Characterization of Structure-Function Correlations of Novel Jak2 Small Molecule Inhibitors and Their Mechanisms of Action

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Characterization of Structure-Function Correlations of Novel Jak2 Small Molecule Inhibitors and Their Mechanisms of Action
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Majumder,Anurima
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Doctorate ( Ph.D.)
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University of Florida
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Medical Sciences, Physiology and Pharmacology (IDP)
Committee Chair:
Sayeski, Peter P
Committee Members:
Law, Brian K
Shiverick, Kathleen A
Rowe, Thomas C
Slayton, William B

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benzothiophene -- calpain -- hematology -- inhibitor -- jak2 -- mpn -- stilbene -- v617f -- vimentin
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
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Abstract:
Jak2 is a cytoplasmic tyrosine kinase that is involved in signaling via a diverse range of ligands, including cytokines and growth factors. Hyperkinetic Jak2 tyrosine kinase has been linked to various neoplastic disorders, including solid tumors and hematological malignancies. In 2005, a Jak2 gain-of function mutation, Jak2-V617F, was identified in a majority of patients with myeloproliferative neoplasms (MPNs). This discovery spurred a great deal of interest in identifying small molecule inhibitors that target Jak2, as effective inhibitors may have significant therapeutic potential for Jak2-mediated pathologies and may also serve as useful research tools to study Jak2-mediated signaling in general. In this dissertation, we first characterize a novel benzothiophene based small molecule Jak2 inhibitor, A46, which we have identified by structure-based drug design. We show that A46 specifically inhibited the proliferation of Jak2-V617F expressing cells in both a time- and dose-dependent manner. Cells exposed to 10 ?M of A46 for 48 hours or more were unable to recover after drug removal. A46 also caused a corresponding decrease in the phosphorylation of Jak2 and STAT3 proteins within these cells. Cell growth inhibition correlated with an induction of cell cycle arrest and promotion of apoptosis. Moreover, we report that this compound inhibited the pathologic growth of primary Jak2-V617F expressing bone marrow cells, ex vivo. Collectively, our data demonstrate that the benzothiophene based compound, A46, suppresses Jak2-mediated pathogenesis, thereby making it a potential candidate drug against Jak2-mediated disorders. Our lab has previously identified a novel Jak2 inhibitor called G6 using structure-based drug design. G6 showed promising results in preclinical studies and inhibited Jak2 tyrosine kinase-mediated pathologic cell growth in vitro, ex vivo and in vivo. Here, we identified a structure-function correlation of this compound. Specifically, we showed that the stilbenoid core in G6 is essential for its ability to i) inhibit Jak2-V617F-dependent cell proliferation, ii) suppress phosphorylation of key signaling molecules involved in the Jak/STAT pathway, such as Jak2, STAT3 and STAT5, iii) induce apoptosis in HEL cells via the intrinsic apoptotic pathway, iv) inhibit pathologic growth of patient-derived Jak2-V617F-positive bone marrow cells, ex vivo and v) form strong docking interactions with the ATP-binding pocket of Jak2 kinase domain. As such, we demonstrated that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential. Having shown that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation, we next wanted to elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation. For this, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 hours with either vehicle control or 25 ?M of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells, but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.
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In the series University of Florida Digital Collections.
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by Anurima Majumder.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
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Adviser: Sayeski, Peter P.
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1 CHARACTERIZATION OF STRUCTURE FUNCTION CORRELATIONS OF NOVEL JAK2 SMALL MOLECULE INHIBITORS AND THEIR MECHANISMS OF ACTION By ANURIMA MAJUMDER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Anurima Majumder

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3 T o my Mom Dad and Arjun for their constant love and support

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4 ACKNOWLEDGMENTS I would like to acknowledge all those people who have made this dissertation possible and because of whom my graduate experience has been one that I will cherish forever. First, I would like to thank m y mentor, Dr. Peter P. Sayeski. I consider myself fortunate to have had Peter as my advisor. His patience, support, encouragement and constant guidance throughout my graduate studies has helped me overcome problems and achieve my goals. He not only taught me research techniques, but also guided me on how to improve my scientific writing and presentation skills. Peter has been more than just a mentor for graduate studies by being readily available to help me out with research problems, career options as well as any other questions that I have had. Second, I would like to thank the members of my supervisory committee, Drs. Kathleen Shiverick, Thomas Rowe, Brian Law and Willia m Slayton for their invaluable guidance, insight and advi ce through the course of my graduate studies. I would also like to thank a few people who have taught me or he lped me with various techniques over these past four years. Special thanks to Andrew Magis and Lakshmanan Govindasamy for helping me with the molecular docking studies I would also like to thank Marjorie Chow and Kanchana Karrupiah The two dimensional ge l electrophoresis studies would not have been possible without them. In addition, I also thank Doug Smith and Steve McClellan for their help with fluorescent microscopy and flow cytometry, respectively. I would next like to thank all past and present mem bers of the Sayeski Lab: Jacqueline Sayyah, Robert Blair, Nicholas Figueroa, Shige haru Tsuda, Dr. Sung Park, Annet Kirabo, Kavitha Gnanasambandan and Rebekah Baskin. They not only p rovided an environment conducive to learning and research but also made la b life a lot more

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5 enjoyable. I am going to miss all the fun stuff we did together both inside and outside lab. Special thanks to my friends for their support and companionship during my stay in Gainesville. Thank you for being there for me when times were stressful. Lastly, I would like thank my family for their unending advice, encouragement, love and support without which I would never have been able to complete my graduate studies. I am deeply indebted to my parents for their constant wisdom and encourag ement. I have come so far in life only because of them and the values that they have instilled in me. I would also like to thank my little sister, Anusmita, for reducing my stress by providing comic relief. I am also grateful to my in laws for their suppor t and encouragement. Finally, thanks to my husband, Arjun, for bearing with me and my mood swings and keeping me sane during times of stress. His love, understanding and support have been invaluable.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Tyrosine Kin ases ................................ ................................ ................................ .... 16 Jak2 Tyrosine Kinase ................................ ................................ .............................. 17 History ................................ ................................ ................................ .............. 17 Structure ................................ ................................ ................................ ........... 18 Jak/STAT Signaling Paradigm ................................ ................................ .......... 20 Jak2 and Cancer ................................ ................................ ................................ ..... 21 Jak2 in Solid tumors ................................ ................................ ......................... 21 Jak2 in Hematological Malignancies ................................ ................................ 22 Jak2 chromosomal translocations and hematological malignancies .......... 23 Jak2 point mutations and hematological malignancies .............................. 23 Myeloproliferative Neoplasms ................................ ................................ ................. 24 Jak2 Mutat ions in Myeloproliferative Neoplasms ................................ .............. 25 Jak2 Inhibitors for the Treatment of Myeloproliferative Neoplasms .................. 27 Rationale for Studies ................................ ................................ ............................... 34 2 A46, A BENZOTHIOPHENE DERIVED COMPOUND, SUPPRESSES JAK2 MEDIATED PATHOLOGIC CELL GROWTH ................................ .......................... 42 Materials and Methods ................................ ................................ ............................ 43 Drug ................................ ................................ ................................ .................. 43 Cell Culture ................................ ................................ ................................ ....... 44 Cell Proliferation Assay ................................ ................................ .................... 44 Enzyme linked Immunosorbent Assay ................................ ............................. 44 Cell Cycle Assay ................................ ................................ .............................. 44 Apoptosis Assay ................................ ................................ ............................... 45 Western Blotting ................................ ................................ ............................... 45 Colony Formation Assay ................................ ................................ .................. 45 Clonogenic Assay ................................ ................................ ............................. 46 Statistical Analysis ................................ ................................ ............................ 46 Results ................................ ................................ ................................ .................... 46

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7 A46 Inhibits Jak2 V617F dependent Cell Prolifer ation ................................ ..... 46 A46 Inhibits the Jak/STAT Signaling Pathway ................................ .................. 48 A46 Induces G1/S Cell Cycle Arrest in HEL Cells ................................ ............ 49 A46 Induces Apoptosis in HEL Cells ................................ ................................ 50 A46 Suppresses Cytokine independent Pathologic Cell Growth of Jak2 V617F Positive Bone Marrow Cells, ex vivo ................................ .................. 52 Discussion ................................ ................................ ................................ .............. 53 3 STRUCTURE FUNCTION CORRELATION OF G6, A NOVEL SMALL MOLECULE INHIBITOR OF JAK2: INDISPENSABILITY OF THE STILBENOID CO RE ................................ ................................ ................................ ..................... 64 Experimental Procedures ................................ ................................ ........................ 65 Drugs ................................ ................................ ................................ ................ 65 Cell Culture ................................ ................................ ................................ ....... 66 Cell Proliferation Assay ................................ ................................ .................... 66 Enzyme linked Immunosorbent Assay ................................ ............................. 66 Cell Lysis and Immunoprecipitation ................................ ................................ .. 66 Western Blotting ................................ ................................ ............................... 67 Apoptosis Assay ................................ ................................ ............................... 67 R eal Time PCR Analysis ................................ ................................ .................. 68 Patient Sample ................................ ................................ ................................ 68 Colony Forming Unit Erythroid Colony Formation Assay ................................ 68 Computational Docking ................................ ................................ .................... 69 Statistical Analysis ................................ ................................ ............................ 70 Results ................................ ................................ ................................ .................... 70 A Stilbenoid Core Is Essential for Time and Dose dependent Inhibition of Jak2 V617F dependent Cell Growth ................................ ............................. 70 Indispensability of the Stilbenoid core in Decreasing Phos phorylation of STAT3 and STAT5 ................................ ................................ ........................ 72 Induction of Apoptosis in HEL Cells by G6 and its Derivatives ......................... 73 Stilbenoid Core Bearing Derivat ives of G6 Suppress Pathologic Cell Growth of Patient derived Bone Marrow Cells, ex vivo ................................ .............. 74 Computational Docking of G6 and its Derivatives into the ATP Binding Pocket of the Jak2 Kinase Domain ................................ ............................... 75 Discussion ................................ ................................ ................................ .............. 77 4 CELL DEATH INDUCED BY THE JAK2 INHIBITOR, G6, CORRELATES WITH CLEAVAGE OF VIMENTIN FILAMENTS ................................ ............................... 90 Experimental Procedures ................................ ................................ ........................ 91 Drugs ................................ ................................ ................................ ................ 91 Reagents ................................ ................................ ................................ .......... 92 Cell Culture ................................ ................................ ................................ ....... 92 2 D Differential In Gel Electrophoresis (2 D DIGE) ................................ .......... 92 Cell Lysis ................................ ................................ ................................ .......... 94 Western Blotting ................................ ................................ ............................... 94

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8 Immunofluorescence ................................ ................................ ........................ 95 Cell Proliferation Assay ................................ ................................ .................... 95 In vivo Animal Model ................................ ................................ ........................ 95 Bone Marrow Immunohistochemistry ................................ ............................... 96 Statistical Analysis ................................ ................................ ............................ 96 Results ................................ ................................ ................................ .................... 96 G6 Treatment Induces Time and Dose Dependent Degradation of Vimentin 96 G6 Treatment Induces Marked Reorganization of Vimentin Intermediate Filaments within Cells ................................ ................................ .................... 98 G6 induced Cleavage of Vimentin is Jak2 mediated ................................ ........ 99 G6 induced Cleavage of Vimentin is Independent of de novo Protein Synthesis and Caspase Activity, but Calpain dependent .............................. 99 The Mobilization of Calcium is Es sential and Sufficient for the Cleavage of the Intermediate Filament Protein Vimentin ................................ ................ 101 Cleavage of Vimentin is Sufficient to Reduce HEL Cell Viability .................... 102 G6 Treatment Decreases the Levels of Vimentin Protein, in vivo ................... 103 Discussion ................................ ................................ ................................ ............ 104 5 CONCLUSIONS AND PE RSPECTIVES ................................ ............................... 116 Importance of Designing/Identifying a Jak2 Specific Inhibitor ............................... 117 Characterization of the Novel Jak2 Inhibitor, A46 ................................ ................. 119 Stilbenes and Benzothiophenes as Potential Scaffolds for the Design of Novel Jak2 Small Molecule Inhibitors ................................ ................................ .......... 120 Comparison of Two Novel Jak2 Inhibitors, G6 and A46 ................................ ........ 122 Potential of Jak2 Inhibitor Therapy for Myeloproliferative Neoplasms .................. 124 Characterization of the Link B etween G6 induced Cel l Death and Clea vage of Vimentin ................................ ................................ ................................ ............. 129 LIST OF REFERENCES ................................ ................................ ............................. 134 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 150

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9 LIST OF TABLES Table page 1 1 Preclinical characterization of Jak2 inhibitors ................................ ..................... 39 1 2 Clinical characterization of Jak 2 inhibitors ................................ .......................... 40 2 1 Chemical characteristics of A46 ................................ ................................ ......... 57 3 1 Percent growth inhibition of G6 and its derivatives ................................ ............. 80 5 1 GI 50 of other Jak2 inhibitors in HEL cells ................................ .......................... 133

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10 LIST OF FIGURES Figure page 1 1 Tyrosine kinase ca talyzed phospho transferase reaction ................................ ... 36 1 2 Schematic representation of the Jak2 protein showing the regions where the somatic mutations identified in myeloproliferative neoplasm (MPN) patients are commonly found ................................ ................................ ........................... 37 1 3 Schematic representation of the Jak/STAT signaling paradigm ......................... 38 2 1 A46 inhibits Jak2 V617F dependent ce ll proliferation ................................ ......... 58 2 2 A46 inhibits the Jak/STAT signaling pathway ................................ ..................... 59 2 3 A46 arrests HEL cells in G1 phase of the cell cycle ................................ ........... 60 2 4 A46 induces apoptosis in HEL cells ................................ ................................ .... 61 2 5 A46 induced apoptosis is mediated by the down regulation/cleavage of Bcl 2 family pr oteins Bim, Bax and Bid ................................ ................................ ........ 62 2 6 A46 suppresses cytokine independent pathologic cell growth of Jak2 V617F positive bone marrow cells, ex vivo ................................ ................................ .... 63 3 1 Time and dose dependent effect of G6 and its derivatives on the HEL cell proliferation and Jak2 phosphorylation ................................ ............................... 81 3 2 Dose dependent effect of G6 and its derivatives on the p roliferation of Ba/F3 EpoR Jak2 V617F cells ................................ ................................ ...................... 82 3 3 Inhibition of phosphorylation of STAT3 and STAT5 by G6 and its derivatives .... 83 3 4 Induction of apoptosis in HEL cells by G6 and its derivatives ............................. 84 3 5 Treatment with G6 or its stilbenoid derivatives leads to HEL cell death via the intrinsic apoptotic pathway ................................ ................................ .................. 85 3 6 Suppression of Jak2 V617F mediated pathologic cell growth in patient derived bone marrow cells by G6 and its derivatives, ex vivo ............................. 86 3 7 Molecular surface representation of the ATP binding site of Jak2 ...................... 87 3 8 Molecular docking of G6 and its derivatives into the ATP binding pocket of Jak2 ................................ ................................ ................................ .................... 88 3 9 Docking of G6 and its derivatives into the ATP binding pocket of Jak2 .............. 89

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11 4 1 Identification of vimentin as a differentially expressed protein between vehicle treat ed and G6 treated HEL cells ................................ ................................ ..... 108 4 2 G6 treatment induces time and dose dependent degradation of vimentin ...... 109 4 3 G6 treatment ind uces marked reorganization of vimentin intermediate filaments within cells ................................ ................................ ......................... 110 4 4 G6 induced cleavage of vimentin is Jak2 mediated ................................ ......... 111 4 5 G6 induced cleavage of vimentin is independent of de novo protein synthesis and caspase activity, but calpain dependent ................................ .................... 112 4 6 Mobilization of calcium is essential and sufficient for the cleavage of vimentin 113 4 7 Cleavage of vimentin is sufficient to reduce HEL cell viability ........................... 114 4 8 G6 treatment decreases t he levels of vimentin protein, in vivo ......................... 115 5 1 Surface representation of the structure of Jak2, indicating possible sites for inhibitor targeting ................................ ................................ .............................. 132

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12 LIST OF ABBREVIATIONS Jak2 Janus Kinase 2 JH1 Jak Homology 1 JH2 Jak Homology 2 SH2 Scr Homology 2 FERM 4.1 Ezrin Radixin Moesin GPCR G protein Coupled Receptor STAT Sign al Transducers and Activators of Transcription MPN Myeloproliferative Neoplasms PV Polycy themia Vera ET Essential Thrombocythemia PMF Primary Myelofibrosis HEL Human Erythroleukemia DMSO Dimethyl Sulfoxide ELISA Enzyme linked Immunosorbent Assay G1/S Gap1/Synthesis DTT Dithiothreitol SDS PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electroph oresis FITC Fluorescein Isothiocyanate IDPN iminodipropionitrile

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Phil osophy CHARACTERIZATION OF STRUCTURE FUNCTION CORRELATIONS OF NOVEL JAK2 SMALL MOLECULE INHIBITORS AND THEIR MECHANISMS OF ACTION By Anurima Majumder August 2011 Chair: Peter P. Sayeski Major: Medical Sciences Physiology and Pharmacology Jak2 is a cyto plasmic tyrosine kinase that is involved in signaling via a diverse range of ligands, including cytokines and growth factors. Hyperkinetic Jak2 tyrosine kinase has been linked to various neoplastic disorders, including solid tumors and hematological malign ancies. I n 2005, a Jak2 gain of function mutation, Jak2 V617F, was identified in a majority of patients with myeloproliferative neoplasms (MPNs). This discovery spurred a great deal of interest in identifying small molec ule inhibitors that target Jak2 as e ffective inhibitors may have significant therapeutic potential for Jak2 mediated pathologies and may also serve as useful research tool s to study Jak2 mediated signaling in general. In this dissertation, we first characterize a novel benzothiophene based small molecule Jak2 inhibitor, A46, which we have identified by structure based drug design. W e show that A46 specifically inhibited the proliferation of Jak2 V617F expressing cells in both a time and dose dependent manner. Cells exposed to 10 M of A46 f or 48 hours or more were unable to recover after drug removal. A46 also caused a corresponding decrease in the phosphorylation of Jak2 and STAT3 proteins within these cells. Cell growth inhibition correlated with an induction of cell cycle arrest and prom otion of apoptosis. Moreover, we report that this compound

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14 inhibited the pathologic growth of primary Jak2 V617F expressing bone marrow cells, ex vivo Collectively, our data demonstrate that the benzothiophene based compound, A46, suppresses Jak2 mediated pathogenesis, thereby making it a potential candidate drug against Jak2 mediated disorders. Ou r lab has previously identified a novel Jak2 inhibitor called G6 using structure based drug design. G6 showed promising results in preclinical studies and inhi bited Jak2 tyrosine kinase mediated pathologic cell growth in vitro ex vivo and in vivo Here, we identified a structure function correlation of this compound. Specifically, we showed that the stilbenoid core in G6 is essential for its ability to i) inhib it Jak2 V617F dependent cell proliferation, ii) suppress phosphorylation of key signaling molecules involved in the Jak/STAT pathway, such as Jak2, STAT3 and STAT5, iii) induce apoptosis in HEL cells via the intrinsic apoptotic pathway, iv) inhibit patholo gic growth of patient derived Jak2 V617F positive bone marrow cells, ex vivo and v) form strong docking interactions with the ATP binding pocket of Jak2 kinase domain. As such, we demonstrated that G6 has a stilbenoid core that is indispensable for maintai ning its Jak2 inhibitory potential. Having shown that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2 mediated cellular proliferation we next wanted t o elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2 mediate d cellular proliferation For this, we treated Jak2 V617F expressing human erythroleukemia (HEL) cells for 12 hours with either vehicle control or 25 M of the drug and compared protein expression profiles using two dimensional gel electrophoresis. One dif ferentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein,

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15 vimentin. It was present in DMSO treated cells, but absent in G6 treated cells. HEL cells treated with G6 showed both time and dose dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2 V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo The G6 induced cleava ge of vimentin was found to be Jak2 dependent and calpain mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filament s, per se is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6 induced inhibition of Jak2 mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describ es a novel pathway for the targeting of Jak2 mediated pathological cell growth.

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16 CHAPTER 1 INTRODUCTION Tyrosine Kinases Tyrosine kinases are enzymes that catalyze the transfer of the phosphate of ATP to tyrosine residues of protein substrates (Fig. 1 1). These enzymes are critical components of important signaling pathways that regulate vital physiological processes such as cell proliferation, survival, differentiation and apoptosis. Abnormal tyrosine kinase activity has however been linked to cance r, immunodeficiency, artherosclerosis, cardiovascular disease etc. Tyrosine kinases can be divided into two broad subfamilies ; receptor tyrosine kinases (RTKs) and non receptor tyrosine kinases (NRTKs). The RTKs usually possess a ligand binding extracellu lar domain, a transmembrane domain and a catalytic intracellular kinase domain. These membrane spanning kinases, therefore, have intrinsic kinase activity and can undergo autophosphorylation upon activation by ligand binding. The RTK family includes the in sulin receptor and several growth factor receptors, such as epidermal growth factor receptor and platelet derived growth factor receptor [ 1 ] The NRTKs are usually cytoplasmic and possess a kinase domain but lack receptor like features such as extracellular and transmembrane domains [ 1 ] However, these tyrosine kinases have additional domains that enable them to interact with other signaling molecules, such as 1) an N terminal myristylation/palmitoylation domain for anchoring the kinase to the plasma membrane, 2) a pleckstrin homology (PH) domain for binding to lipids, 3) an SH2 domain to bind phospho tyrosines on other proteins, and Portion of this chapter has been reproduced from Jak2 Inhibitors for the treatment of Myeloproliferative Neoplasms. Drugs of the Future 2010; 35(8): 651 660 with permission from 2010 P rous Science, S. A. U. or its licensors.

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17 4) an SH3 domain to bind to proline rich sequences on other proteins [ 2 ] Unlike growth factor receptors, cytokine receptors do not have an intrinsic kinase dom ain. However, these receptors associate with non receptor tyrosine kinases and can thereby induce tyrosine phosphorylation of the receptors and a variety of cellular proteins [ 3 ] The NRTK family includes the Src, Abl, Csk and FAK kinase subfamilies among many others. The Janus kinases form a distinct family of cytoplasmic tyrosine kinases that have a critical role to play in diverse signaling pathways that are essential for the normal physiology and function. Jak2 Tyrosine Kinase History Jak2, a cytosolic protein of approximately 130 kDa in mass, is a member of the Janus family of cytoplasmic tyrosine kinases. Other known members of this family are Tyk2, Jak1 and Jak3. These kinases are ubiquitously expressed in a variety o f different cell types with the exception of Jak3, which is predominantly expressed i n cells of hematopoietic origin [ 4 ] Tyk2 (Tyrosine kinase 2), the first memb er of the Janus tyrosine kinase family to be cloned, was identified by screening a T cell cDNA library using the sequence of the c fms tyrosine kinase domain via a low stringency hybridization technique [ 5 ] The kinase domains of Jak1 and Jak2 were cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides complementary to highly conserved kinase domains of the Src family members [ 6 ] These kinase domain sequences were then used to clone the full length Jak1 and Jak2 proteins [ 7 8 ] Jak3 was also cloned using similar PCR based techniques [ 9 10 ] Initially, this family was mology studies revealed that a unique feature of all the members of this family of tyrosine kinases is

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18 that they have two potential kinase domains at their C ter minus. Thus, this family was renamed as Janus Kinase after Janus the Roman God with two opposite faces. Structure The unique structure of the Jak kinases clearly distinguishes them from other members of the protein tyrosine kinase family. Simil ar to other members of the Jak family, Jak2 has seven highly conserved Jak homology (JH) domains, JH1 through JH7, numbered from the C to the N terminus, respectively [ 1 1 ] (Fig. 1 2 ). A hallmark feature of all Janus kinases is the presence of two domains (JH1 and JH2) in the carboxyl half of the protein that both have extensive homology to the kinase domain of other protein tyrosine kinases. However, only the JH1 domain has functional kinase activity whereas the JH2, or pseudokinase domain, has an important role in limiting the activity of the kinase domain. The JH1 domain is a highly conserved kinase domain which contains the activation loop, the critical sites of prima ry phosphorylation (Tyr1007 and Tyr 1008), and the ATP binding region. Phosphorylation of Tyr1007 residue in the activation loo p is essential for maximal Jak2 kinase activity [ 12 ] The JH2 domain which is adjacent to the JH1 domain, negatively regulates both the basal as well as the ligand induced levels of phosphotransferase activity [ 13 14 ] by physically interacting with the JH1 domain [ 15 ] Even though thi s domain is structurally homologous to the JH1 domain, it lacks certain critical amino acid residues that are essential for functional kinase activity and hence is not catalytically active [ 16 ] The amino half of Jak2 c onsists of domains JH3 through JH7. The JH3 JH4 region weakly resembles the canonical Src Homology 2 (SH2) domain [ 17 ] and is hence called the SH2 like domain, but its function is yet to be elucidated [ 18 ] The JH4 JH7 domains a re N terminal to the putative SH 2 domain and are collectively called the FERM (4.1,

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19 Ezrin, Radixin, Moesin) domain This domain is primarily involved in the binding to and crosstalk with other cellular proteins [ 19 ] For example, regions within the JH6 and JH7 domains mediate i nteractions with cell surface receptors by associating with conserved Box 1 structural motifs on the cytokine receptors [ 20 21 ] The three dimensional crystal structure of the entire Jak2 molecule has not yet been resolved. However, a portion of the Jak2 kinase domain has been crystallized recently [ 22 ] This crystal structure gives important insights into the precise atomic architecture of the Jak2 tyrosine kinase domain. Structurally, the kinase domain is highly conserved among all tyrosine kinases thereby making it challeng ing to specifically target Jak2 over the other kinases. However, this paper reports that the Jak2 kinase domain has some unique features that can help distinguish it from the kinase domains of other tyrosine kinases and thus help in achieving specificity. Firstly, Jak family members possess an extra loop structure between amino acids 1056 and 1078, called the Jak2 insertion loop, which is relatively mobile and solvent accessible [ 22 ] The serine at position 1056 is conserved between all members of the Jak family and is solvent exposed, suggesting that this residue might have an important role to play in phosphorylation dependent regulatory role in Jak2 function [ 22 ] Analysis of the Jak2 kinase domain crystal structure shows that the swing of the N terminal lobe towards the C terminal lobe markedly narrows the active site of Jak 2, thereby making it more constricted and closed when compared to the active site of other kinases [ 22 ] Comparisons of the electrostatic surface potentials around the ATP binding sites suggest that Jak2 is more positively charged than Jak1, whereas Jak3 is less charged overall [ 23 ] These structural differences between Jak2 and other closely related

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20 kinases, though subtle, might prove useful in achieving selectively for Jak2 over other kinases. Recent crystal s tructures of the Jak2 kinase domain have undoubtedly improved our understanding of the structure, function and regulation of Jak2 kinase. However, future crystal structure analysis of the entire Jak2 molecule will be critical in further developing our know ledge about these kinases and in attaining selectivity for them. Jak/STAT Signaling Paradigm Jak2 is an important downstream signaling molecule activated by a variety of cytokines, growth factors and G protein coupled receptor (GPCR) ligands. An overview of the canonical Jak/STAT signaling pathway as activated by cytokines and growth factors, is shown in Fig. 1 3 Briefly, signaling is initiated by binding of a ligand to its cognate cell surface receptor resulting in receptor dimerization. Receptor dimeri zation brings the receptor associated Jak proteins in close proximity to each other allowing them to transphosphorylate one another on specific tyrosine residues; Tyr 1007/1008 in the case of Jak2 [ 12 ] An activated Jak can in turn phosphorylate specific tyrosine residues on the cytoplasmic tails of the receptors, thereby creating docking sites for SH2 domain containing proteins such as the Signal Transducers and Activators of Transcription (STAT) proteins. Receptor bound STAT monomers are then phosphorylated by Jak2 on specific tyrosine residues. Phosphorylated STATs form homo or hetero dimers which translocate to the nucleus where they bind to gene promoter elements to modulate gene transcription Thus, Jak/STAT signaling results in a signal cascade from extracellular binding and activation of a cell surface receptor to changes in gene transcription in the nucleus.

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21 The important role that Jak kinases play in mediating cytokine signaling was first characterized using mutant cell lines that did not respond to interferons. Analysis of this particular mutant cell line showed that it lacked the tyrosine kinase Tyk2 and expressing this kinase exogenously restore d the ability of this cell to respond to i nterferons [ 24 ] Specific Jak kinases, either alone or in combination with other Jak kinases, is preferentially activated depending on the type of re ceptor that is being stimulated. For example, binding of erythropoietin (Epo) to its cognate receptor activates Jak2 [ 25 ] whereas binding of interleukin 3 (IL 3), interleukin 5 (IL 5) or granulocyte macrophage colony stimulating factor (GM CSF) to their associated receptors activate both Jak2 and Jak1 [ 26 28 ] Thus, it was shown that Jak2 can associate w ith diverse cytokine and growth factor receptors. Subsequently, it was shown that Jak2 is a versatile signaling molecule that can also be activated by G protein coupled receptors, such as the AT 1 receptor [ 29 ] and by non conventional ligands, such as reactive oxygen species [ 30 ] The Jak/STAT signaling pathway can modulate cell growth, survival, differentiation and death and is therefore critical for maintaining normal physiology. Deregulation of this signal transduction pathway has been implicated in various disease states. On one hand, Jak2 knockout mice die embryonically at day 12.5 due to lack of definitive erythropoiesis [ 31 32 ] suggesting that this kinase has a non redund ant role to play in the erythropoiesis process. On the other side, hyperactive Jak2 signaling promotes cell proliferation and prevents apoptosis, thereby leading to various neoplastic disorders, including solid cancers and hematological malignancies. Jak2 and Cancer Jak2 in Solid tumors

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22 A deregulated Jak/STAT signaling pathway has been linked to several solid tumors, including breast, prostate, ovarian, head and neck cancers [ 33 38 ] Jak2 can cause constitutive tyrosine phosphorylation of ErbB2, an oncogene over expressed in human breast cancer, leading to tumor progression and metastasis [ 39 ] It is also known that overexpression of BRCA1 leads to a constitutively active Jak2 STAT3 signaling in human prostate cancer cells [ 4 0 ] Epidermal growth factor stimulation constitutively activates STAT3, thereby leading to head and neck carcinogenesis by activation of anti apoptotic mechanism [ 37 ] Additionally it has been demonstrated that hyperactivation of the Jak/STAT3 signal transduction pathway contributes directly to ovarian cancer growth and invasiveness [ 38 ] Jak2 in Hematological Malignancies Apart from its role in promoting growth and metastasis of solid tumors, a constitutively active Jak kinase signaling has also been implicated in various hematological malignancies. Hematopoiesis, the process of generating blood cells from a self renewing population of multipotent hematopoietic stem cells, is regulated by a group of soluble factors called cytokines. The binding of cytokines to their cognate receptors initiates signal transduction pathwa ys that govern survival, proliferation, differentiation, and apoptosis of the hematopoietic cells. A deregulation of these signaling pathways can lead to the development of different hematological disorders, such as leukemias and myeloproliferative neoplas ms. Cytokine receptors lack an intergral cytoplasmic kinase domain and hence signal via association with cytoplasmic tyrosine kinases, such as the Jak kinase family members [ 3 41 ] Jak2 is widely involved in cytokine signaling. Cytokines, such as erythropoietin, thrombopoietin, growth

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23 hormone, prolactin and GM CSF, signal exclusively through Jak2 [ 42 43 ] Hence, it is unsurprising that hyper activation of Jak2 kinase activity can cause overproduction of blood cells, there by leading to the development of neoplastic hematological malignancies. Jak2 chromosomal translocations and hematological malignancies Several studies have linked specific Jak2 chromosomal translocations to human neoplastic growth. For example, an abnor mal fusion protein that has the helix loop helix dimerization domain of TEL, an ETS family transcription factor, fused to the catalytic domain of Jak2 was detected in a patient with early B precursor acute lymphobastic leukemia and another diagnosed with a typical chronic myeloid leukemia [ 44 45 ] The phenotypes of these two patients were diverse because of th e fact that the TEL Jak2 fusion proteins had resulted from two distinct translocation events; a t(9:12)(p24;p13) in the former case and an a t(9:15:12)(p24;q15;p13) in the latter. However, in both cases, the translocation event resulted in constitutive act ivation of the Jak2 kinase domain and its downstream signaling pathways [ 46 ] Recent studies have also reported the presence of a BCR Jak2 fusion protein arisi ng from a t(9:22)(p24;q11.2) translocation in patients with typical and atypical chronic myelogenous leukemia [ 47 49 ] In other instances, a t(8:9) translocatio n event results in a PCM1 Jak2 fusion protein that contains the coiled coil domains of PCM1 and the tyrosine kinase domain of Jak2 [ 50 52 ] This fusion protein h as been implicated in several hematological disorders including acute erythroid leukemia, atypical chronic myeloid leukemia, T cell lymphoma, and myeloproliferative neoplasms. Jak2 point mutations and hematological malignancies

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24 In addition to chromosomal t ranslocations, point mutations in the Jak2 allele also cause constitutive Jak2 activation and subsequent activation of its downstream signaling pathways. Substitution and deletion mutations in Jak2 have been detected in many patients with different hematol ogical disorders. For example, Jak2 amino acids 682 686 were found to be deleted (Jak2 in a patient with Down syndrome and B cell precursor acute lymphoblastic leukemia [ 53 ] In another instance, a novel Jak2 T875N mutation was identif ied in an actue megakaryoblastic leukemic cell line using a combination of mass spectrometry and growth inhibition assays [ 54 ] Jak2 K607N [ 55 ] and Jak2 L611S [ 56 ] are other Jak2 point mutations that have been identified in patients with acute myeloid leukemia and acute ly mphoblastic leukemia, respectively. Overall, these studies demonstrate that the Jak2 locus is susceptible to diverse genetic altrerations that hyper activate the catalytic activity of Jak2 kinase, thereby leading to the development of hematological maligna ncies. Myeloproliferative Neoplasms Myeloproliferative Neoplasms (MPNs) are a group of heterogeneous diseases arising from a transformed hematopoietic stem cell and characterized by the presence of excessive numbers of one or more terminally differentiate d blood cells of the myeloid lineage such as erythrocytes, thrombocytes or white blood cells. These differentiated cells spread and accumulate in the bone marrow as well as in the peripheral blood and other sites of extramedullary hematopoiesis due to exce ss proliferation and/or decreased apoptosis of hematopoietic progenitors. MPNs have a high propensity to progress to malignant leukemias. Excess production of red blood cells is a hallmark of polycythemia vera (PV) and too many platelets is a classical cha racteristic of essential

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25 thrombocythemia (ET) whereas primary myelofibrosis (PMF) is characterized by bone marrow fibrosis and impaired hematopoietic stem cell function. Jak2 Mutations in Myeloproliferative Neoplasms The year 2005 was a landmark year for the field of MPNs as five different groups, working independently, identified a somatic Jak2 Val 617 to Phe substitution mutation (Jak2 V617F ) in a large number of patients with myeloproliferative neoplasms (MPNs) [ 57 61 ] It is interesting to note that each group used a different approach to arrive at the result. The group led by William Vainchenker [ 57 ] began by culturing erythroid cells from PV patients in the absence of exogenous cytokines. The cells were then treated with different small molecule inhibitors in an effort to identify the factors and signaling pathways requ ired for PV erythroid cell proliferation. It was observed that Jak2 inhibition can suppress PV erythroid proliferation and that siRNA mediated silencing of Jak2 can also inhibit erythroid cell proliferation. This led them to sequence JAK2 in PV patient sam ples and hence the presence of the Jak2 V617F mutation was discovered [ 57 ] Robert Kralovics and his group [ 59 ] [ 62 ] followed up on the important discovery that loss of heterozygosity at the 9p24 chromosomal locus as a result o f uniparental disomy (UPD) is present in a large number of PV patients [ 62 ] Genes in this region of UPD were sequenced in MPN patient samples and the Jak2 V617F allele was detected [ 59 ] Two other groups used candidate gene approaches to identify the Jak2 V617F mutation. Anthony Green and coworkers [ 58 ] were sequencing genes involved in signaling pathways known to be implicated in oncogenic transformations when they discovered this mutant allele in MPN. On the other hand, Gary [ 60 ] detected the presence of the Jak2 V617F mutation while sequencing conserved domains of all tyrosine kinases on a large set of MPN patients who had submitted

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26 samples via an internet based collection protoco l 61 ] performed a large scale DNA sequencing analysis of candidate tyrosine kinases and phosphatases and thereby identified the presence of the gain of function Jak2 muta tion in a majority of PV patients. These five groundbreaking studies, as well as other subsequent studies, reported the presence of the Jak2 V617F mutation in over 90% of patients with PV and about 50% of patients with ET and PMF. A guanine to thymine muta tion acquired in the hematopoietic stem cells results in a substitution of valine to phenylalanine at codon 617 in the exon 14 of the JH2 pseudokinase domain of Jak2. It is believed that this mutation at position 617 in the autoinhibitory pseudokinase doma in of Jak2 allows the kinase to evade inhibition and leads to a constitutively active Jak STAT signaling pathway [ 62 63 ] and induces cytokine inde pendent growth of cells [ 57 60 ]. The co expression of Jak2 V617F and an ectopic erythropoietin receptor (EpoR) in the IL 3 dependent hematopoietic cell line Ba/F3 transforms these cells to cytokine independent growth [ 57 60 [ 63 ] Subsequently, it has also been shown that expression of this mutation in both murine bone marrow transplant models [ 64 65 ] as well as transgenic models [ 66 71 ] is sufficient for the development of MPN like phenotypes in recipient mice. These reports strongly suggest that the Jak2 V617F mutation plays a critical role in the pathogenesis of myeloproliferative neoplasms. Although the Jak2 V617F mutation on exon 14 is the predominant dise ase associated allele in myeloproliferative neoplasms, several other Jak2 exon 14 mutations, such as C616Y [ 72 ] and D620E [ 73 ] have been identified in V617F negative, MPN patients. Moreover, about sixteen different Jak2 exon 12 mutations have

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27 been detected to date in Jak2 V617F negative PV patients [ 74 ] These exon 12 mutations are usually insertions, deletions or substitutions in the Jak2 sequence spanning amino acids 538 5 43 (Fig. 1 2 ). This region is highly conserved and links the SH2 like and the pseudokinase domains of Jak2. A recent study suggests that this linker region acts as a switch in relaying signals from receptor binding to Jak2 kinase activation by flexing the pseudokinase domain hinge [ 75 ] Finally, a subset of patients with Jak2 V617F negative ET and PMF were found to carry MPLW515L/K mutations in the thrombopoietin re ceptor, which also leads to the deregulation of the Jak/STAT signaling pathway via activation of wild type Jak2 protein [ 76 77 ] Collectively, these studies clearly demonstrate that hyperkinetic Jak2 has a crucial role to play in the pathogenesis of MPNs as well as in a number of hematological malignancies. Therefore, the clinical development of Jak2 inhibitor s is of potential value for patients with these hematological disorders and is currently an area of active research. Jak2 Inhibitors for the Treatment of Myeloproliferative Neoplasms Currently available treatment options for MPN patients are limited. For P V/ET patients, the available therapies aim to reduce myeloproliferation and thrombotic/hemorrhagic complications associated with it. As such, a PV/ET patient is mostly treated with phlebotomy/plateletpheresis or myelosuppressive agents such as hydroxyurea [ 78 80 ] On the other hand, in PMF patients, treatment is directed towards management of symptoms such as anemia and splenomegaly. Therefore, agents that can imp rove cytopenia (erythropoietin, androgens) or induce myelosuppression (such as hydroxyurea, thalidomide) are routinely used for treating these individuals [ 80 81 ] In certain cases, more invasive techniques, such as splenectomy or radiation, are used to treat severe splenomegaly in MPN patients. Unfortunately, these treatment strategies

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28 are more palliative a nd not curative in nature. The only therapy that has been shown to be curative in patients with myeloproliferative neoplasms is stem cell transplantation. Although this approach is potentially curative, risks associated with it, such as graft versus host d isease and nonrelapse mortality, severely limit its application for therapy [ 81 ] Given the critical role that constitutive Jak2 tyrosine kinase activity play s in the pathophysiology of myeloproliferative neoplasms, it became an attractive molecule for therapeutic targeting and prompted researchers to start the search for potent and selective Jak2 small molecule inhibitors. Interestingly, the first widely avail able Jak2 inhibitor dates back to 1995 when Meydan et al identified AG490 via a high throughput screen of potential tyrosine kinase inhibitors [ 82 ] However, it was subsequently reported that although AG490 was a potent Jak2 inhibitor, it had poor specificity [ 83 ] The discovery of the Jak2 V617F mutation in 20 05 and its identification in a vast majority of patients with MPNs provided a new thrust to the search for identification of small molecule inhibitors that specifically target Jak2. Given the fact that the Jak2 V617F mutation leads to sustained kinase sign aling, similar to the effect of the BCR ABL fusion protein in chronic myelogenous leukemia (CML), it was expected that a Jak2 inhibitor would be as effective in MPN therapy as imatinib is in CML [ 84 ] Moreover, in 2006, the resolution of a crystal structure encoding a portion of the Jak2 kinase domain provided a valuable tool for designing inhibitors that could be highly specific for Jak2 [ 22 ] Several pharmaceutical companies have developed Jak2 inhibitors that are currently under study for the treatment of MPNs. The clinical trials conducted thus far have focused on pati ents with PMF because it is the most serious condition among the

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29 different MPNs. PMF patients are characterized by fibrous scar tissue in their bone marrow, impaired hematopoiesis and high levels of pro inflammatory cytokines and growth factors. These symp toms significantly reduce the quality of life of these patients and as mentioned above, effective therapies are limited for these patients. The results from four clinical trials in these patients are publicly available; the inhibitors tested were INCB01842 4, CEP 701, TG101348, and XL019 (Fig. 4) INCB018424 is a potent Jak2 inhibitor which has an in vitro IC 50 of 2.8 nM [ 85 ] In primary cultures, this inhi bitor has an ability to suppress erythroid progenitor colony formation from Jak2 V617F positive PV patients with an IC 50 of 67 nM vs. healthy donors (IC 50 > 400 nM). In a mouse model of Jak2 V617F mediated MPN, treatment with 180 mg/kg/day of INCB018424 si gnificantly decreased splenomegaly, reduced levels of some circulating cytokines, preferentially eliminated neoplastic cells, and increased animal survival without myelosuppressive or immunosuppressive effects. In clinical phase I/II trials, this drug was generally well tolerated; the maximum tolerated dose (MTD) was 25 mg PO BID [ 86 ] INCB018424 treatment resulted in reduced splenomegaly in over 93% of the patients irrespective of Jak2 mutational status [ 87 ] Treatment with this drug inhibited production of pro i nflammatory cytokines in all PMF patients [ 88 ] There was also significant improvement in quality of life and weight of the patients with treatment [ 86 ] However, the Jak2 V617F allele burden showed only a modest decrease of 13% in the marrow and 9% in the peripheral blood [ 8 9 ] suggesting that this drug might be inhibiting downstream Jak/STAT signaling in cells with hyperkinetic Jak2 rather than altering the mutant allele burden. There were also no reports of improvement or normalization of either the diseased bone marrow o r the

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30 spleen tissues in any of the treated patients. No significant changes in fibrosis score, bone marrow cellularity or circula ting CD34+ cells were observed [ 90 ] Finally, thrombocytopenia and myelo suppression were the most common side effects of drug treatment [ 86 ] Phase III clinical studies for this drug have recently been initiated ( www.clinicaltrials.gov ). CEP 701 (also cal led lestaurtinib), an indolocarbazole alkaloid, is an orally available tyrosine kinase inhibitor that has anti Jak2 tyrosine kinase activity. This drug is a known inhibitor of Fms like tyrosine kinase 3 (FLT3) and is currently being evaluated in clinical t rials for treating acute myelogenous leukemia patients with FLT3 mutations [ 91 92 ] Lestaurtinib inhibits Jak 2 in vitro ex vivo and in vivo [ 93 ] It potently inhibits Jak2 kinase activity in vitro with an IC 50 of 1 nM and suppresses ex vivo erythroid colony formation f rom primary CD34+ cells isolated from MPN patients at a concentration of 100 nM. CEP 701 treatment is also able to reduce phosphorylation of STAT3, STAT5 and other downstream signaling molecules of the Jak/STAT signaling pathway in vitro With respect to i n vivo studies, this drug was able to inhibit the proliferation of Jak2 V617F bearing HEL 92.1.7 cells xenografted into nude mice at a dose of 30 mg/kg BID. The results of a phase II clinical study of CEP 701, with twenty two Jak2 V617F positive PMF patien ts, were recently published [ 94 ] These patients were treated with an oral dose of 80 mg twice a day. 27% of the patients (6 out of 22) showed clinical improvemen t with this treatment (i.e. the lowest level of response as per the International Working Group for Myelofibrosis Research and Treatment criteria). The responding patients included three who only showed a reduction in spleen size, two who achieved transfus ion independency, but only one who had a reduction in spleen

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31 size in conjunction with improvement in cytopenia [ 94 ] Median time for response was about three mont hs and the duration of the response was fourteen months or higher. There was an observed decrease in the levels of phosphorylated STAT3 from baseline levels in the responding patients [ 94 ] Most critically, no improvements were seen either in the marrow fibrosis or in the Jak2 V617F allele burden in any of the treated patients. Finally, 36% of the treated patients (8 out of 22) experienced toxic effects; principall y, myelo suppression (anemia and thrombocytopenia) and gastrointestinal problems such as diarrhea and nausea [ 94 ] TG101348 is a potent and selective inhibitor of Jak2. It is an ATP competitive inhibitor that shows a high selectivity for Jak2 over other Jak family members [ 65 95 ] Among all the Jak2 inhibitors that are currently in clinical trials, TG101348 seems to be the most selective for Jak2. Previous studies with this compound have shown that it inhibits Jak2 kinase activity with an IC 50 of 3 nM in vitro [ 65 95 ] Furthermore, it was found to suppress erythroid colony formation from primary progenitor cells with an IC 50 of about 300 600 nM [ 96 ] The in vivo therapeutic efficacy of TG101348 in a murine model of Jak2 V617F mediated PV was also evaluated in a study which found that mice treated with 120 mg/kg BID showed a decrease in Jak2V617F mutant allele burden, hematocrit values, leukocyte counts and spleen sizes [ 65 ] TG101348 was also evaluated in a phase I/II dose escalation study with twenty eight myelofibrosis patients [ 97 98 ] The maximum tolerable dose was found to be 680 mg/day, taken once daily. 64% of the patients showed a decrease in spleen size of more than 50%. Fourteen patients, who initially had leukocytosis, experienced improvement in their WBC counts post treatment. Mutant allele burden was found to decrease in 32% of the Jak2 V617F

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32 positive patients enrolled in the study. The most frequent toxicities observed were nausea/vomiting (64%) and diarrhea (50%) [ 97 ] No changes in levels of plasma cytokines were detected in p atients treated with this drug [ 98 ] Thrombocytopenia, neutr openia and anemia were the other side effects that correlated with drug treatment [ 97 ] XL019 inhibits Jak2 kinase activity with an IC 50 of 2 nM. In EPO stimulated primary erythroid ex vivo cultures, STAT5 phosphorylation was inhibited with an IC 50 of 64 n M [ 99 ] In in vivo studies using the HEL cell xenograft model, treatment with this inhibitor reduced tumor growth in a dose dependent manner; 60% inhibition at a dosage of 200 mg/kg BID and 70% inhibition at a dosage of 300 mg/kg BID relative to vehicle control treated animals [ 99 ] The effect of XL019 administration on human subjects was teste d in a phase I/II clinical study in patients with PMF post PV or post ET [ 100 ] To date, 21 patients have been studied over multiple doses ranging from 25 mg to 300 mg, using different schedules of administration (either once daily or three times weekly). Ad verse neurotoxicity was observed in patients receiving doses >100 mg ( 65 ). However, the drug was well tolerated at the lower doses of 25 to 50 mg daily or 25 mg three times weekly. A greater than 50% reduction in spleen size was achieved in 42% of the pati ents. Reduction in leukocytosis and circulating blast cells and improvement of anemia, pruritis, and fatigue were also observed [ 100 ] The drug associated toxicities were mostly related to adverse neurological effects such as peripheral neuropathy, formicati on, balance disorder and confusion [ 99 ] Adve rse hematological side effects were however not seen in treated patients. Although XL019 showed some beneficial effects, it has been withdrawn from clinical trials due to the neurotoxicity concerns.

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33 A summary of the preclinical data pertaining to INCB0 18424, CEP 701, TG101348, and XL019 is shown in Table 1 1 and a summary of the clinical trial regimens for the same four drugs is displayed in Table 1 2. Data from these initial reports about the clinical trials of Jak2 inhibitors show that the good precli nical efficiencies demonstrated by these compounds in murine models of myeloproliferative neoplasms did not translate to clinical trials in human patient populations. However, t here are also a number of other Jak2 inhibitors that are currently being evalua ted in early clinical trials, such as SB1518 and CYT387 (www.clinicaltrials.gov). SB1518 is a potent, selective, orally active, ATP competitive Jak2 inhibitor that inhibits Jak2 V617F activity with an IC 50 of 19 nM (66). This compound was found to inhibit the proliferation of Ba/F3 cells that had been tra n sfected with erythropoietin receptor (EpoR) and mutant Jak2 V617F with an IC 50 of 81 nM [ 101 ] In a mouse model of MPD, established by injection of Ba/F3 JAK2 V617F cells into nude mice, SB1518 was found to produce therapeutic effects such as normalization of elevated white blood cel l count and reduction of GFP labeled BaF3 cells in the peripheral blood, resolution of hepatosplenomegaly, reduction of phospho STAT5 in diseased organs, prolonged survival and alleviation of terminal stage anemi a and thrombocytopenia [ 101 ] CYT387, an aminopyrimidine derivative, inhibits Jak1/Jak2 with an IC 50 o f 11 and 18 nM respec tively [ 102 ] It inhibits the growth of Ba/F3 JAK2V617F and human erythroleukemia (HEL) cells (IC 50 ~1500 nM) The drug selectively suppressed the in vitro gr owth of erythroid colonies harboring JAK2 V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin [ 102 ] In a mu rine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and

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34 restored normal level s of inflammatory cytokines Furthermore, treatment with this drug was able to reduce the Jak2 V617F allelic burden, but not eliminate the mutant cells [ 103 ] With a number of other putative Jak2 inhibitors currently in various stages of preclinical studies, it is hopeful that the list of Jak inhibitors in clinica l trials will grow significantly in the years to come and a potent and specific small molecule inhibitor that is able to cure Jak2 mediated pathologies will soon be identified. Rationale for Studies Jak2, a cytoplasmic tyrosine kinase, has a critical role to play in hematopoiesis and activating mutations in this kinase are associated with a number of hematological disorders. Recently, several somatic point mutations, such as the Jak2 V617F, that constitutively activate the Jak/STAT signaling pathway have b een identified in the majority of patients with myeloproliferative neoplasms (MPNs). This discovery made Jak2 an attractive molecule for therapeutic targeting in MPNs and has been the driving force behind the development of small molecule inhibitors that s pecifically target Jak2. Since then, many different Jak2 inhibitors have been identified using a variety of screening techniques and have been characterized preclinically as well as clinically. However, as we have discussed in the previous section, initial reports from the clinical trials of Jak2 inhibitors have not lived up to the expectations. These inhibitors were able to alleviate symptoms in patients but failed to actually reverse the progression of the disease. On the basis of these reports from earl y clinical trials of Jak2 inhibitors, we can conclude that there is still an unmet clinical need for Jak2 inhibitors that can successfully cure patients with myeloproliferative neoplasms. The goal of the study presented in the following chapters is to ide ntify and characterize the structure function correlation of novel Jak2 inhibitors. Here, we first

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35 present a study in which we identify and characterize a novel benzothiophene based Jak2 inhibitor, called A46. U sing structure based virtual screening, our g roup recently identified a novel small molecule inhibitor of Jak2 named G6 [ 104 ] We showed that G6 has a specific inhibitory effect on Jak2 kinase activity as me asured by in vitro enzyme assays and an immunoassay ELISA [ 104 ] We also demonstrated that this compound exhibits exceptional therapeutic efficieny, in vivo [ 105 ] Here we perform structure function analysis of this novel Jak2 inhibitor, G6. Additionally, we also attempt to elucidate the molecular and biochemical mechanis ms by which G6 exerts its anti J ak2 activity. The overall aim of the study presented here is to better understand the mechanism of action of novel Jak2 inhbitors both from a structural as well as a functional point of view.

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36 Figure 1 1. Ty rosine kinase catalyze d phospho transferase reaction. Tyrosine kinases catalyze the phosphate of ATP to tyrosine residues of protein substrate. Protein Substrate Protein Substrate ATP ADP Tyrosine Kinase

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37 Figure 1 2. Schematic representation of the Jak2 protein showing the regions where the somatic mutations identified in myeloproliferative neoplasm (MPN) patients are commonl y found. Jak2 has seven highly conserved Jak homology or JH domains, JH1 JH7. The Jak2 V617F point mutation, the predominant disease associated allele in MPNs, is on exon 14 of the JH2 or pseudokinase domain of Jak2. Other exon 14 mutations have also been identified in MPN patients who lack the V617F mutation. Additionally, about sixteen different exon 12 mutations have been detected in Jak2 V617F negative PV patients. These mutations cluster in a region spanning from amino acids 538 543.

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38 Figure 1 3. Schematic representation of the Jak/STAT signaling paradigm. (a) Binding of the ligand to its cognate cell surface receptor causes receptor dimerization. (b) Receptor dimerization brings the receptor associated Jak molecules in close proximity, such that they can trasphosphorylate one another on specific tyrosine residues (c) Active Jaks can now phosphorylate tyrosine residues on the cytoplasmic tail of the receptor. (d) STAT proteins bind to these phosphorylated tyrosine residues on the receptor and are a lso phosphorylated by receptor associated active Jaks. (e) Phosphorylated STAT proteins form dimers which translocate to the nucleus. (f) STAT dimers bind to the promoter region of specific target genes and modulate gene transcription, producing biological effects such as cell proliferation, differentiation, survival and growth.

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39 Table 1 1. Preclinical characterization of Jak2 inhibitors. The Jak2 IC 50 was derived from cell free assays using recombinant Jak2 protein. The HEL cell IC 50 was determined using in vitro cultures of the Jak2 V617F expressing HEL cells. The ex vivo IC 50 values were measured in primary cultures isolated from MPN patients. The in vivo drug dosage (mg/kg) is shown along with the animal model that was employed for that specific study. Drug Jak2 IC 50 (nM) HEL cell IC 50 (nM) Ex Vivo IC 50 (nM) In Vivo Dosage (mg/kg) (Mouse model used) INCB018424 2.8 186 67 180 daily (Ba/F3 Jak2V617F xenograft) CEP 701 1 30 100 100 30 twice daily (HEL xenograft) TG101348 3 300 300 600 120 twice daily (Jak2V617F induced PV bone marrow transplant) XL019 2 60 200 64 200 300 twice daily (HEL xenograft)

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40 Table 1 2. Clinical characterization of Jak2 inhibitors. Shown is a list of the four drugs tested in clinical trials and an overview of the tr eatment regimen. PMF Primary myelofibrosis, post PV/ET MF post polycythemia vera (PV)/essential thrombocythemia (ET) myelofibrosis. Drug Company Phase of Development Clinical Trial Dose (mg) Route of Administration Patient Population Studied Number of Patients Clinical/Molecular Responses Observed Toxicities INCB018424 Incyte Phase I / II Phase III 25 twice daily Oral PMF and post PV/ET MF >100 Decrease in splenomegaly irrespective of Jak2 mutational status, improved quality of life and weight, reduce levels of inflammatory cytokines, modest decrease in Jak2 V617F allele burden, no change in bone marrow fibrosis Thrombocytopenia, myelosuppression CEP 701 Cephalon Phase II 80 twice daily Oral Jak2V617F positive PMF 22 Reduced spleen size, Imp rovement in cytopenia, two patients achieved transfusion independency, decrease in phospho STAT3, no improvement in bone marrow fibrosis, no significant improvement in allele burden Myelosuppression (Anemia and thrombocytopenia), gastrointestinal problems (diarrhea and nausea)

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41 Table 1 2 Continued Drug Company Phase of Development Clinical Trial Dose (mg) Route of Administration Patient Population Studied Number of Patients Clinical/Molecular Responses Observed Toxicities TG101348 TargeGen Phase I / II 680 once daily Oral PMF and post PV/ET MF 28 More than 50% decrease in spleen size, reduction in Jak 2 V617F allele burden, marked reduction in leukocytosis, improvement in constitutional symptoms like pruritus, no change in levels of plasma cytokines Nau sea / vomiting, diarrhea, thrombocytopenia, neutropenia, anemia XL019 Exelixis Phase I / II (Discontinued) 25 50 once daily Oral Primary or post PV/ET MF 21 Decrease in splenomegaly, reduction in leukocytosis and circulating blast cells, improveme nt of anemia, pruritus and fatigue Neurotoxicities such as peripheral neuropathy, formication, balance disorder and confusion

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42 CHAPTER 2 A46, A BENZOTHIOPHEN E DERIVED COMPOUND, SUPPRESSES JAK2 MEDIATED PATHOLOGIC CELL GROWTH Jak2, a member of the Janus f amily of cytoplasmic tyrosine kinases, is ubiquitously expressed and is a key mediator of signal transduction and gene transcription. A variety of cytokines, growth factors and GPCR ligands can activate Jak2, trigger the canonical Jak/STAT signaling cascad e and cause physiological effects such as regulation of cell survival, development, growth, and proliferation. The critical role of Jak2 in embryonic development has been demonstrated by gene deletion studies in which Jak2 knock out mice were found to be e mbryonic lethal due to absence of definitive erythropoiesis that led to profound anemia [ 31 32 ] Aberrant Jak2 activity has, however, been associated with several different pathological conditions. Constitutive activation of the Jak STAT pathway promotes aberrant cell proliferation and can lead to the development of hematological malignancies and myeloprolifer ative neoplasms (MPNs) [ 44 45 48 ] MPNs include three pathogenetically related disorders; polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). These disorders arise from a transformed hematopoietic stem cell and are characterized by increased production of blood cells of the myeloid origin. A somatic Jak2 mutation (Jak2 V617F) was identified in patients with myeloproliferative neoplasms including 90% of polycythemia vera (PV) patients and about 50% of patients with essential thrombocythemia (ET) and primary myelofibros is (PMF) [ 57 61 ] A valine to phenylalanine substitution at codon 617 (V617F) in the pseudokinase domain of Jak2 in hematopoietic stem cells results in a constitut ively active Jak STAT signaling pathway that induces cytokine hypersensitivity and growth factor independent growth of these cells. The presence of this mutation has also been

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43 detected in other hematological malignancies such as acute myeloid leukemia, chr onic myelomonocytic leukemia and chronic neutrophilic leukemia [ 106 ] The co expression of Jak2 V617F and an ectopic erythropoietin receptor (EpoR) in the IL 3 dependent hematopoietic cell line Ba/F3, transforms these cells to cytokine inde pendent growth [ 63 ] Subsequently, it has also been shown that expression of this mutation in both murine bone marrow transplant models [ 64 107 ] as well as transgenic models [ 66 67 70 71 ] is sufficient for the development of MPN like p henotypes in recipient mice. These reports strongly suggest that the Jak2 V617F mutation plays a causative role in the pathogenesis of myeloproliferative neoplasms. Unfortunately, as of present, there are no effective treatment options available for these patients. Specifically, current treatment options are directed towards management of symptoms and are palliative rather than being curative. The presence of the Jak2 V617F mutation in a majority of MPN patients suggests that identification of Jak2 specific inhibitors is an important step towards developing an effective targeted therapy for MPNs. Our laboratory has been actively involved in the identification of Jak2 inhibitors and has previously reported three small molecules with anti Jak2 activity [ 104 105 108 109 ] Here, we used structure based drug design to identify a benzothiophene based structure, 1 benzothiophen 2 yl (4 dimethylaminophenyl)methanol (termed as A46), and show that this compound suppresses Jak2 mediated pathological cell growth in vitr o and ex vivo Materials and Methods Drug The small molecule A46 was obtained from the National Cancer Institute/Developmental Therapeutics Program (NCI/DTP). The drug was solubilized in

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44 dimethyl sulfoxide (DMSO) at a concentration of 10 mM The drug wa s stored at 20 C in small aliquots to avoid repeated freeze thaw cycles Cell Culture HEL and Raji cells were purchased from the American Type Culture Collection (ATCC) and CMK cells from the German Collection of Microorganisms and Cell Cultures DSMZ. SET 2 cells were a kind gift from Dr. Gary Reuther at the Moffitt Cancer Center and Research Institute, Tampa, FL. These cells were cultured in RPMI 1640 (Mediatech) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and L glutamine at 3 7 C and 5% CO 2 Cell Proliferation Assay HEL, Raji, CMK and SET 2 cells were plated in 96 well plates at a concentration of approximately 5 10 4 cells per well The cells were then treated with either 0.25% DMSO or A46 for the indicated periods of time or concentrations. Cell viability was assessed by trypan blue exclusion staining or an MTS assay as indicated. Enzyme linked Immunosorbent Assay HEL cells, treated with either 0.25% DMSO or increasing doses of A46 for 48 hrs, were lysed and analyzed by Enzym e linked I mmunosorbent A ssay ( ELISA ) for detection of phospho Jak2 and phospho STAT5 protein levels. For this, Jak2 [pY1007/pY1008] protocol. Cell C ycle Assay The CycleTES T TM PLUS DNA Reagent Kit (BD Biosciences) was used to analyze the DNA obtained from HEL cells suspensions. The cells were first incubated with

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45 0.25% DMSO or increasing doses of A46 for 48 hrs and then analyzed using a FACSCalibur flow cytometer (BD Bioscie Apoptosis Assay Induction of apoptosis in HEL cells was determined with the FITC AnnexinV were incubated with either 0.25% DMSO o r increasing doses of A46 for 48 hrs, stained with AnnexinV and Propidium Iodide (PI) and then analyzed using a FACSCalibur flow cytometer (BD Biosciences) to determine the percentage of cells undergoing apoptosis. Western Blotting HEL cells were treated w ith the indicated concentrations of A46 for 24 hrs. The cells (~ 1 x 10 7 ) were then lysed in 0.8 ml of ice cold RIPA buffer and protein concentration was determined using a Bradfor d assay (Bio Rad). Protein samples (~50 transferred to nitrocellulose membranes and blotted with the indicated antibodies. All the antibodies used were purchased from Cell Signalin g Technology except for Cyclin D1 which was from Santa Cruz Biotechnology. Western blots were visualized using the enhanced chemi luminescence system (Perkin Elmer). C olony Formation Assay Following an Institutional Review Board approved protocol, a residu al bone marrow aspirate was obtained from a de identified Jak2 V617F positive female diagnosed with Essential Thrombocythemia. The marrow derived mononuclear cells human m ethylcellulose complete medium lacking erythropoietin and thrombopoietin (R&D Systems). Cells were plated in 35 mm petri dishes at a concentration of

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46 approximately 4 10 5 cells/ml in the absence or presence of increasing doses of A46. Thrombopoietin (50 n g/ml) was added to the indicated samples. The dishes were incubated at 37 C and 5% CO2 in a humidified atmosphere for 14 days following which the number of colony forming units megakaryocytes (CFU Megs) were counted. C lonogenic Assay Bone marrow cells fro m Jak2 V617F transgenic mice [ 67 ] were harvested, M o f A46 for the indicated periods of time. Post treatment, the drug was washed away and the cells (approximately 3 x 10 4 cells/ 35 mm dish) were plated in MethoCult medium lacking cytokines. Five days later, the number of granulocyte/macrophage and erythroid colony forming units were counted and plotted as a function of treatment group. Statistical Analysis Results are expressed as mean +/ SEM. For statistical evaluation of time dependent responses to A46, a two way analysis of variance (ANOVA) was used. For the analysis of inhibition of phosphorylation, induction of cell cycle arrest, induction of apoptosis and suppression of pathologic cell growth and clonogenic potential ex vivo a test was employed. Data were assumed to be statistically signi ficant when p < 0.05. Results A46 Inhibits Jak2 V617F dependent Cell P roliferation Using an in silico structure based drug design approach, we screened a library of small molecules from the NCI/DTP repository as described previously [ 109 ] and identified a small molecule, termed A46, which bound the ATP binding pocket of Jak2

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47 with a favorable energy score. Table 2 1 lists the chemical name, NSC number, chemical structure, and molecular weight of this compound. We next wanted to test the ability of this compound to suppress Jak2 V617 mediated cell proliferation, in vitro For this, we used the human erythroleukemia (HEL 92.1.7) cell line, which is homozygous for the Jak2 V617F mutation [ 110 ] and has a transformed proliferative phenotype that is driven by constitutively active Jak2 V617F signaling [ 111 ] HEL cells were treated either with DMSO or with 10 M of A46 for increasing periods of time. Viable cell numbers for each treatment were determined using trypan blue exclusion and hemocytometer. When compared to vehicle treated cell s, we found that A46 significantly reduced viable cell numbers in a time dependent manner (Fig. 2 1 A). We next wanted to determine if the suppressive effects of A46 treatment on HEL cell growth were reversible. For this, HEL cells were first exposed to 1 0 M of A46 for 0, 8, 12, 24, 48 and 72 hrs. At the end of each time point, the drug was washed away and the cells were resuspended in fresh growth medium. They were then allowed to grow for an additional 72 hrs in the absence of the inhibitor. Viable cell numbers were determined at the end of the 72 hour recovery period. We found that ~19 hrs of initial exposure to A46 prevented 50% of the cells from recovering from the treatment (Fig. 2 1B). For cells that had been treated with A46 for 48 hrs or more, few er than 20% were able to recover after removal of the drug (Fig. 2 1B), suggesting that after a finite time of initial treatment, the effect of the drug on HEL cell growth is largely irreversible. To determine the specificity of A46 for Jak2 V617F depende nt cell growth, the drug was applied to four different cell lines and cell viability was assessed. The cell lines were (i) HEL (erythroleukemia) cells that are homozygous for the activating Jak2

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48 V617F mutation, (ii) Raji (Burkitt lymphoma) cells that have a translocation between the c Myc gene on chromosome 8 and the heavy chain locus on chromosome 14, (iii) SET 2 (essential thrombocythemia) cells that are heterozygous for the Jak2 V617F mutation, and (iv) CMK (acute megakaryocytic leukemia) cells that har bor a Jak3 A572V mutation. Each of these cell lines was treated with increasing doses of A46. After 72 hrs of treatment, we found that A46 markedly inhibited the growth of the Jak2 V617F expressing HEL (Fig. 2 1C & D) and SET 2 (Fig. 2 1C) cells in a dose dependent manner with a GI 50 of ~ 400 nM for both cell types. The viability of Raji cells (Fig. 2 1C & D) was, however, not affected by A46 treatment (GI 50 > 25,000 nM). On the other hand, the Jak3 A572V expressing CMK cells had intermediate susceptibility to the drug (Fig. 2 1D) with a GI 50 of ~2,500 nM, suggesting that A46 is selective for Jak2 V617F expressing cell lines over Jak3 and c Myc mutated cell lines. Collectively, the data in Fig. 2 1 demonstrate that the novel small molecule inhibitor A46 sele ctively inhibits Jak2 V617F dependent in vitro cell growth, and this inhibitory effect of the drug on HEL cell growth is largely irreversible after a determinate period of initial drug exposure. A46 Inhibits the Jak/STAT Signaling P athway Phosphorylation o f Jak2 on tyrosine residues 1007/1008 is concomitant with increased Jak2 kinase activity [ 12 ] Activated Jak2 can in turn phosphorylate and activate downstream signaling targets such as STAT5. Hence, we next wanted to see if the A46 mediated reduction in HEL cell numbers directly correlated with the suppression of the Jak/STAT signaling pathway. This study is important in order to eliminate the possibility that A46 exerts its cell growth inhibitory effect via mechanisms that are independent of the Jak/STAT signaling pathway. For this, we treated HEL cells with increasing concentrations of A46 for 48 hours and then measured the levels of

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49 phospho Jak2 and phospho STAT5 proteins in these cells using an ELISA base d assay. It is important to note that the phospho protein levels were measured 48 h after drug exposure rather than the 72 h used in Fig. 2 1 because far fewer viable cells exist at the longer time point. We observed that A46 significantly reduced levels o f both phospho Jak2 (Fig. 2 2A) and phospho STAT5 (Fig. 2 2B) proteins in a dose dependent manner. These reductions in levels of phosphorylated proteins of the Jak/STAT signaling pathway correlated well with the A46 mediated reductions in HEL cell numbers. Thus, the data in Fig. 2 2 indicate that A46 inhibits HEL cell growth by directly down regulating the phosphorylation of key signaling molecules of the Jak/STAT pathway; namely, Jak2 and STAT5. A46 Induces G1/S Cell C ycle Arrest in HEL C ells To determine the mechanism of A46 mediated suppression of HEL cell growth, we first analyzed the cell cycle distribution of cells as a function of drug treatment. Here, we treated HEL cells with increasing doses of A46 for 48 hours and then performed cell cycle analysi s via flow cytometry. Again, analysis was performed 48 h after drug exposure rather than the 72 h used in Fig. 2 1 because far fewer viable cells exist at the longer time point. We found that exposure to A46 increased the percentage of cells in G1 phase an d correspondingly decreased the percentage of cells in S phase, and this effect was dose dependent (Fig. 2 3A). Cell cycle progression is driven and regulated by the cyclic expression of cyclin/CDK complexes [ 112 ] Cyclin D1 levels play a critical role in promoting the progression of the cell cycle from G1 to S phase [ 113 ] and is also known to be a downstream target of STAT5 transcriptional regulation [ 114 ] Therefore, in order to confirm the induction of G1 phase arrest in these cells, we performed anti cyclin D1

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50 western blot analysis on HEL cells that had been tre ated with increasing doses of A46 for 24 hours. We performed our western blot analysis 24 hrs after drug exposure rather the 48 hrs used in Figs. 2 3A because we wanted to ensure there were sufficient numbers of viable cells and hence, sufficient levels of cellular protein to analyze. We found that A46 treatment decreased the levels of cyclin D1 protein in a dose dependent manner (Fig. 2 3B, upper panel), thereby confirming the A46 induced arrest of HEL cells in the G1 phase of the cell cycle. Analysis of t he same samples with an anti actin antibody was used as a loading control (Fig. 2 3B, bottom panel). Overall, the data in Fig. 2 3 demonstrate that A46 treatment induces a G1/S cell cycle arrest in HEL cells via the down regulation of cyclin D1. A46 I ndu ces Apoptosis in HEL C ells The Jak/STAT signaling pathway is known to have direct effects on cell survival and apoptosis ( 28 ). Having shown that A46 inhibited HEL cell growth (Fig 2 1) and induced cell cyc le arrest (Fig. 2 3), we next wanted to determine if this drug causes apoptotic cell death in HEL cells. For this, we used Annexin V/Propidium Iodide (PI) double staining and flow cytometry. The treatment conditions were the same as in Fig. 2 3A. The data in Fig. 2 4A shows representative apoptosis assay staining profiles from one experiment while Fig 2 4B is a quantitative graph of two independent experiments showing the percentage of cells in early apoptosis (Annexin V positive/PI negative) plotted as a f unction of treatment condition. These data show that A46 induces apoptosis in HEL cells in a dose dependent manner. Cleavage of procaspases converts these proteins into their functionally active forms. Active caspases can then act on and cleave their subs trates, such as the DNA repair enzyme PARP, thereby affecting the integrity of the cell and triggering apoptosis

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51 [ 115 ] Cleavage of procaspases and PARP are therefore considered as hallmarks of apoptotic induction. In order to confirm the induction of apoptosis by A46, we analyzed the effect of A46 treatment on the cleavage of procaspase 3 and its downstream substrate, PARP. HEL cells were treated with increasing doses of A46 for 24 hours and procaspase 3 and PARP protein levels were measured via western blot analysis (Fig. 2 actin antibody to demonstrate equal protein loading across all lanes (Fig. 2 5A). We found that A46 induced a dose dependent cleavage of PARP and a decrease in procaspase 3 expression, thereby corroborating the data from the Annexin V/PI apoptosis assay ( Fig. 2 4A & 2 4B). Having confirmed the ability of A46 to induce apoptosis in HEL cells, we next wanted to determine the mechanism by which this drug causes apoptotic cell death. Apoptosis is regulated by members of the Bcl 2 family, many of which are dir ect downstream gene targets of the Jak/STAT signaling pathway ( 28 ). Therefore, we next monitored the expression of Bcl 2 family members in HEL cells exposed to different doses of A46 for 24 hours via weste rn blot analysis. We observed an A46 induced dose dependent decrease in expression Bim EL a pro apoptotic member of the Bcl 2 family (Fig. 2 5B). Although Bim is a pro apoptotic protein, its disappearance/down regulation has been shown to be associated wit h the generation of cleaved pro apoptotic forms that positively regulate and amplify apoptotic signaling [ 116 117 ] Another pro apoptotic Bcl 2 family protein, Bax, was also down regulated dose dependently in response to A46 treatment, although to a much lesser extent (Fig. 2 5B). Cleavage of Bax in response to apoptotic cues is not uncommon and is known t o enhance its cell death function [ 118 119 ] Additionally, we found that A46 decreased

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52 the levels of the inactiv e precursor form of Bid (Fig. 2 5B), which is yet another pro apoptotic protein that is activated upon cleavage [ 120 ] Levels of anti apoptotic Bcl 2 protein did not change with A46 treatment (Fig. 2 5B). Lastly, the sampl es were blotted actin antibody to demonstrate equal protein loading across all lanes (Fig. 2 5B). In summary, the data in Figs. 2 4 and 2 5 indicate that A46 induces apoptotic cell death in HEL cells via the down regulation/cleavage of Bcl 2 family proteins Bim, Bax and Bid. Overall, from the data in Figs. 2 3, 2 4, and 2 5, we can conclude that A46 inhibits HEL cell proliferation by arresting the cells at G1/S transition and inducing apoptosis. A46 Suppresses Cytokine independent P atho logic Cell Growth of Jak2 V617F Positive Bone Marrow Cells ex vivo We next wanted to determine the ability of A46 to inhibit the pathologic cell growth of patient derived Jak2 V617F positive bone marrow cells, ex vivo For this, mononuclear cells isolated from the bone marrow of a female essential thrombocythemia (ET) patient, who was Jak2 V617F positive, were cultured in medium lacking thrombopoietin (TPO) and in the absence or presence of increasing doses of A46. Hematopoietic progenitor cells isolated from a normal individual will be unable to grow in the absence of cytokine. However, the V617F positive progenitor cells from the patient will be able to grow under such conditions because the Jak2 V617F mutation confers thrombopoietin independent growth. Addit ionally, bone marrow cells from MPN patients are known to be hypersensitive to cytokine stimulation [ 121 ] Hence, the ET patient derived bone marrow cells were also cultured in the presence or absence of the exogenously added thrombopoietin. The results obtained show that treatment of the mutant bone marrow cells with A46 significantly suppressed pathologic cell growth in a

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53 dose dependent manner (Fig. 2 6A). We a lso observed that exposure of these patient derived marrow cells to exogenous thrombopoietin significantly increased the number of colonies formed (Fig. 2 6A), suggesting that these mutant cells from the marrow of an ET patient are indeed hypersensit ive to cytokine We also carried out a clonogenic assay to determine if A46 could inhibit the cytokine independent colony forming ability of bone marrow cells obtained from Jak2 V617F transgenic mice [ 67 ] Jak2 V617F positive bone marrow cells were harvested from these transgenic animals, cultured ex vivo and then exposed to 25 M of A46 for the indicated periods of time. At the end of this initial treatment period, the drug was washed away and the cells were plated in semi solid MethoCult medium lacking cytokines. Five days later, the number of granulocyte macrophage (GM) and erythroid (E) colony forming units in each treatment group were counted and plotted as a function of time of initial exposure to the drug. We found that A46 significantly reduced the number of CFU GMs and CFU Es in a time dependent manner (Fig. 2 6B) suggesting that this drug can suppress the cytokine independent colony forming ability of Jak2 V617F expressing murine bone marrow cells. As such, data in Fig. 2 6 demonstrate that A46 inhibits Jak2 V617F dependent pathologic cell growth of patient derived bone marrow cells and also suppresses the clonogenic growth potential of Jak2 V617F positive bone marrow cells from MPN mice, ex vivo Discussion Aberrant Jak2 kinase activity has been linked to human disorders including several hematological malignancie s and the MPNs [ 44 45 48 ] A Jak2 gain of functi on mutation (Jak2 V617F) has been detected in over 90% of patients with PV and about 50% of patients with ET and PMF [ 57 61 ] Jak2 V617F negative MPN patients ofte n

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54 carry other activating mutations either in Jak2 [ 72 74 ] or in upstream signaling molecules, such as the thrombopoietin receptor, MPL [ 76 77 ] The critical role of Jak2 in the pathogenesis of these disorders coupled with the fact that there are no curative treatment o ptions currently available for MPN patients, has generated considerable amount of interest in developing Jak2 inhibitors as potential therapeutics for MPNs. In this study, we characterized a novel benzothiophene inhibitor of Jak2 that was identified by i n silico structure based drug screening. We show that this compound inhibits Jak2 V617F mediated cell proliferation both time and dose dependently (Fig. 1A, C & D) and this inhibitory effect of the drug on HEL cell growth is largely irreversible after ~48 hrs of initial exposure (Fig. 1B) An important parameter used in evaluating the effectiveness and safety of a drug is its specificity since more selective drugs can be safely administered at higher doses without causing severe side effects. In the past, Jak2 inhibitors have been reported to have non specific off target effects. AG490, one of the first Jak2 inhibitors identified, was a potent Jak2 inhibitor, but had poor specificity [ 82 83 122 ] Our data here show that the Jak2 V617F positive cell lines, HEL and SET2, are significantly more sensitive to inhibition by A46 (GI 50 ~300 nM, Fig. 1C) when compared to Jak3 dependent CMK cells (GI 50 ~2,500 nM, Fig. 1D) or c myc dependent Raji cells (GI 50 > 25,000 nM, Fig. 1C & D), thereby suggesting that A46 selectively inhibits the proliferation of Jak2 V617F d ependent cell lines while having little to no effect on other cells lines whose proliferation is driven by Jak2 independent mechanisms. We have also shown that the A46 induced cell growth inhibition correlates with direct suppression of the Jak/STAT sign aling (Fig. 2A & B) which eliminates the

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55 possibility that A46 might be exerting its cell growth inhibitory effect via mechanisms that are independent of the Jak/STAT signaling pathway. Our results indicate that the mechanism by which A46 suppresses Jak2 V6 17F mediated HEL cell proliferation is via the induction of both G1/S cell cycle arrest (Fig. 3) and apoptosis (Fig. 4). Deregulation of the Jak/STAT signaling promotes cell proliferation and blocks apoptosis resulting in disease pathogenesis. Hence, a bet ter understanding of the mechanism of Jak2 inhibition induced cell death may lead to the development of more effective therapeutic strategies for treating MPN patients, including combination therapies of Jak2 inhibitor and apoptotic modulator mimetics. Her e, we show that A46 induces apoptotic cell death in HEL cells via the down regulation/cleavage of Bcl 2 family proteins Bim, Bax and Bid. There are conflicting reports on the effect of apoptosis induction on the expression of the proapoptotic protein Bim; some studies suggest that Bim is up regulated during apoptosis [ 123 124 ] whereas others suggest that Bim is c leaved during apoptosis generating active and proapoptotic cleaved fragments (30, 31). Jak2 small molecule inhibitors currently represent a diverse number of chemical structures including pyrazines, pyrimidines, azaindoles, aminoindazoles, deazapurines, s tilbenes, benzoxazoles, and quinoxalines [ 125 ] Our work here is significant in that it is the first report indicating that benzothiophene ba sed compounds, such as A46, possess Jak2 inhibitory potential. Benzothiophenes are heterocyclic structures known to have pro and anti estrogenic [ 126 ] ant i lipoxygenase [ 127 ] and anti fungal properties [ 128 ] Not surprisingly, several approved pharm aceuticals including raloxifene, zileuton, and sertaconazole have benzothiophene based chemical structures. Molecules with a benzothiophene core have also been reported to inhibit tubulin, cysteine and serine

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56 proteases (such as cathepsins K and L and throm bin), herpes simplex virus type I replication and opioid receptor analgesics [ 129 ] These heterocyclic structures are relatively stable and their reactive site allows for subsequent derivatization, suggesting that A46 is quite amenable to future lead optimization. As such, benzothiophenes may be a new class of scaffolds for Jak2 small molecule inhibitors. In summary, our data collectively show that the novel benzothiophene small molecule inhibitor of Jak2, A46, inhibits Jak2 V617F mediated pathol ogical cell growth in vitro and ex vivo As such, this compound may perhaps serve as a lead therapeutic agent for the treatment of Jak2 V617F mediated pathogenesis.

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57 Table 2 1. Chemical characteristics of A46. A46 is shown here along with its chemical nam e, NSC #, chemical structure, and molecular weight. Compound Name Chemical Name NSC # Structure Molecular Weight A46 1 benzothiophen 2 yl (4 dimethylaminophenyl)methanol 40282 283.3874

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58 Figure 2 1. A46 inhi bits Jak2 V617F dependent cell proliferation. A ) HEL cells were numbers for each treatment were determined by trypan blue exclusion staining using a hemocytometer. Each sample was measured in triplicate. Shown is one of two set s of representative results. *p < 0.05 with respect to DMSO. B ) hours. At the end of each treatment, the cells were washed, placed in fresh medium and cultured in the absence of the inhibito r for an additional 72 hours. The number of viable cells in each sample was then determined. Each sample was measured in triplicate. Shown are the means S.E. from two independent experiments. HEL (C & D), SET 2 (C), Raji (C & D) and CMK (D) were treated with increasing doses of A46 for 72 hours. The percent viable cells from each condition were determined either by trypan blue exclusion staining (Fig. 1C) or via an automated cell viability assay using the MTS reagent and a plate reader (Fig. 1D). Each sam ple was measured in triplicate.

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59 Figure 2 2. A46 inhibits the Jak/STAT signaling pathway. HEL cells were treated with 0, analyzed by ELISA for the measurement of phospho J ak2 [pY1007/pY1008] (A) and phospho STAT5 [pY699] (B) protein levels. Each condition was run in triplicate. Shown are the means S.D. of two independent experiments.

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60 Figure 2 3. A46 arrests HEL cells in G1 phase of the cell cycle. HEL cells were tre ated A) After 48 hrs of treatment, cell cycle distribution of treated cells was determined as a function of drug treatment using flow cytometry. Shown are the means S.E. of two independent experiments. B ) Fol lowing 24 hrs of treatment with the indicated concentrations of A46, cells were lysed and analyzed by immunoblotting with an anti cyclin D1 (top) or an anti actin antibody (bottom). Shown is one of two representative results.

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61 Figure 2 4. A46 indu ces apo ptosis in HEL cells. HEL cells were treated with 0, 0.1, 0.3, FITC and propidium iodide and then analyzed by flow cytometry to determine the level of apoptosis in the treated cells. A ) S hown are representative flow cytometry profiles from one of two independent experiments. B ) Quantification of the percentage of cells in early apoptosis as a function of drug treatment. Shown are the means S.E. of two independent experiments.

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62 Figur e 2 5. A46 induced apoptosis is mediated by the down regulation/cleavage of Bcl 2 family proteins Bim, Bax and Bid. HEL cells were treated with 0, 0.1, 0.3, 1, analyzed by western blotting with a serie s of antibodies as indicated: A) anti poly(ADP ribose) polymerase (PARP), ant i caspase 3 and anti actin, B) anti Bim, Bax, Bid, Bcl 2 and actin. Shown are the results from one of two independent experiments.

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63 Figure 2 6. A46 suppresses cytokine independent pathologic cell growth o f Jak2 V617F positive bone marrow cells, ex vivo A ) Patient derived bone marrow mononuclear cells were cultured in semisolid medium in the presence of increasing doses of A46, with or without thrombopoietin (TPO). At the end of 14 days, the numbers of meg akaryocyte colony forming units (CFU Megs) were counted and plotted as a function of treatment condition. Each condition TPO), #p < vs. ( TPO). B ) Jak2 V617F positive bone marrow cells from transgenic mice were treated ex vivo At the end of each treatment, the drug was washed away and the cells were cultured in drug free semi solid medium lacking cytokines for another 5 days. The numbers of granulocyte macrophage (GM) and erythroid (E) colony forming units in each sample were counted and plotted as a function of treatment condition. Each sample was measured in duplicate. Shown is one of two repres entative experiments. *p < 0.05 vs. 0 hr for CFU GM, #p < 0.05 vs. 0 hr for CFU E.

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64 C HAPTER 3 STRUCTURE FUNCTION CORRELATION OF G6, A NOVEL SMALL MOLECULE INHIBITOR OF JAK2: I NDISPENSABILITY OF T HE STILBENOID CORE Jak2 plays a critical role in animal deve lopment, as mice that are devoid of a functional Jak2 allele die during embryonic development due to a lack of hematopoiesis [ 31 32 ] Deregulation of the Jak/STAT signaling pathway promotes cell growth and prevents apoptosis in a variety of solid tumors and hematological malignancies such as acute lymphoid leukemia and chronic myeloid leukemia [ 45 48 52 130 ] Additionally, a somatic Jak2 mutation (Jak2 V617F) is found in a high number of myeloproliferative neoplasm (MPN) patients including 90% of polycythemia vera (PV) patients and approximately 50% of patients with essential thrombocythemia (ET) and p rimary m yelofibrosis (PM) [ 57 61 ] MPNs are a group of heterogeneous diseases arising from a transformed hematopoietic stem cell and characterized by excessive numbers of one or more terminally differentiated blood cells of the myeloid lineage such as erythrocytes, thrombocytes or white blood cells. A guanine to thymine mutation in hematopoietic stem cells results in a substitution of valine to phenylalanine at codon 617 in exon 14 of the JH2 pseudokinase domain of Jak2. It is believed that this mutation allows the kinase to evade negative feedback inhibition thereby leading to a constitutively active Jak/STAT signaling pathway characterized by growth factor independent cell growth [ 57 60 ] Given the critical role that Jak2 plays in the pathophysiology of MPNs, identification of s pecific Jak2 inhibitors has become an important step towards the development of an effective targeted therapy for these disorders. U sing structure based This research was originally published in the Journal of Biological Chemistry. Structure function correlation of G6, a novel small molecule inhibitor of Jak2: indispensability of the stilbenoid core J Biol Chem. 20 10; 285(41): 31399 407. The American Society for Biochemistry and Molecular Biology.

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65 virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6 [ 104 ] We showed that G6 has a specific inhibitory effect on Jak2 kinase activity as measured by in vitro enzyme assays and an immunoassay ELISA [ 104 ] Examination of the chemical structure of G6 revealed the presence of a central stilbenoid core. Stilbenoids are a group of naturally occurring compounds having a wide range of b trimethoxy trans stilbene, are all stilbenoids and are known to have cytotoxic, anti proliferative, pro apoptotic, anti angiogenic or tumour suppressive e ffects [ 131 135 ] We hypothesized that the central stilbenoid core in the G6 structure has a critical role to play in mediating its Jak2 inhibitory potential. To test this, five derivative compounds of G6, namely D21, D23, D25, D28 and D30, having structural similarity to the original lead compound, were procured from the National Cancer Institute. Two of these compounds, D28 and D30, have a stilbenoid core pre sent in their chemical structure similar to G6 whereas the three other compounds, D21, D23 and D25, lack the stilbenoid core. We report here that the core stilbenoid structure present in G6 is essential for maintaining its ability to inhibit Jak2 kinase ac tivity. Experimental Procedures Drugs G6 and its five structurally related derivative compounds (D21, D23, D25, D28 and D30) were obtained from the National Cancer Institute/Developmental Therapeutics Program (NCI/DTP) which maintains a repository of appro ximately 140,000 compounds. Each compound was solublized in dimethyl sulfoxide at a concentration of 10 mM and stored at 20 C.

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66 Cell Culture Human Erythroleukemia (HEL) cells were purchased from the American Type Culture Collection. Ba/F3 EpoR Jak2 V617F c ells were created as described before [ 136 ] Both cell lines were cultured in RPMI 1640 (Mediatech) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and L glutamine at 37 C and 5% CO 2 Cell Proliferation Assay HEL cells or Ba/F3 EpoR Jak2 V617F cells were plated in 96 well pla tes and treated with either 0.25% DMSO or varying concentrations of G6 and its derivatives for the indicated periods of time. Cell viability was assessed either by trypan blue exclusion staining or by [3 (4,5 dimethylthiazol 2 yl) 5 (3 carboxymethoxy phen yl) 2 (4 sulfophenyl) 2H protocol. Enzyme linked Immunosorbent Assay compounds for 48 hrs and the cell lysates were analyzed by E LISA for detection of phoshpho Jak2, phospho STAT3 and phospho STAT5. Jak2 [pY1007/pY1008], STAT3 [pY705] and STAT5b [pY699] ELISA kits were purchased from Invitrogen and used Cell Lysis and Immunoprecipitation HEL cells were treated with the different drugs for the indicated periods of time. Cells (~ 10 7 ) were then lysed in 0.8 ml of i ce cold RIPA buffer and protein concentration was determined using a Bradford assay (Bio Protein A/G beads (Santa Cruz Biotechnology ) for 4 hrs at 4 C with constant shaking.

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67 The protein complexes were washed thrice with IP wash buffer (25 mM Tris pH 7.5, 150 mM NaCl, and 0.1% Triton X 100) and then resuspended in SDS sample buffer. Immunoprecipitated proteins were separated by SDS PAGE and then transferred onto nitrocellulose membranes. The anti STAT3 and anti STAT5 antibodies used for immunoprecipitation were from Santa Cruz Biotechnology. For whole cell protein PAGE and then tra nsferred to nitrocellulose membranes for analysis by western blotting. Western Blotting Nitrocellulose membranes were first blocked with 5% milk/TBST solution and then probed with the different primary antibodies. The immuno reactive bands were then visual ized using the enhanced chemi luminescence system (Western Lightning Ultra, Perkin Elmer). The following antibodies were used at the indicated dilutions: phospho STAT3 (Santa Cruz Biotechnology and Cell Signaling at 1:500 each), STAT3 (Santa Cruz Biotechno logy, 1:1000), phospho STAT5 (Cell Signaling, 1:500), STAT5 (Santa Cruz Biotechnology, 1:1000), and STAT1 (Santa Cruz Biotechnology, 1:1000). The following were all obtained from Cell Signaling and used at a 1:500 dilution; poly (ADP ribose) polymerase (PA RP), Bcl 2, Bax, Bim, and Bid. Apoptosis Assay Induction of apoptosis in HEL cells was determined with the FITC AnnexinV incubated with 0.25% D FACSCalibur flow cytometer (BD Biosciences).

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68 Real Time PCR Analysis The mRNA levels of Bcl xL and GAPDH were measured by quantitative real time PCR analysis. Total RNA was extracted from HEL c ells, treated with 25 different drugs for 8 and 24 hours, using the RNeasy Mini Kit (Qiagen) as per A Reverse Transcription Kit (Applied Biosystems). TaqMan Gene Expression Assays (Applied Biosystems) Hs02758991_g1 (GAPDH) and Hs00236329_m1 (Bcl xL) were used to detect the levels of expression of these genes. GAPDH gene expression was used as an internal loading control. The real time polymerase chain reaction (PCR) was then performed with TaqMan universal PCR Master Mix (Applied Biosystems) in a final reaction volume of Patient Sampl e Bone marrow aspirates consisting of mononuclear cells were obtained from a de identified Jak2 V617F positive female diagnosed with polycythemia vera (WHO criteria) at the UF & Shands Teaching Hospital as per an Institutional Review Board approved protoco l. Colony Forming Unit Erythroid Colony Formation Assay Marrow derived mononuclear cells were washed in IMDM and cultured in human methylcellulose complete medium without Epo (R&D Systems) at a concentration of approximately 4 10 5 cells/ml in the presenc e or absence of G6 or its structurally related derivatives 0.9 mL of the cultures was placed in 35 mm petri dishes and incubated at 37 C and 5% CO 2 in a humidified atmosphere for 14 days following which the number of erythroid colony forming units (CFU E) were counted.

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69 Computational Docking The molecular docking program DOCK6 [ 137 138 ] was u sed to study the interactions of Jak2 with ATP, the ATP analog ACP (adenosine methylene] triphosphate), G6, and the structurally related derivative compounds. The template used was the crystal structure of Jak2 kinase domain in complex with the Ja k2 i nhibitor 5B3 (PDB ID: 3E64) [ 139 ] The coordinates for ATP, ACP and each of the small molecules were generated and e ner gy minimized using PRODRG [ 140 ] Su bsequently, CHIMERA [ 141 ] was used to add atomi c charges to these small molecules. After removing the 5B3 molecule, the coordinates of the Jak2 kinase domain were saved in PDB format. To prepare the protein for docking, hydrogen atoms and partial electrostatic charges were first added to the molecule. A molecular surface of the Jak2 kinase domain was then generated using the DMS tool in CHIMERA. The program SPHGEN was used to generate spheres around the active site for identification of the target pocket on the protein for small molecule docking. The g rid file, necessary for rapid grid based energy score evaluation, was then generated around the identified docking site using the GRID module in DOCK6. Each compound was docked in 1000 different orientations using flexible dock and a net energy GRID score was generated for each, based on the van der Waals and electrostatic interactions between the compound and the residues in the binding pocket. The favorable binding orientations were selected on the basis of the energy GRID scores generated for each orient ation, wherein a favorable binding orientation would have a more negative score. Finally, the docking results were a nalyzed visually using COOT [ 142 ] and CHIMERA. Surface electrostatic potentials were calculated using APB S [ 143 ] and PYMOL [ 144 ] was used to generate the figures.

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70 Statistical Analysis For statistical evaluation of time dependent responses to the different inhibitor compounds, a two way analysis of variance was used. For analysis of inhibition of phosphorylation, induction of apoptosis, modulation of gene expression and suppression of pathologic cell growth ex vivo a St t test was employed. Data were assumed to be statistically significant when p < 0.05. Results A Stilbenoid Core Is Essential for Time and Dose dependent Inhibition of Jak2 V617F dependent Cell Growth The human erythroleukemia (HEL 92.1.7) cell li ne is homozygous for the Jak2 V617F mutation and this gain of function mutation is responsible for its transformed phenotype [ 110 145 ] Proliferation of HEL cells is mediated by the constitutively active Jak2 V617F signaling which promotes a G1/S phase transition, thereby leading to incr eased cellular proliferation [ 111 ] G6 and its five structurally related derivatives were therefore first analyzed for their ability to inhibit the Jak2 V617F dependent proliferation of HEL cells. Viable cell numbers were determined b y trypan blue exclusion and hemocytometer after 72 hrs. Each sample was measured in triplicate. Inhibition by G6 was arbitrarily set at 100% and the percent inhibition for all the other compounds relative to G6 was defined as 1.00 ( drug / 3 1 summarizes the percent growth inhibition for each of the six compounds. We found that the stilbene containing derivatives (D28 and D30) had high growth inhibition potentials whereas those compounds lacking the stilben oid core (D21, D23 and D25) had low growth inhibition potentials.

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71 To determine the ability of each of these compounds to inhibit Jak2 V617F mediated HEL cell proliferation, cells were treated either for varying periods of time or w ith increasing concentrations of G6 or its derivatives. Viable cell numbers for each treatment were determined. When compared to vehicle treated cells, we found that G6 and its stilbenoid derivatives (D28 and D30) significantly reduced viable cell numbers in a time dependent manner whereas the non stilbenoid derivatives (D21, D23 and D25) did not (Fig. 3 1A). We also found that G6 and the two stilbenoid derivatives (D28 and D30) markedly inhibited the growth of HEL cells in a dose dependent manner (Fig. 3 1 B) whereas the three non stilbenoid derivatives (D21, D23, and D25) showed no growth inhibition of HEL cells (Fig. 3 1C). Phosphorylation of Jak2 at tyrosine residues 1007/1008 is concomitant with higher kinase activity and increased cellular proliferatio n [ 61 ] Therefore, we next wanted to determine whether the presence of the stilbenoid core is critical for reduction of phospho Jak2 levels within treated cells. Ph ospho Jak2 levels were measured 48 hrs after drug exposure rather the 72 hrs used in Figs. 3 1A C as far fewer viable cells exist at the longer time point. Exposure of HEL cells to stilbenoid core bearing compounds (G6, D28, and D30) significantly decrea sed the levels of phospho Jak2 (Fig. 3 1D) when compared to those derivatives that lack the stilbenoid core (D21, D23, and D25). We next wanted to determine if the ability of G6 to inhibit Jak2 V617F mediated cell proliferation was valid for other cell li nes with constitutively active Jak2. For this, we studied the ability of these compounds to inhibit Ba/F3 cells stably expressing Jak2 V617F. The introduction of the Jak2 V617F cDNA into these cells via retroviral

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72 transduction confers cytokine independent growth that is en tirely Jak2 V617F dependent [ 1 36 ] Cells were treated with various doses of the different drugs and viable cell numbers were assessed after 72 hrs using an MTS assay. G6 and its stilbenoid derivatives (D28 and D30) showed significant inhibition of cell growth in a dose dependent mann er (Fig. 3 2A) when compared to the non stilbenoid derivatives (D21, D23 and D25) (Fig. 3 2B). Collectively, these data demonstrate that the stilbenoid core is essential for maintaining the ability of G6 to inhibit Jak2 V617F dependent cell growth and the reduced cell growth correlates with significantly reduced levels of phospho Jak2. Indispensability of the S tilbenoid core in D ecreasing P hosphorylation of STAT3 and STAT5 In the canonical Jak/STAT signalling pathway, an active Jak2 phosphorylates STAT prot eins such as STAT3 and STAT5, which ultimately translocate to the nucleus and modulate gene transcription [ 60 ] Hence, we wanted to determine if the stilbenoid co re is critical for the inhibition of STAT phosphorylation. HEL cells were treated for 48 hrs with the different compounds and phospho STAT levels were determined. G6 and its stilbenoid derivatives (D28 and D30) significantly decreased phospho STAT3 levels as measured by ELISA (Fig. 3 3A) and western blot analysis (Fig. 3 3B). Similarly, only the stilbenoid containing compounds (G6, D28 and D30) significantly decreased phospho STAT5 levels as measured by ELISA (Fig. 3 3C) and western blot analysis (Fig. 3 3D ). As such, these data confirm the ability of G6 and its stilbenioid derivatives to downregulate the phosphorylation of key signaling molecules involved in Jak2 dependent pathological cell growth; namely, STAT3, and STAT5.

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73 Induction of A poptosis in HEL C el ls by G6 and its D erivatives Deregulation of the Jak/STAT signaling pathway is known to promote cell proliferation and prevent apoptosis i n several different cancers [ 146 ] Given that G6 and its stilbenoid core containing derivatives inhibited Jak2 V617F dependent cell proliferation and Jak/STAT activation, we next wanted to determine whether these drugs induce apoptotic death. HEL cells treated with G6 and the stilbeniod derivatives (D28 and D30) exhibited a significant increase in the percentage of cells in early apoptosis when compared to the DMSO or the non stilbenoid (D21, D23, and D25) treated cells (Fig. 3 4A). Fig 3 4B is a quantitative gr aph of four independent experiments showing the amount of apoptosis plotted as a function of treatment condition. We observed that the percentage of cells in early apoptosis increased from 7.45% in the DMSO treated control to 27.8% in G6 treated, 31.3% in D28 treated and 34.2% in D30 treated HEL cells, whereas it remained almost unchanged for the non stilbenoid treated cells (Fig. 3 4B). Jak2/STAT signaling is known to positively regulate cell growth by directly increasing expression of the anti apoptotic marker, Bcl xL, via STAT binding elements present in its promoter region [ 114 147 ] To determine if the presence of the stilbenoid core correlates with reduced levels of Bcl xL, we measured Bcl xL mRNA levels in cells treated with the different compounds. The stilbenoids (G6, D28 and D30), significantly decreased Bcl xL expression in HEL cells when compared to DMSO or the non stilbenoids (D21, D23 and D25) both at 8 hrs (Fig. 3 5A) and 24 hrs (Fig. 3 5B) of treatment. As such, these data indicate that the presence of the stilbenoid core does in fact correlate with markedly reduced levels of the proliferative marker, Bcl xL.

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74 The intrinsic apoptotic pathway is regulated by members of the Bcl 2 family [ 148 ] Therefore, w e next monitored the expression of Bcl 2 family members in HEL cells derivates (G6, D28 and D30) induced greater cleavage of PARP, a marker of apoptosis, when compared to the non stilbenoids (D21, D23 and D25) (Fig. 3 5C). We also observed a stilbenoid dependent increase in the protein levels of Bim, a pro apoptotic member of the Bcl 2 family (Fig. 3 5D). The pro apoptotic protein, Bid, is present in the cytosol as an i nactive precursor. Upon proteolytic cleavage, its active form is generated, which translocates to the mitochondria and induces mitochondrial damage and destabilization [ 120 ] We found that the stilbenoid compounds decreased the levels of the inactive precursor form of Bid, a hallmark of Bid cleavage and subsequent apoptosis. We found that the protein levels of Bax and Bcl 2 did not change with any treatment (Fig. 3 5D). Lastly, the samples were blotted with an anti STAT1 antibody to demonstrate equal protein loading across all lanes (Fig. 3 5D). Overall, the data in Figs. 3 4 and 3 5 indicate that the stilbenoid core of G6 is critical for its ability to induce apopt otic cell death in HEL cells via the intrinsic apoptotic pathway by downregulation of anti apoptotic Bcl xL, up regulation of pro apoptotic Bim, and cleavage of Bid. Stilbenoid C ore B earing D erivatives of G6 S uppress P athologic C ell G rowth of P atient d eriv ed B one M arrow C ells, ex vivo We next wanted to determine whether the stilbene core is also essential for its ability to inhibit pathologic cell growth of patient derived bone marrow cells, ex vivo For this, we used a colony formation assay which measures the number of erythroid colony forming units (CFU E) that are produced when marrow derived stem cells are cultured,

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75 ex vivo Stem cells isolated from a normal individual will be unable to grow in the absence of exogenously added cytokine. However, the J ak2 V617F mutation confers cytokine independent, cell growth. Here, mononuclear cells isolated from the bone marrow of a Jak2 V617F positive female polycythemia vera patient, were cultured in dded as indicated. Our previous work has shown that 5 M of G6 inhibits ~50% of the cytokine independent growth of Jak2 V617F expressing, marrow derived stem cells [ 104 ] The results here show that treatment of the primary cultures with either G6 or the stilbenoids (D28 and D30) significantly blocked cytokine independent pathologic cell growth when compared to the DMSO and non stilbenoid (D21, D23, and D25) treate d samples (Fig. 3 6). As such, data in Fig. 3 6 demonstrate that the stilbenoid core of G6 is essential for reducing Jak2 dependent pathologic cell growth of human bone marrow mononuclear cells cultured ex vivo. Computational D ocking of G6 and its D erivati ves into the ATP B inding P ocket of the Jak2 K inase D omain Using the known structure of the Jak2 kinase domain [ 139 ] ATP, t he ATP analog ACP, G6, and each of its five structurally related derivatives, were docked into the ATP binding pocket. The goal was to analyze the potential interactions of these compounds with amino acids in this binding region. The ATP binding pocket of Jak2 and the important residues clustered in this pocket have been described previously [ 16 22 ] Generation o f the electrostatic surface potential for the Jak2 kinase domain showed the predicted presence of acidic and basic patch residues clustered at the ATP binding pocket (Fig. 3 7 ). We found that both ATP and ACP docked extremely well into this pocket (Fig. 3 8 A) with GRID scores of 76.81 kcal/mole and 62.37 kcal/mole,

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76 respectively. Furthermore, the docked molecules showed excellent correlation with the known crystal structures of ACP and ATP complexed with kinase domains of other proteins [ 149 152 ] We found that the stilbenoids had higher binding affinities when compared to the non stilbenoids as indicated by their more negative energy scores. G6, D28, D30, D21, D23 and D25 had energy scores of 75.46, 63.24, 72.26, 27.69, 38.90 and 48.91 kcal/mole, respectively. The docking orientations of G6, D28 and D30 into the pocket (Fig. 3 8 B) were very similar to that of ATP/ACP ( Fig. 3 7 ) and distinctly different fro m that of the non stilbenoids (Fig. 3 8 C). The docking conformation of each of the stilbenoid inhibitors into the ATP binding pocket also correlated well (r.m.s.d < 2) with the previously reported structure of another Jak2 inhibitor, 5B3, in complex with the Jak2 kinase domain (Fig. 3 8 D). Examination of the most favorable docking orientations for G6 and each of its derivatives showed the presence of hydrogen bonds (< 3.5) and van der Waals interactions between the stilbenoid derivatives and the ATP bind ing pocket of Jak2 (Fig. 3 9 ). Specifically, the stilbenoid core containing derivatives exhibited van der Waals interactions with many of the hydrophobic residues in the binding pocket such as V863, L855, L983, L932, Y931, A880 and V911. The stilbenoid con taining derivatives also formed hydrogen bond interactions with several important surrounding residues including D994, R980, E930 and L932. The non stilbenoid derivatives however, did not show any such hydrogen bond interactions with these critical residue s of the Jak2 ATP binding pocket. Overall, these data demonstrate the importance of the stilbenoid core

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77 structure for stronger docking interactions of the drug with the ATP binding pocket of Jak2. Discussion Somatic Jak2 mutations, such as the Jak2 V617F, which result in deregulated Jak/STAT signaling, have been identified in numerous patients with myeloproliferative neoplasms and are known to play a primary role in the path ogenesis of these diseases [ 57 61 ] Therefore, identification of novel compounds having inhibitory effects against hyperkinetic Jak2 has become an attractive therapeutic strategy in MPN. Using structure based virtual screening, our group recently identified a novel Jak2 small molecule inhibitor called G6 [ 104 ] In this report, we describe a structure function correlation of this inhibitor compound. We demo nstrate that it has a central stilbenoid core in its structure which is indispensible for maintaining its ability to inhibit Jak2 kinase activity. G6 and its stilbenoid core containing derivatives (D28 and D30) effectively inhibit Jak2 V617F mediated HEL c ell proliferation in a time and dose dependent manner. Correspondingly, they are also capable of suppressing the phosphorylation of Jak2, STAT3 and STAT5 proteins. Furthermore, these stilbenoids significantly induce apoptosis in treated cells via the intr insic pathway and possess the ability to block ex vivo pathologic cell growth of bone marrow cells isolated from a Jak2 V617F positive MPN patient. Stilbenes are a group of compounds with a wide range of diverse biological activities. Stilbenoids, such as resveratrol, piceatannol and diethylstilbestrol, are reported to have anti proliferative, anti oxidative, anti neovascularization and t umor suppressive effects [ 131 135 ] Resveratrol has beneficial cardiovascular effects [ 153 ]

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78 whereas diethylstilbestrol is know n to have estrogen a ctivity [ 154 ] Piceatannol, a naturally occurring phenolic stilbenoid, is the only stilbenoid that is a known protein tyrosine kinase inhibitor It inhibits LMP2A, a viral tyrosine kinase implicated in diseases associated with the Epstein Barr virus as well as the non receptor tyrosine kinases Syk and Lck [ 155 156 ] Here, we report that G6, which is also a stilbene, has anti Jak2 tyrosine kinase activity. More importantly, the trans stilbenoid core identified in all the active compounds is now being subjected to bioisosteric replacements [ 157 ] that would result in new Jak2 chemotypes with improved druglike properties. Computational docking of G6 and its structurally related derivatives into the ATP binding pocket of Jak2 revealed important interactions between the inhibitors and specific residues within Jak2. For example, E930 and L932 are part of the important hinge region of the Jak2 kin ase domain and are known to be involved in adenine binding. We found that these residues were occupied by the stilbenoid containing compounds, but not the non stilbenoids. Another amino acid that interacted with the docked stilbenoid inhibitors was D994, located in the highly conserved DFG motif. This motif is part of the critical activation loop of the kinase and our group was the first to identify the importance of this residue as it relates to Jak2 kinase inhibition [ 104 ] We show here that the stilbenoid core bearing inhibitors also had a hydrogen bond interaction with R980, which is a part of the catalytic loop and is known to be one of the residues involved i n the coordination of magnesium ions [ 16 22 ] Thus, the ability of the stilbenoid derivatives to simultaneous ly interact, in silico with the hinge region, the DFG motif, and the catalytic loop suggests that they may interfere with both ATP binding function and activation loop phosphorylation, thereby making them potent Jak2

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79 inhibitors. In summary, our data colle ctively show that the central stilbenoid core structure is indispensable for maintaining anti Jak2 kinase activity of the small molecule inhibitor G6.

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80 Table 3 1. Percent growth inhibition of G6 and its derivatives. G6 and its derivatives are shown along with their NSC #, chemical structure, molecular weight and % growth inhibition. Inhibition by G6 was arbitrarily set at 100% and the inhibition potential of the other compounds relative to G6 was evaluated using the relation 1.00 ( drug / vehicle control). Compound Name NSC # Structure MW a % Growth Inhibition G6 33994 438.652 100 .00 D21 10618 227.3054 5 .00 D23 47911 241.3322 1.8 0 D25 13109 379.5844 0.61 D28 27647 339.4766 124.16 D30 600567 384.5606 108.32

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81 Figure 3 1. Time and dose dependent effect of G6 and its derivatives on the HEL cell proliferation and Jak2 phosphorylation. ( A) HEL cells we re treated with either number of viable cells in each sample was measured by trypan blue exclusion. Each sample was measured in triplicate. D23 vs. G6 (p = 1.22 10 10 ). ( B) & ( C ) HEL cells were treated for 72 hours either with DMSO or with viable cell numbers for each treatment were determined in triplicate. Shown is one of two representative results. ( D) HE the different drugs for 48 h. Cells were then analyzed by ELISA for the detection of phospho Jak2 (pY1007/pY1008). Each experiment was run in triplicate. Shown is one of two representative results. p<0.05 with respect to DMSO, # p<0.05 with respect to non stilbenoids.

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82 Figure 3 2. Dose dependent effect of G6 and its derivatives on the proliferation of Ba/F3 EpoR Jak2 V617F cells. ( A) & ( B) Ba/F3 EpoR Jak2 V617F cells were treated for 72 hours either with DMSO or with 0 .0 1, 0.03, 0.1, 0.3, 1, 3, 10 treatment were determined in triplicate using an MTS assay. Shown is one of two representative results.

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83 Figure 3 3 Inhibition of phosphorylation of STAT3 and STAT5 by G6 and its derivatives. Cells were then analyzed for the detection of phospho STAT3 and phospho STAT5 by both ELISA (A) & ( C) and western blot analysis ( B) & ( D). Stilbe noid containing compounds (G6, D28 and D30) effectively inhibited phosphorylation of STAT3 (A) & (B) and phosphorylation of STAT5b (C) & (D). Shown is one of two representative results for each. p<0.05 with respect to DMSO, # p<0.05 with respect to non s tilbenoids.

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84 Figure 3 4. Induction of apoptosis in HEL cells by G6 and its derivatives. HEL cells Annexin V FITC and propidium iodide followed by flow cytometric analysis. (A) Shown are representative flow cytometry profiles from one of four independent results. (B) Quantification of the number of cells in early apoptosis (i.e. annexin V positive and propidium iodide negative). Data shown are the mean +/ SD from four independent experiments. p<0.05 with respect to DMSO, # p<0.05 with respect to non stilbenoids.

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85 Figure 3 5. Treatment with G6 or its stilbenoid derivatives leads to HEL cell death via the intrinsic apoptotic pathway. different drugs for either 8 hours (A) or 24 hours (B). Bcl xL mRNA levels were normalized to those of GAPDH and plotted as the fold change over DMSO control. Each sample was run in duplicate. Shown is a one of two representative results. p <0.05 with respect to DMSO. (C) Cells were treated lysates were then analyzed by western blotting with an anti PARP antibody. Shown is one of three rep resentative results. (D) Cells were treated with were then serially analyzed by western blot analysis for the indicated proteins. Shown is one of three representative results.

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86 Figure 3 6. Suppression of Jak2 V617F mediated pathologic cell growth in patient derived bone marrow cells by G6 and its derivatives, ex vivo Marrow derived mononuclear cells were cultured in semisolid media in the presence or structurally related derivatives. At the end of 14 days of culture, the numbers of CFU E were counted. Each condition was measured in duplicate. p<0.05 with respect to DMSO, # p<0.05 with respect to non stilbenoids.

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87 Figure 3 7. Molecular surface repr esentation of the ATP binding site of Jak2. Molecular surface representation of Jak2 in complex with ATP and its analog ACP, shown here as stick models. ATP is colored yellow and ACP is green.

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88 Figure 3 8. Molecular docking of G6 and its derivatives i nto the ATP binding pocket of Jak2. Molecular docking of G6 and its derivatives into the ATP binding pocket of Jak2. ATP, the ATP analog ACP, G6, and each of its structurally related derivatives were docked into the ATP binding pocket of the known crystal structure of Jak2 kinase domain (PDB ID: 3E64). (A) Coiled representation of the structure of Jak2 with ATP and ACP docked into the ATP binding pocket. The nucleotide binding loop is blue, the hinge region is cyan, the catalytic loop is magenta, and the a ctivation loop is red. Residues within this pocket that are critical for interactions with ATP and other docked drugs have been represented as spheres. Phosphorylation of Y1007 within the activation loop is necessary for the activation of kinase activity o f Jak2. ATP (yellow) and ACP (green) had strong interactions with the pocket as indicated by their highly negative GRID scores. (B) G6 (pink), D28 (grey) and D30 (yellow) docked at the ATP binding pocket of Jak2. (C) D21 (orange), D23 (magenta) and D25 (g reen) docked at the ATP binding pocket of Jak2. (D) Comparison of the docking of ATP (yellow), ATP analog ACP (green) and G6 (pink) with the crystal structure of a Jak2 inhibitor (5B3) (blue) in complex with Jak2 kinase domain showed good correlation (r.m. s.d. < 2) between the structures.

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89 Figure 3 9. Docking of G6 and its derivatives into the ATP binding pocket of Jak2. G6 (pink) and its stilbenoid derivative D28 (grey) and D30 (yellow) showed hydrogen bond interactions (< 3.5) with several critical residues (E930, L932, R980 and D994) that make up the ATP binding pocket of Jak2. However, the nonstilbenoid derivatives D21 (magenta), D23 (orange) and D25 (yellow) show no such hydrogen bond interactions.

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90 CHAPTER 4 CELL DEATH INDUCED B Y THE JAK2 INHIBIT OR, G6, CORRELATES WITH CLEAVAGE OF VIMENTIN FILAMENTS Jak2 is a member of the Janus family of cytoplasmic tyrosine kinases. Other members of this family include Jak1, Jak3 and Tyk2. Jak2 is activated by a variety of cytokines, growth factors and GPCR lig ands, resulting in signaling cascades that regulate cell growth, proliferation and death. Upon binding of the ligand to its specific receptor, the receptor associated Jak proteins are activated via a phosphorylation event. An activated Jak can in turn pho sphorylate and activate the Signal Transducers and Activators of Transcription (STAT) family of transcription factors. Phosphorylated STATs dimerize and translocate to the nucleus where they modulate gene transcription. Thus, the Jak/STAT pathway results i n a signal cascade from binding and activation at the plasma membrane to changes in gene transcription in the nucleus. Hyperkinetic Jak2 promotes aberrant cell growth and prevents apoptosis. Hence, constitutively active Jak/STAT signaling pathway has been implicated in a variety of neoplastic disorders. Jak2 can become constitutively active by several different gene alterations including specific chromosomal translocations and point mutations. Jak2 chromosomal translocations such as TEL Jak2, REL Jak2, BCR Jak2 and PCM1 Jak2 lead to the development of a variety of leukemias, lymphomas and myelomas [ 44 45 130 158 161 ] Additionally, an activating Jak2 point mutation (Jak2 V617F) has been linked to the myeloproliferative neoplasms (MPN) such as polycythemia ve ra (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) [ 57 61 ] This valine to phenylalanine substitution mutation present in codon 617 of the aut oinhibitory pseudokinase domain of Jak2 allows the kinase to evade negative regulation thereby making it constitutively active. MPN patients bear this mutation in their marrow derived

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91 stem cells and are characterized by the overproduction of terminally di fferentiated blood cells of the myeloid lineage such as red cells or platelets. Current therapies for MPN patients include phlebotomy and hydroxyurea. While these treatments alleviate some disease symptomologies, they are not curative in any way. Therefor e, there is an unmet clinical need for Jak2 inhibitors. U sing structure based virtual screening, our group recently identified a novel stilbenoid small molecule inhibitor of Jak2 named G6 [ 104 ] We subsequently showed that G6 specifically inhibits Jak2 mediated human pathologic cell growth in vitro ex vivo and in vivo [ 105 162 ] We also demonstrated that G6 inhibits Jak2 mediated cell proliferation via the suppression of key signaling molecules of the Jak/STAT pathway; the consequence of this inhibition is G1/S ce ll cycle arrest and apoptosis [ 105 162 ] Here, we sought to elucidate the molecular and biochemical mech anisms by which G6 inhibits Jak2 mediated cellular proliferation. For this, we compared protein expression profiles between vehicle treated and G6 treated cells using two dimensional gel electrophoresis. The intermediate filament protein, vimentin, was one protein that was differentially expressed between the two conditions. We therefore hypothesized that the mechanism by which G6 inhibits Jak2 dependent cell proliferation involves modification of this protein. In this study, our data supports this hypothes is as we show that G6 induced inhibition of Jak2 med iated pathogenic cell growth correlates with the specific cleavage and cellular reorganization of vimentin. Experimental Procedures Drugs G6, obtained from the National Cancer Institute/Developmental Ther apeutics Program (NCI/DTP) The drug was solublized in dimethyl sulfoxide (DMSO) at a

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92 concentration of 10 mM G6 was stored at 20 C in small aliquots to avoid repeated freeze thaw cycles Reagents AG490, Jak Inhibitor I, PD98059 and PP2 were purchased from Calbiochem. Cycloheximide was purchased from Fisher Scientific. Caspase Inhibitor I (Z VAD (OMe) FMK), Calpain Inhibitor V (Mu V al HPh CH2F, Mu = morpholinoureidyl; HPh = homophenylalanyl), Verapamil, BAPTA imino dipropion i trile (IDPN) were purchased from Calbiochem. Cell Culture Human Erythroleukemia (HEL) cells were purchased from the American Type Culture Col lection and maintained in RPMI 1640 (Mediatech) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and L glutamine at 37 C and 5% CO 2 2 D Differential In Gel Electrophoresis (2 D DIGE) HEL cells were treated either with vehicle control DMSO or 25 M G6 for 12 hours. The cell pellets were resuspended in ice cold buffer containing 0.3% SDS, 20 mM Tris pH 8.0, 100 mM DTT, 10 l protease inhibitor cocktail III (Calbiochem), 5 mM MgCl 2 and 250 units of benzonase. The cell suspension was homogenized by sonication. Protein was precipitated with 9 volumes of ice cold 10% trichloroacetic acid (TCA) in acetone overnight at 4C. Prec ipitated proteins were then dissolved in solubilization buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.2% SDS and 20 mM Tris, pH 8.0). After centrifugation at 43,000 rpm for 30 min, solubilized protein in the supernatant was quantified using the EZQ Protein A ssay Kit (Invitrogen). Proteins (100 g per sample) An internal standard, which is loaded on every gel, was created by mixing equal

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93 amounts of protein from all samples. P roteins from the DMSO treated samples were labeled with Cy3 (green) and the G6 treated samples were labeled with Cy5 (red). The internal standard was labeled with Cy2 (blue). 100 g of Cy2 labeled internal standard, 100 g of Cy3 labeled sample, 100 g of Cy5 labeled sample were mixed with 200 ug unlabeled internal standard. The mixture was used to rehydrate a 24 cm pH 3 to 11 nl IPG strip (GE Healthcare) overnight in a rehydration buffer (solubilization buffer with 100 nM DTT containing Orange G as track ing dye) in the dark at room temperature. Three independent replicates of each sample were run on three strips. IEF was carried out in IPGphor3 unit (GE Healthcare) throughout focusi ng. After completion of IEF, strips were first reduced in 15 ml of 50 mM Tris HCl pH 6.8, 6 M Urea, 30% (v/v) glycerol 2% (w/v) SDS 100 mM DTT for 20 min in the dark at room temperature, then alkylated in 15 ml of 50 mM Tris HCl pH 6.8 6 M Urea 30% (v /v) glycerol 2% SDS 2.5% idoacetamide for 20 min. After equilibration, strips were transferred and mounted on an 8% 16% precast Tris Glycine polyacrylamide gel (Jule). Electrophoresis was carried out initially at 12C at 10 mA/gel for one hour and th en at constant current overnight at 12 mA/gel and a limit of 150 V until dye front reached the bottom of the plate. Gels were then scanned with Typhoon 9400 Variable Mode Imager (GE Healthcare). The excitation/emission wavelengths for Cy2, Cy3 and Cy5 wer e 488/520, 532/580 and 633/670 nm, respectively. For each gel, images for the internal standard as well as the control and experimental conditions were acquired. The digital images

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94 were then analyzed with DeCyder 2D version 7.0 ( GE Healthcare). Informati on from replicate gels was analyzed with BVA Module (Biological Variation Analysis). Spots were selected by setting the fold difference threshold to 1.6 fold. Statistical significance t test. Protein identification using elect rospray mass spectroscopy was done at the Scripps Research Institute. Cell Lysis Cells (~ 10 7 ) were washed with two volumes of ice cold PBS and then lysed in 0.8 ml of ice cold RIPA buffer (20 mM Tris pH 7.5, 10% glycerol, 1% Triton X 100, 1% deoxycholic a cid, 0.1% SDS, 2.5 mM EDTA, 50 mM NaF, 10 mM Na 4 P 2 O 7 4 mM benzamidine, and 10 g/mL aprotinin). Protein concentrations in the whole cell lysates were determined using a Bradford assay (Bio Rad). Cell lysates were then resuspended in SDS sample buffer. Who PAGE and then transferred onto nitrocellulose membranes for analysis by western blotting. Western Blotting Nitrocellulose membranes were first blocked with 5% milk/TBST solution at room temperature and then p robed first with the different indicated primary antibodies overnight at 4C followed by the respective secondary antibodies (1:4000, GE Healthcare). The immuno reactive bands were then visualized using the enhanced chemi luminescence system (Western Light ning Ultra, Perkin Elmer). The following antibodies were used at the indicated dilutions: vimentin (Abcam and BD Biosciences, actin (Cell Signaling, 1:500).

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95 Immunofluorescence HEL cells were cultured in RPMI in 100 mm dishe the indicated periods of time. Following treatment, the cells were centrifuged, washed and resuspended in 1X PBS. Cells were then plated onto poly L lysine coated 8 chamber slides (Santa Cruz Biotechnology) and fixed at 20 C in a mixture of 50% methanol and 50% acetone for 10 minutes. The fixed cells were then permeabilized with 0.2% Triton X 100 and blocked with 5% goat serum for 30 minutes at room temperature. The samples were incubated overnight at 4C with a primary anti body of mouse anti vimentin (BD Biosciences, 1:100) and washed 4X with PBS the following morning. The samples were then incubated with a FITC conjugated anti mouse secondary antibody (Santa Cruz Biotechnology) for one hour at room temperature. The cells we re again washed with PBS, mounted with UltraCruz DAPI containing mounting media (Santa Cruz Biotechnology) and sealed with a cover slip. These cells were imaged using a 100X objective on an inverted fluorescence microscope (Olympus). Cell Proliferation Ass ay HEL cells were plated in 96 well plates and treated with either 0.25% DMSO, 30 each sample by trypan blue exclusion staining and hemocytometer. In vivo Animal Model Th e xenograft model of Jak2 V617F expressing HEL cells in NOD/SCID mice ha s been described previously [ 105 ] Briefly, 3 months old NOD/SCID mice were randomized in to 5 groups (n=6 per group). One group consisted of naive animals that did not receive any treatment. All other groups received a single tail vein injection of 2 x 10 6 Jak2 V617F positive HEL cells. Three weeks after HEL cell injection, the mice

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96 developed symptoms of a fully penetrant bone marrow malignancy. The mice then began receiving intraperitoneal injections of either vehicle control (DMSO) or G6 at doses of 0.1, 1, and 10 mg/kg/day for the next 21 days. At the end of the three week treatment period, all groups of mice were euthanized and bone marrow tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Bone Marrow Immunohistochemistry Paraffin embedded bone marrow sections from each treatment group were analyzed by anti vimenti n immunohistochemistry. Antigen retrieval was carried out first by microwaving at 95C for 20 min in 1mM EDTA NaOH solution, pH 8.0. The section were then cooled, blocked with Protein Block (DAKO), and incubated with anti vimentin antibody (Abcam, 1:100) f or 2 hours at room temperature. Antigen antibody complexes were detected using biotinylated secondary antibodies and streptavidin peroxidase substrate (DAKO). Stained sections were then analyzed via a standard light microscope (Nikon) at 40X and 100X magni fications. Statistical Analysis For statistical evaluation of time dependent response of HEL cell viability to G6 and IDPN, a two way analysis of variance was used. For analysis of differential expression of proteins in 2 D DIGE and G6 induced degradation of vimentin using t test was employed. Data were assumed to be statistically significant when p < 0.05. Results G6 T reatment I nduces T ime and D ose D ependent D egradation of V imentin The human erythroleukemia (HEL 92.1.7) cell lin e is homozygous for the Jak2 V617F mutation [ 110 145 ] The presence of this mutation induces constituti vely active

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97 Jak/STAT signaling and promotes a G1/S phase transition thereby driving increased cellular proliferation [ 111 ] We previously demonstrated that the Jak 2 inhibitor, G6, inhibits Jak2 V617F mediated HEL cell proli feration and induces apoptosis [ 105 162 ] How ever, the specific mechanisms by which G6 does this are not known. To gain some insight into the mechanism by which G6 reduces cell viability, HEL The protein expression profiles of these two treatment conditions were compared using two dimensional gel electrophoresis (Fig. 4 1A, 4 1B, 4 1C). One spot in particular was present in the DMSO treated cells, but significantly reduced in the G6 treated cells (Fig. 4 1D & 4 1E; marked by arrows). That spot was excised and identified using electr o spray mass spectrometry as vimentin. In a separate two dimensional gel electrophoresis study, we compared the protein expression profiles between HEL cells time point, t he spot representing vimentin was still significantly reduced in the G6 treated cells when compared to the DMSO treated cells (data not shown). To confirm that vimentin protein levels were decreasing with G6 treatment, protein samples from both conditions were subjected to western blot analysis with an anti vimentin antibody. Consistent with the mass spectroscopy data, treatment of HEL cells with G6 resulted in the disappearance of full length vimentin (Fig. 4 1F). Of note, we also observed the appearance of low molecular weight fragments of vimentin in the G6 treated cells (Fig. 4 1F). To determine whether this effect was time and dose dependent, HEL cells were

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98 G6 for 24 hours. Whole cell lysates were then separated on SDS PAGE and examined by immunoblotting with an anti vimentin antibody. Fig. 4 2A is a representative blot showing that full length vimentin was cleaved into low molecular weight fragments with G6 treat ment as a function of time. The same samples were then reprobed with an anti actin antibody to confirm equal protein loading and also to demonstrate the specificity actin. Quantification of al l blots using densitometry confirmed the total loss of full length vimentin protein in response to G6 treatment over time (Fig. 4 2B). Similarly, Fig. 4 2C is a representative blot showing a dose dependent cleavage of full length vimentin in response to G6 and Fig. 4 2D is a quantification of all blots. Collectively, the data in Figs. 4 1 and 4 2 demonstrate the ability of G6 to induce specific cleavage of the intermediate filament protein vimentin. Furthermore, this effect is both time and dose dependent. G6 T reatment I nduces M arked R eorganization of V imentin I ntermediate F ilaments within C ells We next wanted to study the effect of G6 treatment on structure and cellular distribution of intracellular vimentin filaments. For this, HEL cells were treated with 25 indirect immunofluorescence For the 0 hr time point, we found that vimentin was distributed uniformly over the cytoplasm (Fig. 4 3A, 4 3D, & 4 3G). However, for the 24 and 36 hr time points, vimentin had a punctate staining pattern in the perinuclear region of the cell (Fig. 4 3B, 4 3E, 4 3H & 4 3C, 4 3F, 4 3I, respectively). As such, the data in Fig. 4 3 indicate that G6 treatment induces cellular redistribution and aggregation of vimentin intermediate filament within HEL cells.

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99 G6 induced C leavage of V imentin is Jak2 mediated Having already demonstrated the ability of G6 to induce specific cleavage of vimentin (Fig. 4 2), we next wanted to determine if this G6 induc ed cleavage was Jak2 dependent. For this, HEL cells were treated for 24 hours with increasing concentrations of three different Jak2 inhibitors; G6, AG490 and Jak Inhibitor I. As a control, HEL cells were also treated with non Jak2 inhibitors; namely, the MAPK inhibitor, PD98059 and Src family kinase inhibitor, PP2. Whole cell lysates were separated by SDS PAGE and immunoblotted with an anti vimentin antibody. We observed that the Jak2 specific inhibitors induced cleavage of vimentin dose dependently (Fig. 4 4A) whereas the non Jak2 inhibitors had no effect on full length vimentin (Fig. 4 4B). The loading of total protein was confirmed by immunoblotting the same cellular lysates with an anti STAT1 antibody (Fig. 4 4A & 4B). Thus, from Fig. 4 4 we conclude th at G6 induced vimentin degradation is Jak2 mediated. G6 induced C leavage of V imentin is I ndependent of de novo P rotein S ynthesis and C aspase A ctivity, but C alpain dependent Given that G6 induces specific cleavage of vimentin (Fig. 4 2), we next wanted to d etermine whether this G6 induced vimentin cleavage is dependent on de novo protein synthesis. To assess this, HEL cells were first pretreated for 4 hours with increasing doses of cycloheximide (CHX), an inhibitor of protein biosynthesis, and then treated w ith increasing concentrations of G6 for 24 hours. Cycloheximide inhibits protein synthesis by interfering with the translation elongation process of protein biosynthesis [ 163 ] Western blot analysis of the cell lys ates from the different treatment groups showed that exposure to increasing doses of G6 induced a dose dependent cleavage of vimentin in HEL cells which was not be blocked by pretreatment with cycloheximide (Fig. 4 5A),

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100 indicating that this G6 induced clea vage process does not require de novo protein synthesis. Vimentin is cleaved in response to G6 treatment into low molecular weight fragments of vimentin (Fig. 4 2) suggesting that this proce ss is mediated by a protease/pr oteolytic enzyme. Caspases are a c lass of intracellular cysteine proteases with roles in cytokine maturation, inflammation and apoptosis [ 164 ] We previously showed that G6 induces caspase 3/7 activation in a time dependent m anner in HEL cells [ 105 ] It has also been reported that vimentin is a caspase substrate and can be cleaved by some caspases in vitro [ 165 ] Therefore, we wanted to determine if G6 induced vimentin cleavage is caspase mediated. For this, we first pretreated HEL cells with the pan caspase inhibitor, Caspase Inhibitor I (zVA D fmk), for 4 hours before G6 dependent vimentin cleavage was then studied by western blotting the cell lysates with an anti vimentin antibody. We found that inhibitio n of caspases by zVAD fmk was unable to prevent G6 induced cleavage of vimentin (Fig. 4 5B) but was able to significantly reduce the G6 induced cleavage of PARP (Fig. 4 5B), a substrate known to be cleaved by caspases [ 115 ] thereby indicating that G6 induced cleavage of vimentin is caspase independent. Calpain, a calciu m dependent neutral cysteine protease [ 166 ] is yet another protease that is known to cleave vimentin [ 167 168 ] Henc e, to examine whether G6 induced vimentin cleavage is calpain mediated, we pretreated HEL cells with a calpain inhibitor, Calpain Inhibitor V (Mu Val HPh CH2F), for 4 hours before exposing them to increasing doses of G6 for 24 hours. Immunoblotting analysi s of the HEL cell lysates

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101 showed that calpain inhibition prevented G6 induced cleavage of vimentin in a dose dependent manner (Fig. 4 5C), demonstrating that the protease involved in the cleavage of vimentin in response to G6 treatment is calpain. Overall, the data in Fig. 4 5 indicate that the G6 induced cleavage of intermediate filament protein vimentin is independent of de novo protein synthesis and caspase activity, but dependent on calpain protease activity. The M obilization of C alcium is E ssential and S ufficient for the C leavage of the I ntermediate F ilament P rotein V imentin Given that calpain is a calcium dependent cysteine protease, we next investigated the role of calcium in the G6 induced vimentin cleavage process. Specifically, we first examined th e effect of inhibiting the flux of extracellular calcium into cells by pretreating the cells with verapamil. Verapamil blocks Ca 2+ channels, principally the L type channel, thereby interfering with the extracellular influx of calcium ions. HEL cells were p Cell lysates were then immunoblotted with an anti vimentin antibody. We found that inhibition of extracellular calcium ion influx into the cell via blockage of L type calc ium channels did not have any effect on G6 induced cleavage of vimentin (Fig. 4 6A). Therefore, we next studied the effect of chelating intracellular calcium on G6 mediated AM for 2 hour s AM is membrane permeable ester form of the calcium chelator BAPTA. Once inside the cell, it is hydrolyzed by cytosolic esterases into its active form and can chelate intracellular calcium. Results from t he western blot analysis showed that chelation of intracellular calcium protected vimentin

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102 from G6 induced cleavage (Fig. 4 6B), indicating that intracellular calcium has a critical role to play in mediating the G6 induced cleavage of vimentin. In the next experiment, we examined the effect of the calcium ionophore, A23187, on vimentin protein levels within HEL cells. A23187 is a mobile ion carrier that forms stable complexes with divalent cations, such as calcium, and can hence be used for increasing intra cellular levels of calcium ions. Accordingly, HEL cells were treated with blotted using an anti vimentin antibody. We found that increasing intracellular calcium levels via exposure to an ionophore is sufficient to induce cleavage of vimentin in HEL cells (Fig. 4 6C), further confirming the essential role that calcium ions play in the vimentin cleavage process. As such, data in Fig. 4 6 demonstrate that mobilization of in tracellular calcium ions is both essential and sufficient for the cleavage of the intermediate filament protein, vimentin Cleavage of V imentin is S ufficient to R educe HEL C ell V iability To determine how critical vimentin is to the viability of cells, we s tudied the effect iminodipropiontrile (IDPN) selectively disrupts vimentin intermediate filaments [ 169 ] Therefore, we treated hours. At each time point, the number of viable cells in each condition was determined and cell lysates from those same conditions were im munoblotted with an anti vimentin antibody in order to correlate decreased cell numbers with increased vimentin cleavage. We found that treatment with both G6 and IDPN time dependently decreased viable cell numbers (Fig. 4 7 A) and this decrease in cell via bility correlated with a corresponding time dependent cleavage of full length vimentin in the G6 and IDPN treated cells (Fig.

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103 4 7B). Overall, the data in Fig. 4 7 demonstrate that the cleavage of vimentin intermediate filaments is sufficient to reduce the viability of Jak2 V617F expressing HEL cells. G6 T reatment D ecreases the L evels of V imentin P rotein, in vivo Our data thus far indicate that treatment of HEL cells with G6 results in the degradation and subsequent loss of vimentin protein, in vitro. To det ermine if this is conserved in vivo HEL cells were injected into the tail vein of NOD/SCID mice and allowed to engraft into the bone marrow over the ensuing 21 days at which time the mice began receiving daily intraperitoneal injections of either vehicle control (DMSO) or G6 at doses of 0.1, 1, and 10 mg/kg/day for the next 21 days. At the end of the three week treatment period, all groups of mice were euthanized and bone marrow was analyzed for vimentin protein levels via anti vimentin immunohistochemistr y. Representative stained sections from each treatment group are shown at 40X (Fig. 4 8A) and 100X (Fig. 4 8B) magnification. We found that when a negative control IgG antibody was used in place of the anti vimentin primary antibody in the immuno histochem ical procedure, only the hematoxylin counter stain was observed. Probing the nave bone marrow with the anti vimentin antibody revealed strong staining in erythroid cells, but not myeloid cells. HEL cell injection followed by DMSO treatment resulted in a d ramatic increase in the expression of vimentin protein when compared to nave animals. Treatment with 0.1 mg/kg/day of G6 did not produce any observable change in the expression level of the protein when compared to DMSO treated mice. However, treatment wi th 1 and 10 mg/kg/day of G6 clearly reduced the levels of vimentin protein to those seen in the completely nave animals. Hence, the data in Fig. 4 8 indicate G6

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10 4 treatment reduces HEL cell induced vimentin expression in a dose dependent manner, in vivo Di scussion Our group recently identified a novel stilbenoid Jak2 small molecule inhibitor named G6 [ 104 ] We subsequently showed that G6 specifically inhibits Jak2 m ediated human pathologic cell growth in vitro ex vivo and in vivo [ 105 162 ] We have also demonstrated that G6 inhibits Jak2 mediated cell proliferation via the suppression of key signaling molecules of the Jak/STAT pathway and with the induction of G1/S cell cycle arrest and apoptosis [ 105 162 ] In this report, our objective was to determine the mechanisms by which G6 exerts its inhibitory actions. To this end, we found that G6 treatment induced a time and d ose dependent cleavage of the intermediate filament protein, vimentin (Fig. 4 2). G6 treatment of HEL cells resulted in the movement of vimentin from a uniform distribution in the cytoplasm to aggregates in the perinuclear region of the cell (Fig. 4 3). T he G6 mediated cleavage of vimentin is Jak2 dependent (Fig. 4 4) and calpain mediated (Fig. 4 5). The mobilization of intracellular calcium is critical for G6 mediated vimentin cleavage (Fig. 4 6) and the cleavage of vimentin, per se is sufficient to redu ce HEL cell viability (Fig. 4 7). Lastly, the ability of G6 to cleave vimentin is conserved in vivo (Fig. 4 8). Taken together, these results describe a novel mechanism by which G6 exerts its inhibitory actions. Vimentin, a member of the Type III intermed iate filament protein family, is a key component of the cytoskeleton of the cell. It plays an important role in maintaining cell shape and integrity as well as stabilizing cytoskeletal interactions such as adhesion, migration and signaling. Apoptosis is as sociated with disruption of the cytoskeletal network and caspase and/or calpain induced cleavage of cytoskeletal proteins, such as

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105 vimentin, is known to occur in response to various inducers of apoptosis [ 170 174 ] However, studies have reported that knockdown of vimentin does not induce significant apoptosis per se but cleavage or degradation of vimentin potentiates the therapeutic effects of a drug [ 175 176 ] These studies also report that higher levels of vimentin expression render cells more susceptible t o drug induced apoptosis. In agreement with these previous reports, we found that Jak2 V617F expressing HEL cells have readily detectable levels of vimentin protein and are very susceptible to drug inhibition and subsequent loss of cell viability. However, in contrast to the earlier reports, we found that the loss of vimentin, either by G6 or by IDPN treatment, was sufficient to significantly decrease cell viability (Fig. 4 7). Possible explanations for these observed differences include the fact that the earlier reports used siRNA to knockdown vimentin mRNA levels whereas we used pharmacological inhibitors that resulted in decreased vimentin protein levels. In addition, G6 also reduces Jak2 kinase activity [ 105 162 ] which presumably vimentin siRNA treatment does not. These differences notwithstanding, our work here is significant in that we demonstrate that G 6 treatment results in vimentin cleavage and the subsequent loss of cell viability. With respect to the signaling pathway that facilitates G6 mediated vimentin cleavage, our data indicates that calcium plays a critical role in this process. For example, the G6 induced cleavage of vimentin is mediated by the calcium dependent protease, calpain (Fig. 4 5C). Furthermore, we show that mobilization of intracellular calcium is both essential and sufficient for cleavage of vimentin. Our data therefore suggest t hat there is a close correlation between Jak2 kinase activity and the levels of intracellular calcium ions. This is supported by previous work which indicates that

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106 erythropoietin (Epo) induced activation of its cognate receptor, EpoR, inhibits calcium indu ced neurotransmitter release via the activation of Jak2 [ 177 ] This report also shows that this Epo induced inhibition of calcium activity can be blocked by treatment with a tyrosine kinase inhibitor, such as genistein, further confirming the importance of Jak2 in mediating calcium induced responses. Overall, these studies demonstrate that Jak2 can modulate intracellular cal cium levels. However, the exact protein target(s) that Jak2 may phosphorylate in the calcium signaling pathway are not known. We have previously reported that G6 treatment potently induces apoptosis in HEL cells via the modulation of various Bcl 2 family proteins, such as Bcl xL, Bim and Bid [ 105 162 ] Previous studies have reported that elevation of intrac ellular calcium levels can trigger apoptosis via the destabilization of the mitochondrial membrane and subsequent activation of calcium dependent calpain proteases [ 178 180 ] Activation of calpains can further lead to the cleavage and activation of apoptotic regulators of the Bcl2 family, such as Bid [ 181 ] Therefore, these studies suggest that calcium and calcium dependent proteases may have an important role to play in the G6 mediated cell death/apoptosis process. The Jak2 V617F mutation is found in a large percentage on MPN patients [ 57 61 ] A previous study, which compared the mRNA expression profiles between healthy individuals and MPN patients, reported vimentin to be one gene that was differentially expressed in these two groups [ 182 ] Specifically, MPN patients were found to have elevated levels of vimentin mRNA when compared to non diseased individuals. This increase in vimen tin gene expression correlated positively with the presence of the Jak2 V617F mutation. In other words, significant over expression of vimentin was only

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107 observed in patients that were homozygous for the Jak2 V617F mutation. Thus, our data here, which descr ibes vimentin as one protein that is down regulated in response to Jak2 V617F inhibition by G6, is noteworthy. Furthermore, our data which show that the cleavage of vimentin is Jak2 dependent (Fig. 4 4), imply that pharmacological inhibition of Jak2 is suf ficient to induce cleavage of vimentin and subsequent loss of cell viability (Fig. 4 7). When taken together with the MPN microarray data [ 182 ] our work her e suggests that there may be a link between hyper activation of Jak2 kinase and over expression of vimentin. Furthermore, vimentin expression might be a potential biomarker for the progression of Jak2 V617F mediated pathogenesis and for disease regression via Jak2 inhibitory therapy. In summary, our data show that G6 induced inhibition of Jak2 mediated cell growth correlates with the cleavage and subsequent loss of intracellular vimentin filaments in vitro and in vivo As such, this work describes a novel mechanistic pathway for the targeting of Jak2 mediated pathological cell growth.

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108 Figure 4 1. Identification of vimentin as a differentially expressed protein between vehicle treated and G6 treated HEL cells. HEL cells were treated with either 0.25% DMS (A), DMSO treated (B) and G6 treated (C) were labeled with Cy2 (blue), Cy3 (green) and Cy5 (red), respectively. (D) An overlay of the three colored images is shown. One protein spot, indic ated by the arrow, was differentially expressed between the vehicle treated and G6 treated samples. (E) Analysis of the images obtained from the 2 D DIGE using DeCyder 2D predicted this differentially expressed protein to be significantly downregulated in the G6 treated samples (p=0.01). The ind icated protein was excised and identified using electro spray mass spectrometry as vimentin. (F) HEL cells were blotting using an anti vimentin antibody. The same samples were also b lotted with an anti STAT1 antibody to confirm equal loading across all lanes. Shown is one of three representative images.

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109 Figure 4 2. G6 treatment induces time and dose dependent degradation of vimentin. G6 for varying lengths of time (A) or with increasing doses of G6 for 24 hours (C). Cell lysates were then separated on SDS PAGE and immunoblottted with an anti vimentin antibody. The same samples were then reprobed with an anti actin antibody to confirm equal protein loading and also to demonstrate the specificity of G6 for actin. Shown is one of three independent results for each. Expression of full length vimentin was quantified using densitometry and plotted as a function of either time (B) or dose (D) of G6 treatment. Data shown are the mean SE from three

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110 Figure 4 3 G6 treatment induces marked reorganization of vimenti n intermediate were analyzed via indirect immunofluorescence for changes in the cellular distribution of vimentin in response to drug treatment. Vimentin was indirectly labeled with a FITC conjugated secondary antibody (A, B, and C). The nuclei were counter stained with DAPI (D, E, and F). The images were then merged (G, H and I). Shown is one of two representative results.

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111 Figure 4 4. G6 induced cleavage of vimentin is Jak 2 mediated. HEL cells were treated for 24 hrs with increasing concentrations of Jak2 specific inhibitors (G6, AG490 and Jak Inhibitor I) (A) or non Jak2 inhibitors (MAPK inhibitor, PD98059 and c Src inhibitor, PP2) (B). Whole cell lysates were separated by SDS PAGE and immunoblotted with an anti vimentin antibody. L oading of protein was confirmed using an anti STAT1 antibody. Shown is one of three representative blots.

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112 Figure 4 5. G6 induced cleavage of vimentin is independent of de novo protein synt hesis and caspase activity, but calpain dependent. HEL cells were pretreated for 4 hours with either cycloheximide (CHX) (A), caspase inhibitor I (zVAD) (B) or calpain inhibitor V (C) and then treated with increasing doses of G6 for 24 hours. Whole cell ly sates from the different treatment groups were then analyzed by western blotting with an anti vimentin antibody. The same lysates were also probed with an anti actin antibody to confirm equal protein loading across all lanes. Shown is one of three repres entative results for each.

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113 Figure 4 6. Mobilization of calcium is essential and sufficient for the cleavage of AM for 2 hours (B) and then treated wi G6 for 24 hours. Post treatment, the cells were lysed, proteins were separated by gel electrophoresis and then immunoblotted with an anti vimentin antibody (upper panel) or an anti actin antibody (lower panel) to confirm equal protein across all A23187 for the indicated periods of time. Cellular lysates were then probed with an anti vimentin antibody (top panel). An anti actin antibody was used as a loading control (bottom panel). Shown is a represe ntative blot from three independent experiments for each.

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114 Figure 4 7. Cleavage of vimentin is sufficient to reduce HEL cell viability. HEL cells 6, 12, 24 or 48 hours. (A) The n umbers of viable cells at each time point were determined and plotted as a function of treatment condition. (B) Cell lysates from each treatment group were collected simultaneously and analysed by immunoblotting with either an anti vimentin antibody or an anti actin antibody. Shown is one of three representative results. *p<0.05 with respect to DMSO.

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115 Figure 4 8. G6 treatment decreases the levels of vimentin protein, in vivo Anti vimentin immuno histochemistry was carried out on bone marrow section s from the indicated groups of animals. Shown are representative stained bone marrow sections from each treatment group at 40X (A) and 100X (B) magnifications.

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116 CHAPTER 5 CONCLUSIONS AND PERS PECTIVES Jak2 was first discovered in 1992 by polymerase chain r eaction (PCR) based techniques. Since then, this protein has been linked to many critical physiological processes as well as several pathophysiological states. Specifically, hyperkinetic Jak2 tyrosine kinase has been implicated in diverse hematological dis orders, including myeloproliferative neoplasms and hence is an emerging therapeutic target for these disorders. In this dissertation we characterize the structure function correlations of two novel Jak2 small molecule inhibitors and their mechanisms of ac tion. We have shown here that A46, a novel benzothiophene based compound identified in our lab, suppresses Jak2 mediated pathogenesis, thereby making it a potential candidate drug against Jak2 mediated disorders. Our lab has previously identified yet anoth er small molecule inhibitor of Jak2, called G6, which shows remarkable therapeutic efficiency against Jak2 mediated cell proliferation in vitro and in vivo [ 105 ] Here, we conduct ed structure function studies to demonstrate that G6 has a stilbenoid core which is indispensable for maintaining its Jak2 inhibitory potential suggesting that naturally occurring compounds, such as stilbenoids, might be a good scaffold for future development of potent Jak2 small molecule inhibitors. In addition, we also investigate the molecular and biochemical mechanisms by which G6 inhibits Jak2 mediated cell proliferation. Specifically, we demonstrate that the intermediate filament pr otein vimentin is cleaved in response to G6 treatment and show data which suggest that G6 induced Portion of this chapter has been reproduced from Jak2 Inhibitors for the treatment of Myeloproliferative Neoplasms. Drugs of the Future 2010; 35(8): 651 660 with permis sion from 2010 Prous Science, S. A. U. or its licensors.

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117 inhibition of Jak2 mediated pathogenic cell growth correlates with the disruption of intracellular vimentin filaments. This work is significant for a number of reasons. Firstly, we identify and characterize a novel small molecule inhibitor of Jak2, A46, which may perhaps serve as a lead therapeutic agent for the treatment of Jak2 V617F mediated pathogenesis. Secondly, we identify stilbenes and benzothiophenes as two new potential scaffolds for designing potent Jak2 small molecule inhibitors. Lastly, this work describes a novel mechanistic pathway for the targeting of Jak2 mediated pathological cell growth via inducing cleavage of vimentin As such, our data sug gest that vimentin expression might be a potential biomarker for the progression of Jak2 V617F mediated pathogenesis and for disease regression via Jak2 inhibitory therapy. The implications and perspectives of the studies presented in this dissertation are discussed in further detail below. Importance of Designing/Identifying a Jak2 Specific Inhibitor More than half a century ago, Dameshek first described myeloproliferative neoplasm (MPN) in an editorial [ 183 ] He recognized that the different MPNs were all characterized by proliferation of cells of the myeloid lineage, which he suggested might pathogenesis of these disorders remained elusive for about five decades after that till 2005, when five different groups, each working independently, identified the presence of a Jak2 V617F mutation in a majority of patients with MPNs [ 57 61 ] These five groundbreaking studies, as well as other subsequent studies, reported the presence of the Jak2 V617F mutation in over 90% of patients with PV and about 50% of patients with ET and PMF. In vivo studies have subsequently demonstrated that expression of this mutation in murine models is

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118 sufficient for the development of fully penetrant MPN in these animals [ 64 66 67, 70 71, 112 ] These data strongly suggest that the Jak2 V617F mutation plays a critical role in the pathogenesis of myeloproliferative neoplasms. There are about 22 cases of PV, 24 cases of ET and 1.46 cases of PMF per 100,000 people [ 184 185 ] Constitutively active Jak/STAT signaling pathway has also been linked to various forms of neoplastic disorde rs, including solid cancers and hematological malignancies, such as leukemias, myelomas and lymphomas. Unfortunately, there are currently no effective treatments available for such Jak2 mediated pathologies. Commonly used treatment strategies for MPNs invo lve the use of phlebotomy or myelosuppressive agents such as hydroxyurea and thalidomide. However, these treatments are only palliative in nature and provide only temporary relief without actually curing the disease. Therefore, there is currently a need to develop molecularly targeted therapies that will be able to cure Jak2 mediated disorders. Identification of the hyperactivating Jak2 mutations associated with these disorders has prompted the search for potent and selective Jak2 small molecule inhibitors and has brought us a step closer to the development of an effective and curative therapeutic strategy for such diseases. Elucidating the role of Jak2 in normal physiology and development is challenging since Jak2 knockout mice die embyonically around E12.5 [ 31 32] Hence, one approach to studying the role of Jak2 in later stages of development and in adults is to use a potent and specific small molecule inhibitor of Jak2. Therefore, identification of a good Jak2 inhibitor will not only be useful as a therap eutic drug for Jak2 mediated disorders but also be an important research tool for investigating the role of Jak2 in regulating critical physiological processes. Yet another approach to understanding the importance

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119 of Jak2 in later stages of development an d in adults is to induce tamoxifen mediated deletion of Jak2 at various stages of development and studying the effects of Jak2 deletion on tissue histology and survivability at each stage. Characterization of the Novel Jak2 Inhibitor, A46 In the chapter 2 of this study, we show that the novel benzothiophene small molecule inhibitor of Jak2, A46, inhibits Jak2 V617F mediated pathological cell growth in vitro and ex vivo Specifically, we show that this compound inhibits Jak2 V617F mediated cell pro liferation both time and dose dependently with a GI 50 of ~300 nM. The GI 50 of A46 is comparable to other Jak2 inhibitors that have moved into clinical trials, such as TG101348 (Table 1 1) and lower than many other Jak2 inhibitors that have been characteriz ed preclinically (Table 6 1), suggesting that this compound can potentially be developed into drug for u se in treatment of MPN patients. We have also demonstrated that A46 selectively inhibits the proliferation of Jak2 V617F dependent cell lines while hav ing little to no effect on other cells lines whose proliferation is driven by Jak2 independent mechanisms Selectivity/specificity is a desirable quality in a drug because it allows the drug to be administered in high er doses without producing non desirabl e side effects. We have also shown that the A46 induced cell growth inhibition correlates with direct suppression of the Jak/STAT signaling Additionally, our data indicate that the mechanism by which A46 suppresses Jak2 V617F mediated HEL cell proliferati on is via the induction of both G1/S cell cycle arrest and apoptosis Moreover, we report that this compound inhibited the pathologic growth of primary Jak2 V617F expressing bone marrow cells, ex vivo Although we have demonstrated that A46 inhibits Jak 2 mediated pathological growth in vitro and ex vivo we next need to demonstrate the in vivo efficacy of this drug.

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120 For this, we can use a transgenic mouse model of Jak2 V617F driven myeloproliferative neoplasia [ 67 ]. These trangenic mice develop fully penetrant MPN with symptoms such as erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen splenomegaly, reduced levels of plasma erythropoietin and thrombopoietin and increase d levels of cytokine independent hematopoietic progenitor cells in the peripheral blood, spleen and bone marrow. We hypothesize that treating MPN mice with A46 will result in reversal of the pathologic symptoms concommitant with suppression of the hyperact ive Jak/STAT signaling in these animals In addition to the in vivo characterization of A46, we also need to study the pharmacologic properties of t his drug including its half life and bioavailability before it can be moved into clinical trials for treatm ent of myeloproliferative neoplasms. Stilbenes and Benzothiophenes as Potential Scaffolds for the Design of Novel Jak2 Small Molecule Inhibitors O ur group recently identified a novel stilbenoid small molecule inhibitor of Jak2 named G6 [ 104 ] We subsequently showed that G6 specifically inhibits Jak2 mediated human pathologic cell growth in vitro ex vivo and in viv o [ 105 ] In chapter 3, we discuss the structure activity relationship properties of this novel Jak2 inhibitor. G6 has a stibenoid core in its chemical structure. We demonstrated that the core stilbenoid stru cture present in G6 is indispensable for maintaining its ability to inhibit Jak2 kinase activity. Specifically, we show that the stilbenoid core in G6 is essential for conserving its ability to i) inhibit Jak2 V617F mediated cell growth, ii) suppress the p hosphorylation of key signaling molecules of the Jak/STAT signaling pathway iii) induce apoptosis in HEL cells iv ) suppress pathologic cell growth of Jak2 V617F expressing human bone

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121 marrow cells, ex vivo and v) form stronger docking interactions with th e ATP binding pocket of Jak2 kinase domain. Since the discovery of the Jak2 V617F mutation as the predominant mutation present in a large proportion of MPN patients, there has been a drive to develop Jak2 specific inhibitors as therapeutics for MPNs and ot her Jak2 mediated pathologies. There have been several Jak2 inhibitors that have been identified and characterized preclini cally and clinically. However, a potent, specific and orally bioavailable Jak2 inhibitor that has the ability to cure Jak2 mediated d isorders is yet to be identified. AG490 was the first reported Jak2 inhibitor [ 82 ] Later, it was shown that even though this compound was a potent inhibitor of J ak2, it was highly non specific and inhibited several other kinases as well [ 83 ] Consequently, it was used as a starting point for designing more pote nt and/or selective Jak2 inhibitors. Specifically, AG490 was used as a lead compound and several derivatives of it were identified For example, the AG490 derivative WP1066 had better potency and specificity for Jak2 than AG490 [ 186 187 ] Similarly, LS104, which is structurally related to AG490 is a non competitive inhibitor of Jak2 and showed promising pr eclinical results and is now being evaluated in clinical trials [ 188 ] Recently, a variety of medicinal chemistry techniques such as library screening, fragment b ased drug discovery, scaffold morphing, molecular docking and lead compound derivatization have been used to study structure function correlations of numerous Jak2 inhibitors. The objective of these studies is to identify core scaffolds and functional grou ps that play crucial roles in potent Jak2 inhibition. Jak2 small molecule inhibitors currently represent a diverse number of chemical structures including pyrazines, pyrimidines, azaindoles, aminoindazoles, deazapurines, benzoxazoles, and

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122 quinoxalines [ 125 ] Data presented in the chapters 2 and 3 of this dissertation demonstrate that stilbenes and benzothiophenes may be a new class of scaffolds for Jak2 small molecule inhibitors. Specifically, stilbenoids or stilbene derivatives are naturally occurring compounds that are known to have a wide range of biological activities including anti proliferative, anti oxidative, anti neovascularization tum or suppressive and anti tyrosine kinase effects [ 131 135 153 154 ]. Moreover, the trans stilbenoid core identified in G6 and its active derivatives is amenable to bioisosteric replacements that could potentially result in new Jak2 chemotypes with improved druglike properties. Similarly, b enzothiophenes relatively stable heterocyclic structu re s, have reactive site s that allow for subsequent derivatization, suggesting that A46 is also quite amenable to future lead optimization. Having demonstrated the importance of the stilbene core in maintaining G6 mediated inhibition of Jak2, we next want to modify the positions and sizes of the hydroxyl and amine functional groups on the core stilbene structure of G6 and study how they impact the properties of the lead compound G6. The objective of such studies is to see if derivatization of G6 can result in improvement of its potency, specificity or its drug like properties, such as bioavailability and solubility. Similar structure activity relationship studies also need to be done for the benzothiophene based Jak2 inhibitor, A46. Comparison of Two Nove l Jak2 Inhibitors, G6 and A46 In this work we have characterized a novel Jak2 inhibitor, A46. Specifically, we show that this compound inhibits Jak2 V617F mediated HEL cell proliferation both time and dose dependently with a GI 50 of ~300 nM. On the other h and, G6, the other novel Jak2 inhibitor, inhibits Jak2 V617F mediated cell proliferation with a GI 50 [ 104

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123 105 ] A lower GI 50 suggests that A46 is a more potent inhibitor of Jak2 mediated pathological cell growth than G6, in vitro However, we observed an interesting characteristic of A46. In spite of its greater potency, this compound never kills all the cells even at the highest dose in vitro The inability of A46 to completely eliminate viable cells was also observed in the ex vivo c lonogenic assay that we performed. In comparison, elimination of viable cells after 72 hours, in vitro [ 105 ] This study also demonstrates that G6 ha s exceptional therapeutic efficacy, in vivo in a mouse model of human erythroleukemia At a therapeutic dose of 1mg/kg/day, G6 is able to specifically remove mutant cells from the bone marrow and cause significant improvement in both the bone marrow and t he spleen of the treated animals [ 105 ]. Like most other Jak2 inhibitors currently known, G6 targets and binds to the ATP binding pocket of Jak2. However, initial studies have shown that G6 is more effica cious in an in vivo setting than all the other Jak2 inhibitors that have been developed so far. This raises an interesting question as to what make s G6 different from the other ATP site targeting Jak2 inhibitors. A possible reason for the higher efficacy o f G6 might be its unique stilbenoid chemical structure. The molecular docking studies presented in the third chapter of this dissertation show that the stilbenoid core in G6 is critical for maintaining its strong binding affinity for the ATP binding pocket of Jak2. We show that G6 has potential hydrogen bond interactions with several critical residues lining the ATP binding pocket, including D994, R980, E930 and L932. Potential hydrogen bond interactions with D994 and R980 are unique to G6 and have not been identified for any of the other Jak2 inhibitors currently available. Yet another possible reason for th e

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124 better efficacy of G6 maybe the pharmacokinetic properties of G6. The pharmacokinetic properties of G6 have not been characterized yet. However, it is possible that G6 might have better bioavailability and/or ADME (Absorption, Distribution, Metabolism and Excretions) characteristics when compared to the other inhibitors, which makes it more efficacious than the others. Potential of Jak2 Inhibit or Therapy for Myeloproliferative Neoplasms The discovery of Jak2 activating mutations in the majority of MPN patients has spurred the development of small molecule inhibitors that specifically target Jak2. To date, a limited number of Jak2 inhibitors hav e been evaluated in clinical trials. Interestingly, while these inhibitors exhibited moderate to good preclinical efficacy, including murine models of human MPNs, those results have not translated well at the level of clinical trials. In these trials, the primary clinical benefits observed have been significant reduction in splenomegaly, patient weight gain and in some cases, reductions in the Jak2 mutant allele burden and/or reduced levels of some inflammatory cytokines. However, none of the inhibitors hav e yet been able to reverse the disease by providing any improvement in the bone marrow fibrosis of the treated patients. The reasons as to why these inhibitors have been disappointing in clinical trials are now an area of investigative research. For exampl e, one theory is that the genetics underlying MPNs are more complex than first thought. For instance, it is unclear how a single point mutation such as Jak2 V617F can lead to the development of three phenotypically distinct disorders; namely, PV, ET, and P MF. Some hypothesize that there are additional genetic and/or epigenetic events that contribute to the pathogenesis of these disorders and in turn determine which of the three disorders a specific patient will manifest [ 189 ]

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125 Recently, three indepen dent groups showed that the presence of a genetic haplotype leads to a predisposition for acquiring the Jak2 V617F mutation and consequently developing MPN [ 19 0 192 ] Results from recent epigenetic studies suggest that hypermethylation of the Suppressors of Cytokine Signaling (SOCS) family proteins, negative regulators of Jak/STAT signaling, may also have a role to play in the pathogenesis of MPNs [ 193 ] The presence of such genetic and epigenetic factors may determine ho w a specific MPN manifests itself in a given individual and in turn, how that individual responds to a specific treatment. This also raises the possibility that we might have to consider the use of combination therapies for effective treatment of these dis orders instead of merely Jak2 monotherapy. Lastly, although this runs contrary to current dogmatic drives as they relate to the development of Jak2 specific inhibitors, perhaps inhibitors of the future need to be less specific for Jak2 and instead have som e off target properties as well. In other words, dirty might be better. Another important factor to be considered when judging the limited success of the Jak2 inhibitor clinical trials is the patient population being studied. These early clinical t rials have all been performed with myelofibrosis patients because it is the most severe of the three classical MPNs. However, it must be kept in mind that a drug that does not show significant improvement in these patients might still be efficacious if giv en to a patient with a less severe MPN, such as PV, ET or even early stage PMF. Hence it will be important to design more appropriate clinical trials that can address this important issue before final conclusions are drawn regarding the efficacy of these d rugs in humans.

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126 Most of the efforts to identify Jak2 inhibitors have been focused on screening for small molecules that target the ATP binding pocket within the Jak2 kinase domain (Fig. 5 1 A ) However, since this pocket is highly conserved amongst all kina ses, developing a Jak2 inhibitor that does not inhibit other kinases is a challenge. The ATP pocket targeted inhibitors have another limitation as well. Specifically, they are unable to distinguish between the wild type and mutant Jak2 kinases. Hence, it i s unsurprising that these inhibitors, due to their potency against wild type Jak2, cause myelosuppression and anemia in treated patients. The occurrence of such adverse side effects imposes an upper limit on the dosage of the drug that can be administered to these patients. Another dilemma is that the Jak2 activating mutations identified in MPN patients, including V617F, are mostly present in the JH2 or pseudokinase domain of Jak2 and not the kinase domain. Unfortunately, the crystal structure of Jak2 enc oding both the JH1 (kinase) and JH2 (pseudokinase) domains has not yet been resolved. As such, it is currently not possible to design inhibitors that specifically target mutant forms of Jak2 over the wild type protein. To complicate matters even further, o ne wonders whether an inhibitor that is specifically designed against Jak2 V617F will be effective against the dozens of other Jak2 mutations that are known to exist. That said, in the future, solving the crystal structure of full length Jak2, or at least the JH1 JH2 domains, will be very critical for the designing future Jak2 kinase inhibitors. Attempts are also being made to design more specific Jak2 inhibitors by exploiting the subtle differences between the kinase domain structures of Jak2 and other p rotein kinases. Crystal structure analyses have shown that the ATP binding site of the Jak

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127 kinases is much more constricted when compared to the other protein tyrosine kinases [ 23 ] It has also been shown that the electrostatic surface potential around the active site within Jak2 is more positively ch arged than other Jak family members, such as Jak3 (74). Moreover, several residues in the hinge region (such as M929, Y931, P934, R938), the glycine loop (such as K857), the catalytic loop (such as I982), and the activation loop (such as E1015) may potenti ally confer inhibitor selectivity to Jak2 over other tyrosine kinases including other Jak family members ( 48 ). That said, only completion of such studies will determine whether these structure activity relationships do in fact exist. Developing allost eric inhibitors to Jak2 might be an alternate approach for specific Jak2 targeting. There are several other possible target sites on Jak2, other than the ATP binding site, against which allosteric inhibitors can be designed. The JH2 domain of Jak2 has an a utoinhibitory effect on the kinase domain [ 13 14 ] The mechanism for the constitutive of Jak2 V617F invol ves the disruption of the inhibitory interactions between the JH1 and JH2 domains at the JH1 JH2 interf ace and linker regions (Fig. 5 1B ; marked by a box ) [ 194 195 ] Hence, this region is a possible target for allosteric inhibition of Jak2. A recent study reported that the linker between the SH2 like domain and the JH2 domain (Fig. 5 1C ; boxed region) fle xes the hinge between the JH1 and JH2, thereby altering the accessibility of the kinase domain and leading to its activation [ 75 ] Incidentally, the exon 12 mutatio ns identified in MPN patients are present in this linker. Therefore, this linker region might also be a plausible site for allosteric targeting of Jak2. The FERM domain negatively regulates the wild type Jak2, but positively regulates Jak2 V617F kinase act ivity [ 196 ] Mutation/deletion of this domain also

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128 increases Jak2 kinase activity, further suggesting the role o f the FERM domain in regulating activity of Jak2 [ 197 199 ] The FERM domain is also known to regulate Ja k 2 receptor association [ 20 21 ] Hence, the FERM domain might be another interesting region to target for allosteric inhibition of Jak2. Overall, allosteric inhib itors that target these alternative regulatory sites on Jak2 may provide more effective inhibitors that specifically target mutant Jak2. In summary, the causative nature of Jak2 somatic mutations in the pathogenesis of MPNs was established in 2005. The fir st clinical trial testing a putative Jak2 small molecule inhibitor for the treatment of MPNs initiated less than three years later. While the initial data from these limited number of clinical trials have not lived up to the expectations, it is evident tha t these inhibitors, even if not curative, can still have great therapeutic benefit for MPN patients due to their ability to reduce some clinical symptoms as well as improve the overall quality of life. However, one of the challenges to implementing these J ak2 inhibitor therapies for MPN patients will be their prohibitive costs. The symptoms of MPN patients are effectively managed by existing therapeutic options, such as venesection/cytoreductive agent. Therefore, a shift towards the use of more expensive Ja k2 inhibitor therapy will be warranted only if these inhibitors show some curative effects as opposed to just palliative ones. The issue of acquired resistance can also become a potential problem with the use of potential Jak2 inhibitors similar to that se en with other types of inhibitor based therapeutics, such as BCR ABL and FLT3 (FMS like tyrosine kinase 3) inhibitors. In this time of rapid expectations, we must be mindful that this area of research is still in its infancy. Current avenues of investigati on continue to expand our knowledge of Jak2 kinase and its potential

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129 inhibitors. With an increased knowledge of 1) the molecular pathogenesis of MPNs, 2) the structural properties of wild type and mutant Jak2 proteins 3) the continued identification of str ucture activity relationships between Jak2 and putative inhibitors and 4) the development of better animal models that more closely resemble the complex nature of MPNs observed in humans, we are confident that one day, Jak2 inhibitors will be a viable ther apy for the treatment of myeloproliferative neoplasms. Characterization of t he L ink Between G6 induced Cell Death and Cleavage of Vimentin Our data in chapter 3 showed that the novel stilbenoid Jak2 inhibitor G6 specifically inhibits Jak2 mediated cell pro liferation in a time and dose dependent manner. Therefore, we next wanted to understand the molecular and biochemical mechanisms by which G6 inhibits Jak2 mediated cellular proliferation For this, we compared protein expression profiles between vehicle t reated and G6 treated cells using two dimensional gel electrophoresis. We identified t he intermediate filament protein, vimentin, as one protein that was differentially expressed between the two conditions. The data in chapter 4 show that G6 induced inhibi tion of Jak2 mediated pathogenic cell growth correlates with the specific cleavage and cell ular reorganization of vimentin. Specifically, we demonstrate that G6 treatment induced a time and dose dependent cleavage of the intermediate filament protein, vim entin We also show that G6 treatment of HEL cells resulted in the change in vimentin filament organization from a uniform distribution in the cytoplasm to aggregates in the perinuclear region of the cell. Our data provides evidence that this G6 mediated c leavage of vimentin is Jak2 dependent and calpain mediated Additionally, we demonstrate that t he mobilization of intracellular calcium is critical for G6 mediated vimentin cleavage and the cleavage of

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130 vimentin, per se is sufficient to reduce HEL cell via bility. Lastly, our data also suggest that the ability of G6 to induce cleavage of vimentin is conserved in vivo Taken together, these results describe a novel mechanism by which G6 exerts its inhibitory effects Having established the critical role that calcium and the calcium dependent protease, calpain, plays in this G6 induced vimentin cleavage process we next need to determine the mechanism by which inhibition of Jak2 via G6 can induce calcium mobilization and activation of calpain. For this, we need to study the effects of G6 treatment on the expression of important calcium regulating genes, such as calmodulin and calmodulin kinase. As of now, there is a missing link between the inhibition of Jak2 by the inhibitor G6 and the subsequent mobilization o f calcium and activation of calpain, which finally causes degradation of vimentin. Our data suggest that there is a close correlation between Jak2 kinase activity and the levels of intracellular calcium ions. However, the exact protein target(s) that Jak2 may phosphorylate in the calcium signaling pathway are yet to be identified. Having demonstrated the correlati on between G6 induced cell death and cleavage of viment in, it is important to next determine if there is a direct causative relation between them. For this, we need to first transfect HEL cells with a mutated vimentin construct that is resistant to calpain cleavage and then treat them with G6. We expect that the cells expressing this calpain resistant vimentin protein will be protected from cell dea th induced by the Jak2 inhibitor, G6. This experiment will help us in determining if the Jak2 inhibitor, G6, induces cell death directly via the cleavage of vimentin f ilaments. However, there are some technical difficulties in conducting this experiment.

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131 C alpain is a calcium dependent protease that can recognize and cleave a wide variety of substrates and a unique substrate identification site has not yet been characterized for this enzyme. Hence, creating a calapin resistant vimentin construc t may be techn ically challenging. Currently, due to this limitation, it was not possible to establish a direct causative link between G6 induced cell death and cleavage of vimentin. Overall, the data presented in this dissertation characterizes the structure activity re lationships of novel Jak2 inhibitors and provide useful insight into the mechanisms by which these inhibitors exert their anti Jak2 effect.

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132 Figure 5 1. Surface representation of the structure of Jak2 indicating possible sites for inhibitor tar geting (A) The ATP binding pocket of Jak2 is marked by a box around it. This pocket has several loops/regions that are critical for the interaction of the Jak2 kinase domain with ATP. Rotating the structure shows the regions behind the ATP binding pocket (B & C). (B) The interface between the JH1 and JH2 domains, including the linker between these two domains, is indicated by the box. This interface is critical for regulating the autoinhibitory effect of the JH2 domain on the JH1/kinase domain. (C) The lin ker region between the FERM domain and the SH2 like domain is indicated by the box. This linker has a critical role to play in regulating the flexing of the hinge between the JH1 and JH2 domains, thereby effecting the interaction s between these domains. COLOR CODE: Pa le green FERM domain Light pink SH2 like domain Light orange JH2 (pseudokinase) domain White JH1 (kinase) domain Cyan Linker between FERM and SH2 like domains Yellow Linker between SH2 like and JH2 domains Purple Linker between JH2 and JH1 do mains Blue Nucleotide loop Green Hinge region Magenta Catalytic loop Red Activation loop

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133 Table 5 1. GI 50 of other Jak2 inhibitors in HEL cells. Other known Jak2 inhibitors in various stages of clinical and preclinical development are listed here along with their respective GI 50 s in HEL cells. Drug HEL cell IC 50 (nM) Reference CEP 701 30 100 [ 93 ] TG101 209 152 [ 200 ] INCB018424 186 [ 85 ] TG101348 300 [ 65 ] P1 510 [ 201 ] CYT387 1804 [ 102 ] WP1066 2000 [ 202 ] G6 4000 [ 105 ] Z3 15000 [ 109 ]

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150 BIOGRAPHICAL SKETCH Anurima Majumder was born in 1984, in the small town of Durgapur, India Even as a child, Anurima was inspired by her role model, her father, to pur sue a career in academics and become a professor like him. She attended Carmel High School, where she was first inspired to pursue a career in b ioscience by her b iology teacher, Mrs. Geeta Menon. Anurima went on to earn her Bac helor of Engineering degree i n b iotechnology from the Ben gal College of Engineering and T echnology, in August 2006. In August 2007, she moved to Gainesville, Florida and joined the Interdisciplinary Program in Biomedical Sciences at the University of Florida in pursuit of a Ph.D. She began her graduate dissertation research under the guidance of Dr. Peter Sayeski in May, 2008. Her research was primarily focused on studying structure activity relationships of novel Jak2 small molecule inhibitors. A nurima graduated in August 201 1 and i s now pursu ing her postdoctoral studies at the University of Iowa.