Antagonistic activities of the immunomodulator and protein phosphatase 2A (PP2A)-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven myeloproliferative neoplasms. DISSERTATION Presented in Partial Fulfillment of the Requirements for the degree Doctor of Philosophy in the Graduate School of The Ohio State University By Joshua John Oaks, B.S. Integrated Biomedical Sciences Graduate Program The Ohio State University 2013 Dissertation Committee Danilo Perrotti MD, PhD Roger Briesewitz, PhD Denis Guttridge, PhD Harold Fisk, PhD Copyright 2013 Joshua John Oaks ABSTRACT FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)- activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in Polycythemia Vera (PV) and other myeloproliferative neoplasms (MPNs) characterized by the expression of the transforming Jak2V617F oncogene. PP2A inactivation occurs in a Jak2V617F dose/kinase- dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET and by modulation of nitric oxide synthase (NOS2)-induced nitrosylation of PP2A. Genetic and/or PAD-mediated PP2A reactivation induces Jak2V617F inactivation/downregulation and impairs clonogenic potential of Jak2V617F cell lines and PV but not normal CD34+ progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2V617F cells, FTY720 anti-leukemic activity neither requires FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we showed that Jak2V617F also utilizes an alternative sphingosine kinase-1 (SPHK1)- mediated pathway to inhibit PP2A, and that FTY720-P, acting as a sphingosine-1- phosphate-receptor-1 (S1PR1) agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC- SET-mediated PP2A inhibition. Thus, PADs (e.g. FTY720) represent suitable therapeutic alternatives for Jak2V617F MPNs. ii ACKNOWLEDGEMENTS This work would not be possible without the help and guidance of many people to whom I am indebted. Firstly, I would like to thank my advisor, Dr. Danilo Perrotti. He has given me the inspiration, direction and opportunity to pursue the research I love. He is the spark that drives all of us who work for him and without his guidance and experience none of this work would be possible. Likewise, all of the current and former members of the Perrotti laboratory I have had the pleasure to work with have been essential making this work possible. I would also like to thank: Dr. J. Van Brocklyn who has served as my “sphingolipid expert” throughout this project. He was always willing to assist me with protocols, discussions, or experiments and was never reluctant to do so despite his busy schedule; Dr. Briesewitz for his technical support but more for his willingness to be involved with my professional development; and, finally, Drs. Landesman and Kalid, and Ms. Shechter, and the crew at Karyopharm for their enthusiasm for our research on pharmacologic development of SET inhibitors and their generosity allowing the continuation of this project. iii Vita et Studiorum Born March 18, 1977 . ………………………………………………………. Dayton, Ohio 2003 ………………………………………………B.S. Dietetics, University of Cincinnati Field of Study Major Field: Integrated Biomedical Sciences Area of Emphasis: Pharmacology iv Table of Contents Abstract…………………………………………………………………..……………..……ii Acknowledgements…………………………………………………………………..…….iii Vita et Studiorum…..………………………………………………………….……..……..iv List of Tables……………………………………………………………………………….vi List of Figures………………………………………………………………………….…..vii Chapters 1. Introduction……...………………………………………………….………………..1 2. Regulation of Jak2- and Jak2V617F-driven erythropoiesis…………...…......…..16 3. Methods…………………..…………………………………………………….…...92 4. Results………………………………………………………..………..……….….109 5. Discussion……………………………………………………………….………...139 6. Future Directions……………………………………………………………….....146 References………………………………………………………………………..…..……158 v List of Tables 1 List of major kinases regulated by PP2A…………………………….……..…42 2 S1P receptor G-protein coupling…………………………………………...….58 3 S1P1 downstream inhibitors……………………………………………...….…93 vi List of Figures 1 Jak2 and common Jak2 mutations ………………...……………………..…………4 2 Myeloid hematopoiesis …..………………………..………………..……...……..……13 3 Wild type and V617F Jak2 signaling ...…….………………..…………………...…14 4 Protein Phosphatase 2a (PP2A) ……………………….………..……………….........17 5 Nitric oxide synthase (NOS) activates PP2A …………………..……………..……….53 6 Sphingolipids ………………………………………………..…………….......…….57 7 FTY720 and FTY720-(S)-Phosphate …….……………………...………………..……84 8 FTY720 Immunosuppressive activity …...……………...…………….…………….85 9 FTY720 anti-cancer activity ………….....……………...…………….…………….89 10 Jak2V617F suppresses PP2A activity in a SET-dependent manner A and B……….110 10 Jak2V617F suppresses PP2A activity in a SET-dependent manner C and D………112 11 Jak2V617F/PI3Kγ/PKC signals inhibit PP2A through SET phosphorylation A.….…114 11 Jak2V617F/PI3Kγ/PKC signals inhibit PP2A through SET phosphorylation B.….…115 11 Jak2V617F/PI3Kγ/PKC signals inhibit PP2A through SET phosphorylation C and D…………………………………………………………………………………………...115 12 In vivo FTY720 anti-leukemic effects and lack of toxicity A and B ……………………………………………………………………………………….…….120 12 In vivo FTY720 anti-leukemic effects and lack of toxicity C…………………………121 12 In vivo FTY720 anti-leukemic effects and lack of toxicity D…………………………123 12 In vivo FTY720 anti-leukemic effects and lack of toxicity E…………………………124 13 FTY720 phosphorylation is dispensible for its anti-leukemic activity A….………125 13 FTY720 phosphorylation is dispensible for its anti-leukemic activity B….………126 vii 13 FTY720 phosphorylation is dispensible for its anti-leukemic activity C….………128 14 FTY720-P promotes Jak2 and suppresses PP2A activity A and B…..………....…131 14 FTY720-P promotes Jak2 and suppresses PP2A activity C…..………………....…132 14 FTY720-P promotes Jak2 and suppresses PP2A activity D and E…..………....…134 15 FTY720-dependent PP2A activation in myeloid cells depends on the SET K209- FTY720 interaction but not on SET dimerization or ceramide levels A and B...….136 15 FTY720-dependent PP2A activation in myeloid cells depends on the SET K209- FTY720 interaction but not on SET dimerization or ceramide levels C and D...….138 16 Antagonizing effect of FTY720 and FTY720-P on leukemogenesis……………….145 17 Novel FTY720 analogs …..…….………..............................................................…150 18 SET inhibitor development strategy……… ……………………..…….………..….…152 19 Molecular docking of FTY720 and SET………..………..……..………….……….…153 20 Novel SET inhibitors ……………………………….……………….......…………..…155 viii CHAPTER 1 INTRODUCTION Polycythemia Vera (PV) is a rare hematopoietic malignancy that involves the erythroid (red blood cell) system. It is currently defined as a myeloproliferative neoplasm (MPN) and affects fewer than 2 people in one million. This disorder was first described by Louis Vaquez in 1892 (1) and by William Osler in 1903 (2) and was originally called “Osler- Vaquez disease”. Initially, the disorder was associated to altitude sickness “the torpor, mental and physical; the sensation of fullness in the head, with headache, vertigo and in some cases nausea and vomiting, remind us of the symptoms to which mountain climbers and aeronauts are subject” (2). By modern definitions, PV typically manifests as uncontrolled erythropoiesis (3-5) with a hematocrit of >52% in Caucasian men and >47% in men of African origin or in women. Splenomegaly and pruritus are also common sympthoms used for diagnosis of PV (6). The high erythroid cell content in the peripheral blood (PB) of PV patients leads to hyperviscosity, which can result in thrombosis (7-12) and, therefore, in high risk for heart attack or stroke. While red blood cell count is still part of the PV diagnostic panel, a polymerase chain reaction (PCR)-based test is now used to assess presence of a mutated Janus Kinase 2 (Jak2) oncogene that in the majority of PV cases represents the hallmark of this hematologic disorder (see below). 1 1.1 The Jak2 receptor tyrosine kinase Janus Kinase 2 (Jak2) is a receptor-bound cytosolic tyrosine kinase consisting of 7 JH (Jak homology) domains: a Src homology 2 domain (SH2), a tyrosine kinase domain (JH1), two c-terminal domains, and a pseudokinase domain (JH2). The JH3-7 region forms a FERM (4.1 protein, ezrin, radixin, moesin-homology) domain, which is required for binding to the erythropoietin (EPO) receptor. Jak2 translates a conformational change caused by ligand [e.g. EPO] binding to cytokine receptors (e.g. EpoR) into an intracellular signaling cascade (13). This is accomplished by the phosphorylation of specific tyrosine residues on the receptor N-terminus (intracellular) that provides a docking site for signal transducer and activator of transcription (STAT) proteins (14, 15), which upon Jak2-dependent phosphorylation dimerize and relocate into the nucleus where they act as modulators of transcription (16-18). Chromosomal alterations and gene mutations have been reported for the Jak2 gene which results in a mutated Jak2 protein (Figure 1). The best described chromosomal abnormality is the gene translocation leading to the formation of chimeric tyrosine kinase protein TEL-Jak although a PCM1-Jak2