Src-Family Kinases Impact Prognosis and Targeted Therapy in Flt3-ITD+ Acute Myeloid Leukemia Title Page by Ravi K. Patel Bachelor of Science, University of Minnesota, 2013 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2019 Commi ttee Membership Pa UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE Commi ttee Membership Page This dissertation was presented by Ravi K. Patel It was defended on May 31, 2019 and approved by Qiming (Jane) Wang, Associate Professor Pharmacology and Chemical Biology Vaughn S. Cooper, Professor of Microbiology and Molecular Genetics Adrian Lee, Professor of Pharmacology and Chemical Biology Laura Stabile, Research Associate Professor of Pharmacology and Chemical Biology Thomas E. Smithgall, Dissertation Director, Professor and Chair of Microbiology and Molecular Genetics ii Copyright © by Ravi K. Patel 2019 iii Abstract Src-Family Kinases Play an Important Role in Flt3-ITD Acute Myeloid Leukemia Prognosis and Drug Efficacy Ravi K. Patel, PhD University of Pittsburgh, 2019 Abstract Acute myelogenous leukemia (AML) is a disease characterized by undifferentiated bone-marrow progenitor cells dominating the bone marrow. Currently the five-year survival rate for AML patients is 27.4 percent. Meanwhile the standard of care for most AML patients has not changed for nearly 50 years. We now know that AML is a genetically heterogeneous disease and therefore it is unlikely that all AML patients will respond to therapy the same way. Upregulation of protein-tyrosine kinase signaling pathways is one common feature of some AML tumors, offering opportunities for targeted therapy. Important examples include activating mutations in the FLT3 receptor or overexpression of SRC-family kinases expressed in myeloid cells (HCK, FGR, LYN). Inhibition of HCK with the pyrrolopyrimidine kinase inhibitor A-419259 reversed AML cell bone marrow engraftment in patient-derived xenograft mice. Here we show that A-419259 inhibits not only HCK but also FGR, LYN and FLT3 bearing an activating internal tandem duplication (ITD). To investigate the relationship of FLT3, HCK and FGR to the A-419259 response, we generated TF-1 human myeloid cell populations expressing FLT3-ITD either alone or in combination with HCK or FGR. FLT3-ITD alone sensitized TF-1 cells to growth arrest by A-419259, supporting direct action on the FLT3 kinase domain. Cells transformed with inhibitor-resistant FLT3-ITD mutants (D835Y, F691L) were insensitive to A-419259, while co-expression of wild-type HCK or FGR with these FLT3 mutants restored inhibitor sensitivity. Expression of HCK or FGR mutants with engineered A-419259 resistance also decreased inhibitor sensitivity of TF-1/FLT3-ITD cells. To investigate how resistance to A-419259 evolves de novo, we developed populations of FLT3-ITD+ AML cell lines via long-term dose escalation. Whole exome sequencing identified only a single FLT3-ITD kinase domain mutation (N676S) among all A-419259 target kinases in each of six independent resistant cell populations. Thus, the anti-AML activity of A-419259 sdcjdsj............. iv involves inhibition of FLT3-ITD, HCK and FGR, suggesting that clinical inhibitors targeting all three kinases may enhance efficacy while reducing the probability of acquired resistance. v Table of Contents Acknowledgements .............................................................................................................. xviii 1.0 Introduction .........................................................................................................................1 1.1 Acute Myeloid Leukemia (AML) ................................................................................1 1.1.1 History of AML ................................................................................................1 1.1.2 Epidemiology of AML ......................................................................................2 1.1.3 Morphology and classification of AML ...........................................................2 1.1.3.1 French-American-British classification ................................................3 1.1.3.2 The World Health Organization (WHO) classification .......................6 1.1.3.3 European LeukemiaNet (ELN) classification .......................................9 1.1.4 Genomic landscape of AML ........................................................................... 10 1.1.4.1 Recurrently translocated transcription factor genes ......................... 15 1.1.4.2 Recurrently mutated myeloid transcription factors .......................... 17 1.1.4.3 NPM1 ................................................................................................... 17 1.1.4.4 Recurrently mutated DNA methylation genes ................................... 18 1.1.4.5 Recurrently mutated tumor suppressor genes ................................... 20 1.1.4.6 Recurrently mutated chromatin modifying genes .............................. 22 1.1.4.7 Spliceosome protein mutations ........................................................... 25 1.1.4.8 Cohesion mutations ............................................................................. 26 1.1.4.9 Recurrently activated signaling pathway mutations .......................... 26 1.1.5 Current treatment paradigms in AML ......................................................... 29 1.1.5.1 Chemotherapy regimens with cytarabine and anthracycline (7+3) .. 30 vi 1.1.5.2 All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) ............. 31 1.1.5.3 Therapies targeting mutant IDH1 and IDH2 ..................................... 32 1.1.5.4 Therapies targeting CD33 ................................................................... 32 1.1.5.5 Brief introduction to FLT3-targeted therapy ..................................... 33 1.2 Aberrant tyrosine kinase signaling in AML ............................................................. 34 1.2.1 FLT3 receptor tyrosine kinase ....................................................................... 34 1.2.1.1 Normal functions of FLT3 linked to AML etiology ........................... 34 1.2.1.2 Structure and activation of FLT3 ....................................................... 35 1.2.1.3 Targeted therapies against FLT3 in AML ......................................... 40 1.2.2 Non-receptor tyrosine kinases in AML and multi-targeted inhibitors ......... 48 1.2.2.1 AXL...................................................................................................... 48 1.2.2.2 SYK ...................................................................................................... 48 1.2.2.3 FES and FER ....................................................................................... 50 1.2.2.4 SRC-family kinases ............................................................................. 50 1.2.3 Serine/Threonine kinases in AML and multi-targeted inhibitors ................ 62 1.2.3.1 MAP kinases ........................................................................................ 62 1.2.3.2 PIM kinases ......................................................................................... 62 1.2.3.3 Cyclin dependent kinases 4 and 6 ....................................................... 63 1.3 Two hit model for leukemogenesis ............................................................................ 63 1.4 Hypothesis and specific aims..................................................................................... 65 1.4.1 Hypothesis ...................................................................................................... 65 1.4.2 Specific aims ................................................................................................... 66 vii 1.4.2.1 Aim 1: Examine the relationship of SRC-family kinase expression on AML patient survival...................................................................................... 66 1.4.2.2 Aim 2: Determine the effect of SRC-family kinase expression on AML cell responses to kinase inhibitors ........................................................ 67 1.4.2.3 Aim 3: Investigate de novo mechanisms of A-419259 resistance in FLT3-ITD+ AML ............................................................................................ 67 1.4.2.4 Aim 4: Determine HCK mutations that are highly resistant to A- 419259 .............................................................................................................. 68 2.0 Expression of myeloid SRC-family kinases is associated with poor prognosis in AML and influences FLT3-ITD+ kinase inhibitor acquired resistance ................................ 69 2.1 Chapter 2 summary................................................................................................... 69 2.2 Introduction ............................................................................................................... 70 2.3 Results ........................................................................................................................ 73 2.3.1 Myeloid SRC-family kinase expression is predictive of patient survival in AML….………………………………………………………………………………73 2.3.2 A-419259 targets multiple AML-associated kinases in vitro and in cells ...... 75 2.3.3 FLT3-ITD is a target for A-419259 in transformed AML cells .................... 81 2.3.4 Mutants of HCK and FGR with engineered resistance reduce AML cell sensitivity to A-419259 ...........................................................................................