Preclinical Evaluation of Synergistic Drug Combinations in Acute Myeloid Leukemia
by
Lianne Emily Rotin
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Institute of Medical Science University of Toronto
© Copyright by Lianne E. Rotin (2016)
Preclinical Evaluation of Synergistic Drug Combinations in
Acute Myeloid Leukemia
Lianne E. Rotin
Doctor of Philosophy
Institute of Medical Science University of Toronto
2016
Abstract
The FDA-approved Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has significantly improved patient outcomes in B-cell malignancies, where BTK signaling is implicated in disease progression. Documented expression of constitutively active BTK in AML cells has generated interest in evaluating the potential therapeutic role of ibrutinib in the treatment of AML. We investigated the role of ibrutinib in AML by screening this drug against libraries of approved drugs in AML cell lines, identifying the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate as a profoundly synergistic hit. This drug combination was preferentially cytotoxic to patient-derived AML cells over normal controls, and synergistic cell death was preceded by reactive oxygen species (ROS) production. Ibrutinib similarly synergized with current first-line AML chemotherapy agent daunorubicin. Interestingly, neither ethacridine, nor daunorubicin’s synergy with ibrutinib appeared to be BTK-dependent. Further study with the epidermal
ii growth factor receptor (EGFR) inhibitor erlotinib—which also has preclinical anti-
AML activity—revealed equally profound EGFR-independent synergy with ethacridine, with an increase in ROS that paralleled that induced by the ibrutinib- ethacridine combination. We determined that erlotinib-mediated potentiation of ethacridine accumulation was responsible for this combination’s synergistic cytotoxicity, and hypothesize that ibrutinib and ethacridine likely synergize via the same mechanism. In summary, we have identified a novel BTK-independent role for ibrutinib in AML, and for the first time, report a potential role for PARG inhibition as a combination candidate for AML therapy.
iii Acknowledgements
I extend my deepest gratitude to my supervisor, Dr. Aaron Schimmer, for his guidance and encouragement throughout my PhD studies. Aaron is a wonderful mentor and a role model for the clinician-scientist I hope to one day become.
I would also like to thank my program advisory committee members, Drs. Mark Minden and Meredith Irwin, for their helpful advice and constructive feedback during and in between committee meetings. Furthermore, I would like to thank Dr. Minden for providing me with the opportunity to attend his weekly leukemia clinic for the better part of two years; observing these patient visits gave me a better understanding of this disease and its impact on patients and their families, as well as a sincere appreciation for the need for new ways to tackle it.
Working in the Schimmer Lab has been a truly incredible experience; it has been a privilege to train alongside a team of such bright, creative, and enthusiastic scientists. I would especially like to thank Marcela Gronda, Rose Hurren, and Neil MacLean: you have taught me countless lab techniques, assisted with many important experiments, and you have helped me become a better researcher.
Finally, this acknowledgements section would be incomplete without mention of my wonderful support team outside of the lab. I’m grateful to my parents, Daniela and Robbie, for instilling in me the importance of education, hard work, and putting your heart into everything you do. I am also thankful for their continued and unwavering support throughout this long educational journey. Lastly, I would like to thank my fiancé Zach, who has patiently sat through every single one of my practice talks, asked some impressively pertinent questions, and who has always had something positive and encouraging to say. Thank you!
iv Contributions
This thesis consists of 3 data chapters. Chapter 3 was published in the journal Oncotarget (Rotin et al., 2016b) and Chapter 4 was published in the journal Leukemia and Lymphoma (Rotin et al., 2016a). Chapter 5 has yet to be published.
The author performed all experiments and analyses outlined in the thesis, except as indicated below:
Mr. Neil MacLean – performed combination high-throughput drug screens against ibrutinib and ethacridine, provided assistance with combination drug screens against erlotinib, analyzed screen data, and prepared lentiviral stocks
Ms. Marcela Gronda – performed immunoblots (BTK, phospho-BTK, BMX, RLK, TEC, ITK, EGFR), olaparib assays, reactive oxygen species measurements depicted in figures 4-10 and 4-11, and provided assistance with the PARG inhibitor assay
Ms. Rose Hurren – conducted in vivo combination ibrutinib-ethacridine studies, radiolabeled daunorubicin uptake studies, and Z-VAD-FMK experiments
Ms. XiaoMing Wang – carried out in vivo combination ibrutinib-ethacridine studies
Dr. Ahmed Aman – mass spectrometry analysis of ethacridine accumulation in AML cell lines
v Dr. Feng-Hsu Lin – designed software program for excess-over-Bliss analysis of drug screen data
Dr. Alessandro Datti – guidance in planning high-throughput drug screening procedures
vi Table of Contents ACKNOWLEDGEMENTS……….………………………………..……………….IV
CONTRIBUTIONS………….………………………………………..……...... V
TABLE OF CONTENTS……………………………………………..……………VII
LIST OF TABLES………………………………………………………………….XI
LIST OF FIGURES………………………………………………….…….……….XII
LIST OF ABBREVIATIONS………………………………………………………XIV
Table of Contents Preface ...... 1 Chapter 1: Literature Review ...... 2 1.1 Acute Myeloid Leukemia ...... 3 1.1.1 Normal Hematopoiesis ...... 3 1.1.2 Acute Myeloid Leukemia ...... 5 1.1.2.1 AML Pathogenesis ...... 5 1.1.2.2 Epidemiology of AML ...... 6 1.1.2.3 AML Classification and Prognostication ...... 6 1.1.2.4 AML Management ...... 7 1.2 Tyrosine Kinase Inhibitor Therapy in AML ...... 10 1.2.1 Targeted Cancer Therapies ...... 10 1.2.1.1 Tyrosine Kinase Inhibitors ...... 10 1.2.2 Oncogenic Tyrosine Kinases in AML ...... 11 1.2.2.1 FMS-Related Tyrosine Kinase 3 ...... 11 1.3 Ibrutinib ...... 14 1.3.1 Bruton’s tyrosine kinase: background & role in signal transduction from the B-cell receptor ...... 14 1.3.1.1 BTK domains ...... 14 1.3.1.2 BTK expression ...... 15 1.3.1.3 BTK: Role in B-cell Maturation ...... 15 1.3.1.4 BTK Signaling in B-Cells ...... 15 1.3.2 A Rationale for Targeting BTK in B-cell Malignancies ...... 16 1.3.2.1 Chronic Lymphocytic Leukemia ...... 17 1.3.2.2 Mantle-Cell Lymphoma ...... 17 1.3.2.3 Waldenström Macroglobulinemia ...... 17 1.3.3 Development of Ibrutinib as a Selective and Irreversible BTK Inhibitor with In Vivo Activity ...... 18 1.3.4 Preclinical and clinical activities of ibrutinib in B-cell cancers ...... 19 1.3.4.1 Chronic Lymphocytic Leukemia ...... 19
vii 1.3.4.2 Mantle Cell Lymphoma ...... 20 1.3.4.3 Waldenström Macroglobulinemia ...... 21 1.3.5 B-cell independent BTK signaling: myeloid-lineage cells ...... 22 1.3.5.1 Mast Cells ...... 23 1.3.5.2 Macrophages ...... 24 1.3.5.3 Erythroid Cells ...... 25 1.3.5.4 Platelets ...... 26 1.3.5.5 Neutrophils ...... 27 1.3.6 A Role for Targeting BTK Beyond B-Cell Cancers ...... 29 1.3.6.1 Rheumatoid Arthritis ...... 29 1.3.6.2 Multiple Myeloma ...... 30 1.3.6.3 Acute Myeloid Leukemia ...... 31 1.3.6.4 Prostate Cancer ...... 32 1.4 EGFR inhibitors in AML: Anti-Leukemic Mechanisms of Action and Preclinical and Clinical Activity ...... 34 1.4.1 Development of Small Molecule EGFR Tyrosine Kinase Inhibitors ...... 34 1.4.2 Expression of EGFR in AML Cells ...... 35 1.4.3 Preclinical EGFR-TKI activity against AML ...... 36 1.4.3.1 Differentiation ...... 36 1.4.3.2 Cell Cycle Arrest and Cell Death ...... 37 1.4.4 Proposed Anti-Leukemic Targets of EGFR-TKIs ...... 37 1.4.4.1 JAK2 Inhibition ...... 37 1.4.4.2 SRC Family Kinase Inhibition ...... 38 1.4.4.3 SYK Inhibition ...... 39 1.4.4.4 Bruton’s Tyrosine Kinase Inhibition ...... 39 1.4.4.5 Inhibition of ATP-Binding Cassette Transporter Efflux Activity ...... 40 1.4.6 Clinical EGFR-TKI Activity Against AML ...... 41 1.4.7 Summary ...... 42 1.5 Ethacridine Lactate ...... 44 1.5.1 Ethacridine Lactate Indications ...... 44 1.5.2 Ethacridine Lactate Mechanisms of Action ...... 45 1.5.2.1 Poly(ADP-ribose) Glycohydrolase Inhibition ...... 45 1.5.2.2 Non-Genotoxic Activation of p53 ...... 46 Chapter 2: Project Rationale and Aims ...... 48 2.1 Thesis Aims ...... 49 2.1.1 Aim I: Identify compounds that synergize with ibrutinib in AML ...... 49 2.1.2 Aim II: Evaluate the mechanism of synergy between ibrutinib and daunorubicin in AML ...... 49 2.1.3 Aim III: Identify compounds that synergize with erlotinib in AML ...... 50 Chapter 3: Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism ...... 51 3.1 Abstract ...... 52 3.2 Introduction ...... 53 3.3 Methods ...... 54 3.3.1 Materials ...... 54
viii 3.3.2 Cell Culture ...... 54 3.3.3 Primary cells ...... 55 3.3.4 In vivo Combination Treatment ...... 55 3.3.5 Immunoblotting ...... 56 3.3.6 Cell Growth and Viability Assays ...... 56 3.3.7 Combination High-Throughput Screen ...... 57 3.3.8 Excess-over-Bliss Additivism for Calculating Synergy ...... 57 3.3.9 Intracellular and Mitochondrial Reactive Oxygen Species Measurement .. 58 3.3.10 shRNA Knockdown Experiments ...... 58 3.3.11 PARG Activity Assay ...... 59 3.3.12 Statistical Analysis ...... 59 3.4 Results ...... 60 3.4.1 BTK is overexpressed and constitutively active in AML cells ...... 60 3.4.2 AML cell lines are insensitive to chemical BTK inhibition with ibrutinib ... 62 3.4.3 A combination chemical screen with ibrutinib in AML cell lines identifies the PARG inhibitor, ethacridine lactate, as an ibrutinib sensitizer ...... 64 3.4.4 The ibrutinib-ethacridine combination is preferentially cytotoxic to a subset of primary AML cells compared to normal hematopoietic cells ...... 70 3.4.5 The combination of ibrutinib and ethacridine delays the growth of AML cells in vivo ...... 74 3.4.6 Ethacridine synergizes with other small molecule BTK inhibitors, but not inhibitors of unrelated kinases ...... 76 3.4.7 Ibrutinib and ethacridine synergize to induce cell death via a ROS- dependent mechanism ...... 80 3.4.8 The chemical PARG inhibitor gallotannin also synergizes with ibrutinib to induce cell death by excessive ROS production ...... 82 3.4.9 The synergy of ibrutinib with ethacridine is independent of the inhibitory effect on BTK ...... 85 3.5 Discussion ...... 88 Chapter 4: Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells...... 91 4.1 Abstract ...... 92 4.2 Introduction ...... 93 4.3 Methods ...... 94 4.3.1 Radiolabelled daunorubicin accumulation assay ...... 94 4.4 Results & Discussion ...... 94 Chapter 5: Erlotinib synergizes with the poly(ADP-ribose) glycohydrolase inhibitor ethacridine in acute myeloid leukemia cells ...... 106 5.1 Abstract ...... 107 5.2 Introduction ...... 108 5.3 Materials and Methods ...... 110 5.3.1 Reagents ...... 110 5.3.2 Cell culture ...... 110 5.3.3 Primary cells ...... 111
ix 5.3.4 Immunoblotting ...... 111 5.3.5 Cell viability assays ...... 111 5.3.6 High-throughput combination drug screening & excess-over-Bliss additivism synergy calculations ...... 112 5.3.7 Reactive oxygen species measurement ...... 112 5.3.8 Mass spectrometry ...... 113 5.4 Results ...... 114 5.4.1 TEX and OCI-AML2 cell line sensitivity to erlotinib ...... 114 5.4.2 A high-throughput combination chemical screen identifies erlotinib sensitizers in TEX and OCI-AML2 cells ...... 116 5.4.3 The erlotinib-ethacridine combination synergizes in primary AML cells and other AML cell lines ...... 119 5.4.4 Combining erlotinib and ethacridine generates lethal levels of reactive oxygen species ...... 122 5.4.5 Ethacridine synergizes with EGFR-targeting kinase inhibitors ...... 125 5.4.6 TEX and OCI-AML2 cell lines do not express EGFR ...... 125 5.4.7 Erlotinib potentiates ethacridine accumulation in TEX and OCI-AML2 cells...... 126 5.4.8 High-dose ethacridine treatment mimics ROS production observed from the erlotinib-ethacridine combination...... 126 5.5 Discussion ...... 131 Chapter 6: General Discussion & Conclusion ...... 134 6.1 Discussion ...... 135 6.1.1 BTK-independent anti-leukemic activity of ibrutinib ...... 135 6.1.1.1 Ibrutinib potentiates ethacridine accumulation ...... 135 6.1.1.2 Synergy between ibrutinib and daunorubicin is mediated by a mechanism unrelated to that of ibrutinib/erlotinib and ethacridine ...... 137 6.1.2 Anti-leukemic activity of ethacridine lactate ...... 138 6.1.3 Clinical relevance of BTK-independent effects of ibrutinib ...... 139 6.2 Conclusion ...... 142 Chapter 7: Future Directions ...... 143 7.1 Future Directions ...... 144 7.1.1 Determining the mechanism of ethacridine accumulation by erlotinib and ibrutinib ...... 144 7.1.1.1 ABC transporters ...... 144 7.1.2 Determining the relevant target of ethacridine ...... 145 7.1.2.1 PARG inhibition ...... 145 7.1.2.2 p53 induction and the ribosomal stress pathway ...... 146 References ...... 148 Appendix 1 ...... 167
x List of Tables
Table 3-1: Patient demographics………………………………….…………………71
Table 5-1: Patient demographics…………………………...………………………121
List of Figures
Figure 1-1: Hematopoiesis: original and revised models...... 4 Figure 1-2: Structure of Ethacridine lactate ...... 44 Figure 1-3: Mechanism of PARG activity ...... 47 Figure 3-1: BTK mRNA levels in AML cell lines are similar to those of B-cell malignancies...... 61 Figure 3-2: AML cell lines express constitutively active BTK, but are insensitive to ibrutinib...... 63 Figure 3-3: The PARG inhibitor ethacridine lactate sensitizes AML cell lines to ibrutinib...... 65 Figure 3-4: Combination chemical screen validation for pentamidine...... 67 Figure 3-5: Cell death caused by ibrutinib-ethacridine combination is caspase independent...... 68 Figure 3-6: The ibrutinib-ethacridine combination is strongly synergistic in HL60, U937, and K562, but not KG1a AML cell lines...... 69 Figure 3-7: The ibrutinib-ethacridine combination is preferentially cytotoxic to primary AML cells over normal hematopoietic cells...... 73 Figure 3-8: Ibrutinib-ethacridine combination displays anti-AML activity in mice. 75 Figure 3-9: Ethacridine synergizes with other small-molecule BTK inhibitors. ... 77 Figure 3-10: Ethacridine does not synergize with inhibitors of unrelated kinases...... 78 Figure 3-11: Dasatinib and imatinib do not synergize with ethacridine in OCI- AML2 cells...... 79
xi Figure 3-12: The ibrutinib-ethacridine combination induces cytotoxic levels of intracellular ROS...... 81 Figure 3-13: The PARG inhibitor gallotannin synergizes with ibrutinib ...... 83 Figure 3-14: Treatment of TEX and OCI-AML2 cells with olaparib in combination with ibrutinib and ethacridine...... 84 Figure 3-15: Ibrutinib’s synergy with ethacridine is independent of BTK...... 86 Figure 3-16: Expression of TEC family kinases in AML cell lines...... 87 Figure 4-1: Ibrutinib and daunorubicin synergize in TEX and OCI-AML2 cells. .. 95 Figure 4-2: Combination ibrutinib-cytarabine treatment of TEX and OCI-AML2 cells...... 96 Figure 4-3: Ibrutinib inhibits BTK phosphorylation...... 96 Figure 4-4: BTK knockdown confirmation...... 98 Figure 4-5: Daunorubicin treatment of BTK-knockdown cells...... 98 Figure 4-6: Ibrutinib treatment of BTK-knockdown TEX cells...... 100 Figure 4-7: Daunorubicin accumulation in the presence or absence of ibrutinib...... 100 Figure 4-8: α-tocopherol rescue of combination-treated TEX and OCI-AML2 cells...... 102 Figure 4-9: Combination ibrutinib-daunorubicin treatment increases intracellular ROS ...... 102 Figure 4-10: Intracellular ROS production following combination ibrutinib- daunorubicin treatment in the presence or absence of α-tocopherol...... 103 Figure 4-11: Mitochondrial ROS production following combination ibrutinib- daunorubicin treatment in TEX and OCI-AML2 cells...... 104 Figure 5-1: AML cell line sensitivity to erlotinib...... 115 Figure 5-2: Erlotinib sensitizers in TEX and OCI-AML2 cells...... 117 Figure 5-3: Validation of synergistic hits...... 118 Figure 5-4: Erlotinib and ethacridine synergize in additional AML cell lines and primary AML blasts...... 120 Figure 5-5: Combination erlotinib-ethacridine treatment induces lethal intracellular ROS production...... 123
xii Figure 5-6: Erlotinib enhances ethacridine accumulation in TEX and OCI-AML2 cells...... 128 Figure 5-7: Imatinib does not synergize with ethacridine in TEX and OCI-AML2 cells...... 130
List of Appendices
Appendix 1: Clinically achievable concentrations of kinase inhibitors………….167
xiii List of Abbreviations
ABC ATP-binding cassette ABC-DLBCL Activated B-cell-like subtype of diffuse large B-cell lymphoma Abl Abelson kinase ADP Adenosine diphosphate ADP-HPD Adenosine diphosphate (hydroxymethyl)pyrrolidinediol AIF Apoptosis inducing factor AML Acute myeloid leukemia APL Acute promyelocytic leukemia ATP Adenosine triphosphate ATRA All-trans retinoic acid Bcl-2 B-cell lymphoma 2 BCR B-cell receptor BCR Breakpoint cluster region BCRP Breast cancer resistance protein BMSC Bone marrow stromal cell BMX Bone marrow kinase in chromosome X BTK Bruton’s tyrosine kinase CAIA Anti-collagen antibody-induced arthritis CFU-L Colony forming unit-leukemia CIA Collagen induced arthritis CLL Chronic lymphocytic leukemia CLP Common lymphoid progenitor CML Chronic myeloid leukemia CMP Common myeloid progenitor CR Complete response/remission CRP C-reactive protein CXCR4 Chemokine (C-X-C Motif) Receptor 4 CXCR5 Chemokine (C-X-C Motif) Receptor 5 DAG Diacylglycerol DiOC2(3) 3,3’-diethyloxacarbocyanine DMSO dimethyl sulfoxide DNA Deoxyribonucleic acid EGFR Epidermal growth factor receptor EOBA Excess-over-Bliss additivism Epo Erythropoietin EpoR Erythropoietin receptor ER Endoplasmic reticulum ERK Extracellular signal related kinase FAB French American British classification FcεRI High-affinity IgE receptor FDA Food and Drug Administration FITC Fluorescein isothiocyanate FL FLT ligand FLT3 FMS-like tyrosine kinase 3
xiv FLT3-ITD FMS-like tyrosine kinase 3 with internal tandem duplication FLT3-TKD FMS-like tyrosine kinase 3 with tyrosine kinase domain mutation fMLP N-Formyl-methionyl-leucyl-phenylalanine GAPDH Glyceraldehyde-3-phosphate dehydrogenase G-CSF Granulocyte-colony stimulating factor GM-CSF Granulocyte macrophage colony-stimulating factor GMP Granulocyte-monocyte progenitor GPCR G-protein coupled receptor GPVI Glycoprotein VI HRP Horseradish peroxidase HSC Hematopoietic stem cell HSCT Hematopoietic stem cell transplant IFN Interferon IgE Immunoglobulin E IgM Immunoglobulin M IκBα Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha IL Interleukin iNOS Inducible nitric oxide synthase IP3 Inositol triphosphate IRAK Interleukin-1 receptor-associated kinase ITAM Immunoreceptor tyrosine-based activation motif ITK Interleukin-2-inducible T-cell kinase JAK Janus kinase LCK Lymphocyte-specific protein tyrosine kinase LPS Lipopolysaccharide LSC Leukemic stem cell M-CSF Macrophage colony-stimulating factor MAPK Mitogen-activated protein kinase MCL Mantle cell lymphoma MDM2 Murine double minute 2 MDS Myelodysplastic syndrome MEP Megakaryocyte erythroid progenitor MM Multiple myeloma MPP Multipotent progenitor mRNA Messenger ribonucleic acid MRP Multidrug resistance-associated protein MRR Major response rate mTOR Mammalian target of rapamycin MYD88 Myeloid differentiation primary response gene 88 NAD Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NFAT Nuclear facor of activated T-cells NFκB Nuclear factor kappa-light-chain-enhancer of activated B-cells NSCLC Non small-cell lung cancer NO Nitric oxide
xv OB Osteoblast OC Osteoclast ORR Overall response rate P-gp P-glycoprotein P53/TP53 Tumor protein 53 PAR Poly(ADP-ribose) PARG Poly(ADP-ribose) glycohydrolase PARP Poly(ADP-ribose) polymerase pBTK Phosphorylated BTK PDAC Pancreatic ductal adenocarcinoma PDGFRA Platelet-derived growth factor receptor alpha PDGFRB Platelet-derived growth factor receptor beta PH Pleckstrin homology PI Propidium iodide PI3K Phosphoinositide 3-kinase PIP2 Phosphatidylinositol 4,5-bisphosphate PIP3 Phosphatidylinositol 3,4,5-triphosphate PKC Protein kinase C PLCγ2 Phospholipase C gamma 2 qPCR Quantitative polymerase chain reaction RA Rheumatoid arthritis RAEB-2 Refractory anemia with excess blasts-2 RANKL Receptor activator of nuclear factor-kappaB ligand RIPA Radioimmunoprecipitation assay RLK/TXK Resting lymphocyte kinase ROS Reactive oxygen species rRNA Ribosomal RNA RT-PCR Reverse-transcriptase polymerase chain reaction SCF Stem cell factor SCID Severe combined immunodeficiency SDF1 Stromal cell-derived factor 1 SFK SRC-family kinase SH2 Src homology domain 2 SH3 Src homology domain 3 shRNA short hairpin RNA siRNA Short-interfering RNA SRB Sulforhodamine-B STAT Signal transducer and activator of transcription SYK Spleen tyrosine kinase T-ALL T-cell acute lymphoblastic leukemia TH TEC homology TK Tyrosine kinase TKI Tyrosine kinase inhibitor TLR Toll-like receptor TNFα Tumor necrosis factor alpha TRAIL TNF-related apoptosis-inducing ligand
xvi VEGFR Vascular endothelial growth factor receptor WHO World Health Organization WM Waldenström macroglobulinemia WT Wild type Xid X-lined immunodeficiency XLA X-linked agammaglobulinemia
xvii Preface
Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of improperly differentiated – and thus nonfunctional – myeloid lineage cells. The mainstay of first-line therapy for this disease is aggressive treatment with chemotherapy, which aims to eradicate leukemic cells and to restore normal hematopoiesis. Unfortunately, this approach is inadequate for the majority of patients: treatment-related toxicities and drug resistance have translated to five-year survival rates of 25%. Thus, there is a great need for novel approaches to AML treatment. One potential strategy for reducing therapy- associated toxicity and improving efficacy is to combine anti-leukemic drugs with synergizing agents in order to enhance AML cell sensitivity to these drugs. We therefore screened drugs with documented preclinical anti-AML activity against chemical libraries in AML cell lines to identify synergistic drug combination candidates.
1
Chapter 1: Literature Review
2 1.1 Acute Myeloid Leukemia
1.1.1 Normal Hematopoiesis
In the traditional model of normal hematopoiesis in humans, maturation of hematopoietic cells follows a hierarchy in which stem cells differentiate to give rise to the lineages that produce mature blood cells: hematopoietic stem cells (HSCs) self-renew or differentiate to multipotent progenitor cells (MPPs), which in turn give rise to the oligopotent common myeloid (CMP) and common lymphoid (CLP) progenitor cells. CLPs differentiate to T- and B-lymphocytes and natural killer cells, while CMPs differentiate into megakaryocyte erythroid progenitors (MEP) and granulocyte monocyte progenitors (GMP). MEPs ultimately give rise to erythrocytes and megakaryocytes (from which platelets form), while GMPs differentiate to infection and pathogen-fighting granulocytes (neutrophils, eosinophils, and basophils) and monocytes. This model is illustrated in Figure 1- 1 (top panel).
Recent work has contested this original model: (Notta et al., 2015) provided evidence to support a two-tier model of hematopoiesis in the adult bone marrow wherein multipotent HSCs give rise to unipotent progenitor cells that mature to form monocytes, granulocytes, erythrocytes, and lymphocytes. Interestingly, megakaryocytes were found to originate from the multipotent tier, and thus do not arise from the same progenitors (CMPs) as the rest of the myeloid lineage, as had previously been thought (Figure 1-1, bottom-right).
3
Figure reproduced from Notta et al. (2015), license #3817030184707
Figure 1-1: Hematopoiesis: original and revised models. Top panel: classical model of hematopoiesis. Bottom panel: revised model of hematopoiesis in the adult bone marrow (right) and fetal liver bone marrow (left). Abbreviations: HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte erythroid progenitor; GMP, granulocyte-monocyte progenitor; Ly, lymphoid cell; Er, erythroid cell; Mk, megakaryocyte; Gran, granulocyte; Mono, monocyte; My, granulocyte/monocyte
4 1.1.2 Acute Myeloid Leukemia
Acute myeloid leukemia (AML) comprises many different hematologic neoplasms with one unifying feature: the presence of proliferative, clonal, myeloid-lineage cells that are improperly differentiated and the ensuing absence of one or more mature myeloid-lineage cell types (reviewed in Döhner et al. (2015)). These improperly differentiated cells—termed blasts—accumulate in the bone marrow and peripheral blood. Generally, when the blast percentage in the blood and bone marrow reaches or exceeds 20%, a diagnosis of AML is made (Vardiman et al., 2002).
1.1.2.1 AML Pathogenesis
AML results from a series of mutations that cooperate to confer a proliferative and survival advantage to the leukemic clone. The cells of origin in AML are leukemic stem cells (LSCs), which develop from alterations in normal hematopoietic stem cells (HSCs) (Bonnet & Dick, 1997; Hope et al., 2004; Lapidot et al., 1994). LSCs, like HSCs, are highly primitive (often possessing the CD34+CD38- immunophenotype) and have the capacity to recapitulate the entire AML cell hierarchy (Bonnet & Dick, 1997; Lapidot et al., 1994). LSCs have the ability to self-renew or give rise to non self-renewing leukemic progenitor cells (also known as CFU-L, or colony-forming unit-leukemia cells) (Bonnet & Dick, 1997; Hope et al., 2004). CFU-L cells actively proliferate and undergo incomplete differentiation to leukemic blasts (reviewed by (Griffin & Löwenberg, 1986). Left untreated, blast accumulation ultimately prevents the formation of mature myeloid cells, such as neutrophils, erythrocytes, and platelets, leading to infection susceptibility, anemia, and hemorrhage, respectively.
5 1.1.2.2 Epidemiology of AML
AML is the most common form of acute leukemia in adults, and the risk of developing the disease increases significantly with age. The incidence in those under the age of 65 is approximately one per 100,000, while this figure climbs to 12 per 100,000 in those over the age of 65. AML is also slightly more prevalent in males than females, with a 5:3 ratio (Siegel et al., 2012).
1.1.2.3 AML Classification and Prognostication
The French American British (FAB) Cooperative Group classification system was an early system used to divide AML into subtypes based on morphology (Bennett et al., 1976). Organized from M0-M7, each subtype reflects the cell type from which the leukemia originated and its degree of differentiation, with M0 representing the most primitive (“undifferentiated acute myeloid leukemia”), and M6 and M7 representing the most mature AML subtypes (“acute erythroid leukemia” and acute megakaryoblastic leukemia”, respectively). Several FAB subtypes have associated somatic cytogenetic abnormalities, and identifying such abnormalities in newly diagnosed AML patients may at times offer useful insights into individualized disease management. For instance, FAB M3 (acute promyelocytic leukemia, APL) is most commonly associated with a t(15;17) translocation, producing the oncogenic PML-RARα fusion protein, which can be successfully targeted through addition of all trans-retinoic acid differentiation therapy in conjunction with an anthracycline-containing chemotherapy regimen or arsenic trioxide (Tallman & Altman, 2009).
For most other AML patients, however, the FAB classification is of limited clinical utility, as it does not take into consideration other, non-morphologic abnormalities that also provide prognostic insight and thus inform AML treatment approaches. These limitations prompted the development of the World Health Organization (WHO) classification of myeloid neoplasms, which in addition to AML blast
6 morphology, utilizes genetic, immunophenotypic, biologic, and clinical information to categorize AML subtypes (Vardiman et al., 2002). The WHO system classifies AML into the following four major subgroups: AML with recurrent genetic abnormalities (which include mutations such as t(8:21)(q22;q22), inv(16)(p13.1q22), t(16;16)(p13.1;q22), and t(15;17)(q22;q12)), AML with myelodysplasia-related changes, therapy-related AML (caused by previous treatment with alkylating agents, radiation, or topoisomerase II inhibitors), and AML, not otherwise specified (Vardiman et al., 2009). The majority of identified recurrent genetic abnormalities associated with AML have been stratified into favourable, intermediate-I, intermediate-II, and adverse prognostic categories (Döhner et al., 2010). Three-year overall survival rates for each category, in both younger and older AML patients, have been reported (Mrózek et al., 2012): patients with alterations such as t(8;21)(q22;q22), inv(16)(p13.1q22) and t(16;16)(p13.1;q22) have “favourable” prognoses, with a 3-year overall survival of 66% and 33% in the under- and over-60 age groups, respectively. Patients with karyotypically normal AML harbouring the FLT3-ITD mutation with or without NPM1 mutation, or karyotypically normal AML without the NPM1 or FLT3-ITD mutations fall under the intermediate-I prognostic classification, with 3-year survival rates of 28% and 11% in the under- and over-60 age group, respectively. The t(9;11)(p22;q23) translocation is classified prognostically as intermediate-II and is associated with 3-year survival rates of 45% in patients under the age of 60, and 16% in patients over the age of 60. Finally, adverse-risk alterations such as inv(3)(q21q26.2), t(3;3)(q21;q26.2), t(6;9)(p23;q34), -5, del(5q), -7, and complex karyotype AML are associated with 3-year survival rates of 12% and 3% in adults under- and over the age of 60, respectively.
1.1.2.4 AML Management
Standard pharmacologic AML therapy aims to eradicate leukemic blasts and restore normal multilineage hematopoietic cell growth. It often consists of two
7 phases: induction and consolidation. During the induction phase, a combination of the chemotherapy drugs, daunorubicin and cytarabine (known as the 3+7 regimen) is administered over a seven-day period. Daunorubicin, an anthracycline antibiotic, likely induces death of AML cells via several proposed mechanisms. Daunorubicin binds to and inhibits topoisomerase II, preventing replication fork progression and creating single- and double-stranded DNA breaks and inducing subsequent cell death. In addition to topoisomerase II inhibition, daunorubicin blocks DNA synthesis via DNA intercalation, induces the generation of free radicals, and may disrupt DNA helicase activity (Gewirtz, 1999).
Cytarabine is a nucleoside analogue. It inhibits DNA polymerases and competes with deoxycitidine for incorporation into newly synthesized DNA. Cytarabine incorporation halts DNA synthesis, inducing subsequent cell death (Grant, 1998; Inagaki et al., 1969).
Induction therapy produces complete remissions (defined as <5% bone marrow blasts, and recovery of absolute neutrophil and platelet counts to >1.0x109/L and >10x109/L, respectively (Döhner et al., 2010) in 60-85% of AML patients under the age of 60, and in 40-60% of those over the age of 60 (Döhner et al., 2015). However, induction therapy alone is inadequate to produce lasting remissions in patients: without further treatment, AML patients relapse within several months (Cassileth et al., 1988). Additional therapy is therefore necessary in order to reduce AML relapse risk and prolong remissions.
The post-remission treatment approach to AML is dependent upon the prognostic classification of the patient’s cytogenetic and/or molecular abnormalities, as well as the patient’s age and presence or absence of comorbidities. In general, pharmacologic consolidation therapy with cytarabine is recommended in patients with favourable-risk AML, with this approach leading to cure in 60-70% of patients aged <60 (Döhner et al., 2015). Allogeneic hematopoietic stem cell
8 transplantation (HSCT) is recommended in patients with intermediate or adverse- risk disease, as these individuals are unlikely to be cured with cytarabine consolidation; however, HSCT carries significant risk of mortality, lifelong morbidity, and requires identification of an appropriately matched donor (Döhner et al., 2015).
9 1.2 Tyrosine Kinase Inhibitor Therapy in AML
1.2.1 Targeted Cancer Therapies
The significant toxicity associated with standard chemotherapy treatment for AML is related to the lack of specificity of these drugs: DNA synthesis and replication are ubiquitous processes and daunorubicin and cytarabine are therefore also lethal to many non-cancerous cell types. Therapies with targets unique—or at least more specific—to cancer cells have thus been investigated as a less toxic therapeutic strategy compared to standard chemotherapies.
1.2.1.1 Tyrosine Kinase Inhibitors
Tyrosine kinase inhibitors (TKIs) are one class of targeted therapy used to treat malignancies. Although the expression of protein tyrosine kinases (TKs) is not exclusive to cancer cells, their critical role in many cell growth, proliferation, and survival signaling pathways (which are frequently aberrantly activated in cancer) make them ideal candidates for therapeutic targeting. Tyrosine kinases catalyze the transfer of a phosphoryl group from ATP to a tyrosine residue on protein substrates. Tyrosine phosphorylation of protein targets may serve as activation or regulatory signals, and the majority of TKIs intercept TK activity by competing with ATP for binding to the ATP-binding site of the TK (reviewed by Roskoski (2015)).
A classic example of the success of TKIs in cancer therapy is that of imatinib. This small-molecule inhibitor of Abelson tyrosine kinase (Abl) has vastly improved the survival of patients with chronic myelogenous leukemia (CML). CML is characterized by the presence of the Philadelphia translocation (t(9;22)(q34;q11.2)), which results in expression of the oncogenic breakpoint cluster region (BCR)-Abl fusion protein. This fusion of BCR and Abl renders Abl
10 constitutively active, which causes leukemic transformation. The small molecule imatinib binds to the inactive conformation of Abl (Capdeville et al., 2002) and produces long-term complete remissions in CML patients: in a long term follow up study of first-line imatinib therapy for this disease, six-year progression-free survival was found to be 87% (with overall survival at 89%) (Castagnetti et al., 2015), compared to a median survival of five to seven years with interferon α therapy (Apperley, 2015).
1.2.2 Oncogenic Tyrosine Kinases in AML
The striking success of imatinib in CML prompted the evaluation of TKs as therapeutic targets in other malignancies, including AML. Given the comparatively heterogeneous nature of AML, however, this approach has proven to be far more challenging for this disease: no one TK target is consistently deregulated in the majority of AML cases. In reality, many different kinases implicated in proliferation and survival pathways may be deregulated in AML and may thus contribute to the pathogenesis of this malignancy (Kelly & Gilliland, 2002). While there are currently no TKIs approved for clinical use in AML, several inhibitors are under clinical investigation, and other kinases have been proposed as potential therapeutic targets.
1.2.2.1 FMS-Related Tyrosine Kinase 3
FMS-related tyrosine kinase 3 (FLT3) is an extensively studied receptor TK that is expressed in normal hematopoietic stem and progenitor cells (Rosnet et al., 1996; Small et al., 1994). FLT3 signaling plays an important role in hematopoiesis: stimulation of this receptor with its ligand, FL, induces proliferation and contributes to differentiation of hematopoietic progenitor cells (Gabbianelli et al., 1995; Lyman et al., 1994). FLT3 is also expressed in primary
11 AML cells (Birg et al., 1992; Carow et al., 1996). FL-mediated FLT3 stimulation was found to induce proliferation and clonogenic growth in a subset of AML cell lines and primary AML blasts, respectively (Dehmel et al., 1996; Stacchini et al., 1996).
Two common oncogenic FLT3 mutations have been identified in AML: an internal tandem duplication (FLT3-ITD), present in approximately 20% of cases (Nakao et al., 1996; Yokota et al., 1997), and a point mutation in the second tyrosine kinase domain (FLT3-TKD), which is present in 7% of AML cases (Yamamoto et al., 2001). Both mutations induce constitutive (FL-independent) FLT3 activity in AML (Hayakawa et al., 2000; Kiyoi et al., 1998; Yamamoto et al., 2001), and FLT3-ITD mutations have been associated with particularly poor AML patient prognoses (Whitman et al., 2001; Yamamoto et al., 2001). Thus, there has been significant interest in investigating the therapeutic potential of FLT3 inhibitors in patients with AML.
Clinical trials for some FLT3 inhibitors have reported some benefit to patients with FLT3-ITD positive AML. The first-generation FLT3 inhibitors sunitinib, sorafenib, midostaurin and lestaurtinib—all multi-receptor TK inhibitors—caused reductions in peripheral blast count, with sunitinib producing complete remissions as a single-agent, and midostaurin inducing remissions when administered in combination with chemotherapy agents (Wander et al., 2014). However, these studies reported transient reductions of peripheral or marrow blasts (sunitinib, midostaurin, lestaurtinib), and lestaurtinib in combination with chemotherapy failed to improve complete remission rates or overall survival in relapsed/refractory AML patients in a Phase III trial (Wander et al., 2014). Subsequent generations of FLT3 inhibitors have produced more promising results in the clinical trial setting. Quizartinib (AC220), which has been shown to target FLT3, KIT, PDGFRA, PDGFRB, and RET kinases in preclinical studies, produced responses in nine of 17 AML patients harboring the FLT3-ITD mutation, five of 37 FLT3-ITD negative patients, and nine of 22 patients with
12 undetermined FLT3-ITD status in a Phase I trial (Cortes et al., 2013). In a Phase II trial of single-agent quizartinib in patients over the age of 60 with relapsed/refractory AML, 54% of FLT3-ITD positive and 32% of FLT3-ITD negative patients achieved a composite complete remission (Cortes et al., 2012). Crenolanib and PLX3397 are newer FLT3 inhibitors with impressive preclinical activity against FLT3-mutant AML cell lines and are currently undergoing evaluation in clinical trial (Wander et al., 2014).
13 1.3 Ibrutinib
Ibrutinib (PCI-32765) is a Bruton’s tyrosine kinase (BTK) inhibitor that is currently clinically approved for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström’s macroglobulinemia (WM) (FDA, 2015a). BTK is an important therapeutic target in these B-cell malignancies, and ibrutinib was designed to inhibit this cytoplasmic kinase with high specificity. In the clinic, ibrutinib is well tolerated by patients and clinical trials for ibrutinib use in these cancers have demonstrated improved patient outcomes. As preclinical research into novel roles for BTK in different diseases continues, the list of FDA-approved ibrutinib indications is likely to rapidly expand.
1.3.1 Bruton’s tyrosine kinase: background & role in signal transduction from the B-cell receptor
1.3.1.1 BTK domains
The BTK gene is located at the Xq21.3-Xq22 locus (Kwan et al., 1986; Malcolm et al., 1987) and encodes a 659 amino acid protein (Vetrie et al., 1993). BTK belongs to the TEC family of cytoplasmic protein kinases, which also includes TEC, ITK, RLK/TXK and BMX. BTK, like all TEC family kinases, is similar in sequence to SRC family kinases (SFKs): it contains Src homology (SH) 2 and SH3 domains, as well as a C-terminal kinase domain (Vetrie et al., 1993). However, BTK and the majority of the other TEC family members are distinguishable from SFKs by the presence of a Tec homology (TH) domain, an N-terminal plasma membrane-targeting pleckstrin homology (PH) domain (as opposed to a myristoylation sequence found in SFKs), as well as the absence of a negative regulatory tyrosine analogous to the C-terminal Y527 of SFKs (Vetrie et al., 1993).
14
1.3.1.2 BTK expression
BTK is a cytoplasmic protein and its expression is restricted to hematopoietic cells. This kinase is expressed throughout B-cell maturation, with BTK mRNA and/or protein detection in pro-B cell, early pre-B cell, late pre-B cell, and mature B cell lines (de Weers et al., 1993; Genevier et al., 1994). BTK is also expressed in many myeloid cell lines, but its expression is downregulated in plasma cells and T-cells (de Weers et al., 1993; Genevier et al., 1994; Smith et al., 1994).
1.3.1.3 BTK: Role in B-cell Maturation
The importance of BTK function during B-cell maturation is illustrated clinically in X-linked agammaglobulinemia (XLA), a human primary immunodeficiency caused by germline BTK mutations (Tsukada et al., 1993; Vetrie et al., 1993). Patients with XLA often have normal pre-B cell levels, but severely reduced or absent levels of B-cells and plasma cells (and thus immunoglobulins), implying that BTK is critical for maturation beyond the pre-B cell stage (de Weers et al., 1993). This disease manifests itself clinically as an increased susceptibility to recurrent bacterial infections in infant males, and is treated with donor-derived immunoglobulin therapy, and antibiotics in the presence of confirmed or suspected infections (Bruton, 1952; Timmers et al., 1991).
1.3.1.4 BTK Signaling in B-Cells
In B-cells, BTK is required for appropriate signal transduction from the B-cell receptor (BCR) upon receptor crosslinking (reviewed in-depth by Dal Porto et al. (2004)). When an antigen binds to the immunoglobulin (IgM) portion of the BCR, the CD79A (Igα) and CD79B (Igβ) components of the BCR are tyrosine-
15 phosphorylated on their immunoreceptor tyrosine-based activation motifs (ITAMs) by LYN and other SRC family kinase (SFK) members. Tyrosine phosphorylation of ITAMs attracts spleen tyrosine kinase (SYK) via SYK’s SRC homology 2 (SH2) domains, and SYK is subsequently phosphorylated and activated by vicinal SFKs. Antigen binding to the BCR also triggers simultaneous activation of phosphoinositide 3-kinase (PI3K), which phosphorylates the membrane phospholipid, PIP2. Phosphorylated PIP2 (also known as PIP3) recruits cytoplasmic BTK via its pleckstrin homology domain to the plasma membrane. Here, proximal SFKs and SYK phosphorylate BTK at Y551 of its kinase domain and BTK undergoes subsequent autophosphorylation at Y223 in its SH3 domain, which stabilizes its active conformation (Rawlings et al., 1996; Wahl et al., 1997). Activated BTK and SYK then phosphorylate and activate phospholipase Cγ2 (PLCγ2) (Takata & Kurosaki, 1996), which cleaves the 2+ membrane-associated PIP2 to form IP3 and DAG. IP3 generation leads to Ca mobilization from intra- and extracellular Ca2+ stores, ultimately triggering NFAT activation. Ca2+ and DAG, together, activate protein kinase Cβ, which causes
NFKB pathway activation. BCR signaling also triggers the activation of MAPK and RAS signaling pathways. The end result of BCR signaling, mediated by BTK, is B-cell survival and proliferation.
1.3.2 A Rationale for Targeting BTK in B-cell Malignancies
BTK emerged as an attractive therapeutic target in B-cell malignancies due to its relatively restricted expression pattern and its role as a signal transducer in several pathways implicated in the pathogenesis and progression of these diseases.
16 1.3.2.1 Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of CD5-expressing mature B cells within the bone marrow and secondary lymphoid tissues. Proliferation and survival of CLL cells is dependent upon stimulatory signals from their respective tissue microenvironments, as well as increased antigen-dependent or independent (“tonic”) BCR signaling (Burger, 2013). Given that BTK is implicated in signal transduction downstream of both the BCR and chemokine receptor (CXCR4 and CXCR5) pathways (Hendriks et al., 2014), BTK inhibition would thus act as a two-pronged approach to interfering with CLL cell proliferation and survival.
1.3.2.2 Mantle-Cell Lymphoma
Mantle-cell lymphoma (MCL) is a type of non-Hodgkin lymphoma that is associated with aberrant cyclin D1 overexpression in mantle-zone B-cells. Cyclin D1 overexpression drives proliferation of these cells. BCR signaling pathway proteins, including BTK, are overexpressed in MCL and this pathway is highly active in MCL cell lines (Cinar et al., 2013; Pighi et al., 2011). BCR signaling contributes to MCL cell proliferation and survival, and inhibiting SYK within this pathway (which is upstream of BTK) has been shown to induce apoptosis and decrease cyclin D1 expression (Pighi et al., 2011; Rinaldi et al., 2006).
1.3.2.3 Waldenström Macroglobulinemia
Waldenström macroglobulinemia (WM) is an indolent lymphoma characterized by the overproduction of IgM-producing lymphoplasmacytic cells in the bone marrow. One commonly identified feature of this disease is the presence of the myeloid differentiating factor 88 (MYD88) L265P mutation, which has been detected in over 90% of WM patients (Treon et al., 2012; Varettoni et al., 2013;
17 Xu et al., 2013). This mutation promotes WM cell growth and survival by increased activation of NF-KB, which is mediated in part by BTK signaling (Yang et al., 2013). In contrast, wild-type MYD88 does not associate with, nor signal through, BTK (Yang et al., 2013). Constitutively active BTK has also been detected in two WM cell lines harbouring the MYD88 L265P mutation (Tai et al., 2012).
1.3.3 Development of Ibrutinib as a Selective and Irreversible BTK Inhibitor with In Vivo Activity
Prior to the development of ibrutinib, there were no existing small molecule BTK inhibitors with in vivo activity. Pan et al. (2007) used a scaffold screening approach to identify compounds with inhibitory activity against BTK, noting that one of these compounds inhibited BTK at a Ki of 8.2nM in enzymatic assays. This compound, however, was not BTK-specific, as it also potently inhibited other TEC and SRC family kinases. To design a compound with enhanced potency and selectivity, this group mapped the predicted binding site of the original compound to within the kinase domain of BTK and LCK, a SRC family kinase. They reported the presence of a nucleophilic residue within the kinase domain of BTK (Cys481) that was absent in LCK, and noting that other kinases with equivalent Cys residues were potently and irreversibly inhibited by small molecules with electrophilic centres, they designed several compounds to analogously inhibit BTK. The most potent of these compounds, “Compound 4”
(later designated PCI-32765, or ibrutinib), inhibited BTK activity at an IC50 of
0.72nM and the activity of the BTK substrate PLCγ1 at an IC50 of 14nM and was more than 500 times more selective for BTK than SYK or the SRC family kinase, LYN. Ibrutinib treatment prevented arthritis development in an anticollagen antibody and LPS-induced murine arthritis model.
18 Further investigation of the activity of ibrutinib by Honigberg et al. (2010) confirmed its selectivity against BTK and provided additional in vivo evidence to support its use as a clinical BTK inhibitor in several B-cell disease models. Oral ibrutinib administration improved renal function in the MRL-Fas(lpr) murine model of lupus (which induces glomerulonephritis), and improved clinical arthritis scores in mouse models of rheumatoid arthritis. Ibrutinib also produced partial responses in a canine model of non-Hodgkin lymphoma.
1.3.4 Preclinical and clinical activities of ibrutinib in B-cell cancers
1.3.4.1 Chronic Lymphocytic Leukemia
BTK inhibition by ibrutinib was found to only modestly induce patient-derived CLL cell apoptosis, but significantly reduced CLL cell proliferation at clinically achievable concentrations (Cheng et al., 2014; Herman et al., 2011; Ponader et al., 2012) and delayed CLL progression in an adoptive transfer TCL1 murine model of CLL (Ponader et al., 2012). Perhaps the most striking effect of ibrutinib treatment CLL cells was the impact of this drug on the interactions between these cells and their microenvironment: ibrutinib counteracted IL-6, IL-10, and TNFα production by T-cells (Herman et al., 2011), abrogated the pro-survival and proliferative effects imparted by Hs5 stromal cell (Herman et al., 2011) and nurse-like cell (Ponader et al., 2012) co-culture, reduced CLL cell migration toward the tissue homing chemokines CXCL12 and CXCL13 (Ponader et al., 2012), and blocked IgM-stimulated CLL cell adhesion to fibronectin and VCAM-1 (de Rooij et al., 2012). The ibrutinib-mediated disruption of CLL cell homing to microenvironment-produced chemokines has been hypothesized as the explanation for the observed transient peripheral blood lymphocytosis following ibrutinib treatment in vivo (Ponader et al., 2012) and in clinical trial participants (Byrd et al., 2013).
19
Clinical trials of ibrutinib in both treatment-naïve and pre-treated CLL patients have yielded impressive responses: in a Phase 1b/2 trial assessing single-agent ibrutinib treatment in relapsed/refractory CLL, the overall response rate was 71% and 26-month progression-free survival was 75% (Byrd et al., 2013). Plasma ibrutinib concentrations in these patients reached ~450nM (see Appendix 1). A three-year follow-up of this study demonstrated the long-term efficacy of ibrutinib, with overall response rates of 90% and 84% in patients with relapsed/refractory and treatment-naïve CLL, respectively (Byrd et al., 2015). In a Phase 1b/2 trial assessing ibrutinib in newly diagnosed CLL in patients over the age of 65, ibrutinib was generally well tolerated and efficacious, with 71% of patients achieving an objective response, and 4/22 responders achieving complete responses (O'Brien et al., 2014). In a Phase 3 trial in relapsed/refractory CLL, ibrutinib was superior to the anti-CD20 monoclonal antibody ofatumumab in extending progression-free and overall survival and overall survival (Byrd et al., 2014). Ibrutinib is now FDA-approved for CLL with 17p deletion and for previously treated CLL (FDA, 2015a).
1.3.4.2 Mantle Cell Lymphoma
In preclinical studies, ibrutinib was found to block BTK activity in primary MCL cells stimulated by IgM or co-culture with stromal cells, and in the MCL cell lines Mino, Jeko, and HBL2 (Chang et al., 2013). This drug only modestly reduced MCL cell line growth and viability at clinically achievable concentrations, and induced apoptosis at supraclinical (10-20 µM) concentrations (Cinar et al., 2013). Ibrutinib has been shown to synergize with the proteasome inhibitor bortezomib, with the combination inducing ER stress, AKT and NFkB inhibition, downregulation of Bcl-2 family proteins, and apoptotic cell death in Granta519 MCL cells (Dasmahapatra et al., 2013). Ibrutinib-mediated downregulation of Bcl-
20 2 family anti-apoptotic proteins was confirmed in Mino, an MCL cell line, by Cinar et al. (2013).
In the context of MCL-microenvironment signaling, ibrutinib reduced chemokine production by MCL cell lines and blocked their adhesion and migration in response to BCR-, CXCL12- and CXCL13-mediated stimulation. Ibrutinib treatment of C57BI/6 mice reduced MCL cell migration toward lymphoid tissues, and reduced MCL cell infiltration of lymph nodes and bone marrow in a murine model of MCL lymphadenopathy (Chang et al., 2013).
A Phase II clinical trial in relapsed/refractory MCL patients demonstrated an overall response rate (ORR) of 68%, with 21% achieving complete responses (CR) (Wang et al., 2013) and led to the drug’s accelerated approval for previously-treated MCL by the FDA. A longer-term follow-up of this trial (median follow-up of 26.7 months) demonstrated similarly impressive outcomes, with an ORR of 67%, CR of 23%, and 31% progression-free survival at 24 months (Wang et al., 2015).
1.3.4.3 Waldenström Macroglobulinemia
In preclinical studies, ibrutinib treatment of the MYD88 L265P BCWM.1 and
MWCL1 WM cell lines reduced IKBα phosphorylation (IKBα phosphorylation permits nuclear translocation and activation of NFKB). In addition, these cell lines were more sensitive to killing by ibrutinib compared to MYD88 WT WM cell lines (Yang et al., 2013). Combining ibrutinib with an inhibitor of interleukin – receptor- associated kinase (IRAK) 1 and 4—the other reported pathway by which MYD88 signalling activates NFKB—profoundly enhanced NFKB inhibition and induced synergistic cell death (Yang et al., 2013).
21 Somatic activating C-terminus CXCR4 mutations are present in approximately 30% of WM patients (Roccaro et al., 2014). WM cells engineered to express CXCR4 C-terminus mutations commonly found in WM patients, exhibited constitutive receptor activity due to impaired CXCR4 internalization following ligand (SDF-1a) binding, as well as sustained ERK and AKT activation (Cao et al., 2015a; Cao et al., 2015b). Mutant CXCR4-mediated ERK and AKT activation were shown to contribute to ibrutinib resistance in WM, which was reversible by CXCR4 inhibitor (AMD3100, plerixafor) administration (Cao et al., 2015a; Cao et al., 2015b).
A clinical trial evaluating the efficacy of ibrutinib in WM provided strong evidence to support targeting BTK as a therapeutic strategy for this disease. In this study, ibrutinib treatment of 63 pre-treated WM patients resulted in an overall response rate of 90.5%, with major responses in 73% of participants. Ibrutinib efficacy was greatest in WM patients harboring MYD88 L265P and wild-type CXCR4 (91.2% major response rate (MRR), 100% overall response rate (ORR)), but still highly effective in patients with both the MYD88 and CXCR4 mutations (61.9% MRR, 85.7% ORR). Ibrutinib was the least effective in patients harbouring both wild type MYD88 and CXCR4 (28.6% MRR, 71.4% ORR) (Treon et al., 2015). The findings of this study led to the FDA-approval of ibrutinib for WM.
1.3.5 B-cell independent BTK signaling: myeloid-lineage cells
BTK is expressed in both primitive and mature myeloid-lineage cells (Schmidt et al., 2004a). Given the observation that individuals with BTK mutations do not appear to have abnormal myeloid cell numbers or defective myeloid cell activity, it was long assumed that BTK played an insignificant or redundant role in the development and function of these cells. However, several groups have
22 demonstrated evidence of the important role of this kinase in both normal and malignant myeloid cells.
1.3.5.1 Mast Cells
In mast cells, BTK is activated upon high-affinity IgE receptor (FcεRI) cross- linking (Kawakami et al., 1994). The mechanism of BTK activation in these cells resembles that of mature B-cells: FcεRI cross-linking in response to antigen binding leads to phosphorylation of receptor-associated ITAMs by LYN. ITAM phosphorylation triggers the recruitment and activation of SYK and additional LYN, which in turn phosphorylate and activate BTK. Btk itself does not associate with FcεRI in murine mast cells (Kawakami et al., 1994), however it is constitutively bound to protein kinase C (PKC) via its PH domain (Yao et al., 1994). PKC phosphorylates Btk and this event is inhibitory: Btk autophosphorylation is decreased and pharmacologic PKC inhibitors enhance tyrosine phosphorylation of Btk upon FcεRI stimulation (Yao et al., 1994).
In contrast to B-cells, BTK does not appear to be required for mast cell development. In two murine models of defective Btk (btk null and xid), mast cell numbers, morphology and expression of important signaling proteins were not different compared to wild type controls (Hata et al., 1998).
BTK does appear to be important, however, for normal mast cell signaling and function: Btk-defective mice exhibited diminished anaphylactic responses relative to wild type mice. Upon FcεRI cross-linking of mast cells cultured from xid and/or btk-null mice, these cells exhibited reduced Ca2+ mobilization, impaired degranulation (as indicated by reduced histamine release), and demonstrated severely compromised TNF-α transcription, and TNF-α, IL-2, IL-6, and GM-CSF secretion (Hata et al., 1998; Setoguchi et al., 1998). Consistent with these findings, transfection of multiple siRNAs directed against Btk in RBL-2H3 rat
23 mast cells induced a 20-25% decrease in histamine release upon FcεRI stimulation (Heinonen et al., 2002). This observation was further corroborated with pharmacologic Btk inhibitors: the leflunomide metabolite LFM-A13 also moderately reduced histamine release in FcεRI-stimulated RBL-2H3 cells (Heinonen et al., 2002) and the quinone epoxide terreic acid decreased TNF-α and IL-2 secretion in antigen-stimulate mouse bone marrow-derived mast cells (Kawakami et al., 1999).
In a more recent study by Soucek et al. (2011), oral administration of ibrutinib in a Myc-driven murine insulinoma model (in which mast cell recruitment is critical to tumor expansion and angiogenesis) was found to block mast cell degranulation and induce tumor regression. Furthermore, in addition to blocking mast cell degranulation and tumor cell proliferation in murine models of pancreatic ductal adenocarcinoma (PDAC), ibrutinib was found to block mast cell-mediated fibrosis of stromal tissue, which is commonly associated with therapy resistance in this tumor type (Massó-Vallés et al., 2015).
1.3.5.2 Macrophages
BTK expression does not appear essential for macrophage development, as bone marrow-derived and splenic macrophages from Btk-deficient mice were found to be phenotypically similar to those of wild-type mice (Schmidt et al., 2006). In addition, macrophages derived from Xid mice exhibited no differences in phagocytic activity (Mangla et al., 2004).
BTK does however appear to be important for other macrophage functions. Macrophages derived from Xid mice demonstrated impaired bactericidal activity (Mukhopadhyay et al., 2002), and cytokine production by Btk-deficient macrophages was altered: TLR stimulation of Btk-deficient macrophages resulted in reduced production of the anti-inflammatory cytokine IL-10 relative to
24 wild-type controls (Schmidt et al., 2006). Moreover, Btk-deficient monocyte/macrophage production of pro-inflammatory cytokines TNFα and IL-1β following LPS stimulation was impaired relative to control macrophages (Horwood et al., 2003; Mukhopadhyay et al., 2002).
BTK also appears to be implicated in reactive oxygen species (ROS) and nitric oxide (NO) generation in response to macrophage stimulation: relative to macrophages derived from wild-type mice, LPS stimulation of macrophages from Xid mice resulted in reduced ROS generation (Mangla et al., 2004). Xid mouse- derived stimulated macrophages were also found to have profoundly reduced NO production, as a result of impaired induction of the STAT1/IFN regulatory factor- 1/iNOS pathway relative to control mice (Mukhopadhyay et al., 1999). Reduced NO production was associated with increased production of IL-12 in these Xid macrophages (Mukhopadhyay et al., 1999), a cytokine that drives T-cells to activate macrophages via IFNγ production.
1.3.5.3 Erythroid Cells
In mouse erythroid progenitor cells, erythropoietin (Epo) and stem cell factor (SCF) stimulation of the erythropoietin receptor (EpoR) and cKit, respectively, results in progenitor cell proliferation, and EpoR stimulation by Epo induces cell differentiation into erythrocytes. Btk expression in erythroblastoid cell lines derived from chickens was first reported by Robinson et al. (1998). Building on this work, Schmidt et al. (2004b) investigated the role of Btk in erythroid progenitor cell signaling. They noted that Epo and/or SCF stimulation of mouse- derived erythroid progenitor cells induced BTK phosphorylation at Y223. Btk- deficient erythroid progenitor cells derived from mice demonstrated reduced sensitivity to Epo and thus impaired phosphorylation of EpoR, Jak2, Stat5, and Plcγ1 following Epo stimulation, relative to wild type-derived controls. In addition, SCF treatment was found to induce Btk association with TNF-related apoptosis-
25 inducing ligand (TRAIL) receptor 1, and Btk-deficient cells were more sensitive to TRAIL-induced apoptosis in the presence of SCF. On a functional and morphologic level, exposure of Btk-deficient cells to physiologic concentrations of Epo and SCF resulted in a block in proliferation and premature terminal differentiation of these cells, relative to cells derived from wild-type littermate controls.
While Btk evidently plays an important role in erythroid progenitor signaling and proliferation in mouse-derived cells, the clinical significance of BTK disruption in human erythroid progenitors and erythrocytes is not known: to our knowledge, there have been no reports of erythroid progenitor or erythrocyte abnormalities in patients with XLA.
1.3.5.4 Platelets
BTK is expressed in platelets (Futatani et al., 2001) and is implicated in platelet activation. Upon collagen (or collagen-related peptide) binding to the FcRγ- associated collagen receptor glycoprotein VI (GPVI), FcRγ chain ITAMs are phosphorylated by the GPVI-bound SFKs LYN and FYN (Watson et al., 2005). These phosphorylated ITAMs serve as docking sites for SYK, which is recruited and tyrosine-phosphorylated, resulting in its activation. BTK is activated downstream of SYK and in turn phosphorylates and activates neighbouring PLCγ2 (Quek et al., 1998). BTK is also activated downstream of platelet stimulation by thrombin; integrin αIIb/β3 and PI3K mediate this activation (Laffargue et al., 1999).
The functional importance of BTK in platelet activation was first reported by Quek et al. (1998), who found that platelets isolated from XLA patients demonstrated significantly reduced aggregation, calcium mobilization, dense granule secretion, and PLCγ2 phosphorylation in response to collagen or CRP, relative to platelets
26 derived from healthy controls. In line with these observations, Rushworth et al. (2013) noted that ex vivo ibrutinib treatment of platelets from CLL and MCL patients impaired platelet aggregation in response to collagen or adenosine diphosphate (ADP) stimulation. Kamel et al. (2015) later confirmed the capacity of ibrutinib to reduce collagen-mediated platelet aggregation in patients undergoing ibrutinib treatment, however they observed no effect of ibrutinib on platelet aggregation in response to ADP stimulation. Kamel et al. (2015) and Levade et al. (2014) also found an association between disruption of collagen- mediated platelet aggregation and likelihood of adverse bleeding events in patients with CLL or MCL.
There have been no clinical reports of platelet dysfunction in patients with XLA, possibly due to the fact that TEC kinase—which is also expressed in platelets— has some functional redundancy with BTK in these cells (Atkinson et al., 2003). However, given that ibrutinib inhibits TEC kinase in addition to BTK (Honigberg et al., 2010), this drug may contribute to adverse bleeding events experienced by patients undergoing ibrutinib therapy. Adverse bleeding events have been reported in ibrutinib clinical trials: in a Phase Ib-II CLL trial carried out by Byrd et al. (Byrd et al., 2013), 16% of patients experienced Grade 1 or 2 bruising, and 5% of patients experienced bleeding that was Grade 3 or higher. Similarly, in a Phase II trial for MCL, 17% of patients experienced Grade 1 or 2 bruising and 5% of patients experienced Grade 3 bleeding (Wang et al., 2013). While CLL itself is associated with diminished collagen-mediated platelet aggregation, ibrutinib was shown to transiently worsen collagen-mediated platelet aggregation in these patients (Lipsky et al., 2015). These findings therefore have implications for CLL patients undergoing concurrent ibrutinib and antiplatelet or anticoagulant therapy (Lipsky et al., 2015).
1.3.5.5 Neutrophils
27 Neutrophils are a major component of the innate immune response. In the presence of invading pathogens, these cells are recruited to the site of infection by chemokines and kill microbes via oxidative burst or phagocytosis.
BTK appears to be important for normal neutrophil development: GM-CSF- and TLR- stimulation of granulocyte-monocyte progenitors (GMPs) isolated from a Btk-deficient murine model of XLA preferentially induced granulopoiesis at the expense of monocytes or undifferentiated myeloid cells (Fiedler et al., 2011). However, resultant neutrophils exhibited maturation defects, as evidenced by a reduced number of granules and expression of granule contents (Fiedler et al., 2011).
In human peripheral blood neutrophils, TEC family kinases (including BTK) are activated downstream of G-protein coupled receptor (GPCR) stimulation and PI3K activation: the chemotactic factor (and GPCR ligand) fMet-Leu-Phe (fMLP) induced tyrosine phosphorylation and membrane translocation of these kinases, and GPCR and PI3K inhibition with pertussis toxin and wortmannin, respectively, disrupted this activation response (Lachance et al., 2002).
Several studies support an important role for BTK in normal neutrophil function. Mangla et al. (2004) and Fiedler et al. (2011) both noted defective neutrophil migration into tissues and reduced tissue edema following inflammatory stimuli in two murine models of XLA. While no significant differences in the phagocytic activities of neutrophils between Xid and wild-type mice were noted, reduced generation of reactive oxygen intermediates and nitric oxide following LPS stimulation were apparent (Mangla et al., 2004). This finding is in contrast to that of Honda et al. (2012), who demonstrated that stimulation of neutrophils isolated from human XLA patients induced exaggerated levels of NADPH oxidase- mediated reactive oxygen species (ROS) production compared to neutrophils from healthy controls, resulting in higher levels of apoptotic cell death. This finding was not reproduced, however, by Broides et al. (2014): this group did not
28 observe increased ROS production following fMLP stimulation of XLA patient- derived neutrophils relative to stimulated neutrophils from healthy volunteers. It is important to note, however, that both groups used differing methods of neutrophil stimulation and ROS quantification for their respective studies.
Clinically, a link between BTK mutations and neutropenia has been reported: in a chart review of 50 XLA patients, Farrar et al. (1996) noted that 26% had experienced severe episodes of neutropenia. Similarly, an XLA database of 201 patients reported neutropenia as the initial clinical presentation in 11% of XLA patients (Winkelstein et al., 2006). The cause of neutropenia in XLA patients has been attributed to abnormally elevated ROS production by neutrophils in response to pathogens (Farrar et al., 1996; Honda et al., 2012) and defective neutrophil function as a result of abnormal maturation (Fiedler et al., 2011).
1.3.6 A Role for Targeting BTK Beyond B-Cell Cancers
1.3.6.1 Rheumatoid Arthritis
Rheumatoid arthritis (RA) is an autoimmune disease initiated by B-cell-mediated production of autoantibodies targeting self-antigens within joints. Autoantibody- autoantigen interactions within the joint trigger immune complex formation, and these immune complexes subsequently activate innate immune cells (predominantly macrophages, but also neutrophils and mast cells) via Fc receptor (FcR) crosslinking. FcR crosslinking of these cells triggers processes such as phagocytosis and inflammatory cytokine production, leading to the cartilage and bone destruction that is characteristic of this disease (Whang & Chang, 2014).
BTK emerged as a promising therapeutic target in RA because of its role as a node in two independent signaling pathways implicated in RA pathogenesis: the
29 BCR signaling pathway in B-cells, and the FcR pathway in myeloid cells (Di Paolo et al., 2011). Blocking BCR signaling with the small molecule BTK inhibitor CGI1746 prevented arthritis development and decreased autoantibody formation in a murine collagen induced arthritis (CIA) model, which are B-cell-dependent (Di Paolo et al., 2011). Similarly, ibrutinib reversed joint inflammation, decreased infiltration of granulocytes and macrophages (and correspondingly decreased synovial fluid proinflammatory cytokines), and was protective against joint destruction in a murine CIA model (Chang et al., 2011; Honigberg et al., 2010).
Meanwhile, blocking FcR (specifically, FcγRIII) signaling with CGI1746 significantly diminished proinflammatory cytokine production by murine macrophages, and prevented arthritis development in a murine anti-collagen antibody-induced arthritis (CAIA) model, which is an FcγR-dependent (and thus myeloid cell-dependent) model of autoantibody-mediated arthritis (Di Paolo et al., 2011). Ibrutinib likewise prevented inflammation, blocked myeloid cell infiltration, and was protective against cartilage and bone destruction in a murine CAIA model (Chang et al., 2011).
Given the impressive preclinical activity of small molecule BTK inhibitors in murine models of RA, several BTK inhibitor compounds are currently being investigated in Phase I (GDC-0834, HM-71224) and II (CC-292) clinical trials for this disease (Whang & Chang, 2014).
1.3.6.2 Multiple Myeloma
Multiple myeloma (MM) is characterized by the clonal proliferation of plasma cells in the bone marrow, as well as the presence of osteolytic bone lesions. MM patients exhibit excessive osteoclast (OC) activity, with inadequate opposing osteoblast (OB) activity and thus significant bone resorption. Interactions between MM cells and the cells of their microenvironment (bone marrow stromal
30 cells (BMSCs), OBs, and OCs) are critical for MM cell proliferation and survival, and BTK signaling mediates some of these interactions.
BTK, which is expressed in primary MM cells and in a subset of MM cell lines (Bam et al., 2013; Tai et al., 2012), appears to be important for MM cell growth, survival, migration, and adhesion to BMSCs: ibrutinib treatment reduced proliferation and induced cytotoxicity in the BTK-expressing MM cell line INA6 stimulated with IL-6, or when cocultured with patient-derived BMSCs or OCs (Tai et al., 2012). Ibrutinib similarly reduced MM burden in SCID-hu mice. BTK in MM cells was activated in response to the chemokine SDF-1 (Bam et al., 2013; Tai et al., 2012), and ibrutinib treatment of SDF-1-stimulated MM cells (both cell lines and primary MM samples) blocked cell migration and adherence to BMSCs (Tai et al., 2012).
BTK and TEC kinase are strongly expressed in OCs (but not OBs) and are required for osteoclast differentiation (osteoclastogenesis), as evidenced by the observation that Tec-/-Btk-/- mice exhibit decreased bone resorption and consequent osteopetrosis (Shinohara et al., 2008). BTK inhibition with ibrutinib blocked osteoclastogenesis and reduced bone resorption in healthy donor- derived precursor OC cells stimulated with M-CSF and RANKL and in SCID-hu mice (Tai et al., 2012).
Ibrutinib is currently under investigation for the treatment of relapsed/refractory multiple myeloma, both as a single agent and as a combination candidate with dexamethasone, or carfilzomib and dexamethasone, in two Phase II clinical trials (NCT01478581 and NCT01962792).
1.3.6.3 Acute Myeloid Leukemia
31 Acute myeloid leukemia (AML) is characterized by excessive proliferation of aberrantly differentiated hematopoietic cells of the myeloid lineage. BTK expression in AML cell lines and primary blasts was first reported in 1993 by de Weers et al. and has been extensively corroborated by other groups in the years since. While the presence of non-mutated BTK expression (Ritis et al., 1998) in AML cells is well established, the role of this kinase in this disease has only recently been investigated. Rushworth et al. (2014) were the first to identify the presence of constitutively active BTK (by detection of Tyr223 phosphorylation) in AML cell lines and primary blasts, noting a positive correlation between the pBTK to total BTK ratio and ibrutinib sensitivity. They also noted that BTK knockdown impaired colony formation by AML progenitor cells in a subset of patients. In a separate study, this group demonstrated the impact of BTK inhibition on AML cell migration, demonstrating that at clinically relevant concentrations, ibrutinib can block SDF1-induced CXCR4-mediated migration of AML cell lines and primary patient blasts (Zaitseva et al., 2014). BTK knockdown yielded a similar, though less dramatic, inhibitory effect on migration, suggesting that BTK is involved in signal transduction from the CXCR4 receptor (Zaitseva et al., 2014).
Another important study investigated the impact of BTK signaling in AML cell survival and proliferation in both FLT3-ITD positive and FLT3-ITD negative AML cell lines. Oellerich et al. (2015) demonstrated that in AML cells harboring the FLT3-ITD mutation, BTK couples FLT3-ITD signaling to STAT5 and MYC activation. Interestingly, in AML cells expressing the FLT3 wild type receptor, BTK does not interact with FLT3, but instead couples TLR9 signaling to STAT5 and NFkB activation.
1.3.6.4 Prostate Cancer
BTK has been reported as a potential therapeutic target in prostate cancer. Guo et al. (2014) were the first to report overexpression of BTK in this disease and
32 observed that the degree of BTK overexpression correlated with tumor grade, a finding that was more recently corroborated by Kokabee et al. (2015). BTK silencing in the prostate cancer cell line PC3 was found to reduce cell proliferation, an effect which was not observed following BTK knockdown in a normal prostate cell line (RWPE1) (Guo et al., 2014). Moreover, treatment of prostate cancer cell lines LNCaP and DU145 with ibrutinib and other small molecule BTK inhibitors was found to reduce cell growth and viability, albeit only modestly at clinically achievable concentrations (Kokabee et al., 2015).
Combined knockdown of BTK and the TEC family member BMX (which has known oncogenic activity in prostate cancer) in PC3 cells additively reduced cell proliferation. A small molecule dual BTK/BMX inhibitor, CTN06, demonstrated potent autophagic and apoptotic activity against several prostate cancer cell lines and in a PC3 xenograft model (Guo et al., 2014).
Interestingly, Kokabee et al. (2015) found that two different BTK isoforms are commonly expressed in prostate cancer tissue samples and cell lines: BTK-A (the isoform expressed in B-cell cancers) and BTK-C, with the latter isoform having higher expression in cell lines of this tumor type.
33 1.4 EGFR inhibitors in AML: Anti-Leukemic Mechanisms of Action and Preclinical and Clinical Activity
There is an unmet need for novel approaches to acute myeloid leukemia (AML) treatment, where five-year patient survival is 25.9% (NIH, 2012) despite aggressive chemotherapy treatment regimens. The need for new therapies is especially great for those older than the age of 65 for two reasons: first-line chemotherapy is less tolerable for these individuals (Döhner et al., 2015), and AML prevalence rises sharply in this age group (NIH, 2012).
Small molecule epidermal growth factor receptor (EGFR) inhibitors are clinically approved for use in non small-cell lung cancer (NSCLC) and pancreatic adenocarcinoma. They are well tolerated in elderly patients, prolonging patient survival often without compromising quality of life. Within the last ten years, several groups have described the activity of EGFR inhibitors—specifically erlotinib and gefitinib—against AML, despite the absence of EGFR expression in these tumor cells. This review will summarize the mechanisms of action that likely account for these desirable off-target effects, and will summarize the current preclinical and clinical evidence for their indicated use in the treatment of AML.
1.4.1 Development of Small Molecule EGFR Tyrosine Kinase Inhibitors
Small-molecule inhibitors of EGFR were developed as a strategy to block signaling from this receptor in solid tumors exhibiting increased activation of this pathway. Activation of EGFR by ligand-receptor binding and subsequent receptor homo- or heterodimerization promotes signal transduction through the
34 Ras/Raf/MAPK and PI3K/Akt/mTOR pathways, which in turn stimulate cell proliferation and survival, respectively (Sharma et al., 2007; Siegelin & Borczuk, 2014). Given the pro-survival and proliferative downstream effects of this pathway, it is not surprising that aberrant EGFR signaling—due to mutations conferring constitutive receptor activation, receptor overexpression, ligand upregulation, or reduced receptor turnover (Ciardiello & Tortora, 2001)—can strongly favor tumorigenesis.
The EGFR tyrosine kinase inhibitors (EGFR-TKIs) erlotinib and gefitinib compete with ATP for binding to the cytoplasmic kinase domain of this kinase, resulting in the inhibition of C-terminal autophosphorylation (Moyer et al., 1997; Wakeling et al., 2002). These EGFR-TKIs therefore prevent the formation of docking sites for downstream effectors of EGFR signaling.
Gefitinib was the first oral EGFR-TKI to receive FDA approval. It is currently approved for first-line use in NSCLC patients with EGFR exon 19 deletions or exon 21 L858R mutations (FDA, 2015b). Erlotinib is also currently indicated for first-line use in EGFR-mutant (exon 19 deletions or exon 21 L858R) NSCLC and in combination with gemcitabine in the treatment of locally advanced, metastatic, or unresectable pancreatic cancer. Erlotinib is also approved for use as maintenance therapy in locally advanced or metastatic NSCLC following first-line treatment with platinum-based chemotherapy, and in NSCLC patients who have failed at least one chemotherapy regimen (FDA, 2013).
1.4.2 Expression of EGFR in AML Cells
To investigate EGFR inhibitor targets in AML, several groups have examined the expression of this receptor in AML cell lines and primary blasts. Sun et al. (2012) noted EGFR mRNA expression by RT-PCR in 48 of 143 AML samples of various
35 French American British subtypes, however the majority of studies are in conflict with these findings, having reported no EGFR expression in this tumor type.
Walz et al. (1993) were the first to report the absence of EGFR expression in the HL60 cell line. Stegmaier et al. (2005) and Boehrer et al. (2008a) confirmed the lack of EGFR expression in HL60, in addition to determining that EGFR expression was absent in Kasumi-1, KG1, and P39 cells. In line with these observations, EGFR was undetectable in primary AML blasts from eight patients (DeAngelo et al., 2014). Finally, in agreement with the above observations, querying EGFR expression using the Cancer Cell Line Encyclopedia revealed lower EGFR mRNA levels in AML cell lines relative to cell lines of other tumor types, except for T-ALL (Barretina et al., 2012).
1.4.3 Preclinical EGFR-TKI activity against AML
1.4.3.1 Differentiation
The differentiating capacities of erlotinib and gefitinib have been examined in several AML cell lines as well as primary AML blasts. Gefitinib treatment was found to induce functional, morphologic, and gene expression changes (Stegmaier et al., 2005) in the AML cell lines HL60, Kasumi-1, and U937, which were consistent with their maturation (Stegmaier et al., 2005). Erlotinib induced morphologic maturation of KG1, HL60, and P39 cell lines, with P39 and HL60 expressing the myeloid differentiation marker CD11b in response to this drug (Boehrer et al., 2008a; Boehrer et al., 2008b). Erlotinib also induced CD11b expression in CD34+ primary AML cells (Boehrer et al., 2008a).
Lainey et al. (2013b) found that as single agents, gefitinib and erlotinib had negligible effects on HL60 and primary AML blast differentiation, however they noted that both drugs potentiated the differentiation capacities of all-trans retinoic
36 acid (ATRA) and vitamin D3 in these cell lines (based on changes in CD11b and
CD14 marker levels), and that erlotinib potentiated ATRA and vitamin D3- mediated differentiation in a subset of primary AML samples.
1.4.3.2 Cell Cycle Arrest and Cell Death
Erlotinib has been shown to induce G1 arrest (Boehrer et al., 2008a) and subsequent apoptosis in KG1 cells (Boehrer et al., 2008a; Boehrer et al., 2008b). Neither erlotinib, nor gefitinib induced apoptosis in HL60, P39, MV4-11, MOLM- 13, or U937 cells (Boehrer et al., 2008a; Boehrer et al., 2008b). Erlotinib induced modest cell cycle arrest in HL60 and P39 cells (Boehrer et al., 2008a).
Erlotinib treatment of SCID mice prevented tumor formation following intraperitoneal KG1 cell inoculation and significantly increased tumor-free survival (Boehrer et al., 2008a). Primary CD34+ AML cells were also more sensitive than normal CD34+ hematopoietic cells to apoptosis induction by erlotinib and gefitinib (Boehrer et al., 2008a; Boehrer et al., 2008b). Likewise, primary AML samples were preferentially sensitive to gefitinib treatment: gefitinib
IC50 in six out of eight primary AML samples was less than 5 µM, while the average IC50 in five peripheral blood mononuclear control samples was greater than 9 µM using the Cell Titer Glo assay (Stegmaier et al., 2005).
1.4.4 Proposed Anti-Leukemic Targets of EGFR-TKIs
1.4.4.1 JAK2 Inhibition
JAK/STAT signaling is increased in AML blasts (Gouilleux-Gruart et al., 1996; Ikezoe et al., 2011) and AML progenitor (CD34+) cells relative to normal hematopoietic stem cells (Cook et al., 2014). JAK2 knockdown by siRNA and
37 treatment with JAK1/2 inhibitors blocked phosphorylation of STAT3/5 and reduced colony formation and increased Annexin V staining in primary AML CD34+ cells, implicating JAK2 as a promising therapeutic target in AML (Cook et al., 2014).
Boehrer et al. (2008a) reported decreased phosphorylation of JAK2 at Tyr1007/1008 and its substrate, STAT5 (Tyr694) in response to erlotinib treatment of KG1 cells. This group also demonstrated the functional importance of erlotinib-mediated inhibition of JAK2-STAT5 signaling using siRNA knockdown of JAK2. KG1 cells transfected with JAK2 siRNA recapitulated the reduced STAT5 phosphorylation and increased apoptosis induction seen with erlotinib treatment.
1.4.4.2 SRC Family Kinase Inhibition
SRC family kinases (SFKs)—particularly the family member LYN—are reportedly overexpressed and constitutively active in primary AML blasts and the primitive CD34+CD38-CD123+ fraction, as evidenced by the presence of SFK tyrosine phosphorylation at Y416 in these cells (Dos Santos et al., 2008). LYN was also found to act as an upstream effector of mTOR signaling by this group. Inhibition of SFKs has been shown to induce G1 arrest and inhibit colony formation in primary AML cells, providing a rationale for targeting these kinases in the treatment of AML (Dos Santos et al., 2008).
Boehrer et al. (2011) found that erlotinib decreased SFK phosphorylation in KG1 cells and AML blasts from one patient, and induced autophagy by inhibition of mTORC1, as demonstrated by reduction of p70S6K phosphorylation. Weber et al. (2012) validated the finding of decreased SFK phosphorylation following erlotinib (and gefitinib) treatment of KG1 cells using quantitative phospho-mass
38 spectrometry. This group also demonstrated—using chemical proteomics and an in vitro kinase assay—that these compounds directly bind SFKs.
1.4.4.3 SYK Inhibition
Spleen tyrosine kinase (SYK) has been proposed as a therapeutic target in AML. It is constitutively phosphorylated at Y525/526, a marker associated with its activation, in multiple AML cell lines and primary blasts (Hahn et al., 2009), and is an upstream effector of mTOR signaling in this disease (Carnevale et al., 2013). Genetic knockdown of SYK has been shown to induce differentiation and reduce proliferation of several AML cell lines, and the small molecule SYK inhibitor R406 reduced tumor burden in KG1- and primary AML-engrafted mice (Hahn et al., 2009). SYK was identified as a target of gefitinib in AML in a phospho-mass spectrometry study carried out by Hahn et al. (2009), in which SYK phosphorylation (and the phosphorylation of SYK substrates) in HL60 cells was reduced in response to gefitinib treatment. SYK was further confirmed as a target of gefitinib (and erlotinib) in a study carried out by Weber et al. (2012). In this phosphoproteomics study, erlotinib and gefitinib reduced SYK-Tyr352 phosphorylation in KG1 cells. Through chemical proteomics and in vitro kinase activity assays, SYK was confirmed as an indirect target of both kinase inhibitors.
1.4.4.4 Bruton’s Tyrosine Kinase Inhibition
Bruton’s tyrosine kinase (BTK) is constitutively active in AML cell lines and primary blasts (Rushworth et al., 2014). It has been proposed as a therapeutic target in AML based on its reported capacity to promote AML cell proliferation and survival by mediating signal transduction from the receptor tyrosine kinase FLT3 in FLT3-mutant (FLT3-ITD) AML cells, and from TLR9 in FLT3-wild type cells (Oellerich et al., 2015). BTK has also been shown to promote AML cell
39 migration (Zaitseva et al., 2014). In a study carried out by Weber et al. (2012), erlotinib and gefitinib were found to reduce phosphorylation of BTK at Tyr551 (phosphorylation at this site is required for BTK activation). This work also demonstrated that these kinase inhibitors target BTK directly.
1.4.4.5 Inhibition of ATP-Binding Cassette Transporter Efflux Activity
Expression and enhanced activity of ATP-binding cassette (ABC) transporter family members such as P-glycoprotein (P-gp) in AML cells is a predictor of poor patient prognosis (Campos et al., 1992; Pirker et al., 1991; Zöchbauer et al., 1994). ABC transporters mediate the extrusion of cytotoxins, such as chemotherapy drugs, from cells and their activity is thus associated with therapy resistance in AML. Inhibiting the activity of one or more of these transporters has been proposed as a strategy for the re-sensitization of AML cells to chemotherapy agents. The addition of the P-gp inhibitor cyclosporine to an AML chemotherapy regimen was found to prolong overall and relapse-free survival in unfavourable-risk AML patients (List et al., 2001). Moreover, P-gp inhibitor quinine addition to AML induction therapy was found to increase complete remission rates in treatment-naïve AML patients with increased rhodamine-123 efflux (Solary et al., 2003). However, subsequent clinical trials investigating the addition of the cyclosporine analog valspodar to AML regimens have not demonstrated benefit to older or younger patient populations (Kolitz et al., 2010; van der Holt et al., 2005)
Multiple small-molecule EGFR inhibitors are known to inhibit ABC transporters in many tumor types (Dai et al., 2008; Kuang et al., 2010; Shi et al., 2007), including AML. Erlotinib was found to inhibit the activities of P-glycoprotein (P- gp), breast cancer resistance protein (BCRP), and multidrug resistance- associated protein (MRP) in KG1 cells, as evidenced by increased retentions of the P-gp substrate 3,3’-diethyloxacarbocyanine (DiOC2(3)), the MRP substrate
40 calcein, and the BCRP substrate Hoechst33342 in response to gefitinib and/or erlotinib treatment (Lainey et al., 2012). Simultaneous inhibition of these transporters with erlotinib and gefitinib led to the accumulation of the chemotherapy agents doxorubicin, etoposide, and mitoxantrone, effectively sensitizing KG1 cells to chemotherapy-induced cell death. Erlotinib and gefitinib have also been found to potentiate the accumulation of the hypomethylating agent azacytidine in AML cells (presumably a consequence of ABC-transporter efflux inhibition). Combining these EGFR inhibitors with azacytidine was synergistically cytotoxic (Lainey et al., 2013a).
1.4.6 Clinical EGFR-TKI Activity Against AML
Clinical evidence for EGFR inhibitor activity against AML was first described in two case reports. In the former, Chan and Pilichowska (2007) reported complete AML remission (less than 3% bone marrow blasts with normal hematopoietic count recovery) in a 68 year-old male diagnosed with concomitant NSCLC and AML and treated with single-agent erlotinib for three months. The patient’s remission was maintained for six months following erlotinib discontinuation. The second case report described a 64 year-old male, also diagnosed with concomitant NSCLC and AML, who received daily erlotinib treatment for three months (Pitini et al., 2008). The patient’s AML remission was maintained for at least seven months.
To date, three clinical trials have investigated the activity of small molecule EGFR inhibitors as single agents in AML, with very modest results. A Phase II trial examining single-agent gefitinib treatment in 18 patients with intermediate-to- poor-risk cytogenetics noted zero objective responses (DeAngelo et al., 2014). A Phase I/II trial assessing response to erlotinib in 30 patients (12 with AML that progressed from myelodysplastic syndrome (MDS) and 18 with high-risk MDS (RAEB-2)) who had previously received azacytidine treatment reported
41 responses in six RAEB-2 patients (median duration of five months), of whom two achieved complete remission (Thepot et al., 2014). Finally, a Phase I trial carried out by Sayar et al. (2015) evaluated erlotinib use in 11 patients with AML, including nine patients with treatment-naïve disease. Peripheral blast counts were reduced greater than 50% in two patients, however both patients, as well as the remaining nine patients, experienced disease progression.
1.4.7 Summary
Several groups have demonstrated convincing evidence of preclinical EGFR- independent erlotinib and gefitinib activity in AML. Collectively, these groups have identified the cellular targets of these EGFR-TKIs that may plausibly account for their effects on differentiation, proliferation, and cell death: JAK2, SFKs, SYK, BTK, mTOR and ABC transporters are all inhibited by erlotinib and gefitinib, and have all been proposed as potential therapeutic targets in AML. To date, the impressive preclinical activity of these drugs has not been observed in the clinical trial setting, where responses to EGFR-TKIs have been modest at best: erlotinib only mildly reduced bone marrow blasts in a small subset of AML patients (Sayar et al., 2015), and produced complete remissions or hematologic improvement in a subset of high-risk MDS patients (Thepot et al., 2014).
While it is difficult to pinpoint the exact reason for the failure of these TKIs in AML clinical trials, it is important to note that none of these trials assessed the pharmacodynamic effects of these TKIs on their reported targets in AML, meaning that it is not known whether these drugs inhibited any of the targets that may account for the anti-AML activity of erlotinib or gefitinib.
Finally, it is also important to note that these trials did not assess the clinical activity of erlotinib or gefitinib in combination with other chemotherapy agents approved for use in AML. Given that erlotinib and gefitinib exhibit profound
42 preclinical synergistic cytotoxicity with chemotherapy agents, further preclinical and clinical investigation of these EGFR-TKIs in combination with AML chemotherapies may be warranted.
43 1.5 Ethacridine Lactate
1.5.1 Ethacridine Lactate Indications
Ethacridine lactate (2-ethoxy-6,9-diaminoacridine monolactate, see Figure 1-2 for molecular structure) is an antiseptic ointment with modest activity in venous leg ulcers (O’Meara et al., 2014). Ethacridine is also used as an extra-amniotic second-trimester abortifacient in China and Cuba (Boza et al., 2008; Hou et al., 2010). When administered in conjunction with tannin albuminate, ethacridine lactate has moderate effectiveness as chemoprophylaxis for travellers’ diarrhea (Ericsson, 2005).
Figure 1-2: Structure of Ethacridine lactate Source: www.sigmaaldrich.com
44 1.5.2 Ethacridine Lactate Mechanisms of Action
1.5.2.1 Poly(ADP-ribose) Glycohydrolase Inhibition
Poly(ADP-ribose)ylation, also known as PARylation, is a post-translational modification of nuclear and cytoplasmic acceptor proteins that contributes to cellular processes such as DNA damage repair, transcriptional regulation, and cell death (reviewed by Feng and Koh (2013)). In the presence of cellular stressors such as DNA damage, activated poly(ADP-ribose) polymerase 1 (PARP1) catalyzes the transfer of PAR moieties to acceptor proteins such as PARP1 itself, histones, DNA repair proteins, transcription factors, and chromatin modulators. The activity of PARP1 is opposed by poly(ADP-ribose) glycohydrolase (PARG), which hydrolyzes PAR polymers (Figure 1-3). Closely coordinated activity between PARP1 and PARG is critical, as unopposed PARP1 activation (and thus PAR accumulation) can lead to necrotic (Feng & Koh, 2013) or apoptosis-inducing factor-mediated cell death (Yu et al., 2006; Yu et al., 2002; Zhou et al., 2011).
Ethacridine lactate is a PARG inhibitor (Bernardi et al., 1997; Boulikas, 1990; Tavassoli et al., 1985). Ethacridine blocks PARG activity by binding directly to PAR polymers, preventing PARG binding and thus PARG-mediated PAR catabolism (Tavassoli et al., 1985).
1.5.2.1.1 Other Chemical PARG Inhibitors
Tannins, which are naturally occurring polyphenolic compounds derived from plants, are also reported PARG inhibitors (Aoki et al., 1993; Tanuma et al., 1989; Tsai et al., 1992). Gallotannin is the best studied of the tannins, and consists of trigalloylglucose, tetragalloylglucose, and pentagalloylglucose compounds, which are cell membrane permeable and inhibit PARG activity at an in vitro IC50 of
45 approximately 18-33 µM (Aoki et al., 1993; Tsai et al., 1992). Tsai et al. (1992) and Aoki et al. (1993) also noted a positive relationship between the number of galloyl groups and the degree of PARG inhibition in vitro.
Adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) is a more potent inhibitor of PARG, relative to tannins. It is an ADP-ribose analogue that inhibits PARG activity through partial noncompetitive inhibition, with an IC50 of 120nM (Slama et al., 1995a; Slama et al., 1995b). The use of this compound is however limited to enzymatic assays, as this compound is not cell membrane permeable (Feng & Koh, 2013).
1.5.2.2 Non-Genotoxic Activation of p53
Ethacridine lactate is a reported inducer of p53 activation via the ribosomal stress pathway. In a recent study by Morgado-Palacin et al. (2014), ethacridine at a concentration of 5 µM increased p53 expression and induced nucleolar disruption—denoted by an increase in the ratio of rounded (disrupted) to irregular (undisrupted) nucleoli—but failed to induce DNA damage, as indicated by the absence of γH2A.X foci. The likely cause of nucleolar disruption-mediated p53 induction by ethacridine was inhibition of rRNA transcription by defective RNA polymerase I: in this study, ethacridine treatment led to degradation of the essential RNA polymerase I component, RP194. This group further demonstrated that non-genotoxic p53 activation by acridine derivatives was sufficient to induce cell cycle arrest and apoptosis in TP53-wild type, but not TP53 knock-out HCT116 (human colon carcinoma) cells.
46
Figure 1-3: Mechanism of PARG activity PARP uses NAD+ as a substrate for PAR synthesis. PAR is assembled as polymers onto proteins. PARG hydrolyzes PAR polymers, releasing ADP-ribose. Reproduced from Feng and Koh (2013), license #3817021464698.
47
Chapter 2: Project Rationale and Aims
48 2.1 Thesis Aims
Given the limited effectiveness of currently approved therapy, novel approaches to the treatment of AML are urgently needed for this disease. Identifying clinically approved drugs that enhance the anti-AML activity of agents that are currently approved for use in – or under investigation for – the treatment of AML would provide a new therapeutic strategy that could be rapidly advanced to the clinic. The central objective of this thesis was to identify synergistically cytotoxic combinations of approved drugs that preferentially kill AML cells, and to uncover the mechanisms by which these drug pairs synergize.
2.1.1 Aim I: Identify compounds that synergize with ibrutinib in AML
Several groups have described a potential role for the Bruton’s tyrosine kinase inhibitor ibrutinib in the treatment of AML. With the goal of enhancing ibrutinib’s anti-AML activity, we used a combination high-throughput screening approach to identify ibrutinib-sensitizing agents. We subsequently investigated the synergistic mechanism between ibrutinib and the top synergistic screen hit, hypothesizing that this synergistic activity was dependent upon ibrutinib-mediated BTK inhibition.
2.1.2 Aim II: Evaluate the mechanism of synergy between ibrutinib and daunorubicin in AML
Ibrutinib was previously reported to synergize with daunorubicin in AML cells. We hypothesized that this synergy was dependent upon ibrutinib-mediated BTK inhibition. We evaluated the role of BTK in ibrutinib-daunorubicin synergy by treating BTK-knockdown AML cell lines with daunorubicin. We further evaluated
49 this synergistic mechanism by measuring combination-induced reactive oxygen species production and ibrutinib-mediated intracellular daunorubicin accumulation.
2.1.3 Aim III: Identify compounds that synergize with erlotinib in AML
The epidermal growth factor receptor (EGFR) inhibitor erlotinib has been reported to exert modest EGFR-independent anti-AML activity in clinical trials. We sought to identify erlotinib combination candidates by carrying out a combination high-throughput chemical screen against this drug in erlotinib- insensitive AML cell lines. We subsequently delineated the synergistic mechanism of action between erlotinib and the top synergistically cytotoxic hit using mass spectrometry.
50
Chapter 3: Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism
This chapter has been published:
Rotin LE, Gronda M, MacLean N, Hurren R, Wang X, Lin F, Wrana J, Datti A, Barber DL, Minden MD, Slassi M, Schimmer AD (2016). Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism. Oncotarget, 7(3): 2765-2779. doi: 10.18632/oncotarget.6409.
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51 3.1 Abstract
Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease.
52 3.2 Introduction
Ibrutinib is a small-molecule Bruton’s tyrosine kinase (BTK) inhibitor approved for clinical use in several B-cell malignancies, including chronic lymphocytic leukemia (CLL). Inhibition of BTK induces cell death by blocking constitutive B- cell receptor (BCR) signaling and impairing tumor-microenvironment interactions in CLL cells (Herman et al., 2011; Ponader et al., 2012). BTK is expressed in almost all B-hematopoietic malignancies, but is also expressed in myeloid cells and myeloid malignancies where it can be activated through mechanisms distinct from BCR signaling. Since BTK is expressed in myeloid cells, we evaluated ibrutinib in acute myeloid leukemia (AML).
AML is a hematologic malignancy characterized by the overproduction of poorly differentiated myeloid-lineage cells (Löwenberg et al., 1999). Previously, other groups reported increased expression and constitutive activation of BTK in AML cell lines and primary AML patient samples (Barretina et al., 2012; de Weers et al., 1993; Oellerich et al., 2015; Rushworth et al., 2014; Wu et al., 2015). BTK mediates signal transduction from the FLT3-ITD, TLR9 and CXCR4 receptors in AML cell lines, thereby promoting leukemic cell survival, growth, and migration (Oellerich et al., 2015; Zaitseva et al., 2014). We further characterized the anti- AML activity of ibrutinib and identified drugs that sensitize AML cells to ibrutinib. Through exploration of the synergistic activity of ibrutinib with other drugs, we uncovered a BTK-independent role for ibrutinib with potential clinical utility in AML.
53 3.3 Methods
3.3.1 Materials
BTK and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA), BTK pTyr-223 antibody was obtained from Abcam (Cambridge, MA), and β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were acquired from GE Healthcare (Buckinghamshire, UK). Kinase inhibitors ibrutinib, CC-292, ONO–4059, PIM1/2, and STO-609 were provided by the Ontario Institute for Cancer Research (Toronto, ON, Canada). Ibrutinib was also obtained from Selleckchem (Houston, TX), as was olaparib. Z-VAD-FMK was purchased from Enzo Life Sciences (Farmingdale, NY). Ethacridine lactate, gallotannin, sulforhodamine-B, hydrogen peroxide, puromycin, and shRNA plasmid-containing bacterial glycerol stocks were purchased from Sigma-Aldrich (St. Louis, MO). The library of internationally prescribed drugs was purchased from MicroSource Discovery Systems, Inc. (Gaylordsville, CT). Alamar Blue was purchased from Life Technologies (Carlsbad, CA) and carboxy-H2DCFDA-FITC and MitoSOX Red were obtained from Molecular Probes/Life Technologies (Eugene, OR).
3.3.2 Cell Culture
TEX cells (Warner et al., 2005) were provided by Dr. John Dick (Ontario Cancer Institute, Toronto, Canada) and grown in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 15% fetal bovine serum (FBS) (Seradigm/VWR, Radford, PA), 100 µg/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine (Life Technologies, Carlsbad, CA), 20 ng/ml SCF (Miltenyi Biotec, San Diego, CA), and 2 ng/ml IL-3 (R&D Systems, Minneapolis, MN). OCI-AML2, NB4, KG1a, Daudi, Thp1, and U937 cells were provided by Dr. Mark Minden (Ontario Cancer
54 Institute, Toronto, Canada). K562 and HL60 cells were provided by Dr. Suzanne Kamel-Reid (Ontario Cancer Institute, Toronto, Canada) and Jurkat D1.1 cells were provided by Dr. Pamela Ohashi (Ontario Cancer Institute, Toronto, Canada). OCI-AML2, K562, Thp1, and NB4 cells were grown in IMDM supplemented with 10% FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin. Daudi, Jurkat D1.1, KG1a, U937, and HL60 cells were grown in RPMI 1640 supplemented with 10% FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin.
All cell lines were maintained at 37°C and 5% CO2.
3.3.3 Primary cells
Bulk AML cells from AML patients and peripheral blood stem cells from healthy G-CSF-treated stem cell donors were isolated by Ficoll density centrifugation and apheresis, respectively. Isolated cells were maintained in IMDM supplemented with 10% FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin, at 37°C and 5%
CO2. All samples were obtained from consenting patients. The collection and use of human tissue for this study were approved by the University Health Network (Toronto, Canada) institutional review board.
3.3.4 In vivo Combination Treatment
Animal studies were carried out with the approval of the Princess Margaret Cancer Centre ethics review board, and in accordance with Canadian Council on Animal Care regulations. SCID mice were subcutaneously injected with 1 × 106 OCI-AML2 cells. Once tumors were palpable (8 days following injection), mice were treated with ibrutinib (300 mg/kg), ethacridine (20 mg/kg), both in combination (300 mg/kg ibrutinib + 20 mg/kg ethacridine), alongside vehicle control once per day, 5 days/week, for a total of 9 treatments. Mice were subsequently sacrificed and tumor volumes were measured.
55
3.3.5 Immunoblotting
Cells were washed twice with 1xPBS and lysed in 1xLaemmli or radioimmunoprecipitation assay (RIPA) buffer. Following quantification with the DC Assay (1xLaemmli) or the Bradford assay (RIPA), protein lysates were resolved by SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked for 1 hour at room temperature in 5% milk-TBST. Blotting with primary antibody was carried out in 5% milk-TBST or 5% BSA-TBST overnight at 4°C. Membranes were then incubated with HRP-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK) for one hour at room temperature. Proteins were detected by HRP chemiluminescence.
3.3.6 Cell Growth and Viability Assays
Cells were treated with drug(s) for 72 hours in a 96-well flat-bottomed, clear microplate. Cell growth and viability was determined by the Alamar Blue assay as per the manufacturers instructions or the sulforhodamine-B (SRB) assay as previously described (LaPointe et al., 2005). Cell viability and apoptosis was measured by staining cells with Annexin V-FITC (BioVision, Milpitas, CA) and propidium iodide-PE (Sigma-Aldrich, St. Louis, MO) as per manufacturer’s instructions. All flow cytometry experiments were carried out using Canto II 96w or Fortessa LSR X20 cytometers (BD Biosciences, San Jose, CA). Flow cytometry data were analyzed with FlowJo version 7.6.5 (TreeStar, Ashland, OR).
56 3.3.7 Combination High-Throughput Screen
TEX and OCI-AML2 cells were treated with ibrutinib at its respective IC10 and
IC25 values, alongside vehicle (DMSO) controls. Ibrutinib- and vehicle-treated cells were also treated with a library of known drugs at concentrations of 0.133 µM, 1.6 µM, 3.3 µM, 6.7 µM, and/or 13.3 µM. Combination-treated TEX and OCI-
AML2 cells were incubated for 72 h at 37°C and 5% CO2. The read-out for this assay was percent growth and viability, measured with the sulforhodamine-B (SRB) assay. These data were then used to calculate synergy according to Excess-over-Bliss additivism criteria.
3.3.8 Excess-over-Bliss Additivism for Calculating Synergy
Excess-over-Bliss additivism (EOBA) (Borisy et al., 2003) provides an estimate of resultant cytotoxicity when two drugs are combined. According to this model, any cytotoxicity unaccounted for by the added effects of both drugs is due to synergy between the two compounds. The formula for excess-over-Bliss is as follows:
EOBA = C − (A + B − (A × B)) where C is equal to the fractional inhibition of both drugs simultaneously, A is equal to the fractional inhibition of drug A, and B is equal to the fractional inhibition of drug B. Fractional inhibition is equal to 1.0 minus the viability (expressed as a value from 0.0–1.0). Positive EOBA values reflect a synergistic combination; the more positive the difference, the greater the synergy. Negative EOBA values reflect an antagonistic drug combination, while near-zero EOBA scores are indicative of an additive drug combination.
57 3.3.9 Intracellular and Mitochondrial Reactive Oxygen Species Measurement
Intracellular reactive oxygen species (ROS) in TEX and OCI-AML2 cells was measured by carboxy-H2DCFDA staining on flow cytometry. Cells were stained with 10 µM carboxy-H2DCFDA (dissolved in 100% ethanol) and incubated for 30 minutes at 37°C and 5% CO2. Dead cells were excluded by propidium iodide (PI) staining. Fold change in intracellular reactive oxygen species production was + − calculated by dividing the geometric mean of H2DCFDA , PI -staining treated + − cells by the geometric mean of H2DCFDA , PI -staining untreated (vehicle- treated) cells. Mitochondrial ROS was evaluated by the same procedure, using 5 µM MitoSOX (dissolved in DMSO) and Annexin V staining for dead cell discrimination.
3.3.10 shRNA Knockdown Experiments
Stable knockdown of BTK in TEX and OCI-AML2 was achieved using lentiviral transduction of short hairpin RNAs (shRNA) delivered by the PLKO.1 vector, which contains a puromycin resistance gene. A 72-hour puromycin selection (2 µg/mL in TEX, and 1 µg/mL in OCI-AML2) was used to select for transduced cells 24 hours after lentiviral infection. Following completion of puromycin selection, knockdown was confirmed by immunoblot. The following shRNA sequences directed against BTK (Accession No. NM_000061) were used: shRNA-BTK_974: 5′- GAAGCAGAAGACTCCATAGAACTCGAGTTCTATGGAGTCTTCTGCTTC-3′, and shRNA-BTK_1066: 5′- AGGAGGTTTCATTGTCAGAGACTCGAGTCTCTGACAATGAAACCTCCT-3′.
58 3.3.11 PARG Activity Assay
The HT Universal Colorimetric PARG Assay Kit (Trevigen, Gaithersburg, MD) was used to measure the PARG inhibitor activity of ethacridine. The assay was carried out as per manufacturer instructions.
3.3.12 Statistical Analysis
All graphed viability data are expressed as mean ± SD. Statistical significance was determined by the unpaired Student’s t test with Holm-Sidak correction for multiple comparisons or a one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. Statistical tests were performed using GraphPad Prism 6.03 software (La Jolla, CA).
59 3.4 Results
3.4.1 BTK is overexpressed and constitutively active in AML cells
To determine the relevance of BTK as a therapeutic target in AML, we examined the protein and mRNA expression of BTK in a panel of AML cell lines. Analysis of the Cancer Cell Line Encyclopedia (Barretina et al., 2012) demonstrated that AML cells expressed levels of BTK mRNA similar to B-cell malignancies (Figure 3-1). Likewise, a subgroup of primary AML patient samples had increased BTK expression (Figure 3-2A). Next, we evaluated the expression of BTK by immunoblotting in a series of AML cell lines. OCI-AML2, THP1, U937, NB4, K562, and the stem cell-like AML cell line TEX all expressed BTK, but this protein was not detectable in KG1a AML cells or Jurkat D1.1 T-ALL cells (Figure 3-2B). Phosphorylation of BTK at Tyr223, a marker of BTK activation (Park et al., 1996; Rawlings et al., 1996; Wahl et al., 1997), was detected in all cell lines expressing BTK (Figure 3-2B), suggesting constitutive BTK activity
60
Figure 3-1: BTK mRNA levels in AML cell lines are similar to those of B-cell malignancies. BTK mRNA expression AML relative to other cancer cell lines, as reported by the Cancer Cell Line Encyclopedia (Barretina et al., 2012).
61 3.4.2 AML cell lines are insensitive to chemical BTK inhibition with ibrutinib
To investigate the effects of BTK inhibition on AML viability and proliferation, we treated AML cells with increasing concentrations of ibrutinib. Phospho-BTK was reduced to undetectable levels at doses as low as 1 µM ibrutinib (Figure 3-2C). Compared to the sensitive B-lymphoblastic leukemia cell line, Daudi, the AML cell lines TEX, OCI-AML2, HL60, and U937 were relatively insensitive to ibrutinib, with IC50s ranging from 4- to 30-fold higher than Daudi cells in the Alamar Blue Assay, which measures cell proliferation and viability (Figure 3-2D) and much higher than the 1 µM concentration required to reduce levels of phospho-BTK. Similar insensitivity to ibrutinib was seen when measuring cell viability with Annexin V and PI staining (Figure 3-2E), which measures apoptosis. Interestingly, KG1a cells lacking detectable expression of BTK were the most sensitive to ibrutinib compared to other AML cell lines (KG1a IC50 = 2.87 µM by Alamar Blue assay).
62
Figure 3-2: AML cell lines express constitutively active BTK, but are insensitive to ibrutinib. (A) BTK mRNA expression in primary AML cells was determined by analysis of microarray gene expression (GEO accession code: GSE1159). BTK mRNA expression was examined in 267 patients with AML (Valk et al., 2004). (B) BTK and pBTK-Y223 expression in AML cell lines was determined by immunoblotting. (C) TEX and OCI-AML2 cells were treated with 1 µM ibrutinib for 1 h or 16 h. pBTK-Y223 and BTK expression in cell lysates was detected by immunoblotting. (D, E) AML cell lines were treated with increasing concentrations of ibrutinib over 72 h and cell growth and viability relative to untreated cells was determined by (D) Alamar Blue or (E) Annexin V and PI staining on flow cytometry. Data depict mean relative viability ± SD from a representative experiment performed in triplicate. Data are representative of three (D) or two (E) independent experiments.
63
3.4.3 A combination chemical screen with ibrutinib in AML cell lines identifies the PARG inhibitor, ethacridine lactate, as an ibrutinib sensitizer
Given the limited single-agent cytotoxicity in AML cell lines, we sought to determine whether we could identify drugs that sensitize AML cells to ibrutinib. To this end, we carried out a high-throughput combination chemical screen with ibrutinib in both TEX and OCI-AML2 cells. The cell lines were co-treated with ibrutinib and increasing concentrations of compounds from an in-house chemical library containing 240 internationally prescribed drugs for a total of 5046 different assays among the two cell lines in this screen. Following 72 hours of incubation, cell growth and viability were measured by the sulforhodamine-B (SRB) assay. Potential synergistic hits were identified using the excess-over-Bliss additivism (EOBA) formula and average positive EOBA scores for each combination were rank ordered (Figure 3-3A). Ethacridine lactate and pentamidine were top synergistic hits common to both TEX and OCI-AML2 cells. We validated both combinations in these cell lines, but pursued ethacridine lactate over pentamidine because of its greater synergy with ibrutinib (EOBA scores of up to 0.58 and 0.47 by Alamar Blue in TEX and OCI-AML2, respectively) (Figure 3-3B & 3-3C, Figure 3-4). The ibrutinib-ethacridine combination induced cell death, as determined by Annexin V and PI staining, but the mechanism of cell death was caspase-independent (Figure 3-5). Ibrutinib and ethacridine also induced strong synergistic cytotoxicity in U937, HL60, and K562 leukemia cells, but not in KG1a cells (Figure 3-6).
Ethacridine lactate is used clinically as a topical antiseptic (O’Meara et al., 2014) and intra-amniotic abortifacient (Mei et al., 2014). It is a DNA intercalator and putative poly(ADP-ribose) glycohydrolase (PARG) inhibitor (Boulikas, 1990; Tavassoli et al., 1985).
64
Figure 3-3: The PARG inhibitor ethacridine lactate sensitizes AML cell lines to ibrutinib.
(A) Ibrutinib was co-treated with 240 drugs in TEX and OCI-AML2 cells for 72h. Growth and viability was determined with the SRB assay and synergy was calculated using the EOBA formula as described in the methods. Compounds were ranked in order of increasing average positive EOBA score. (B, C) TEX and OCI-AML2 cells were combination-treated with ibrutinib and ethacridine for 72 h and cell growth and viability relative to untreated cells was determined by Alamar Blue. (B) Data represent mean
65 growth and viability ± SD from a representative experiment performed in triplicate. (C) EOBA synergy scores (shown) were calculated for each of the combinations tested. EOBA values > 0.1 (lightest grey) denote a synergistic combination, while values > 0.5 (darkest grey) denote a strongly synergistic combination. Data represent mean EOBA scores from a representative experiment performed in triplicate.
66
Figure 3-4: Combination chemical screen validation for pentamidine. TEX and OCI-AML2 cells were subjected to 72h treatment with concentrations of ibrutinib and pentamidine similar to those tested during the combination chemical screen. Cell growth and viability was measured with the SRB assay, and calculated relative to untreated cells. Data represent mean percent growth and viability ± SD and mean EOBA scores from a single experiment performed in triplicate.
67
Figure 3-5: Cell death caused by ibrutinib-ethacridine combination is caspase independent. Top: TEX and OCI-AML2 cells were subjected to combination ibrutinib (4 µM)- ethacridine (6 µM) treatment in the presence and absence of 50 µM Z-VAD-FMK (caspase inhibitor) for 48h. Viability was subsequently measured with Annexin V and PI staining on flow cytometry and calculated relative to vehicle-treated cells. Bottom: TEX and OCI-AML2 cells were treated at the indicated concentrations of ibrutinib and/or ethacridine for 48h, and induction of apoptosis was measured by Annexin V staining on flow cytometry.
68
Figure 3-6: The ibrutinib-ethacridine combination is strongly synergistic in HL60, U937, and K562, but not KG1a AML cell lines. AML cell lines were treated with increasing concentrations of ibrutinib and ethacridine for 72h. Relative growth and viability was measured with the Alamar Blue assay. Data depict mean growth and viability ± SD and mean EOBA scores from a representative experiment performed in triplicate.
69 3.4.4 The ibrutinib-ethacridine combination is preferentially cytotoxic to a subset of primary AML cells compared to normal hematopoietic cells
Having identified the combination of ethacridine and ibrutinib as synergistic in AML cell lines, we tested the combination in primary AML cells (n = 9) (see Table 3-1 for patient characteristics) and normal hematopoietic cells obtained from consenting donors of G-CSF mobilized stem cells for allotransplantation (n = 9). Primary cells were incubated with increasing concentrations of ethacridine and ibrutinib for 48 hours in Iscove’s Modified Dulbecco’s Medium supplemented with 10% fetal bovine serum, without additional growth factors, and viability was subsequently measured with Annexin V/PI staining and flow cytometry (Figure 3- 7). Similar to the AML cell lines, ibrutinib had minimal single-agent cytotoxicity, with IC50s exceeding 8 µM in all primary cells. We noted that primary AML cells, on average, were more sensitive to single-agent ethacridine and combination ibrutinib-ethacridine treatment compared to normals: a subset of 6 of 9 AMLs demonstrated greater than 70% cell death from the combination, while only 1 of 9 normals (Normal 2) exhibited similar sensitivity. However, in some normal samples, the drug combination induced ≥ 50% cell death, suggesting that the ibrutinib-ethacridine combination may also have toxicity towards some normal hematopoietic cells.
70
Table 3-1: Patient demographics.
Patient Characteristics Age at Sample Gender Cytogenetics Molecular ID Diagnosis Diagnosis 130794 AML 73 Male 46,XY[20] Not done 130819 AML, M5a 63 Female 46,XX[20] NPM1+, FLT3-ITD-, FLT3-TKD- 130826 AML, M4 58 Male 46,XY[20] NPM1+, FLT3-ITD+, FLT3-TKD- 130874 AML 69 Male 49,XY,+12,+16,+21[10] Not done 130877 AML 75 Male 48,XY,+9,+13[4]/46,XY[16] Not done 140994 AML 67 Male 45,XY,-7[10] NPM1-, FLT3-ITD-, FLT3-TKD- 141130 AML, M5b 80 Female Inconclusive CBFB-MYH11- 42~46,XX,- 2,der(3)add(3)(p21)?del(3)(q21q26),del(5)(q12), 150177 AML 53 Female der(7)t(7;?11)(p13;q13),del(8)(p21),add(11)(q13), Not done -18, add(20)(p13),+3mar[4]
150256 AML 24 Male 45,XY,der(6;7)t(6;7)(p21;q22)del(6)(q13q21)[17]/ Not done 46,XY[3]
71
72
Figure 3-7: The ibrutinib-ethacridine combination is preferentially cytotoxic to primary AML cells over normal hematopoietic cells. (Preceding page) Primary AML and normal hematopoietic cells (G-CSF mobilized peripheral blood stem cells) were treated with ibrutinib, ethacridine, or both in combination for 48 h. Viability was determined by Annexin V and PI staining. Data represent mean percent viability ± SD from a single experiment performed in triplicate. Ibru = ibrutinib, Ethac = ethacridine.
73 3.4.5 The combination of ibrutinib and ethacridine delays the growth of AML cells in vivo
To assess the in vivo efficacy and toxicity of ibrutinib in combination with ethacridine, we evaluated this combination in a mouse model of leukemia. SCID mice were injected subcutaneously with OCI-AML2 cells. When tumors were palpable, mice were treated with ibrutinib, ethacridine, or the combination of both drugs. The combination of ibrutinib and ethacridine decreased the growth of OCI- AML2 cells more than either drug alone (*P < 0.001 and **P < 0.0001). Of note, no toxicity from combination treatment was detected as measured by changes in body weight, behavior or gross examination of the organs at the end of the experiment (Figure 3-8).
74
Figure 3-8: Ibrutinib-ethacridine combination displays anti-AML activity in mice.
1 × 106 OCI-AML2 cells were subcutaneously injected in SCID mice. Eight days after injection, mice were treated with 300 mg/kg of ibrutinib by oral gavage, 20 mg/kg of ethacridine by i.p. injection, a combination of two drugs, or vehicle control (5% DMSO, 20% Cremophor, 0.9% NaCl) by oral gavage on the indicated days. Tumor volume (A) and body weight (B) were monitored over time. Mean ± SEM for tumor volume and mean ± SD for body weight, n = 7. *P < 0.001 and **P < 0.0001 from a two-way ANOVA with Tukey’s posttests comparing all treatment groups at day 18 and 20.
75 3.4.6 Ethacridine synergizes with other small molecule BTK inhibitors, but not inhibitors of unrelated kinases
We sought to investigate whether the observed synergy with ethacridine was specific to ibrutinib or a property common to other BTK inhibitors. We therefore tested ethacridine in combination with two other BTK inhibitors currently in clinical trials: CC-292 and ONO-4059. Cell growth and viability was measured 72 hours after incubation by the Alamar Blue assay and EOBA scores were calculated. CC-292 and ONO-4059 synergized with ethacridine in TEX and OCI- AML2 cells with efficacy similar to ibrutinib (Figure 3-9).
To further examine the specificity of the synergistic activity of ethacridine, we sought to determine whether this compound generally sensitized AML cells to kinase inhibitors. We therefore selected inhibitors of kinase targets bearing minimal sequence similarity to BTK. Specifically, we tested PIM1/2 and STO-609, inhibitors of Calcium/calmodulin-dependent protein kinase family members PIM 1/2 and CaMKK, respectively. TEX cells were treated with these compounds in combination with ethacridine. Synergy was assessed by EOBA calculation following viability determination at 72 hours with Annexin V and PI staining on flow cytometry. Neither PIM1/2, nor STO-609 synergized with ethacridine in TEX cells, with EOBA scores not exceeding 0.03 for either combination (Figure 3- 10A).
We also tested the combination of ethacridine with the ABL kinase inhibitor imatinib and the ABL and SRC family kinase inhibitor, dasatinib. Of note, ibrutinib is reported to inhibit SRC family kinases (Honigberg et al., 2010) as they share sequence homology to the TEC kinases. TEX and OCI-AML2 cells were combination-treated with ethacridine and these kinase inhibitors. Following a 72- hour incubation, cell growth and viability was determined by the Alamar Blue assay. The combinations produced primarily additive effects as calculated by the
76 EOBA formula (Figure 3-10B & Figure 3-11). Thus, the observed synergy with ethacridine appears specific for TEC family kinase inhibitors.
Figure 3-9: Ethacridine synergizes with other small-molecule BTK inhibitors. TEX and OCI-AML2 cells were treated with increasing concentrations of ethacridine and (A) CC-292 or (B) ONO-4059 for 72 h. Growth and viability was measured by Alamar Blue and EOBA synergy scores were calculated. Data depict mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of three independent experiments.
77
Figure 3-10: Ethacridine does not synergize with inhibitors of unrelated kinases. (A) TEX cells were combination-treated with ethacridine and PIM1/2 or STO-609 for 72 h. Viability was measured by Annexin V/PI staining and EOBA scores were generated. Combination ibrutinib-ethacridine treatment of TEX cells was included as a positive synergy control for this method of cell viability determination. (B) TEX cells were combination-treated with ethacridine and dasatinib or imatinib for 72 h. Growth and viability was measured by Alamar Blue and EOBA synergy scores were calculated. Data depict mean percent viability (A) or growth and viability (B) ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of three independent experiments.
78
Figure 3-11: Dasatinib and imatinib do not synergize with ethacridine in OCI- AML2 cells. OCI-AML2 cells were combination-treated with ethacridine and dasatinib (left) or imatinib (right) for 72 h. Cell growth and viability was measured with the Alamar Blue assay, and calculated relative to untreated cells. Synergy was calculated with the EOBA formula. Data represent mean percent growth and viability ± SD and mean EOBA scores from a single experiment performed in triplicate. Data are representative of three independent experiments.
79 3.4.7 Ibrutinib and ethacridine synergize to induce cell death via a ROS-dependent mechanism
To investigate the mechanism of synergy between ethacridine and ibrutinib, we examined ROS (reactive oxygen species) production in AML cells treated with the drug combination. Using carboxy-H2DCFDA staining and flow cytometry, we measured total intracellular ROS production after TEX and OCI-AML2 treatment with ibrutinib, ethacridine or the drug combination. At concentrations that were associated with synergistic cell death, neither drug alone markedly increased intracellular ROS production. However, ROS production in live cells was increased with the drug combination as early as two hours following treatment in both TEX and OCI-AML2 cells (Figure 3-12A). Moreover, the increased ROS production was functionally important for the observed cell death, as the addition of the anti-oxidant α-tocopherol abrogated cytotoxicity from the combination in both cell lines (Figure 3-12B). The observed increase in ROS following combination treatment did not appear to be mitochondrial in origin, as MitoSOX staining did not increase following combination treatment relative to single-agent treatment (Figure 3-12C). Thus, these findings suggest that the synergistic cytotoxicity caused by the ibrutinib-ethacridine combination is due to excessive intracellular, but not mitochondrial, ROS production.
80
Figure 3-12: The ibrutinib-ethacridine combination induces cytotoxic levels of intracellular ROS. (A) TEX and OCI-AML2 cells were treated with ibrutinib, ethacridine, or both in combination for 2, 6 or 24 h. Intracellular ROS was measured by carboxy-H2DCFDA staining and dead cells were excluded by PI staining on flow cytometry. Fold increase in ROS was calculated relative to the geometric means of carboxy-H2DCFDA (FITC) staining in untreated cells. H2O2 treatment served as a positive control for intracellular ROS generation. (B) TEX and OCI-AML2 cells were pre-treated with α-tocopherol prior to a 48 h incubation with ibrutinib and/or ethacridine. Viability was measured by Annexin V and PI staining, and calculated relative to respective untreated controls (+ or − α- tocopherol). (C)TEX and OCI-AML2 cells were treated with ibrutinib, ethacridine, or both drugs in combination for 2, 6 or 24 h. Mitochondrial ROS was measured by MitoSOX Red staining, with dead cell exclusion by Annexin V staining on flow cytometry. Fold increase in mitochondrial ROS was calculated relative to the geometric means of carboxy-H2DCFDA (FITC) staining in untreated cells. Antimycin A (50 µM) treatment served as a positive control for mitochondrial ROS generation.
81 Data represent mean fold increase in ROS production ± SD (A, C) or mean viability ± SD (B) from representative experiments performed in triplicate. Data are representative of two (A, C) or three (B) independent experiments. In all panels, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 as determined by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons (A), or unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%) (B).
3.4.8 The chemical PARG inhibitor gallotannin also synergizes with ibrutinib to induce cell death by excessive ROS production
Ethacridine is a putative PARG inhibitor (Boulikas, 1990; Tavassoli et al., 1985) and we demonstrated that ethacridine inhibited PARG (Figure 3-13A). Therefore, we evaluated the combination of ibrutinib and gallotannin, another reported PARG inhibitor (Aoki et al., 1993; Formentini et al., 2008; Tsai et al., 1992). We treated TEX and OCI-AML2 cells with increasing concentrations of ibrutinib and gallotannin over 48 hours and then measured viability with Annexin V and PI staining. The ibrutinib-gallotannin combination was also profoundly synergistic, yielding EOBA values of up to 0.60 and 0.72 in TEX and OCI-AML2 cells, respectively (Figure 3-13B). Likewise, pre-treatment with α-tocopherol abrogated ibrutinib-gallotannin cytotoxicity (Figure 3-13C). Pretreatment with the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib did not rescue combination-induced cytotoxicity (Figure 3-14). However, olaparib was directly toxic to the cells, thus potentially obscuring any protective effects.
82
Figure 3-13: The PARG inhibitor gallotannin synergizes with ibrutinib (A) Ethacridine’s inhibitory activity against PARG was determined using a cell-free colorimetric assay that measures levels of biotinylated PAR attached to histones in the presence of PARG enzyme. A loss of absorbance at 450 nm correlates with increased PARG activity. Relative PARG activity was calculated by comparing the loss of absorbance at 450nm in the presence of ethacridine to that of no PARG control (maximal absorbance at 450 nm). Data represent mean PARG activity ± SD from a single experiment performed in triplicate. (B) TEX and OCI-AML2 cells were treated with increasing concentrations of ibrutinib and gallotannin for 48 h. Viability was measured by Annexin V and PI staining and EOBA scores were calculated. Data represent mean EOBA scores from a representative experiment performed in triplicate. Data are representative of two (OCI-AML2) or three (TEX) independent experiments. (C) TEX cells were pre-treated with α-tocopherol and subjected to 48 h treatment with ibrutinib and gallotannin. Viability was measured by Annexin V and PI staining and calculated relative to untreated controls. Data represent mean viability ± SD from a representative experiment performed in triplicate. Data are representative of three independent experiments. In all panels, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 as determined by unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%) (C).
83
Figure 3-14: Treatment of TEX and OCI-AML2 cells with olaparib in combination with ibrutinib and ethacridine. TEX and OCI-AML2 cells were pre-treated with the PARP inhibitor olaparib 4 hours prior to a 72 h incubation with ibrutinib, ethacridine or both in combination at the indicated concentrations. Growth and viability was measured by the Alamar Blue assay and then calculated relative to untreated controls.
84 3.4.9 The synergy of ibrutinib with ethacridine is independent of the inhibitory effect on BTK
To determine whether synergy between ibrutinib and ethacridine was due to BTK inhibition by ibrutinib, we knocked down BTK with shRNA in TEX and OCI-AML2 cells. The reduction of BTK expression was confirmed by immunoblotting (Figure 3-15A) and qPCR (not shown). Knockdown cells were then treated with increasing concentrations of ethacridine and cell growth and viability was assessed by Alamar Blue. Despite substantial levels of BTK knockdown by shRNA, ethacridine treatment of BTK-knockdown cells was no more cytotoxic than ethacridine treatment of shRNA control cells (Figure 3-154A). These observations suggest that synergy of ibrutinib with ethacridine is independent of its inhibitory effect on BTK.
To further examine whether synergy of ibrutinib with ethacridine is due to targets beyond BTK, we tested the drug combination in Jurkat D1.1 cells, a T-acute lymphoblastic leukemia cell line that does not express BTK (Figure 3-2B). The ibrutinib-ethacridine combination synergized in Jurkat D1.1 cells, reaching EOBA values of 0.25 (Figure 3-15B), further supporting a synergistic mechanism for ibrutinib and ethacridine beyond AML cell lines analyzed in this study.
85
Figure 3-15: Ibrutinib’s synergy with ethacridine is independent of BTK. (A) TEX and OCI-AML2 cells were transduced with 2 different shRNAs targeting BTK or a non-targeting shRNA control in lentiviral vectors. On day 4 post-transduction, cells were treated with ethacridine at concentrations previously shown to synergize with ibrutinib. Growth and viability at 72 h post-ethacridine treatment was determined by Alamar Blue and calculated relative to untreated control. BTK knockdown was confirmed by immunoblotting. (B) Jurkat cells were treated with increasing concentrations of ibrutinib and ethacridine for 72 h. Cell growth and viability was determined by the Alamar Blue assay and synergy was calculated using the EOBA formula. Data depict mean percent growth and viability ± SD from a representative experiment performed in triplicate. Data in (A) and (B) are representative of two independent experiments. In all panels, ns = not significant, based on the results of an unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%).
86
Figure 3-16: Expression of TEC family kinases in AML cell lines. Expression of kinases BMX, TLK, TEC, and ITK in a panel of AML cell lines, Jurkat D1.1 and Daudi cells was determined by immunoblotting.
87 3.5 Discussion
The small molecule BTK inhibitor ibrutinib has demonstrated exceptional efficacy with minimal toxicity in several B-cell cancers. Ibrutinib is currently approved for clinical use in CLL, mantle-cell lymphoma (MCL) and Waldenström’s macroglobulinemia (WM), owing to its impressive patient response rates during recent clinical trials. With widespread BTK expression across B-cell malignancies and its role as a node in several oncogenic signaling pathways, this cytoplasmic kinase has emerged as an attractive therapeutic target for B-lymphoid cancers. Multiple clinical trials investigating ibrutinib alone and in combination for these diseases are currently underway.
In addition to its expression in B-cell malignancies, BTK is also expressed in myeloid cell lines and can be activated through mechanisms independent of the B-cell receptor (Doyle et al., 2007; Kawakami et al., 1994; Oellerich et al., 2015). Thus, targeting BTK with ibrutinib may have efficacy in myeloid malignancies such as AML.
In concordance with previous work by Rushworth et al. (2014) and Oellerich et al. (2015), we demonstrated the expression of constitutively active BTK in several AML cell lines. However, in contrast to these other studies, in the cell lines we tested, the cytotoxicity from ibrutinib was likely independent of its effects on BTK. Supporting this contention, of the tested AML cell lines, KG1a cells were the most sensitive to ibrutinib and yet lacked detectable BTK by immunoblot.
Moreover, the IC50 in TEX and OCI-AML2 leukemia cells were over 10-fold higher than the concentration of ibrutinib required to completely repress BTK phosphorylation and higher than the pharmacologically achievable concentrations in humans (Appendix 1). Consistent with these observations, ibrutinib has been reported to induce AML cell death via a BTK-independent mechanism (Wu et al., 2015).
88 Although ibrutinib was largely inactive as a single agent in the tested AML cells, we successfully sensitized two of the more resistant AML cell lines (TEX, IC50 =
13.01 µM and OCI-AML2, IC50 = 27.44 µM) to ibrutinib by combining the drug with the putative PARG inhibitor ethacridine. However, the observed synergistic cytotoxicity was independent of the inhibitory effect of ibrutinib on BTK. These findings suggest that ibrutinib may also have anti-AML activity that extends to targets beyond BTK.
In addition to inhibiting BTK, ibrutinib cross-reacts with TEC and SRC family kinases with similar efficacy (Honigberg et al., 2010). We noted that the SRC inhibitor dasatinib did not recapitulate ibrutinib’s synergy with ethacridine, however the BTK inhibitors ONO-4059 and CC-292, which have reported inhibitory activity against TEC family kinases (Akinleye et al., 2013; Ariza et al., 2013; Evans et al., 2013; Hendriks et al., 2014; Yoshizawa et al., 2012), did synergize with ethacridine. We therefore favor the TEC family kinases as likely targets of ibrutinib in its synergy with ethacridine in AML. To date, 5 TEC family members have been identified: BTK, BMX, TEC, ITK, and RLK (Schmidt et al., 2004a). ITK is expressed selectively in T cells and its inhibition by ibrutinib leads to decreased STAT6, IkBa, JUNB, and NFAT activity, as well as decreased intracellular calcium release (Dubovsky et al., 2013). BMX is expressed in hematopoietic progenitor cells and myeloid leukemias (Kaukonen et al., 1996; Weil et al., 1997) and has been found to mediate STAT3 activation and subsequent transformation by Src (Tsai et al., 2000). Interestingly, BMX, TEC, and RLK are all expressed in AML and Jurkat D1.1 cell lines (Figure 3-16). However, further investigations will be required to determine whether these TEC family members—or different kinases altogether—are targets of ibrutinib that explain its synergy with ethacridine. One possible strategy for ibrutinib target determination is a synthetic-lethal human kinome shRNA array, which would uncover kinases that when individually knocked down induce AML cell line sensitivity to ethacridine.
89 To our knowledge, this work is the first to report on the activity of PARG inhibitors such as ethacridine and gallotannin in AML. While further work is required to fully determine the impact of PARG inhibition in AML, it is interesting to speculate whether some of the single agent activity of ibrutinib in primary AML might be observed in patients with the lowest basal PARG expression.
The addition of poly(ADP-ribose) (PAR) moieties to target proteins alters their structure, function, and localization. PAR-ylation of target proteins is mediated by the PARP (Poly(ADP-ribose) polymerase) family of enzymes, of which PARP-1 is the most abundant and best characterized (Durkacz et al., 1980; Luo & Kraus, 2012; Virág et al., 2013). PARP adds PAR groups to target proteins, and these moieties are removed by PARG (Feng & Koh, 2013). Thus, genetic or chemical inhibition of PARG leads to the accumulation of excess PAR-ylated proteins. Through the accumulation of excess PAR-ylated proteins, PARG inhibition reduces the proliferation of malignant cells (Erdélyi et al., 2009; Pan et al., 2012), sensitizes cells to genotoxic stress (Cortes et al., 2004; Koh et al., 2004; Shirai et al., 2013) and inhibits cell signaling pathways including NFκB, p38 and ERK (Pan et al., 2012). Through these and other mechanisms, increased levels of PAR- ylated proteins may also promote ROS generation (Krenzlin et al., 2012), which is relevant to our observed mechanism of action of the drug combination.
Though the ibrutinib-PARG inhibitor combination produced striking synergistic cytotoxicity in AML cell lines, it is important to note that this combination also induced cytotoxicity in a subset of normal hematopoietic cells (Figure 3-7). This observation highlights a potential toxicity that would need to be assessed in the context of clinical trials of these agents.
Thus in summary, through identification of an ibrutinib combination that sensitizes resistant AML cell lines to this kinase inhibitor, we uncovered a novel BTK-independent role for ibrutinib in AML. Moreover, we present a potential role for PARG inhibition as a novel target for combination therapy in AML.
90
Chapter 4: Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells.
This chapter has been published as a Letter to the Editor:
Rotin LE, Gronda M, Hurren R, Wang X, Minden MD, Slassi M, Schimmer AD (2016). Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells. Leuk Lymphoma. Feb 17:1-5. Doi: 10.3109/10428194.2016.1138292 [Epub ahead of print]
Leukemia & Lymphoma © 06 Jan 2016 available: http://www.tandfonline.com/10.3109/10428194.2016.1138292
91 4.1 Abstract
Bruton’s tyrosine kinase (BTK) is a potential therapeutic target in acute myeloid leukemia (AML) and the small molecule BTK inhibitor ibrutinib has been found to potentiate the cytotoxic activity of the anthracycline daunorubicin in AML cells (Rushworth et al., 2014). We sought to determine whether synergy between ibrutinib and daunorubicin was mediated by the anti-BTK activity of ibrutinib. Our findings highlight the possibility that this synergistic mechanism is BTK- independent and unrelated to the reported capacity of ibrutinib to enhance cellular accumulation of other drugs. Reactive oxygen species (ROS) production may be functionally important for synergistic cell death.
92 4.2 Introduction
Ibrutinib is a well-tolerated BTK inhibitor that is used in the treatment of several B-lymphoid malignancies, particularly chronic lymphocytic leukemia. The TEC family kinase member BTK is expressed in most hematopoietic cell types, including myeloid-lineage cells, and is constitutively active in primary AML and AML cell lines (Oellerich et al., 2015; Rushworth et al., 2014). Both ibrutinib treatment and BTK knockdown have been shown to induce cell cycle arrest (Oellerich et al., 2015), apoptosis (Oellerich et al., 2015; Rushworth et al., 2014), and to block migration of AML cells (Zaitseva et al., 2014). Moreover, combining ibrutinib with first-line AML chemotherapy agents daunorubicin and cytarabine enhanced cell killing and reduced colony formation in AML bulk cells (Rushworth et al., 2014). Thus, ibrutinib has emerged as a possible combination therapy candidate for this disease. The purpose of the present study was to investigate the mechanism by which ibrutinib and daunorubicin synergize in AML cells.
93 4.3 Methods
4.3.1 Radiolabelled daunorubicin accumulation assay
Radioactivity of lysed cells was quantified with a Beckman LS6000IC liquid scintillation counter, and counts were normalized to cellular protein content.
All other experiments were carried out using materials and procedures previously described in Chapter 3.3 (pgs 56-61).
4.4 Results & Discussion
We first validated the previously reported synergistic cytotoxicity resulting from ibrutinib-daunorubicin and ibrutinib-cytarabine combinations in AML cell lines. We treated the AML cell lines OCI-AML2 with wild type FLT3 (Quentmeier et al., 2003) and TEX whose FLT3 mutation status is not known with increasing concentrations of ibrutinib and daunorubicin or cytarabine for 72 hours and measured cell viability using Annexin V and PI staining on flow cytometry. We determined the extent of any resultant synergy for each of the combinations tested with the excess-over-Bliss additivism (EOBA) formula, as previously described (Borisy et al., 2003). The ibrutinib-daunorubicin combination yielded synergistic EOBA scores of up to 0.33 in TEX and 0.25 in OCI-AML2 (Figure 4- 1). These findings are in line with a previous report describing synergistic killing activity between ibrutinib and doxorubicin, another anthracycline, in activated B- cell-like subtype of diffuse large B-cell lymphoma cells (ABC-DLBCL) (Mathews Griner et al., 2014). Meanwhile, the ibrutinib-cytarabine combination was only borderline synergistic, producing EOBA scores no greater than 0.11 in TEX and OCI-AML2 (Figure 4-2). Given that the ibrutinib-daunorubicin combination was more profoundly synergistic, we decided to investigate this combination further.
94
Figure 4-1: Ibrutinib and daunorubicin synergize in TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”) and daunorubicin alone and in combination for 72h. Viability was measured by Annexin V and PI staining on flow cytometry, then calculated relative to untreated cells (histograms). Excess-over-Bliss additivism (EOBA) scores for each tested combination are shown (tables); values >0.10 (grey) denote a synergistic combination, with higher EOBA values (darker grey) indicating greater synergy. Data represent mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of two independent experiments.
95
Figure 4-2: Combination ibrutinib-cytarabine treatment of TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”) and cytarabine alone and in combination for 72h. Viability was measured by Annexin V and PI staining on flow cytometry and then calculated relative to untreated cells (histograms). Excess-over-Bliss additivism (EOBA) scores are shown (tables). Data represent mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate.
Figure 4-3: Ibrutinib inhibits BTK phosphorylation. TEX and OCI-AML2 cells were incubated with 1 µM ibrutinib (+) or vehicle (-) for 1h. Cells were lysed and probed for BTK-pY223, a marker of BTK activation, on immunoblot.
96 To determine whether the synergy between ibrutinib and daunorubicin was dependent upon BTK inhibition by ibrutinib, we treated BTK-knockdown TEX and OCI-AML2 cells with daunorubicin to reproduce the sensitization observed in combination with ibrutinib. Both cell lines express constitutively active BTK, and treatment with ibrutinib inhibited BTK phosphorylation (Figure 4-3). We lentivirally transduced TEX and OCI-AML2 cells with five different BTK-targeting shRNAs, alongside one non-targeting shRNA control (GFP). On Day 3 following puromycin selection, transduced cells were treated with increasing concentrations of daunorubicin for 72 hours, and viability relative to respective untreated controls was subsequently measured with Annexin V and PI staining.
Despite knockdown of BTK to undetectable levels (Figure 4-4), little to no sensitization to daunorubicin was seen among five independent shRNA clones targeting BTK (Figure 4-5). While sensitization to daunorubicin was observed at single doses of daunorubicin in two shRNA clones targeting BTK, for the majority of clones and doses of daunorubicin tested, no increased sensitization was observed despite undetectable levels of BTK. Thus, these findings do not convincingly support BTK inhibition as the target of ibrutinib that would explain synergy with daunorubicin. Given that ibrutinib is known to inhibit other kinases, including SRC family kinases and other TEC family members (Honigberg et al., 2010), it is possible that off-target kinase inhibition by ibrutinib may explain its synergy with daunorubicin. Possible BTK-independent explanations for profound daunorubicin sensitization in clones shRNA-BTK508 and shRNA-BTK2490 include off-target shRNA effects (Manjunath et al., 2009).
97
Figure 4-4: BTK knockdown confirmation. BTK knockdown was confirmed by immunoblotting in TEX and OCI-AML2 cells.
Figure 4-5: Daunorubicin treatment of BTK-knockdown cells. TEX and OCI-AML2 cells were transduced with 5 shRNAs targeting BTK alongside a non-targeting shRNA control (GFP) via the PLKO.1 lentiviral vector, which contains a puromycin resistance gene. On day 3 post-puromycin selection, transduced cells were treated with increasing concentrations of daunorubicin for 72h, and viability relative to respective untreated controls was determined by Annexin V/PI staining. Data depict mean percent viability ± SD from representative experiments performed in triplicate. Data are representative of two independent knockdown experiments.
98 To address the possibility that a subpopulation of BTK-knockdown cells was highly BTK-dependent and thus killed off prior to treatment with daunorubicin, we treated BTK-knockdown TEX cells with ibrutinib and measured viability with Annexin V and PI staining. Compared to shRNA control, neither shRNA-BTK974, nor shRNA-BTK1066 was more resistant to ibrutinib at the concentrations evaluated (Figure 4-6), suggesting that the overall observed lack of daunorubicin sensitization in BTK knockdown cells was not the result of having selected for a subpopulation of BTK-independent cells.
One of the mechanisms by which kinase inhibitors have been shown to synergize with antineoplastic agents is through inhibition of ATP-binding cassette (ABC) transporter-mediated drug efflux activity (Lainey et al., 2012). Ibrutinib is a reported inhibitor of the MRP1 (ABCC1) transporter and sensitized MRP1- overexpressing cell lines to the MRP1 substrate vinblastine by enhancing its accumulation (Zhang et al., 2014). We sought to determine whether potentiation of daunorubicin accumulation by ibrutinib might explain the synergistic killing by this combination. We treated TEX cells with increasing concentrations of radiolabelled daunorubicin in the presence and absence of ibrutinib, comparing daunorubicin counts from lysed cells normalized to total protein content. Ibrutinib failed to increase daunorubicin accumulation (Figure 4-7), suggesting that inhibiting daunorubicin efflux is unlikely to be the mechanism by which ibrutinib and daunorubicin synergize in TEX cells.
99
Figure 4-6: Ibrutinib treatment of BTK-knockdown TEX cells. TEX shRNA-BTK974 and shRNA-BTK1066 cells were treated with ibrutinib alongside shRNA control and parental TEX cells for 48 hours (starting on day 3 post-puromycin selection). Viability was measured with Annexin V/PI staining and calculated relative to respective untreated controls. Data depict mean percent viability ± SD from representative experiments performed in triplicate. Data are representative of two independent knockdown experiments.
Figure 4-7: Daunorubicin accumulation in the presence or absence of ibrutinib. TEX cells were incubated with radiolabelled daunorubicin in the presence and absence of 8 µM ibrutinib for 3h. Data depict mean radioactive counts ± SD relative to protein content from an experiment performed in triplicate. Data are representative of two independent experiments.
100 We have previously noted that ibrutinib synergizes with reactive oxygen species (ROS)-inducing agents to cause cell death mediated by excessive ROS production (Rotin et al., 2014). Because daunorubicin is also a ROS-inducer (Gewirtz, 1999), we sought to determine whether cell death after treatment with the ibrutinib-daunorubicin combination was ROS-dependent. TEX and OCI-AML2 cells were treated with daunorubicin and ibrutinib with and without the antioxidant α-tocopherol. Pre-treatment with α-tocopherol dramatically rescued viability in TEX and OCI-AML2 cells treated with ibrutinib, daunorubicin, or the combination (Figure 4-8). We also tested whether the ibrutinib-daunorubicin combination increased total intracellular ROS production by staining cells with carboxy-
H2DCFDA. Mildly increased carboxy-H2DCFDA staining was observed following a 6-hour combination treatment, and this change was statistically significant (Figure 4-9). α-tocopherol pre-treatment abrogated the slight increase in intracellular ROS production (Figure 4-10). Examination of mitochondrial ROS production with MitoSOX Red staining in combination-treated TEX and OCI- AML2 cells revealed a small increase in mitochondrial ROS production following ibrutinib treatment; however no further increases were noted when ibrutinib was combined with daunorubicin (Figure 4-11).
101 Figure 4-8: α-tocopherol rescue of combination-treated TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with 3mM α-tocopherol for 24h prior to a 72h incubation with ibrutinib and daunorubicin (“DNR”) alone and in combination, with α- tocopherol maintained at a concentration of 2.4mM. Viability relative to respective untreated controls was measured with Annexin V and PI staining. Data depict mean percent viability ± SD from an experiment performed in triplicate. Data are representative of two independent experiments.
Figure 4-9: Combination ibrutinib-daunorubicin treatment increases intracellular ROS
TEX and OCI-AML2 cells were treated with ibrutinib and daunorubicin (“DNR”) for 6h, then stained with carboxy-H2DCFDA and PI to detect intracellular ROS in live cells by flow cytometry. Fold increase in ROS was calculated relative to the geometric mean fluorescence intensities (GMFI) of carboxy-H2DCFDA staining in untreated live cells. Hydrogen peroxide treatment was included as a positive carboxy-H2DCFDA staining control. Data depict average GMFIs ± SD and are representative of a single experiment performed in triplicate. Data are representative of two independent experiments. In all panels, ns = not significant, *P<0.05, **P<0.01; ***P<0.001; ****P<0.0001, as determined by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.
102
Figure 4-10: Intracellular ROS production following combination ibrutinib- daunorubicin treatment in the presence or absence of α-tocopherol. TEX and OCI-AML2 cells were treated with 3mM α-tocopherol prior to a 6h incubation with ibrutinib (“Ibru”), daunorubicin (DNR), or both drugs in combination. Cells were subsequently stained with carboxy-H2DCFDA and PI to measure intracellular ROS production in live cells on flow cytometry. Increases in ROS were calculated relative to the average geometric mean fluorescence intensity (GMFI) of carboxy-H2DCFDA staining in untreated TEX or OCI-AML2 cells. Hydrogen peroxide (H2O2) was included as a positive control for intracellular ROS. Data depict mean fold increase in ROS ± SD from an experiment performed in triplicate.
103
Figure 4-11: Mitochondrial ROS production following combination ibrutinib- daunorubicin treatment in TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”), daunorubicin (DNR), or both drugs in combination for 6h. Cells were stained with MitoSOX Red and Annexin V to measure mitochondrial ROS on flow cytometry. Fold increase in mitochondrial ROS production was calculated relative to the average GMFI of MitoSOX+, Annexin V- staining untreated cells. Antimycin A was included as a positive control for mitochondrial ROS staining. Results depict the mean fold increase in mitochondrial ROS ± SD from an experiment performed in triplicate. Data are representative of two independent experiments.
104 A possible explanation for the discrepancy between the degree of viability rescue by α-tocopherol and induction of ROS following combination treatment is the length of incubation with drug (6 hours) prior to ROS measurement: previous studies in ABC-DLBCL cells demonstrated highest ROS production by ibrutinib at 1-2 hours post-treatment, with levels significantly decreasing by four hours (Dasmahapatra et al., 2013). Alternatively, it is possible that the modest increase in ROS production following ibrutinib-daunorubicin treatment does not fully account for the extent of antioxidant-mediated cell rescue: the observed cytoprotection by α-tocopherol may have been due to an off-target effect. In support of this possibility, a recent study reported reversal of kinase inhibitor- mediated apoptosis and cell cycle arrest by α-tocopherol, which is independent of its antioxidant activity (Pédeboscq et al., 2012).
In conclusion, ibrutinib potentiates the AML cell-killing activity of daunorubicin via a mechanism that is potentially BTK-independent, and unrelated to enhancement of intracellular daunorubicin accumulation. Incorporating ibrutinib into treatment regimens for AML patients, regardless of BTK expression and constitutive activation status, may be warranted.
105
Chapter 5: Erlotinib synergizes with the poly(ADP-ribose) glycohydrolase inhibitor ethacridine in acute myeloid leukemia cells
106 5.1 Abstract
Erlotinib is a small-molecule epidermal growth factor receptor (EGFR) inhibitor that has demonstrated significant EGFR-independent activity against acute myeloid leukemia (AML) cell lines and primary AML blasts in preclinical studies, however these findings have not been reproducible in the clinical trial setting. Combining erlotinib with other antineoplastic agents has been proposed as a strategy for improving the clinical activity of erlotinib. With the goal of identifying erlotinib-sensitizing drugs, we screened erlotinib against several chemical libraries in the erlotinib-insensitive AML cell lines TEX and OCI-AML2, identifying the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate as the top synergistic hit common to both cell lines. The erlotinib-ethacridine combination induced synergistic cell death, which was preceded by a profound and lethal increase in intracellular reactive oxygen species (ROS) production. Using mass spectrometry, we determined that erlotinib synergized with ethacridine by potentiating ethacridine accumulation in TEX and OCI-AML2 cells. This synergistic mechanism of action was confirmed by demonstrating that high-dose ethacridine treatment mimics the significant increases in ROS observed following combination erlotinib-ethacridine treatment. Thus, we have identified that erlotinib promotes the accumulation of select drugs, thereby leading to synergism. In addition, the potential anti-AML activity of PARG inhibitors warrants further study.
107 5.2 Introduction
Erlotinib is a small-molecule epidermal growth factor receptor (EGFR) inhibitor that reversibly blocks autophosphorylation of C-terminal EGFR tyrosine residues, inhibiting cell proliferation and inducing apoptosis (Moyer et al., 1997). It is used in the clinical treatment of non small-cell lung cancer (NSCLC), where EGFR is often over-expressed or constitutively active, and thus promotes tumor cell survival and proliferation via PI3K/AKT/mTOR, STAT, and Ras/Raf/MAPK signaling pathways (Sharma et al., 2007; Siegelin & Borczuk, 2014).
Erlotinib has well-documented preclinical activity against acute myeloid leukemia (AML) cells, where it induces differentiation (Boehrer et al., 2008a), cell cycle arrest (Boehrer et al., 2008a; Boehrer et al., 2011; Lainey et al., 2011), and apoptosis (Boehrer et al., 2008a; Boehrer et al., 2008b), yet EGFR expression in these cells is absent (Boehrer et al., 2008a; Chan & Pilichowska, 2007; Stegmaier et al., 2005). Several erlotinib targets have been proposed or reported to account for its anti-leukemic effects: erlotinib was shown to inhibit SRC family kinases (SFKs) (Boehrer et al., 2011; Weber et al., 2012), which are constitutively active in primary AML cells and AML cell lines and mediate mTOR complex 1 (mTORC1) signaling in these cells (Dos Santos et al., 2008). In line with these findings, erlotinib was found to inhibit phosphorylation of mTORC1 targets and to induce autophagy in the AML cell line KG-1 (Boehrer et al., 2011).
Erlotinib has also been found to bind directly to Bruton’s tyrosine kinase (BTK) and to decrease phosphorylation at Y551 (Weber et al., 2012); Y551 phosphorylation is required for activation of this kinase. BTK has been proposed as a potential therapeutic target in AML because of its role as a mediator of FLT3-ITD and TLR9 signaling in FLT3-ITD-positive and FLT3-ITD-negative AML cell lines, respectively (Oellerich et al., 2015).
108 Finally, erlotinib was shown to inhibit JAK2 Tyr1007/1008 phosphorylation and downstream STAT5 phosphorylation at Tyr694 in KG-1 cells (Boehrer et al., 2008a). JAK2-STAT3/5 signaling has been implicated in AML CD34+ cell survival and colony formation (Cook et al., 2014).
Clinically, the anti-AML activity of erlotinib has been more modest. While two case reports described AML remissions in two patients with concomitant NSCLC and AML (Chan & Pilichowska, 2007; Pitini et al., 2008), subsequent clinical trials examining the single-agent activity of erlotinib in AML have not yielded equally remarkable results, with a small minority of patients exhibiting decreased peripheral blast counts and zero patients achieving complete remission (Sayar et al., 2015; Thepot et al., 2014). The authors of both studies suggested that erlotinib may have better clinical utility when administered in combination with other anti-leukemic agents (Sayar et al., 2015; Thepot et al., 2014).
In light of the limited clinical single-agent activity of erlotinib in AML, its favorable safety profile (Gordon et al., 2005; Sayar et al., 2015; Soulieres et al., 2004; Thepot et al., 2014), and previous reports of synergistic interactions with other antineoplastic agents (Lainey et al., 2013a; Lainey et al., 2013b; Landriscina et al., 2010), we sought to identify erlotinib combination candidates in AML using a high-throughput drug screening approach.
109 5.3 Materials and Methods
5.3.1 Reagents
Anti-EGFR (#2232) and anti-GAPDH (#2118) antibodies were obtained from Cell Signaling Technology (Danvers, MA). HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were acquired from GE Healthcare (Buckinghamshire, UK). Erlotinib was obtained from Cayman Chemical (Ann Arbor, MI). Ethacridine lactate, sulforhodamine-B, α-tocopherol, and hydrogen peroxide were purchased from Sigma-Aldrich (St. Louis, MO). Imatinib was obtained from AK Scientific, Inc. (Union City, CA). Drug libraries were obtained from MicroSource Discovery Systems, Inc. (Gaylordsville, CT), and Sequoia Research Products (Pangbourne, UK). The kinase inhibitor library was obtained from the Ontario Institute for Cancer Research, Toronto, Canada.
5.3.2 Cell culture
TEX cells (Warner et al., 2005) were provided by Dr. John Dick (Ontario Cancer Institute, Toronto, Canada) and maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 15% fetal bovine serum (FBS) (Seradigm/VWR, Radford, PA), 100µg/ml penicillin, 100 U/ml streptomycin, 2mM L-glutamine (Life Technologies, Carlsbad, CA), 20ng/ml SCF (Miltenyi Biotec, San Diego, CA), and 2ng/ml IL-3 (R&D Systems, Minneapolis, MN). OCI-AML2 and U937 cells were provided by Dr. Mark Minden (Ontario Cancer Institute, Toronto, Canada). K562 cells were provided by Dr. Suzanne Kamel-Reid (Ontario Cancer Institute, Toronto, Canada). OCI-AML2 and K562 cells were grown in IMDM supplemented with 10% FBS 100 µg/ml penicillin, and 100 U/ml streptomycin. U937 cells were grown in RPMI 1640 supplemented with 10% FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin. MDA-468 cells were grown in RPMI-1640 supplemented with 10% FBS. All cell lines were maintained at
110 37°C and 5% CO2.
5.3.3 Primary cells
Primary bulk AML cells and peripheral blood stem cells (PBSCs) from stem cell donors treated with G-CSF were collected from consenting patients, with the approval of the University Health Network (Toronto, Canada) institutional review board. Primary AML cells were isolated by Ficoll density centrifugation and PBSCs were isolated by apheresis. Cells were maintained in IMDM supplemented with 10% FBS, 100µg/ml penicillin, and 100U/ml streptomycin, at
37°C and 5% CO2.
5.3.4 Immunoblotting
PBS-washed cells were lysed in 1xLaemmli buffer and protein from whole cell lysates was quantified with the DC Protein Assay (Biorad Laboratories, Mississauga, ON, Canada). Lysates were resolved by SDS-PAGE and transferred to a PVDF membrane. Following blocking in 5% milk-TBST for 1 hour at room temperature, membranes were incubated with primary antibody on a rocker overnight at 4°C. Following a 1-hour incubation with secondary antibody (GE Healthcare, Buckinghamshire, UK), proteins were detected by chemiluminescence.
5.3.5 Cell viability assays
Cells were treated with drugs alongside vehicle control (DMSO) in 96- or 384- well plates over 48 or 72 hours. Cell growth and viability was measured with the
111 sulforhodamine-B assay, performed as previously described (LaPointe et al., 2005), or the Alamar Blue assay, as per manufacturer’s instructions (Life Technologies, Carlsbad, CA). Cell viability was also measured by Annexin V and PI staining on a BD CantoII 96w (BD Biosciences, San Jose, CA) flow cytometer, and analyzed using FlowJo version 7.6.5 (TreeStar, Ashland, OR). For both methods, cell viability was calculated relative to that of vehicle controls.
5.3.6 High-throughput combination drug screening & excess- over-Bliss additivism synergy calculations
High-throughput combinatorial drug screening was carried out as previously described (Rotin et al., 2016b). The excess-over-Bliss additivism formula (Borisy et al., 2003), which calculates the degree of cell killing unaccounted for by the added cytotoxicities of each individual drug, was applied as previously described (Rotin et al., 2016b).
5.3.7 Reactive oxygen species measurement
Intracellular ROS production in live drug-treated cells was measured by staining cells with 10 µM carboxy-H2DCFDA (Molecular Probes/Life Technologies,
Eugene, OR) for 30 minutes at 37°C and 5% CO2 and subsequent staining with propidium iodide. ROS production in stained live cells was detected with a BD Fortessa LSR X20 (BD Biosciences, San Jose, CA) flow cytometer, and calculated relative to the geometric mean fluorescence intensity of untreated live cells.
112 5.3.8 Mass spectrometry
Chromatographic separations were carried out on an Acquity UPLC BEH C18 (2.1 X 50 mm, 1.7 µm) column using ACQUITY UPLC II system. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). A gradient starting at 95% solvent A going to 5% in 4.5 min., holding for 0.5 min., going back to 95% in 0.5 min. and equilibrating the column for 1 min. was employed. A Waters Synapt G2S QTof mass spectrometer equipped with an atmospheric pressure ionization source was used for mass spectrometric analysis. MassLynx 4.1 was used for data analysis.
113 5.4 Results
5.4.1 TEX and OCI-AML2 cell line sensitivity to erlotinib
To ascertain erlotinib sensitivity in the AML cell lines TEX and OCI-AML2, we subjected these cells to treatment with increasing concentrations of erlotinib for 72 hours, and subsequently measured relative growth and viability using the sulforhodamine-B (SRB) assay. The average erlotinib IC50s were 8.99µM and
15.61µM in TEX and OCI-AML2, respectively (Figure 5-1). These erlotinib IC50 values are significantly higher than clinically achievable concentrations
(Appendix 1) and far greater than the nanomolar-range IC50 values reported in the NSCLC cell lines HCC827, HCC4006, HCC4011, H3255, and H292, using the same assay (Gao et al., 2014).
114
Figure 5-1: AML cell line sensitivity to erlotinib. (A) TEX and OCI-AML2 cells were treated with increasing concentrations of erlotinib for 72h. Growth and viability was measured using the SRB assay and calculated relative to vehicle-treated cells. Results depict average percent growth and viability ± SD from a representative experiment performed in triplicate. Data are representative of three independent experiments.
115 5.4.2 A high-throughput combination chemical screen identifies erlotinib sensitizers in TEX and OCI-AML2 cells
With the goal of identifying synergistic combination candidates for erlotinib, we screened this drug against three chemical libraries—the Microsource Discovery Systems International Drug and Natural Product (“NatProd”) collections, and a 312-compound library from Sequoia Research Products—in TEX and OCI-AML2 cells. Erlotinib at its IC10 and IC25 was combined with increasing concentrations of 1,352 drugs for a total of 16,230 different assays in two cell lines. Treated cells were incubated for 72 hours and relative growth and viability was measured with the SRB assay. These viability data were then used to determine synergy based on the excess-over-Bliss additivism (EOBA) formula (Borisy et al., 2003), which calculates the difference between observed and predicted killing by a given drug combination, with a greater positive difference indicating stronger synergy. Compounds were plotted in order of increasing positive EOBA scores for each drug library, in both cell lines (Figure 5-2). The most profoundly synergistic hits from each library were individually validated using a broader range of concentrations to more thoroughly evaluate synergy. Ethacridine lactate, a poly(ADP-ribose) glycohydrolase (PARG) inhibitor (Bernardi et al., 1997; Boulikas, 1990; Tavassoli et al., 1985) and abortifacient (Hou et al., 2010), was validated as the top synergistic hit common to both TEX and OCI-AML2 cells, generating EOBA scores of up to 0.79 in TEX and 0.69 in OCI-AML2 (Figure 5- 3, top). Validation of two other screen hits, apigenin and berberine, are included for comparison (Figure 5-3, bottom).
116
Figure 5-2: Erlotinib sensitizers in TEX and OCI-AML2 cells. Erlotinib was screened against 1,352 drugs from three chemical libraries (Sequoia, International, and Natural Products A, B and C). Following a 72h incubation, EOBA synergy scores were calculated from relative growth and viability values determined by the SRB assay. Compounds were ranked in order of increasing positive EOBA score.
117
Figure 5-3: Validation of synergistic hits. Synergistic hits (shown: ethacridine, apigenin, and berberine) were validated in TEX and OCI-AML2 cells using broader concentration ranges. Cells were combination-treated for 72h and percent growth and viability was measured with the SRB assay (graphs). Synergy was calculated according to EOBA criteria (tables), with EOBA values >0.1 (lightest grey) denoting a synergistic combination, while values >0.5 (darkest grey) denoting a profoundly synergistic combination. Results depict mean percent growth and viability ± SD (graphs) or mean EOBA scores (tables) from a single experiment performed in triplicate.
118 5.4.3 The erlotinib-ethacridine combination synergizes in primary AML cells and other AML cell lines
To determine whether the observed synergy between erlotinib and ethacridine extended beyond TEX and OCI-AML2 cells, we combination-treated U937 and K562 leukemia cells over 72 hours and measured relative growth and viability with the SRB assay. Combination erlotinib-ethacridine treatment yielded EOBA scores in the profoundly synergistic range (>0.50) at concentrations of erlotinib that were clinically relevant (Figure 5-4A). We also examined this combination in 5 primary AML samples and 2 samples of normal hematopoietic cells derived from healthy volunteers donating G-CSF-mobilized peripheral blood stem cells for allogeneic transplant (denoted as “Normal”) (see Table 5-1 for patient demographics). For this assay, cells were incubated with increasing concentrations of erlotinib, ethacridine, or both in combination for 48 hours. Viability relative to untreated (vehicle) controls was measured with Annexin V and propidium iodide (PI) staining on flow cytometry (Figure 5-4B). None of the primary samples or normals, with the exception of AML130183, were sensitive to single-agent erlotinib treatment. Single-agent ethacridine sensitivity was variable amongst primary AML cells, however all were more sensitive to ethacridine killing compared to normals. The erlotinib-ethacridine combination was synergistic in all 5 primary AML samples, most strikingly in AML130208 and AML130237, which yielded EOBA scores in the >0.30 range (Figure 5-4C). In contrast, the combination was not synergistic in either of the normals; the highest EOBA score calculated was 0.11, which was only observed in one pairing (Figure 5-4C).
119
Figure 5-4: Erlotinib and ethacridine synergize in additional AML cell lines and primary AML blasts. U937, K562, primary AML blasts, and PBSCs were combination-treated with erlotinib and ethacridine for 72h (A) or 48h (B). Viability was determined by the SRB assay (A) or Annexin V and PI staining (B). Graphs depict (A) mean percent growth and viability or (B) mean percent viability ± SD from a single experiment performed in triplicate. Tables (A, C) represent mean EOBA values.
120
Table 5-1: AML patient demographics.
Patient Demographics Sample Age at Diagnosis Cytogenetics Molecular ID Diagnosis Gender CBFB-MYH11 130177 AML, M4Eo 62 Male 46,XY,inv(16)(p13.1q22)[8]/46,XY[2] +,KIT- NPM1+, FLT3- 130183 AML 69 Male 46,XY[20] ITD+, FLT3- TKD-
45,XY,der(1)t(1;?21)(p13;q11.2),del(5)(q13q33),- 7,+8,-10, add(12)(p11.2),-15,add(19)(q13.3),- 130185 AML 69 Male 21,+2mar[5]/45,XY,der(1)t(1;?21)(p13;q11.2),-5,- Not done 7,+8,-10,add(12)(p11.2), -15,add(19)(q13.3),- 21,+3mar[5]
NPM1+, FLT3- 130208 AML, M1 21 Male 46,XY[20] ITD+, FLT3- TKD- 130237 AML 66 Male 46,XY,inv(3)(q21q26.2)[3]/45,idem,-7[8] Not done
121 5.4.4 Combining erlotinib and ethacridine generates lethal levels of reactive oxygen species
To investigate the mechanism of synergistic cytotoxicity by the erlotinib- ethacridine combination, we quantified intracellular reactive oxygen species (ROS) production in TEX and OCI-AML2 cells following a 24-hour incubation with erlotinib, ethacridine, and both drugs in combination. ROS was measured using carboxy-H2DCFDA staining on flow cytometry, with dead cell exclusion by PI staining. Fold change in ROS was calculated relative to the geometric mean fluorescence intensity of carboxy-H2DCFDA (FITC) staining in vehicle-treated cells. We observed a striking increase in ROS production: up to a 2-fold increase in TEX and a 4-fold increase in OCI-AML2 cells, respectively (Figure 5-5A). ROS induction appeared functionally important for combination-induced synergistic cytotoxicity, as pre-treatment with the antioxidant α-tocopherol significantly increased viability by Annexin V and PI staining following a 48-hour treatment with the erlotinib-ethacridine combination (Figure 5-5B).
Because of the reported activity of ethacridine against PARG, we sought to determine whether gallotannin, another known PARG inhibitor (Aoki et al., 1993; Tsai et al., 1992), synergized with erlotinib. Using Annexin V and PI staining on flow cytometry, we observed profound synergistic activity between erlotinib and gallotannin in TEX cells (Figure 5-5C).
122 Figure 5-5: Combination erlotinib-ethacridine treatment induces lethal intracellular ROS production. (A) TEX and OCI-AML2 cells were combination-treated with erlotinib and ethacridine for 24h. Intracellular ROS was measured with carboxy-H2DCFDA staining and dead cells were excluded by PI staining. Fold increase in ROS production was calculated relative to the geometric mean fluorescence intensity (GMFI) of vehicle-treated cells. H2O2 was included as a positive intracellular ROS control. Results depict mean fold increase in GMFI ± SD from a representative experiment performed in triplicate. Data are representative of two independent experiments. (B) TEX and OCI-AML2 cells were treated with 3mM α-tocopherol for 24h, then treated with the erlotinib-ethacridine combination for the following 48h (with α-tocopherol maintained at 2.4mM). Viability was measured with Annexin V and PI staining on flow cytometry, and calculated relative to respective (± α-tocopherol) controls. Data represent mean percent viability ± SD from an experiment performed in triplicate. These data are representative of two independent experiments. (C) TEX cells were combination-treated with erlotinib and gallotannin for 48h. Viability was determined by Annexin V and PI staining. Graph depicts mean percent
123 viability ± SD from a single experiment performed in triplicate. Table represents mean EOBA values from the same experiment. Data are representative of at least three independent experiments.
124 5.4.5 Ethacridine synergizes with EGFR-targeting kinase inhibitors
Given that erlotinib has been proposed to inhibit other kinases in its activity against AML cells (Boehrer et al., 2008a; Boehrer et al., 2011; Weber et al., 2012), we sought to determine whether off-target kinase inhibition by erlotinib was responsible for its synergy with ethacridine. We therefore screened ethacridine against an in-house kinase inhibitor library, comprising 480 kinase inhibitors with a broad range of kinase targets and varying degrees of kinase specificity. OCI-AML2 cells were incubated with ethacridine along with two different concentrations of each kinase inhibitor for 72 hours, for a total of 2,883 different assays. Relative growth and viability was subsequently measured with the SRB assay and synergy scores were again calculated with the EOBA formula and plotted in order of increasing synergy score (Figure 5-6A). Erlotinib was the second most synergistic hit, which served as further validation for our initial combination screen. Interestingly, 4 of the 5 top synergistic hits (GW583340, erlotinib, GW2974, and WHI-P 154) were reported EGFR inhibitors. Furthermore, the clinically approved EGFR inhibitors gefitinib and lapatinib were also identified as synergistic hits, with EOBA values reaching 0.18 and 0.14, respectively.
5.4.6 TEX and OCI-AML2 cell lines do not express EGFR
The observed synergy between ethacridine and multiple EGFR inhibitors prompted us to investigate whether erlotinib might inhibit EGFR to synergize with ethacridine in TEX and OCI-AML2 cells. We evaluated total EGFR expression in these cell lines by immunoblot and were unable to detect expression of this kinase in either cell line (Figure 5-6B). Likewise, EGFR expression was absent in K562 and U937 cells, which also demonstrated erlotinib-ethacridine synergy (Figure 5-4A). Thus, these chemical EGFR inhibitors likely synergize with ethacridine via a common, EGFR-independent mechanism.
125 5.4.7 Erlotinib potentiates ethacridine accumulation in TEX and OCI-AML2 cells.
One property common to several small molecule EGFR inhibitors is their potent inhibition of ATP-binding cassette (ABC) transporter efflux activity, and thus their ability to enhance the cellular accumulation of—and sensitivity to—their substrates. We therefore investigated whether erlotinib potentiates ethacridine accumulation in AML cells. We treated TEX and OCI-AML2 cells with 5 µM ethacridine in the presence and absence of erlotinib for one hour. Cells were lysed and contents were quantified by LC-MS/MS. Ethacridine concentrations increased nearly 2-fold in the presence of as little as 1 µM erlotinib in both cell lines (Figure 5-6C). In contrast, imatinib (which does not synergize with ethacridine (Figure 5-7)) did not potentiate ethacridine accumulation in either cell line (Figure 5-6C).
5.4.8 High-dose ethacridine treatment mimics ROS production observed from the erlotinib-ethacridine combination.
To determine whether erlotinib-mediated intracellular ethacridine accumulation could be responsible for excessive ROS production, we treated TEX and OCI- AML2 cells with high concentrations of ethacridine and measured changes in ROS production. Cells were incubated for 24 hours with 15 µM and 20 µM ethacridine to mimic the increase in ethacridine accumulation observed in the presence of 3 µM erlotinib, alongside combination-treated cells. ROS production in live cells was measured by carboxy-H2DCFDA and PI staining on flow cytometry, and calculated relative to vehicle-treated controls. High-dose ethacridine increased ROS production more than 2-fold in TEX cells, and greater than 3-fold in OCI-AML2 cells (Figure 5-6D), which was comparable to the increase in ROS production observed with combination erlotinib-ethacridine treatment (Figure 5-5A). These findings suggest that erlotinib-mediated
126 ethacridine accumulation is the mechanism that explains synergistic cell death caused by excessive ROS production.
127
Figure 5-6: Erlotinib enhances ethacridine accumulation in TEX and OCI-AML2 cells. (A) Ethacridine was screened against a 480-compound kinase inhibitor library in OCI- AML2 cells. Cells were treated for 72h. Growth and viability was measured with the SRB assay and synergy scores were calculated with the EOBA formula. Compounds were ranked in order of increasing positive synergy score. (B) EGFR expression in a panel of AML cell lines was detected by immunoblotting, with the MDA-468 cell line included as an EGFR-positive control. (C) TEX and OCI-AML2 cells were treated with ethacridine in the presence and absence of erlotinib for 1h. Cells were lysed and analyzed by LC-MS. Imatinib was included as a negative (non-synergizing) control. Data depict mean ethacridine accumulation ± SD from an experiment performed in triplicate. Data are representative of three (TEX) or two (OCI-AML2) independent experiments. (D) TEX and
128 OCI-AML2 cells were treated with high-dose ethacridine or erlotinib and ethacridine in combination for 24h. Intracellular ROS was measured with carboxy-H2DCFDA staining (with PI exclusion of dead cells) and calculated relative to GMFI of vehicle-treated cells. Data depict mean fold increase in GMFI ± SD from a representative experiment performed in triplicate, and are representative of two independent experiments.
129
Figure 5-7: Imatinib does not synergize with ethacridine in TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with increasing concentrations of imatinib and ethacridine alone and in combination. Relative growth and viability following a 72h incubation was measured with the Alamar Blue assay. Synergy was calculated with the EOBA formula. Graphs depict mean percent growth and viability ± SD from an experiment performed in triplicate. Tables depict mean EOBA scores from the same experiment. Data are representative of three independent experiments.
130 5.5 Discussion
The impressive EGFR-independent antileukemic activity of erlotinib reported in preclinical studies has not translated to significant responses in the clinical setting, with two clinical trials reporting negligible or no single-agent erlotinib activity against AML. The first, a Phase I/II clinical trial evaluating erlotinib use in 30 high-risk MDS and AML patients who had previously failed azacytidine treatment, reported no responses to erlotinib treatment in those with AML (n=12) (Thepot et al., 2014). The second study, which assessed erlotinib treatment in nine patients with treatment-naïve AML and two patients with relapsed or refractory AML, reported disease progression in all 11 patients, however two patients demonstrated a significant decrease in peripheral blasts (Sayar et al., 2015). Failure of erlotinib as a single-agent therapeutic strategy for this disease in clinical trials has prompted interest in the investigation of this tyrosine kinase inhibitor (TKI) as a combination candidate for AML therapy.
With the goal of identifying novel erlotinib combination candidates, we screened this TKI against several drug libraries in two non-EGFR expressing AML cell lines, which had erlotinib sensitivities in the micromolar range. The PARG inhibitor ethacridine lactate was the most synergistic hit common to both TEX and OCI-AML2 cells. We determined that synergy was due to erlotinib potentiation of intracellular ethacridine accumulation, as evidenced by the fact that high-dose ethacridine treatment recapitulated the lethal increase in ROS production observed following combination erlotinib-ethacridine treatment.
Erlotinib has been previously shown to synergize with other drugs in AML cell lines and primary AML blasts: erlotinib potentiated ATRA- and vitamin D3- mediated differentiation of HL60 cells, an effect attributed to erlotinib’s inhibition of SRC and p38MAPK phosphorylation (Lainey et al., 2013b). Erlotinib and gefitinib, another EGFR inhibitor, were also found to synergistically block cell
131 proliferation and induce apoptosis when combined with the hypomethylating agent azacytidine, owing to erlotinib- and gefitinib-mediated potentiation of intracellular azacytidine accumulation (Lainey et al., 2013a). Finally, erlotinib and gefitinib were shown to sensitize KG-1 AML cells to antineoplastic agents such as etoposide and doxorubicin by enhancing their accumulation through simultaneous inhibition of P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP)-mediated drug efflux activity (Lainey et al., 2012). EGFR inhibitors have been extensively shown to enhance the cytotoxicity of antineoplastic drugs through inhibition of ABC transporter-mediated drug efflux (Dai et al., 2008; Kuang et al., 2010; Shi et al., 2007).
While our study did not address the mechanism of intracellular ethacridine uptake or the mechanism by which it is extruded from AML cells, the fact that the concentrations of erlotinib required for synergy with ethacridine are in line with those required for erlotinib synergy with other antineoplastic agents (1 to 10 µM range (Lainey et al., 2012; Lainey et al., 2013a)) would strongly suggest that erlotinib inhibition of one or more ABC transporters accounts for lethal levels of ethacridine accumulation in TEX and OCI-AML2 cells.
Given the capacity of erlotinib to promote the accumulation of other drugs, it is surprising that the combination chemical screen against erlotinib did not yield a greater number of validated synergistic hits common to both TEX and OCI-AML2 cells. This observation may suggest that in these AML cell lines, erlotinib may inhibit a more restricted number of transporters. This observation may also highlight the potential therapeutic relevance of PARG inhibition in AML. PARG hydrolyzes poly(ADP-ribose) (PAR) polymers, which are synthesized by poly(ADP-ribose) polymerases (PARPs) in response to DNA damage and other cellular stresses. PARG therefore quenches PARP-elicited signals, which drive processes such as DNA repair or cell death.
132 With the exception of our own previous report describing the synergistic cytotoxicity between chemical PARG inhibitors and the Bruton’s tyrosine kinase inhibitor ibrutinib (Rotin et al., 2016b), the role of PARG inhibition in AML has not been investigated. PARG inhibition has been found to sensitize other cancer cell lines to DNA-damaging and oxidative stress-inducing agents by failing to reduce cellular PAR levels following PARP activation. Excessive PAR accumulation induces cell death by necrosis (NAD+ is a substrate for PAR synthesis, thus cellular ATP stores become depleted), or parthanatos (excess PAR triggers nuclear translocation of apoptosis-inducing factor). We therefore hypothesize that erlotinib-mediated ethacridine accumulation induces lethal levels of PAR accumulation due to profound PARG inhibition.
In summary, we have identified the PARG inhibitor ethacridine as a novel combination candidate for erlotinib in AML. Erlotinib synergizes with ethacridine by potentiating its intracellular accumulation. The impact of PARG inhibition as a therapeutic strategy in AML warrants further investigation.
133
Chapter 6: General Discussion & Conclusion
134 6.1 Discussion
The goal of this project was to further elucidate the role of BTK as a therapeutic target in AML, through the identification of drugs that sensitize AML cells to killing by ibrutinib. We determined that both ethacridine lactate and daunorubicin synergized with ibrutinib, however neither synergistic interaction was dependent upon ibrutinib-mediated BTK inhibition. Further work investigating the striking synergy between ethacridine and the kinase inhibitor erlotinib, a known inhibitor of ATP-binding cassette (ABC) transporter-mediated drug efflux, shed light on the possibility that ibrutinib also likely synergizes with ethacridine via this mechanism. The mechanism by which ibrutinib and daunorubicin synergize is not yet known.
6.1.1 BTK-independent anti-leukemic activity of ibrutinib
In demonstrating that ethacridine or daunorubicin treatment of BTK-knockdown AML cell lines does not recapitulate the synergy observed when these drugs are combined with ibrutinib, and that ibrutinib and ethacridine synergized in cells lacking BTK protein expression, we provided evidence to suggest that ibrutinib has anti-AML activity that extends beyond BTK inhibition.
6.1.1.1 Ibrutinib potentiates ethacridine accumulation
Given that ibrutinib has well-known inhibitory activity against other kinases such as those from the TEC and SRC families (Dubovsky et al., 2013; Honigberg et al., 2010), and because the ibrutinib concentrations at which synergy was observed were significantly greater than those required for in vitro BTK inhibition (Honigberg et al., 2010), we postulated that ibrutinib was inhibiting additional
135 kinases, and that inhibition of the TEC family kinases in particular was responsible for the observed synergy between this drug and ethacridine (Chapter 3.5). However, determining that the synergy between erlotinib and ethacridine (which was as striking as that of ibrutinib and ethacridine) was due to erlotinib potentiation of intracellular ethacridine accumulation (Chapter 5.4) shed light on the possibility that ibrutinib and ethacridine may synergize via this same mechanism.
Many tyrosine kinase inhibitors (TKIs)—in particular those inhibiting ABL, EGFR, and VEGFR kinases—have well-described activity as both substrates and, at higher concentrations, inhibitors of ATP-binding cassette (ABC) transporters (Deng et al., 2014). At micromolar concentrations, erlotinib has been shown to sensitize AML cells to chemotherapy agents by blocking P-gp, MRP1, and BCRP-mediated drug efflux and thus increasing intracellular concentrations and cytotoxicity of these drugs, which are substrates of these transporters (Lainey et al., 2012). In line with these observations, we demonstrated that concentrations as low as 1 µM erlotinib were sufficient to nearly double intracellular concentrations of ethacridine in TEX and OCI-AML2 cells, which would suggest that ethacridine is a substrate of one or more of these ABC transporters. Moreover, treating AML cell lines with high-dose ethacridine recapitulated the ROS increases observed in response to combination erlotinib-ethacridine treatment, indicating that ethacridine accumulation is responsible for the observed synergistic cytotoxicity.
Further supporting the possibility that the mechanism accounting for ibrutinib’s synergy with ethacridine is the same as that of erlotinib’s synergy with ethacridine, Zhang et al. (2014) demonstrated that ibrutinib, via MRP1 inhibition, potently sensitized MRP1-overexpressing HL60 leukemia cells to the MRP1 substrate drugs vincristine and doxorubicin, which was accompanied by an increase in intracellular chemotherapy concentrations. We therefore anticipate that like erlotinib, ibrutinib is potentiating ethacridine accumulation to induce ROS
136 and synergistic cell death. Indeed, preliminary mass spectrometry data generated by our group (not shown) would suggest that this hypothesis is correct: ibrutinib enhances intracellular ethacridine accumulation and intracellular ROS induction following high-dose ethacridine treatment parallels that observed in response to combination treatment.
6.1.1.2 Synergy between ibrutinib and daunorubicin is mediated by a mechanism unrelated to that of ibrutinib/erlotinib and ethacridine
Ibrutinib and daunorubicin very likely synergize via a mechanism that differs from that of the TKI-ethacridine combination. Several lines of evidence point to this conclusion. First, the patterns of synergy were notably different: synergy between ibrutinib and daunorubicin was far less striking, with EOBA values reaching the 0.20-0.30 range, whereas ibrutinib-ethacridine combinations reached well beyond 0.50 in the same cell lines, at the same ibrutinib concentrations. Furthermore, although the ibrutinib-daunorubicin combination induced statistically significant increases in ROS, this increase was modest relative the increase observed following ibrutinib-ethacridine treatment. Finally, and perhaps most importantly, ibrutinib was not found to potentiate TEX cell accumulation of daunorubicin, a mechanism that was central to the TKI-ethacridine synergistic mechanism. This finding is difficult to reconcile, because anthracyclines, which include daunorubicin, are reported substrates for P-gp and MRP1 (Deng et al., 2014). However, it is important to note that the technique used to measure drug accumulation in the presence and absence of ibrutinib was different for both drugs (radiolabeling of daunorubicin, versus mass spectrometric measurement of ethacridine). Thus, these findings cannot be directly compared, however it is likely that potentiation of daunorubicin accumulation is not a significant contributor to ibrutinib-daunorubicin synergy.
Kinase inhibitor synergy with cytotoxic agents is a commonly reported observation in in vitro preclinical studies (Eriksson et al., 2012; Landriscina et al.,
137 2010; Li et al., 2014; Pichot et al., 2009). Ibrutinib has been found to synergize with the anthracycline doxorubicin in activated B-cell-like subtype of diffuse large B-cell lymphoma (ABC-DLBCL) cells (Mathews Griner et al., 2014). The hypothesized mechanism to explain this synergistic effect in ABC-DLBCL cells is related to NFKB inhibition by ibrutinib: DNA damage induced by chemotherapeutic agents stimulates NFKB activation, and ibrutinib blocks this cytoprotective response (Mathews Griner et al., 2014). Likewise, NFKB lies downstream of BTK signaling in FLT3-ITD negative AML cells, and ibrutinib treatment downregulates this transcription factor (Oellerich et al., 2015), which in turn leads to decreased expression of survival genes downstream of NFKB
(Rushworth et al., 2014). Thus, a similar NFKB-mediated mechanism may underlie the observed synergistic cytotoxicity between ibrutinib and daunorubicin in FLT3-ITD negative AML cells, such as OCI-AML2.
6.1.2 Anti-leukemic activity of ethacridine lactate
Mass spectrometry, intracellular ROS, and antioxidant rescue experiments confirmed that intracellular ethacridine accumulation resulted in increased ROS production, which was functionally important for the synergistic cell death observed following combination erlotinib-ethacridine (and ultimately, ibrutinib- ethacridine) treatment. However, our work did not address the mechanism responsible for the lethal effects of ethacridine.
Ethacridine lactate has several reported cellular targets with potential antineoplastic activity. Because we also observed synergy between erlotinib or ibrutinib and gallotannin, another reported poly(ADP-ribose) glycohydrolase (PARG) inhibitor, we reasoned that PARG inhibition was the relevant target of ethacridine responsible for AML cell death. In the setting of PARP1 activation, PARG inhibition causes poly(ADP-ribose) accumulation leading to cell death due to apoptosis-inducing factor activation or necrosis. The finding that TKI-mediated
138 ethacridine accumulation was responsible for cell killing does not preclude PARG inhibition as the relevant target; rather, increased intracellular ethacridine may suggest more potent PARG inhibition.
However, given that our work did not address whether on-target PARG inhibition was responsible for ethacridine cytotoxicity, other possible reported ethacridine targets cannot be ruled out as the cause of this drug’s lethality. Ethacridine, among other acridine compounds, impairs RNA polymerase 1 complex formation by degrading the RP194 subunit, which inhibits ribosomal RNA transcription and subsequently induces ribosomal stress, leading to p53 induction (Morgado- Palacin et al., 2014). This mechanism of p53 induction is independent of DNA damage (Morgado-Palacin et al., 2014). Preliminary work (not shown) investigating ethacridine-mediated p53 induction as its cytotoxic mechanism in TEX and OCI-AML2 cells does not rule out this possibility: ethacridine concentrations similar to those used by Morgado-Palacin et al. (2014) induced p53 expression by immunoblot. In line with our observation that ibrutinib potentiates ethacridine accumulation, incubation with ibrutinib and ethacridine in combination further induced p53 expression in both cell lines, while single-agent ibrutinib treatment did not induce p53. Interestingly, p53 induction in both cell lines was not accompanied by increased phosphorylation of histone γH2A.X, a marker of DNA damage. Thus, ethacridine may exert its cytotoxic effect via ribosomal stress-mediated p53 induction.
6.1.3 Clinical relevance of BTK-independent effects of ibrutinib
The findings of this work, and of that of Zhang et al. (2014) suggest that ibrutinib may have a role in the reversal of multidrug resistance in AML. However, there may be some limitations to exploiting ibrutinib for its activity against this particular target in the clinical setting. First, the ibrutinib concentrations required for synergy
139 in our cell lines (4-8 µM) and those used by Zhang et al. (2014) (1-5 µM) are greater than what is clinically achievable in patients: 420 mg/day orally resulted in maximum steady-state concentrations of 100 ng/mL, or ~230 nM (Sukbuntherng et al., 2013). However, the concentrations achieved in in vivo studies carried out by our group and Zhang et al. (2014) did demonstrate a mild- to-moderate sensitization effect by ibrutinib, which would imply that at clinically relevant concentrations, ibrutinib has some effect on drug sensitization.
Another reason for the limited clinical utility of this particular application of ibrutinib is that previous efforts to sensitize AML cells to chemotherapy agents using drug efflux pump modulators have failed to improve AML outcomes in the clinical setting. Leukemic stem cells and blasts have well-documented expression of P-gp, MRP1 and BCRP, and other ABC transporter proteins, and expression of these transporters (particularly P-gp) on leukemic cells is correlated with chemotherapy resistance and poor patient prognosis (van der Kolk et al., 2002). This observation prompted extensive clinical evaluation of P- gp inhibitors as adjuncts to chemotherapy in AML: the addition of the second- generation P-gp modulator valspodar to a chemotherapy regimen consisting of mitoxantrone, etoposide, and cytarabine did not increase complete response rates or overall survival in patients with relapsed/refractory AML or high-risk myelodysplastic syndrome (Greenberg et al., 2004). Similarly, adding valspodar to AML induction regimens with daunorubicin and cytarabine in treatment-naïve patients over the age of 60 did not improve complete response, event-free survival, disease-free survival, or overall survival rates regardless of P-gp expression status (van der Holt et al., 2005). Finally, valspodar did not improve complete remission, disease-free survival, or overall survival in treatment-naïve AML patients under the age of 60 when added to cytarabine-daunorubicin- etoposide induction regimens (Kolitz et al., 2010). The failure of valspodar to improve treatment efficacy in AML may be related to the presence of compensatory chemotherapy resistance mechanisms: BCRP and MRP1 also mediate extrusion of daunorubicin, etoposide, and/or mitoxantrone (Shaffer et al.,
140 2012; van der Kolk et al., 2002). While ibrutinib may overcome some of these compensatory chemotherapy resistance mechanisms by potentially inhibiting multiple ABC transporters (as opposed to P-gp alone), transporter-mediated drug efflux is not the only cause of treatment resistance in AML (Funato et al., 2004). Thus, the use of ibrutinib as a drug efflux modulator may have limited efficacy against treatment resistance in AML.
Finally, perhaps the most important barrier to clinical translation of ibrutinib for this purpose is the potential for significant hematologic toxicity. As was demonstrated in Chapter 3.4, while combination ibrutinib-ethacridine treatment was preferentially cytotoxic to primary AML blasts, this combination was also cytotoxic in a subset of peripheral blood stem cell samples from healthy volunteers. Ibrutinib combinations would therefore require careful safety evaluation prior to its investigation as a combination candidate in clinical trials.
141 6.2 Conclusion
Using a high-throughput combination screening approach to identify drugs that synergize with ibrutinib, we have demonstrated that ibrutinib has activity against AML cells that is not limited to its inhibitory effect against BTK. While we did not elucidate the exact mechanism by which ibrutinib synergizes with chemical PARG inhibitors in our study, the observation that erlotinib synergizes with ethacridine by enhancing intracellular ethacridine accumulation sheds light on the strong possibility that ibrutinib relies on this same mechanism to synergize with ethacridine. Furthermore, we have demonstrated a potential role for PARG inhibition in AML. Both the mechanism by which ibrutinib mediates ethacridine accumulation in AML cells, and the impact of PARG inhibition in AML, warrant further study.
142
Chapter 7: Future Directions
143 7.1 Future Directions
7.1.1 Determining the mechanism of ethacridine accumulation by erlotinib and ibrutinib
7.1.1.1 ABC transporters
Given that erlotinib is known to inhibit drug efflux by P-gp, MRP1, and BCRP in its synergy with chemotherapy agents (Lainey et al., 2012) and that both erlotinib and ibrutinib potentiate ethacridine accumulation, it would be worthwhile to determine whether ethacridine accumulation is mediated by the inhibition of one or more of these transporters. To test this possibility, one could first assess expression of P-gp, MRP1, and BCRP by immunoblot in all of the cell lines in which ibrutinib and ethacridine were found to synergize (TEX, OCI-AML2, HL60, K562, U937, Jurkat D1.1). One possible method for narrowing down which ABC transporter(s) to investigate would be to compare expression of these transporters in cell lines exhibiting synergy to those expressed in KG1a, a cell line in which ibrutinib and ethacridine did not synergize: transporters common to KG1a and the other cell lines would likely not be responsible for mediating ethacridine accumulation.
After determining which transporters are exclusively expressed in synergizing cell lines, one could then assess the capacity of ibrutinib and erlotinib to functionally inhibit the relevant transporters by measuring substrate extrusion in the presence or absence of erlotinib or ibrutinib treatment. This would be carried out using the approach undertaken by Lainey et al. (2012): flow cytometry would be used to measure intracellular accumulation of fluorescent dyes that are preferentially extruded by a single ABC transporter in the presence or absence of TKI treatment. Rhodamine 123 and 3,3-diethyloxacarbocyanine iodide would be used to assess P-gp, Hoechst 33342 would be used to assess BCRP, and calcein and 5-(and-6)-carboxy-2',7' dichlorofluorescein diacetate would be used to assess
144 MRP function.
An alternate or complementary method for assessing whether ethacridine accumulation is caused by erlotinib or ibrutinib-mediated inhibition of these ABC transporters would be to assess whether substitution of erlotinib/ibrutinib with specific chemical ABC transporter inhibitors alone or in combination could recapitulate the synergy observed with ethacridine. Cyclosporine A (P-gp inhibitor), KO-143 (BCRP inhibitor), verapamil (P-gp and BCRP inhibitor), and MK-571 (MRP family inhibitor) could all be used alone or in combination with ethacridine in the synergizing AML cell lines to confirm whether inhibition of one or more of these transporters is responsible for ethacridine accumulation by erlotinib/ibrutinib.
To further confirm whether erlotinib/ibrutinib inhibits one or more ABC transporters to potentiate ethacridine accumulation, one could carry out lentiviral- mediated shRNA knockdown of ABC transporter genes in AML cell lines. Enhanced sensitization of these cells to ethacridine, relative to parental cells, would provide further confirmation that TKI-mediated ABC transporter efflux inhibition is the mechanism by which these drugs synergize with ethacridine.
7.1.2 Determining the relevant target of ethacridine
7.1.2.1 PARG inhibition
To determine whether PARG inhibition is the mechanism by which ethacridine is lethal to AML cells, one could first assess the effects on cell proliferation and viability following shRNA knockdown of PARG in TEX and OCI-AML2 cells. Increased PAR accumulation accompanied by reduced proliferation and viability in clones where complete knockdown is achieved would suggest that PARG
145 activity is essential for AML cell survival, and thus a possible lethal target of ethacridine.
One could further investigate the impact of PARG inhibition in AML cell lines by measuring resultant PAR accumulation by immunoblotting following combination TKI-ethacridine treatment, and then measuring nuclear translocation of apoptosis-inducing factor (AIF) by subcellular fractionation and subsequent immunoblotting. PAR accumulation—due to excess PARP1 activation or inhibited PARG activity—is necessary for nuclear translocation of apoptosis-inducing factor (AIF) from the mitochondria, which triggers nuclear condensation and subsequent cell death (Yu et al., 2006; Yu et al., 2002; Zhou et al., 2011). Thus, the observation that increased PAR accumulation leads to nuclear AIF translocation in response to combination TKI-ethacridine treatment would further support PARG inhibition as a relevant mechanism of ethacridine-mediated cell death.
Finally, PARG inhibition could be further ruled in or out as the lethal target of ethacridine by combination TKI-ethacridine treatment of PARP1-knockdown TEX and OCI-AML2 cells: reduced susceptibility of PARP1 knockdown cells to ibrutinib/erlotinib-ethacridine-induced synergistic cytotoxicity would favour PARG inhibition as the relevant target of ethacridine, as PARP1 inhibition impairs PAR polymer synthesis, thereby reducing the toxicity associated with PARG inhibition. Alternatively, one could overexpress PARG in synergizing cell lines and treat these cells with the TKI-ethacridine combination: reduced synergistic cytotoxicity relative to parental cell lines would also implicate PARG as the relevant target of ethacridine.
7.1.2.2 p53 induction and the ribosomal stress pathway
146
Given our preliminary data suggesting that ethacridine treatment induces p53 expression in TEX and OCI-AML2 cells, and a previous report demonstrating the capacity of acridine compounds (including ethacridine) to induce p53 activation via the ribosomal stress pathway (Morgado-Palacin et al., 2014), it would be worthwhile to investigate the potential involvement of this pathway in ethacridine- mediated AML cell death.
One could first determine whether single-agent ethacridine induces nucleolar disruption—and whether combination ibrutinib/erlotinib-ethacridine further potentiates nucleolar disruption—in TEX and OCI-AML2 cells by anti- nucleophosmin staining and immunofluorescence microscopy: relocalization of the nucleolar protein nucleophosmin to the nucleoplasm is one marker of nucleolar disruption.
If ethacridine (or the TKI-ethacridine combination) is found to induce nucleolar disruption in TEX and OCI-AML2 cells, one could then investigate whether this effect of ethacridine is linked to its induction of p53: ribosomal stress causes formation of the preribosomal complex RPL11/RPL5/5SrRNA, which binds MDM2 and thus blocks MDM2-mediated ubiquitination of p53 (Horn & Vousden, 2008). Morgado-Palacin et al. (2014) demonstrated that the acridine compound CID-765471 caused p53 activation by inducing RPL11 binding to MDM2. This group further demonstrated that knockdown of RPL11 abrogated p53 induction following CID-765471 treatment. To determine whether ethacridine induces p53 by this same mechanism, one could perform an immunoprecipitation assay to determine whether ethacridine and/or TKI-ethacridine treatment induces RPL11- MDM2 binding. Furthermore, reduced p53 induction by immunoblot following ethacridine treatment of RPL11-knockdown TEX and OCI-AML2 cells (relative to p53 induction in shRNA control TEX and OCI-AML2 cells) would also support ribosomal stress pathway-mediated induction of p53 as a mechanism of ethacridine toxicity.
147
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166 Appendix 1
Clinically achievable concentrations of tyrosine kinase inhibitors
Approximate steady-state Drug Dose (oral) Reference plasma concentration Ibrutinib 840mg/d 450nM (peak) Byrd et al. (2013) 5.3µM (peak) Imatinib 400mg/d Peng et al. (2005) 2.4µM (trough) 140nM (peak) Dasatinib 140mg/d (70mg BID) Demetri et al. (2009) 20nM (trough) Erlotinib 150mg/d 3.1µM (trough) Hidalgo et al. (2001)
167