of ARQ 531 was similar for wild-type and C481S BTK (0.85 nM and
0.39 nM respectively). We next evaluated ARQ 531 in a BTK-/- HEK293T cell line transfected with wild-type or C481S BTK. ARQ 531 inhibited both wild type and
C481S mutated BTK in this system, whereas ibrutinib demonstrated efficacy only against wild-type BTK (Figure 14A). Next, we evaluated ARQ 531 in vitro in CLL cells isolated from a patient with C481S BTK acquired following ibrutinib therapy.
We found that ARQ 531 inhibited activation of Src family members, BTK, AKT, and
ERK similarly in baseline and relapse samples (Figure 14B). Finally, we
71 determined the cytotoxicity of ARQ 531 against ibrutinib resistant primary CLL cells expressing C481S BTK. At 72 hours ARQ 531 mediated significant cytotoxicity
(28% decrease compared to vehicle, p=0.002) whereas there was no cytotoxicity following ibrutinib treatment (p=0.906) (Figure 14C).
3.3.7 ARQ 531 Effectively Inhibits Downstream Signaling in Ibrutinib Resistant
PLCγ2 Mutations
Previous work has shown that PLCγ2 can be directly activated by LYN and
SYK in ibrutinib refractory models possessing mutant PLCγ2119, effectively bypassing the role of BTK in PLCγ2 activation. Due to its direct inhibition of LYN and MEK1 and its indirect inhibition of SYK, we hypothesized that ARQ 531 may provide effective inhibition of downstream BCR signaling in patients with mutations in the autoinhibitory domain of PLCγ2. To test this hypothesis, we utilized a PLCγ2
-/- DT40 cell line transfected with wild-type PLCγ2, R665W PLCγ2, or L845F
PLCγ2. We found that ARQ 531 successfully inhibited the downstream activation of ERK in these cell lines, whereas ibrutinib was ineffective (Figure 15A). To further validate this finding, we tested ARQ 531 in vitro in CLL cells from a patient with an
R665W PLCγ2 mutation acquired following ibrutinib treatment. We found that ARQ
531 preserved BTK, SYK, and ERK inhibition post-relapse, whereas ibrutinib was only able to inhibit BTK (Figure 15B).
72
3.4 Discussion
Due to its unique BTK binding affinity and distinct kinase profile, ARQ 531 is an attractive molecule to study in ibrutinib resistant CLL which progresses due to acquired mutations in BTK and PLCγ2 or Richter’s transformation114. Herein, we demonstrate that ARQ 531 maintains inhibitory pressure on the BCR pathway in situations of C481S BTK and autoactivating PLCγ2 mutations. Furthermore, ARQ
531 was found to significantly prolong survival over ibrutinib in a murine model of
Richter’s transformation. The clinical success of ibrutinib has established that BCR signaling is vital to CLL progression and that inhibition of BTK is a successful therapeutic paradigm159. While previous work with the XID mouse model suggests that BTK inhibition alone is sufficient to delay disease progression94, the extent to which inhibition of additional kinases may affect disease progression is unknown.
It is reasonable that combining additional inhibitory pressure on the BCR pathway via kinases both upstream and downstream of BTK might improve efficacy, as is suggested by our in vivo experiment using the Eμ-TCL1 mouse model. These data challenge the convention that highly selective inhibition of a single target should serve as the model of drug development for CLL patients who are resistant to current therapies.
Patients who relapse on ibrutinib progress rapidly and have limited therapeutic options112. While venetoclax has been shown to be effective in this setting160, not all patients respond and long-term remissions are uncommon. Given
73 the importance of BCR signaling in CLL progression, it is unsurprising that CLL progression in patients receiving ibrutinib is overwhelmingly mediated by mutation of BTK at its ibrutinib binding site or in the autoinhibibtory domain of BTK’s immediate downstream signaling partner PLCγ2112. Because the BCR pathway remains active in patients who relapse on ibrutinib161, continuing to inhibit BTK and associated kinases through alternate mechanisms is of great clinical interest and may provide an alternative strategy for long-term disease control. Unlike ibrutinib,
ARQ 531 reversibly inhibits BTK and does not require stabilization from C481 to block BTK mediated signaling. We therefore hypothesized, and found, that ARQ
531 would be effective in models of ibrutinib resistance containing the recurrent
C481S BTK mutation. ARQ 531 was also found to inhibit downstream BCR signaling in models expressing activating PLCγ2 mutations. This is likely due to inhibition of SYK and LYN, which have been shown to directly activate PLCγ2 in the setting of activating PLCγ2 mutations119. Activity against both C481S BTK and mutated PLCγ2 distinguishes ARQ 531 from other BTK inhibitors currently in preclinical and clinical development.
Another critical area of unmet need in CLL is Richter’s transformation, where survival is uniformly poor and current therapies are inadequate. Based on our data in the Eµ-MYC/TCL1 mouse, ARQ 531 may be an effective therapy in patients with aggressive lymphomas, including those with Richter’s transformation.
Considering that ibrutinib does not directly inhibit members of the RAS/RAF pathway it is possible that the superior efficacy of ARQ 531 in the setting of 74
Richter’s transformation may be due to its inhibition of distal targets including
MEK1, which has been shown to be an effective target in diffuse large B-cell lymphomas162. Inhibiting a broader spectrum of targets may lead to deeper and more durable remissions while delaying the emergence of resistance.
Ibrutinib induces durable responses and extends disease remission in malignancies including CLL, mantle cell lymphoma, and Waldenström’s macroglobulinemia in part through its inhibition of BTK. Because ARQ 531 inhibits
BTK with comparable potency, it is expected that ARQ 531 may also be effective in these B cell malignancies. Furthermore, its inhibition of additional targets and its efficacy in the Eµ-MYC/TCL1 mouse model suggests that ARQ 531 may possess activity in malignancies ineffectively treated by ibrutinib or other BTK inhibitors.
Among the most intriguing additional targets of ARQ 531 are RAF1 and MEK1, constituents of the ERK signaling pathway which are upregulated in ibrutinib treated CLL cells107. ERK signaling is of general interest in hematological malignancies as well as solid tumors due to its ability to influence proliferation and its high frequency of mutation in cancer. RAF1 and MEK1 have been shown to be important targets in the setting of RAS mutations163, which suggests that ARQ 531 may be applicable to RAS mutated malignancies including multiple myeloma164,165, melanoma166, and others167. Moreover, indirect inhibition of SYK by ARQ 531 may allow this molecule to be utilized in the setting of acute myeloid leukemia where
SYK inhibition has been shown to impair leukemia progression168-170. Rationale therefore exists to explore the effect of ARQ 531 in additional malignancies. 75
Our preclinical studies with ARQ 531 demonstrate that this compound is an effective ATP-competitive kinase inhibitor in the context of ibrutinib naïve CLL as well as ibrutinib resistant disease in the form of acquired mutations in BTK and/or
PLCγ2 or Richter’s transformation. Especially in post-ibrutinib patients who tend to have genomically complex disease, a multi-targeted agent like ARQ 531 may be ideal. The improved survival associated with ARQ 531 treatment in multiple murine models suggests that less discriminating kinase inhibitors may deserve additional consideration in the development of novel therapies for this purpose and potentially other malignancies. Our findings justify continued preclinical work on ARQ 531 and investigation in human clinical trials for CLL and Richter’s transformation.
76
3.5 Figures Figure 8. Inhibition of BCR mediated signaling by ARQ 531. A) Chemical structure of ARQ 531. B) 1.1 angstrom resolution crystal structure of ARQ 531 in complex with BTK. ARQ 531 occupies the ATP binding pocket and the solvent exposed side chain forms water mediated hydrogen bond network C) Rosette-Sep isolated primary CLL cells were treated with escalating concentrations of ARQ 531 for 1 hour and then stimulated with anti-IgM for 15 minutes. Immunoblotting was then performed to show that ARQ 531 inhibits phosphorylation of BTK, AKT, and
ERK. D) Immunoblot data from five CLL patients treated with ARQ 531 were quantitated using densitometry software. E) Rosette-Sep isolated primary CLL cells were assessed via immunoblot for transphosphorylation of Y551 following
ARQ 531 treatment for one hour and anti-IgM stimulation for 15 minutes. For all experiments, differences were assessed using linear mixed-effects models. (**,
0.01≥p≥0.001; ***, p<0.001)
A
ARQ 531 (Continued on next page)
77
B
K430 A428 Cl
H2O
C481
(Continued on next page)
78
+ anti-IgM C
DMSO DMSO 0.1µM ARQ531 1.0µM ARQ531 10µM ARQ531 1.0µM Ibrutinib BJAB
pBTK (Y223)
Total BTK
pAKT (S473)
Total AKT pERK (T202/Y204)
Total ERK
GAPDH
D
(Continued on next page)
79
E + anti-IgM
M ARQ531 M Ibrutinib
μ μ
DC9 1 DMSO DMSO 1 pBTK (Y551) pBTK (Y223) Total BTK GAPDH
80
Table 1. ARQ 531 inhibits multiple kinases including members of the TEC and Src families and RAS/RAF pathway. Kinase selectivity was assessed against 236 kinases at 200 nM concentration of ARQ 531, the percent inhibition was determined relative to DMSO control. Off-target kinases demonstrating greater than 50% inhibition at 200 nM ARQ 531 were subjected to IC50 determination at a physiological 1 mM ATP concentration.
81
Figure 9. Kinome tree illustrating the selectivity profile of ARQ 531. IC50 values from Supplemental Table 1 were plotted in a representation of the human kinome. Representation was generated using KinMap. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).
82
Figure 10. ARQ 531 is cytotoxic to CLL cells and more potently inhibits proximal BCR pathway kinases than ibrutinib. A) Primary CLL cells were continuously treated with increasing concentrations of ARQ 531 for 48 hours to assess cytotoxicity via annexin V/PI staining and flow cytometry (n=9). B) Primary
CLL cells were treated daily with ARQ 531 for two hours followed by drug washout and media replacement in order to simulate clearance of the reversible BTK inhibitor ARQ 531 (n=9). C) Primary CLL cells from three patients were treated in vitro with ARQ 531 and ibrutinib to contrast the effect on proximal BCR associated kinases including Src family members and SYK as well as downstream kinases including MEK and ERK. Differences were assessed using linear mixed-effects models. (**, 0.01≥p≥0.001; ***, p<0.001)
100 100 *** e
e *** v
A v B *** i
i *** t
t ** **
a
a g
g 80 e
e 80
N
N
I
I
P
P
/
/ V
V 60 60
n
n
i
i
ex ex
40 40 nn
nn
A
A
e
e
l l
20 20 oub
oub
D
D % % 0 0
O ib O ib S S n 531 531 531 in 531 531 531 ti M t M Q Q Q u D Q Q Q ru D r b b AR AR AR I AR AR AR I M M M M M M M M 1 1 1 1 0.1 10 0.1 10
(Continued on next page)
83
C
Pt 00250071 Pt 00252283 Pt 00252502
Anti IgM - - + + + - - + + + - - + + + DMSO - + + - - - + + - - - + + - - 1 µM ARQ 531 - - - + - - - - + - - - - + - 1 µM Ibrutinib - - - - + - - - - + - - - - + DC9 + - - - - + - - - - + - - - - pSRC Family Lyn pSyk Syk pBTK Y223 BTK
pPLCγ2
PLCγ2 pAKT AKT pMEK
MEK pERK ERK GAPDH
84
Figure 11. Changes in NF-κB function, activation, and migration due to ARQ
531. A) Transcriptional expression of MYC, CD40, and MCL-1 in CLL cells was assessed via real-time PCR following 72 hours of treatment with 1 µM ARQ 531
(n=7 for MYC, n=8 for MCL1 and CD40). B&C) The NF-κB dependent activation markers CD40 and CD86 were measured via flow cytometry 48 hours following treatment with 1 µM ARQ 531 and 3.2 µM CpG in CLL B cells (n=9). D&E) The number of CLL cells migrating through an 8.0 micron transwell insert towards the chemokines CXCL12 and CXCL13 were counted by flow cytometry (n=8 for
CXCL12, n=9 for CXCL13). Differences were assessed using linear mixed-effects models. (*, 0.05≥p>0.01; **, 0.01≥p≥0.001)
(Continued on next page)
85
on next page) next on
Continued (
86
Continued on next page)Continued next on (
87
b
i
n i
t
u
r
b
I
1 M
3
5
1 page)Continued next on
Q
+ (
** R
3
A 1
3
M
1
CL
X
1
CL C
X +
C 3
1
O
CL S
X
M
C D
8 6 4 2 0 0
1
n o i t a r g i M d ce ndu I 13 L C X C
b
i
n i
t
u
r
b
I
1 M
3
5
1
Q +
** R 2
A 1
2
M
1
CL
X
1
CL C
X +
C 2
1
O
CL S
X
M
C D
5 4 3 2 1 0
n o i t a r g i M d ce ndu I 12 L C X C
88
Figure 12. ARQ 531 improves survival in the Eμ-TCL1 engraftment model compared to ibrutinib. A) C57BL/6 mice engrafted with Eμ-TCL1 leukocytes via tail vein injection were treated with ARQ 531 for 60 days via daily oral gavage and monitored for survival and drug induced toxicity. Survival curves were estimated using the Kaplan-Meier method and curve differences among groups assessed using the log-rank test. B) White blood cell counts were measured weekly by microscopic examination. C) A cohort of mice were sacrificed after two weeks of treatment and spleens were weighed to measure disease progression (n=3 in each group). Analysis of variance (ANOVA) was used to compare spleen weight among groups. (*, 0.05≥p>0.01; **, 0.01≥p≥0.001; ***, p<0.001)
(Continued on next page)
89
80
(Day 74) (Day
Treatment Cessation Treatment
Continued on next page)Continued next on (
60
40
Days Post Engraftment Post Days
rank rank -
value
-
<.001
<.001
0.671
0.003
0.009
0.044
p Log
531 20
75mg/kg ARQ ARQ 75mg/kg
vs. Vehicle vs.
531 vs. Vehicle vs. 531
531 vs. Vehicle vs. 531
531 vs. 531 (Day 14) (Day
Survival B6 Engraftment to TCL1
Treatment Initiation Treatment
75mg/kg 531 ARQ (n=13)
50mg/kg 531 ARQ (n=14)
25mg/kg (n=6) Ibrutinib
Vehicle (n=14)
75mg/kg ARQ ARQ 75mg/kg
50mg/kg ARQ ARQ 50mg/kg
50mg/kg ARQ ARQ 50mg/kg
25mg/kg Ibrutinib Ibrutinib 25mg/kg
25mg/kg Ibrutinib vs. 75mg/kg ARQ 531 ARQ 75mg/kg vs. Ibrutinib 25mg/kg
25mg/kg Ibrutinib vs. 50mg/kg ARQ 531 ARQ 50mg/kg vs. Ibrutinib 25mg/kg
Comparison
0
0
20
40
60
80
100
Percent Survival Percent
A
90
B
C
91
Figure 13. ARQ 531 improves survival in the Eμ-MYC/TCL1 model compared to ibrutinib. A) C57BL/6 mice engrafted via tail vein injection with Eμ-MYC/TCL1 leukocytes were treated with ARQ 531 via daily oral gavage starting 10 days after engraftment and monitored for survival. B) Percent CD19+ leukocytes collected from weekly bleeding were measured via flow cytometry. Survival curves were estimated using the Kaplan-Meier method and curve differences among groups assessed using the log-rank test.
A
(Continued on next page)
92
93
Figure 14. C481S BTK is inhibited by ARQ 531. A) A HEK293T cell line stably transfected with WT or C481S BTK was treated with ARQ 531 or ibrutinib for one hour followed by SDS-PAGE to determine efficacy against C481S BTK. B) CLL cells isolated at baseline and time of progression from an ibrutinib resistant patient who acquired a C481S BTK mutation were treated with ARQ 531 for one hour followed by SDS-PAGE. C) Primary CLL cells isolated from ibrutinib resistant patients who all possessed BTK C481S mutations were treated with ARQ 531 for
72 hours to determine cytotoxicity (n=9). Differences were assessed using linear mixed-effects models. (**, 0.01≥p≥0.001)
A
DMSO µM1 ARQ531 µM1 Ibrutinib DMSO µM1 ARQ531 µM1 Ibrutinib BJAB 1 µM1 Ibrutinib DMSO µM1 ARQ531 Parental WT BTK C481S BTK
pBTK (Y223)
BTK
GAPDH
(Continued on next page)
94
(Continued on next page)
95
100 C
80
e
v
i
t
a
g
e
N I
60
P
/
V
n
i ex
nn 40
A
e
l
oub D
% 20 ** **
0 1 b 3 i 5 in MSO t D Q ru AR Ib M M 1 1
96
Figure 15. ARQ 531 inhibits BCR signaling in cells with ibrutinib-resistant
PLCγ2 mutations. A) A DT40 cell line stably transfected with WT, R665W, or
L845F PLCγ2 and treated with ARQ 531 was assessed via SDS-PAGE to determine the effect on activation of the downstream kinases ERK and AKT. B)
CLL cells isolated from an ibrutinib resistant patient harboring an R665W PLCγ2 mutation were treated with ARQ 531 followed by SDS-PAGE and assessed for distal BCR signaling.
PLCγ2 WT PLCγ2 R665W PLCγ2 L845F A
DT40 PLCg2-/- w/ LV125 Vector
-
/
-
2 2
γ
IgM + 1 µM Ibrutinib 1 µM + IgM
IgM
IgM + 1 µM Ibrutinib 1 µM + IgM
IgM + 1 µM ARQ 531 ARQ 1 µM + IgM
IgM IgM + 1 µM ARQ 531 ARQ 1 µM + IgM
IgM + 1 µM Ibrutinib 1 µM + IgM IgM + 1 µM ARQ 531 ARQ 1 µM + IgM
IgM
DC9
α α
α α
α α
α α
α α DMSO
α α
α α
α α
PLC
DMSO
α DMSO pPLCγ2 (Y1217)
PLCγ2
pERK (T202/Y204) ERK pAKT (S473) AKT GAPDH
(Continued on next page)
97
B Pre Relapse Post Relapse +αIgM +αIgM R665W PLCg2 Primary Patient
DC9
DMSO
DMSO DMSO
1 µM Ibrutinib 1 1 µM 1 Ibrutinib
DMSO
1 µM ARQ 531 µM 1 ARQ 1 µM ARQ 531 µM ARQ1 pBTK (Y223) BTK pSyk (Y525/526) light pSyk (Y525/526) dark Syk pERK (T202/Y204) ERK
GAPDH
98
CHAPTER 4
Concluding Remarks
It is hard to overstate the impact of BTK inhibitor therapy on the treatment of CLL and other hematologic malignancies. Patients treated with ibrutinib enjoy durable responses which often last for years with manageable drug related toxicities. Importantly, ibrutinib is effective in patients with high risk features including early age of disease onset, del(17p13.1), and unmutated IgVH status, although these risk factors predispose patients to early relapse. Resistance to ibrutinib is an emerging clinical concern with a currently unmet need. There is strong rationale to develop novel therapeutics capable of reestablishing BCR pathway blockade, especially for patients who develop resistance to ibrutinib via
C481S BTK mutations. Maintaining BTK inhibition in these patients would be expected to restore the therapeutic responses observed prior to relapse.
Here I have demonstrated that reversible BTK inhibitors may be a viable treatment option to address acquired resistance to ibrutinib. Both GDC-0853 and
ARQ 531, due to their reversible mechanism of action, inhibit C481S BTK in biochemical and cellular models. The unique inhibitory profiles of these two molecules provide an interesting comparison of selective and nonselective BTK 99 inhibition. Selective inhibition of BTK by GDC-0853 spares NK cell mediated ADCC in combination with immunotherapy, unlike combinations of immunotherapy with ibrutinib which inhibits ITK and abrogates ADCC. Conversely, promiscuous kinase inhibition by ARQ 531 likely contributes to its observed efficacy in two additional mechanisms of ibrutinib resistance, autoactivating PLCγ2 mutations and Richter’s transformation.
In an ATP competitive biochemical assay including 286 kinases, GDC-0853 was found to inhibit only three kinases in addition to BTK170. A recent study investigating the safety of GDC-0853 in 111 healthy volunteers found no dose limiting adverse events and no statistical differences in the frequency of adverse events compared to placebo171. Its exquisite specificity allows GDC-0853 to circumvent the off-target effects of ibrutinib associated with treatment related adverse effects in patients. Although the adverse effects of ibrutinib are generally mild and low grade in severity, the daily dosing schedule of ibrutinib means that patients must tolerate these toxicities indefinitely. A BTK inhibitor with similar efficacy to ibrutinib which lacked deleterious side effects such as rash and diarrhea would be ideal.
The results shown in Figure 6 of Chapter 2 demonstrate that GDC-0853 preserves the efficacy of NK cell mediated ADCC against CLL cells targeted with immunotherapy while ibrutinib renders these therapies ineffective. Clinically, utilizing a selective BTK inhibitor which spares ITK may facilitate synergistic combinations with immunotherapy. By inhibiting migration and promoting CLL 100 egress from protective microenvironments, BTK inhibition may potentiate NK cell mediated ADCC. To test this hypothesis, I would propose an engraftment study using Eµ-TCL1 donor B cells and C57/BL6 recipient with the following cohorts: no treatment, GDC-0853 alone, ibrutinib alone, rituximab alone, GDC-0853 + rituximab, and ibrutinib + rituximab. I would expect the GDC-0853 + rituximab cohort to produce the greatest survival benefit. Comparing the survival of the GDC-
0853 + rituximab cohort to the GDC-0853 alone and the rituximab alone cohorts would inform whether the observed effect is additive or synergistic. Additionally, I would expect the ibrutinib and the ibrutinib + rituximab cohort to overlap in terms of survival. A phase III trial evaluating the efficacy of acalabrutinib in combination with bendamustine and rituximab is currently ongoing (NCT02972840), though the results of this study have yet to be reported.
Although the effect of ITK inhibition in the context of immunotherapy is relatively straightforward, the immunomodulatory effects of ITK inhibition on T cell function are complex. Preclinical work has shown that ibrutinib can polarize helper
T cell populations to an antitumor Th1 phenotype134. The results of this work indicate that ibrutinib inhibits Th2 cell subsets, however evidence of Th1 potentiation is lacking and polarization towards Th1 subtypes have not been observed clinically172. However, CD4+ and CD8+ T cell populations do increase in patients receiving ibrutinib. Interestingly, increases in T cell number are not observed in patients receiving acalabrutinib, a BTK inhibitor which lacks ITK activity172. The proposed mechanism for increased T cell numbers in patients
101 receiving ibrutinib is inhibition of ITK mediated activation induced cell death172.
However, myeloid derived suppressor cell (MDSC) populations, which directly antagonize the development and function of immune effector cells, are depleted by BTK inhibition, further obfuscating the relative roles of BTK and ITK in T cell development173. Clinically, ibrutinib decreases the expression of the immune checkpoint molecules PD-1 and CTLA-4, although acalabrutinib also decreases their expression indicating that this is not an ITK mediated response172.
Additionally, it has been shown that ibrutinib enhances the response of chimeric antigen receptor (CAR) T cell therapies although the mechanism for this has yet to be described174. It is reasonable that ibrutinib mediated decreases in immune checkpoint molecules may be responsible for the increase in CAR T cell efficacy.
To characterize the relative effects of BTK and ITK inhibition on T cell function, I would propose a drug study in the Eµ-TCL1 mouse model with ibrutinib and GDC-0853 treatment cohorts measuring absolute and relative T cell populations, expression of checkpoint molecules, cytokine expression, and MDSC function. If, as expected based on previous results with acalabrutinib, both the
GDC-0853 and ibrutinib cohorts demonstrate comparable decreases in checkpoint molecule expression, I would then propose an OSU-CLL donor NSG recipient xenograft study combining CAR T cell therapy with either GDC-0853 or ibrutinib to elucidate the mechanism of ibrutinib’s synergy with CAR T cells. If checkpoint molecule inhibition is responsible for CAR T cell expansion then GDC-0853 and ibrutinib will demonstrate comparable efficacy. However, if checkpoint molecule
102 expression insufficiently explains ibrutinib’s improvement of CAR T cell expansion differences will be observed between the two groups.
In contrast to GDC-0853, ARQ 531 inhibits multiple kinases at physiologic concentrations with surprising consequences. The multi-kinase inhibition of ARQ
531 is likely responsible for its ability to improve survival over ibrutinib in both the
Eµ-TCL1 engraftment model of CLL and the Eµ-MYC/TCL1 engraftment model of
Richter’s transformation. ARQ 531 inhibits both proximal components of the BCR pathway as well as distal B cell kinases upon which multiple survival pathways converge, essentially imposing two separate blockades to survival signaling.
Inhibition of SRC and TEC family kinases prevents the generation of BCR signaling in CLL cells while inhibition of RAF1 and MEK prevents the distal activation of ERK in response to non-BCR signaling. Considering that ibrutinib does not directly inhibit members of the RAS/RAF pathway it is possible that the superior efficacy of ARQ 531 in the setting of Richter’s transformation may be due to its inhibition of distal targets including MEK, which has been shown to be an effective target in diffuse large B-cell lymphomas162. I propose that this could be tested by comparing the efficacy ARQ 531 against a MEK inhibitor like trametinib in a Eµ-MYC/TCL1 engraftment model. Comparable survival of mice receiving ARQ 531 and trametinib would suggest that ARQ 531 prolongs survival in the Eµ-MYC/TCL1 engraftment model via inhibition of MEK.
103
Although inhibiting a broader spectrum of targets may lead to more durable responses, elucidating the exact mechanisms by which ARQ 531 is superior to ibrutinib in murine models of CLL and Richter’s transformation is difficult precisely due to the many kinases it inhibits. However, I would propose that the most efficient means of determining which targets of ARQ 531 are most critical for its activity would be genomic and transcriptome comparisons of mice prior to and subsequent to the development of resistance. Correlating resistance to ARQ 531 with the acquisition of mutations or changes in gene expression may elucidate which targets of ARQ 531 are most important for preserving drug action.
The efficacy of ARQ 531 in models of PLCγ2 mediated ibrutinib resistance is also likely mediated by its potent inhibition of proximal BCR signaling and concurrent inhibition of downstream ERK pathway signaling. LYN and SYK have been shown to bypass BTK in R665W PLCγ2 mutations leading to hyperactive signaling and ibrutinib resistance119. Figures 10C and 15B in Chapter 3 demonstrate that ARQ 531 more effectively inhibits SYK activation than does ibrutinib, suggesting that ARQ 531 is able to disrupt the BTK bypass pathway in these patients. Although this signaling data is informative, further experiments to characterize the functional effects of ARQ 531 in PLCγ2 mutations are warranted.
I propose that ARQ 531 should be tested for efficacy in primary patient CLL cells possessing PLCγ2 mutations as a result of acquired resistance to ibrutinib via a variety of functional assays measuring cytotoxicity, migration, activation, CCL3 production, and calcium release. 104
Reversible inhibition of C481S BTK is effectively achieved by GDC-0853 and ARQ 531, but like with any cancer therapy, resistance to these molecules is expected to emerge. Due to their inhibitory profiles, resistance to GDC-0853 and
ARQ 531 may develop in different ways. GDC-0853 specifically targets BTK so it is probable that resistance to this molecule would be facilitated by similar mechanisms as that of ibrutinib, i.e. mutations in BTK which decrease the potency of GDC-0853 or autoactivating mutations in PLCγ2. Conversely, ARQ 531 is less likely to develop mutations in PLCγ2 due to its efficacy in these mutants. Both ARQ
531 and GDC-0853 could induce resistance via mutations in the gatekeeper amino acid of BTK Thr474, which would prevent these inhibitors from entering the ATP binding pocket of BTK113. In order to determine likely mechanisms of resistance to
GDC-0853 and ARQ 531, I propose to treat the OSUCLL cell line with gradually escalating doses of these BTK inhibitors over several months to induce resistance.
Cells which have survived the selective pressure imposed by either GDC-0853 or
ARQ 531 will then be compared founders from the start of the experiment by RNA- seq and exome sequencing to determine changes in RNA expression and potential resistance mutations, respectively.
Acquired resistance to ibrutinib is an emerging complication to prolonged
BTK inhibitor therapy in CLL which manifests as mutations in BTK, PLCγ2, or
Richter’s transformation. Patients whose disease progresses during ibrutinib therapy have limited therapeutic options and poor survival following relapse. The observation that acquired resistance to ibrutinib is most frequently facilitated by
105 mutation of BTK or its immediate downstream partner, PLCγ2, strongly implies that the progression of CLL is contingent upon BCR signaling. Therefore, reestablishing inhibitory pressure on BTK in the context of ibrutinib resistance is an attractive therapeutic strategy. C481S BTK mutations disrupt the irreversible action of ibrutinib and decrease its potency leading to disease progression. GDC-
0853 and ARQ 531 are reversible inhibitors which can recognize and bind the ATP binding domain of BTK at low nanomolar concentrations in both WT and C481S
BTK thereby blocking BCR induced survival signaling in CLL. In addition to its ability C481S BTK, ARQ 531 also prevents downstream signaling in models of ibrutinib resistance harboring PLCγ2 mutations and prolongs survival in a murine model of Richter’s transformation. Reversible inhibitors such as GDC-0853 and
ARQ 531 represent an exciting advancement in the realm of targeted cancer therapy and validate reversible BTK inhibition as a strategy to circumvent acquired resistance to ibrutinib.
106
Works Cited
1. Murphy K, Weaver C. Janeway’s Immunobiology (ed. 9th). New York: Garland Science; 2017.
2. Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007;449(7164):819-26.
3. Kondo M. Lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors. Immunol Rev. 2010;238(1):37-46.
4. Loder F, Mutschler B, Ray RJ, et al. B Cell Development in the Spleen Takes Place in Discrete Steps and Is Determined by the Quality of B Cell Receptor-Derived Signals. JEM. 1999;190(1):75
5. LeBien TW, Tedder TF. B lymphocytes: how they develop and function. Blood. 2008;112(5):1570-80.
6. Martensson IL, Almqvist N, Grimsholm O, et al. The pre-B cell receptor checkpoint. FEBS Letters. 2010;584(12):2572-9.
7. Pelanda R, Torres RM. Central B-Cell Tolerance: Where Selection Begins. Cold Spring Harbor Perspectives in Biology. 2012;4(4):a007146.
8. Allman DM, Ferguson SE, Lentz VM, et al. Peripheral B cell maturation. II. Heat-stable antigen(hi) splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells. J Immunol. 1993;151:4431-44.
9. Melchers, Rolink A, Grawunder TH, et al. Positive and negative selection events during B lymphopoiesis. Curr Opin Immunol. 1995;7:214-27.
10. Methot SP, Di Noia JM. Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination. Adv Immunol. 2017;133:37-87.
11. Larson ED, Maizels N. Transcription-coupled mutagenesis by the DNA deaminase AID. Genome Biol.2004;5(3):211.
107
12. Teng G, Papavasiliou FN. Immunoglobulin somatic hypermutation. Annu Rev Genet.2007;41:107-20. 13. Klasse PJ,Sattentau QJ. Occupancy and mechanism in antibody- mediated neutralization of animal viruses. J Gen Virol. 2002;83:2091- 108.
14. Rus H, Cudrici C, Niculescu F. The role of the complement system in innate immunity. Immunol Res. 2005;33(2):103-12.
15. Ravetch J, Bolland S. IgG Fc receptors. Annu Rev Immunol. 2001;19(1):275-90.
16. Wang W, Erbe AK, Hank JA. NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy. Front Immunol. 2015;6:368.
17. American Cancer Society. Cancer Facts & Figures 2016. Atlanta: American Cancer Society; 2016.
18. Parikh SA, Rabe KG, Kay NE, et al. Chronic lymphocytic leukemia in young (≤ 55 years) patients: a comprehensive analysis of prognostic factors and outcomes. Haematologica. 2014;99(1):140-7.
19. Kikushige Y, Ishikawa F, Miyamoto T, et al. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell. 2011;20(2):246-59.
20. Chiorazzi N, Ferrarini M. Cellular origin(s) of chronic lymphocytic leukemia: cautionary notes and additional considerations and possibilities. Blood. 2011;117(6):1781-91.
21. Klein U, Tu Y, Stolovitzky GA, et al. Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med. 2001;194:1625-38
22. Speedy HE, Sava G, Houlston RS. Inherited susceptibility to CLL. Adv Exp Med Biol. 2013;792:293-308.
23. Hoffman R, Benz EJ, Shattil SJ, et al. Hematology Basic Principles and Practice. (ed. 5th). Philadelphia;Elsevier:2009.
108
24. Matutes E, Owusu-Ankomah K, Morilla R, et al. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia. 1944;8:1640-5.
25. Hallek M. Chronic lymphocytic leukemia:2017 update on diagnosis, risk stratification, and treatment. Ammerican journal of hematology. 2017;92(9):946-65.
26. Bain BJ. Blood Cells: A Practical Guide. Blackwell Publishing Limited. 2006;p439.
27. Kaushansky K, Lichtman M, Beutler E, et al. Williams Hematology (ed. 8th). McGraw-Hill:2010.
28. Tsimberidou AM, Keating MJ. Richter syndrome biology, incidence, and therapeutic strategies. Cancer. 2005;103(2):216-28.
29. Rossi D, Gaidano G. Richter syndrome: molecular insights and clinical perspectives. Hematol Oncol. 2009;27(1):1-10.
30. Dohner H, Stilgenbauer S,Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343(26):1910-6.
31. Haferlach C, Dicker F, Schnittger S, et al. Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping. Leukemia. 2007;21(12):2442-51.
32. Liu Y, Hermanson M, Grander D, et al. 13q deletions in lymphoid malignancies. Blood. 1995;86(5):1911-5.
33. Yi S, Li H, Li Z, et al. The prognostic significance of 13q deletions of different sizes in patients with B-cell chronic lymphoproliferative disorders: a retrospective study. Int J Hematol. 2017;106(3):418-25.
34. Ouillete P, Collins R, Shakhan S, et al. Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes f deletion 13q14. Cancer Res. 2008;68(4):1012-21.
35. Bonci D, Coppola V, Musumeci M, et al. The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat Med. 2008;14(11):1271-7.
109
36. Fegan C, Robinson H, Thompson P, et al. Karyotypic evolution in CLL: identification of a new sub-group of patients with deletions of 11q and advanced or progressive disease. Leukemia. 1995;9(12):2003-8.
37. Lee JH, Paull TT. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene. 2007; 26(56):7741-8.
38. Neilson JR, Auer R, White D, et al. Deletions at 11q identify a subset of patients with typical CLL who show consistent disease progression and reduced survival. Leukemia. 1997;11(11):1929-32.
39. Wattel E, Preduhomme C, Hecquet B, et al. p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies. Blood. 1994;84(9):3148-57.
40. Rosenwald A, Chuang EY, Davis RE, et al. Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53-dependent gene expression response. Blood. 2004;104(5):1428-34.
41. Dicker F, Herholz H, Schnittger S, et al. The detection of TP53 mutations in chronic lymphocytic leukemia independently predicts rapid disease progression and is highly correlated with a complex aberrant karyotype. Leukemia. 2009;23(1):117-24.
42. Danilov AV, Danilova OV, Klein AK, et al. Molecular pathogenesis of chronic lymphocytic leukemia. Curr Mol Med. 2006;6:665-75.
43. Vroblova V, Smolej L, Vrbacky F, et al. Biological prognostic markers in chronic lymphocytic leukemia. Acta Medica. 2009;52:3-8.
44. Hamblin TJ, Davis Z,Gardiner A, et al. Unmutated IgVH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood. 1999;94:1848-54.
45. Damle JN, Wasil T, Fais F, et al. IgV gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 1999;94:1840-47.
46. Laurenti L, Tarnani M, De Pude L, et al. Oral fludarabine and cyclophosphamide as a front-line chemotherapy in patients with chronic lymphocytic leukemia. The impact of biological parameters in the response duration. Ann Hematol. 2008;87:-891-8.
110
47. Dreger P, Montserrat e. Autologous and allogeneic stem cell transplantation for chronic lymphocytic leukemia. Leukemia. 2002;16:985-92.
48. Lin TS. What is the optimal initial treatment for chronic lymphocytic leukemia? Oncology. 2007;21:1641-49.
49. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111(12):5446.
50. Keating MJ, O’Brien S, Albitar M, et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. J Clin Oncol. 2005;23(18):4079-88.
51. Tam CS, O’Brien S, Wierda W, et al. Long-term results of the fludarabine, cyclophosphamide, and rituximab regimen as initial therapy of chronic lymphocytic leukemia. Blood. 2008;112(4):975.
52. Wu M, Akinleye A, Zhu X. Novel agents for chronic lymphocytic leukemia. J Hematol Oncol. 2013;6:36
53. Hutchinson CV, Dyer MJS. Breaking good: the inexorable rise of BTK inhibitors in the treatment of chronic lymphocytic leukemia. Br J Haematol. 2014;166(1):12-22.
54. Fruman DA, Chiu H, Hopkins BD, et al. The PI3K Pathway in Human Disease. Cell. 2017;170(4):605-35.
55. Mihalyova J, Jelinek T, Growkova K, et al. Venetoclax: a new wave in haemato-oncology. Exp Hematol. 2018;[Epub ahead of print].
56. Smith CIE, Islam TC, Mattsson PT, et al. The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species. Bioessays. 2001;23:436-46.
57. Ekman N, Arighi E, Rajantie I, et al. The Bmx tyrosine kiniase is activated by IL-3 and G-CSF in a PI-3K dependent manner. Oncogene. 2000;19:4151-58.
111
58. Berg LJ, Finkelstein LD, Lucas JA, et al. Tec family kinase in T lymphocyte development and function. Annu Rev Immunol. 2005;23:549- 600.
59. Hu Q, Davidson D, Schwartzberg PL, et al. Identification of Rlk, a novel protein tyrosine kinase with predominant expression in the T cell lineage. J Biol Chem. 1995;270(4):1928-34.
60. Lewis CM, Broussard C, Czar MJ, et al. Tec kinases: modulators of lymphocyte signaling and development. Curr Opin Immunol. 2001;13:317-25.
61. Woyach JA, Johnson AJ, Byrd JC. The B-cell receptor signaling pathway as a therapeutic target in CLL. Blood. 2012;120(6):1175-84.
62. Russel RB, Breed J, Barton GJ. Conservation analysis and structure prediction of the SH2 family of phosphotyrosine binding domains. FEBS Lett. 1992;304(1):15-20.
63. Park H, Wahl MI, Afar DE, et al. Regulation of Btk function by a major autophosphorylation site within the SH3 domain. Immunity. 1996;4(5):515-25.
64. Bruton OC. Agammaglobulinemia. Pediatics. 1952;9(6):722-8.
65. Vetrie D, Vorechovsky I, Sideras P, et al. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature. 1993;361(6409):226-33.
66. Thomas JD, Sideras P, Smith CIE, et al. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science. 1993;261:355-8.
67. Ellmeier W,Jung S, Sunshine MJ, et al. Severe B cell deficiency in mice lacking the tec kinase family members Tec and Btk. J Exp Med. 2000;192(11):1611-24.
68. Yamamoto T, Yamanashi Y, Toyoshima K. Association of Src-family kinase LYN with B-cell antigen receptor. Immunol Rev. 1993;132:187- 206.
69. Rolli V, Gallwitz M, Wossning T, et al. Amplification of B cell antigen receptor signaling by a SYK/ITAM positive feedback loop. Mol Cell.2002;10(5):1057-69. 112
70. Johnson SA, Pleiman CM, Pao L, et al. Phosphorylated immunoreceptor signaling motifs (ITAMs) exhibit unique abilities to bind and activate LYN and SYK tyrosine kinases. J Immunol. 1995;155:4596-603. 71. Kurosaki T, Johnson SA, Pao L, et al. Role of the SYK autophosphorylation site and SH2 domains in B cell antigen receptor signaling. J Exp Med. 1995;182:1815-23.
72. Rowley RB, Burkhardt AL, Chao HG, et al. SYK protein-tyrosine kinase is regulated by tyrosine-phosphorylated Ig alpha/Ig beta immunoreceptor tyrosine activation motif binding and autophosphorylation. J Biol Chem. 1995;270:11590-4.
73. Shiue L, Zoller MJ, Brugge JS. SYK is activated by phosphotyrosine- containing peptides representing the tyrosine-based activation motifs of the high affinity receptor for IgE. J Biol Chem. 1995;270:10498-502.
74. O’Rourke LM, Tooze R, Turner M, et al. CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav. Immunity. 1995;8(5):635-45.
75. Fuc, Turck CW, Kurosaki T, et al. BLNK: A central linker protein in B cell activation. Immunity. 1998;9(1):93-103.
76. Dal Porto JM, Gauld SB, Merrell KT, et al. B cell antigen receptor signaling 101. Mol Immunology. 2004;41(6):599-613.
77. Hashimoto A, Okada H, Jiang A, et al. Involvement of guanosine triphosphates and phospholipase C-γ2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor. J Exp Med. 1998;188(7):1287-95.
78. Shinohara H, Maeda S, Watari H, et al. IkappaB kinase-induced phosphorylation of CARMA1 contributes to CARMA1 Bcl10 MALT1 complex formation in B cells. 2007;204(13):1414-19.
79. Lazarus AE, Kawauchi K, Rapoport MJ, et al. Antigen-induced B lymphocyte activation involves the p21ras and ras.GAP signaling pathway. J Exp Med. 1993;178(5):1765-9.
80. Manning BD, Toker A. AKT/PKB Signaling: Navigating the Network. Cell. 2017;169(3):381-405.
113
81. Gold MR, Scheid MP, Santos L, et al. The B cell antigen receptor activates the Akt (protein kinase B/glycogen synthase kinase-3 signaling pathway via phosphatidylinositol 3-kinase. J Immunol. 1999;163:(4):1894-1905.
82. Nishizumi H, Horikawa K, Mlinaric-RascanI, et al. A double edged kinase LYN: a positive and negative regulator for antigen receptor-mediated signals. J Exp Med. 1998;187:1343-48.
83. Mohammad DK, et al. Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3zeta, regulates shuttling, and attenuates both tonic and induced signaling in B cells. Mol Cell Biol. 2013;33(16):3214-26.
84. Singh SP, Dammeijer F, Hendriks RW. Role of Bruton’s tyrosine kinase in B cells and malignancies. Mol Cancer. 2018;17(57)
85. de Gorter DJ, Beuline EA, Krsseboom R, et al. Bruton’s tyrosine kinase and phospholipase Cgamma2 mediate chemokine-controlled B cell migration and homing. Immunity. 2007;(26):93-104.
86. Okada T, Ngo VN, Ekland EH, et al. Chemokine requirements for B cell entry to lymph nodes and Peyer’s patches. J Exp Med. 2002;10:819-30.
87. Tsukada S, Simon MI, Witte ON, et al. Binding of beta gamma subunits of heterotrimeric G proteins the the PH domain of Bruont tyrosin kinase. Proc Natl Acad Sci USA. 1994;91:11256-60.
88. Lowry WE, Huang XY. G protein beta gamma subunits act on the catalytic domain to stimulate Bruton’s agammaglobulinemia tyrosine kinase. J Biol Chem. 2002;277:1488-92.
89. Koopman G, Keehnen RM, Lindhout E, et al. Adhesion through the LFA- 1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells.J Immunol. 1994;152(8):3760-7.
90. Weber ANR, Bittner Z, Liu X, et al. Bruton’s Tyrosine Kinase: An Emerging Key Player in Innate Immunity. Front Immunology. 2017;8:1454.
91. Rawlings DJ, Schwartz MA, Jackson SW, et al. Integration of B cell responses through toll-like receptors and antigen receptors. Nat Rev Immunol. 2012;12:282-94. 114
92. Gray P, Dunne A, Brikos C, et al. MyD88 adapter-like (mal) is phosphorylated by Bruton’s tyrosine kinase during TLR2 and TLR4 signal transduction. J Biol Chem. 2006;281:10489-95.
93. Herman SE, Gordon AL, Hertlein E, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. 2011;117(23):6287-96.
94. Woyach JA, Bojnik E, Ruppert AS, Stefanovski MR, Goettl VM, Smucker KA, et al. Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL). Blood 2014; 123(8):1207-13.
95. Kil LP, Marjolein J. W. de Bruijn, Menno van Nimwegen, et al. BTK levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice. Blood. 2012;119(16):3744-56.
96. Ponader S, Chen SS, Buggy JJ, Balakrishan K, Gandhi V, Wierda WG, et al. The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo. Blood 2012;119(5):1182-89.
97. de Rooij MF, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ, et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;119(11):2590-4.
98. Advani RH, Buggy JJ Sharman JP. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies. J Clin Oncol. 2013;31:88-94.
99. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med. 2013;369(1):32- 42.
100. Burger JA, Tedeschi A, Barr PM, et al. Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia. N Engl J Med. 2015;373(25):2425-37.
101. Byrd JC, Brown JR, O’Brien S, et al. Ibrutinib versus ofatumumab in previously treated chronic lymphocytic leukemia. N Engl J Med. 2014;371(3):213-23. 115
102. Chanan-Khan A, Cramer P, Demirkan F, et al. Ibrutinib combined with bendamustine and rituximab compared with placebo, bendamustine, and rituximab for previously treated chronic lymphocytic leukaemia or small lymphocytic lymphoma (HELIOS): a randomized, double-blind, phase 3 study. Lancet Oncol. 2016;17(2):200-11.
103. Burger JA, Sivina M,Ferrajoli A, et al. Randomized Trial of Ibrutinib Versus Ibrutinib Plus Rituximab (Ib+R) in Patients with Chronic Lymphocytic Leukemia (CLL). Oral Abstract #427: ASH 59th Annual Meeting and Exposition, December 2017, Atlanta, GA.
104. Roit FD, Engelberts PJ, Taylor RP, et al. Ibrutinib interferes with the cell- mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy. Haematologica. 2015;100(1):77-86.
105. Byrd JC, Murphy T, Howard RS, et al. Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity. J Clin Oncol. 2001;19(8):2153-64.
106. Byrd JC, Rai K, Peterson BL, et al. Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. Blood. 2005;105(1):49-53.
107. Woyach JA, Smucker K, Smith LL, et al. Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy. Blood 2014;123(12):1810-7.
108. Harandi A, Zaidi AS, Stocker AM, et al. Clinical Efficacy and Toxicity of Anti-EGFR Therapy in Common Cancers. J Oncol. 2009;2009:567486.
109. Wang Y, Zhang LL, Champlin RE, et al. Targeting Bruton’s tyrosine kinase with ibrutinib in B-cell malignancies. Clin Pharmacol Ther. 2015;97(5):455-68.
110. Wiczer TE, Levine LB, Brumbaugh J, et al. Cumulative incidence, risk factors, and management of atrial fibrillation in patients receiving ibrutinib. Blood Adv. 2017;1(20):1739-48.
116
111. O’Brien SM, Furman RR, Coutre SE, et al. Five-year experience with single-agent ibrutinib in patients with previously untreated and relapsed/refractory chronic lymphocytic leukemia/small lymphocytic leukemia. Blood 2016;128(22):233.
112. Woyach JA, Ruppert AS, Guinn D, et al. BTKC481S-mediated resistance to ibrutinib in chronic lymphocytic leukemia. J Clin Oncol 2017;35(13):1437- 43.
113. Maddocks KJ, Ruppert AS, Lozanski G, et al. Etiology of ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia. JAMA Oncol 2015;1(1):80-87.
114. Ahn IE, Underbayev C, Albitar A, et al. Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia. Blood 2017;129(11):1469-79.
115. Parikh SA, Chaffee KR, Call TG, et al. Ibrutinib therapy for chronic lymphocytic leukemia (CLL): an analysis of a large cohort of patients treated in routine clinical practice. Blood 2015;126(23):2935.
116. Woyach JA, Guinn D, Ruppert AS, et al. The development and expansion of resistant subclones precedes relapse during ibrutinib therapy in patients with CLL. Blood 2016;128(22):55.
117. Woyach JA, Furman RR, Liu TM, et al. Resistance mechanisms for the Bruton’s tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014 June 12;370(24): 2286-94.
118. Furman RR, Cheng S, Lu P, et al. Ibrutinib resistance in chronic lymphocytic leukemia. N Engl J Med 2014;370(24):2352-4.
119. Liu TM, Woyach JA, Zhong Y, et al. Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation. Blood 2015;126(1):61-8.
120. Zhou Q, Lee GS, Brady J, et al. A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am J Hum Genet. 2012;91(4):713-20.
121. Alizadeh AA, Eisen MB, Davis RE. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503- 11. 117
122. Staudt LM. Targeting pathological B-cell receptor signaling therapeutically. In: American Society of Hematology: Meeting on Lymphoma Biology. (ed. By ASH), pp. 14-15. ASH, Colorado Springs, CO.
123. Wilson W, Gerecitano J, Goy A, et al. The Bruton’s Tyrosine Kinase (BTK) inhibitor ibrutinib (PCI-32765), has preferential activity in the ABC subtype of relapsed/refractory de novo diffuse large B-cell lymphoma (DLBCL):interim results of a multicenter, open-label, phase 2 study. Blood. 2012;120:686.
124. Packham G, Stevenson F. The role of the B-cell receptor in the pathogenesis of chronic lymphocytic leukaemia. Semin Cancer Biol. 2010;20(6):391-9.
125. Jain N, O’Brien S. Targeted therapies for CLL: Practical issues with the changing treatment paradigm. Blood. 2016;30(3):233-44.
126. Buggy JJ, Elias L. Bruton tyrosine kinase (BTK) and its role in B-cell malignancy. Int Rev Immunol. 2012;31(2):119-32.
127. Lee KG, Xu S, Wong ET, et al. Bruton’s tyrosine kinase separately regulates NFkappaB p65RelA activation and cytokine interleukin (IL)- 10/IL-12 production in TLR9-stimulated B Cells. J Biol Chem. 2008;283(17):11189-98.
128. Lougaris V, Baronio M, Vitali M, et al. Bruton tyrosine kinase mediates TLR9-dependent human dendritic cell activation. J Allergy Clin Immunol. 2014;113(6):1644-50.
129. Chen SS, Chang BY, Chang S, et al. BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia. Leukemia. 2016;30(4):833-43.
130. Honigberg LA, Smith AM, Sirisawad M, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci U S A. 2010;107(29):13075-80.
131. Herman SE, Mustafa RZ, Gyamfi JA, et al. Ibrutinib inhibits BCR and NF- κB signaling and reduces tumor proliferation in tissue resident cells of patients with CLL. Blood. 2014;123(21):3286-95.
118
132. Byrd JC, Harrington B, O’Brien S, et al. Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia. N Engl J Med. 2016;374(4):323- 32.
133. Lynch TJ Jr, Kim ES, Eaby B, et al. Epidermal growth factor receptor inhibitor-associated cutaneous toxicities: an evolving paradigm in clinical management. Oncologist. 2007;12(5):610-21.
134. Dubovsky JA, Beckwith KA, Natarajan G, et al. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. 2013;122(15):2539-49.
135. Khurana D, Arneson LN, Schoon RA, et al. Differential regulation of human NK cell-mediated cytotoxicity by the tyrosine kinase Itk. J Immunol. 2007;178(6):3575-82.
136. Burger JA, Keating MJ, Wierda WG, et al. Safety and activity of ibrutinib plus rituximab for patients with high-risk chronic lymphocytic leukaemia: a single-arm, phase 2 study. Lancet Oncol. 2014;15(10):1090-9.
137. Jaglowski SM, Jones JA, Nagar V, et al. Safety and activity of BTK inhibitor ibrutinib combined with ofatumumab in chronic lymphocytic leukemia: a phase 1b/2 study. Blood. 2015;126(7):842-50.
138. Young W and Crawford J. Discovery of GDC-0853: A highly potent, selective and non-covalent BTK Inhibitor. American Chemistry Society. San Diego, CA.
139. Erickson RI, Schutt LK, Tarrant JM, et al. Bruton’s Tyrosine Kinase Small Molecule Inhibitors Induce a Distinct Pancreatic Toxicity in Rats. J Pharmacol Exp Ther. 2017;360(1):226-38.
140. Crawford JJ, et al. Discovery of GDC-0853, a Potent, Selective, and Non-Covalent Inhibitor of Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development J. Med. Chem., invited paper in prep.
141. Johnson AR, Kohli PB, Katewa A, et al. Battling BTK Mutants With Noncovalent Inhibitors That Overcome Cys481 and Thr474 Mutations. ACS Chem Biol. 2016;11(10):2897-907.
142. Byrd JC, Smith S, Wagner-Johnston N, et al. First-in-human phase 1 study of the BTK inhibitor GDC-0853 in relapsed or refractory B-cell NHL and CLL. In Review
119
143. Blomberg KEM, Boucheron N, Lindvall JM, et al. Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells. BMC Genomics. 2009;10:223
144. Fisher K, Bahlo J, Fink AM, et al. Long-term remissions after FCR chemoimmunotherapy in previously untreated patients with CLL: updated results of the CLL8 trial. Blood. 2016;127(2):208-15.
145. Goede V, Fisher K, Busch R, et al. Obinutuzumab plus Chlorambucil in Patients with CLL and Coexisting Conditions. N Engl J Med. 2014;370(12):1101-10.
146. Xu L, Tsakmaklis N, Yang G, et al. Acquired mutations associated with ibrutinib resistance in Waldenstrom Macroglobulinemia. Blood. 2017;129(18):2519-25.
147. Stephens DM, Spurgeon SE. Ibrutinib in mantle cell lymphoma patients: glass half full? Evidence and opinion. Ther Adv Hematol. 2015;6(5):242- 52.
148. Advani RH, Buggy JJ, Sharman JP, et al. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies. J Clin Oncol 2013;31(1):88-94.
149. Walter HS, Rule SA, Dyer MJ, et al. A phase 1 clinical trial of the selective BTK inhibitor ONO/GS-4059 in relapsed and refractory mature B-cell malignancies. Blood 2015;127(4):411-19.
150. Tam C, Grigg AP, Opat S, et al. The BTK inhibitor, Bgb-3111, is safe, tolerable, and highly active in patients with relapsed/ refractory B-cell malignancies: initial report of a phase 1 first-in-human trial. Blood 2015;126(23):832.
151. Herman SE, Sun X, McAuley EM, et al. Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy. Leukemia 2013;27(12):2311-21.
152. Rogers KA, El-Gamal D, Harrington BK, et al. The Eu-Myc/TCL1 transgenic mouse as a new aggressive B-cell malignancy model suitable for preclinical therapeutics testing. Blood 2015;126(23):2752.
153. Bichi R, Shinton SA, Martin ES, et al. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc Natl Acad Sci U S A 2002;99(10):6955-60. 120
154. Bender AT, Gardberg A, Pereira A, et al. Ability of Bruton’s tyrosine kinase inhibitors to sequester Y551 and prevent phosphorylation determines potency for inhibition of Fc receptor but not B-cell receptor signaling. Mol Pharmacol 2017;91(3):208-19.
155. Browning RL, Mo X, Muthusamy N, Byrd JC. CpG oligodeoxynucleotide CpG-685 upregulates functional interleukin-21 receptor on chronic lymphocytic leukemia B cells through an NF-κB mediated pathway. Oncotarget 2015;6(18):15931-9.
156. Hinz M, Loser P, Mathas S, et al. Constitutive NF-kappaB maintains high expression of a characteristic gene network, including CD40, CD86, and a set of antiapoptotic genes in Hodgkin/Reed-Sternberg cells. Blood 2011;97(9):2798-807.
157. Herman SE, Mustafa RZ, Gyamfi JA, et al. Ibrutinib inhibits BCR and NF- κB signaling and reduces tumor proliferation in tissue-resident cells of patients with CLL. Blood 2014;123(21):3286-95.
158. Herman SEM, Montraveta A, Niemann CU, Mora-Jensen H, Gulrajani M, Krantz F, et al. The Bruton tyrosine kinase (BTK) inhibitor acalabrutinib demonstrates potent on-targets effects and efficacy in two mouse models of chronic lymphocytic leukemia. Clin Cancer Res 2017;23(11):2831-41.
159. Hendriks RW, Yuvaraj S, Kil LP. Targeting Bruton's tyrosine kinase in B cell malignancies. Nat Rev Cancer 2014;14(4):219-32.
160. Jones J, Choi MY, Mato AR, et al. Venetoclax (VEN) monotherapy for patients with chronic lymphocytic leukemia (CLL) who relapsed after or were refractory to ibrutinib or idelalisib. Blood 2016;128(22):637.
161. Cheng S, Guo A, Lu P, et al. Functional characterization of BTKC481S mutation that confers ibrutinib resistance: exploration of alternative kinase inhibitors. Leukemia 2015;29(4):895-900.
162. Bhalla S, Evens AM, Dai B, et al. The novel anti-MEK small molecule AZD6244 induces BIM-dependent and AKT-independent apoptosis in diffuse large B-cell lymphoma. Blood 2011;118(4):1052-61.
163. Lamba S, Russo M, Sun C, et al. RAF suppression synergizes with MEK inhibition in KRAS mutant cancer cells. Cell Rep 2014;8(5):1475-83.
121
164. Bezieau S, Devilder MC, Avet-Loiseau H, et al. High incidence of N and K-Ras activating mutations in multiple myeloma and primary plasma cell leukemia at diagnosis. Hum Mutat 2001;18(3):212-24.
165. de la Puente P, Muz B, Jin A, et al. MEK inhibitor, TAK-733 reduces proliferation, affects cell cycle and apoptosis, and synergizes with other targeted therapies in multiple myeloma. Blood Cancer J 2016;6:e399.
166. Vu HL, Aplin AE. Targeting mutant NRAS signaling pathways in melanoma. Pharmacol Res 2016;107:111-6.
167. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res 2012;72(10):2457-67.
168. Carnevale J, Ross L, Puissant A, et al. SYK regulates mTOR signaling in AML. Leukemia 2013;27(11):2118-28.
169. Boros K, Puissant A, Back M, et al. Increased SYK activity is associated with unfavorable outcome among patients with acute myeloid leukemia. Oncotarget 2015;6(28):25575-87.
170. Crawford JJ, Johnson AR, Misner DL, et al. Discovery of GDC-0853: A Potent, Selective and Noncovalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development. J Med Chem. 2018 [Epub ahead of print].
171. Herman AE, Chinn LW, Kotwal SG, et al. Safety, Pharmacokinetics, and Pharmacodynamics in Healthy Volunteers Treated with GDC-0853, a Selective Reversible Bruton’s Tyrosine Kinase Inhibitor. Clin Pharmacol Ther. 2018 [E pub ahead of print].
172. Long M, Beckwith K, Do P, et al. Ibrutinib treatment improves T cell number and function in CLL patients. J Clin Invest. 2017;127(8):3052-64.
173. Stiff A, Trinkha P, Wesolowski R, et al. Myeloid-Derived Suppressor Cells Express Bruton’s Tyrosine Kinase and Can Be Depleted in Tumor- Bearing Hosts by Ibrutinib Treatment. Cancer Res. 2016;76(8):2125-36.
174. Fraietta JA, Beckwith KA, Patel PR, et al. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia. Blood. 2016;127(9):1117-27.
122