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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-05-31.

Permanent Link: http://ufdc.ufl.edu/UFE0024295/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-05-31.
Physical Description: Book
Language: english
Creator: Sayyah, Jacqueline
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Jacqueline Sayyah.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Sayeski, Peter P.
Electronic Access: INACCESSIBLE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024295:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024295/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-05-31.
Physical Description: Book
Language: english
Creator: Sayyah, Jacqueline
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Jacqueline Sayyah.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Sayeski, Peter P.
Electronic Access: INACCESSIBLE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024295:00001


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1 CHARACTERIZATION OF NOVEL JAK2 TYROSINE KINASE SMALL MOLECULE INHIBITORS AND SITES OF JAK2 PHOSPHO REGULATION By JACQUELINE SAYYAH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL F ULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Jacqueline Sayyah

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3 This dissertation is dedicated to my family for the support, love, advice and enco uragement they have given me.

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4 ACKNOWLEDGMENTS First and foremost, I would like to express my sincere gratitude to my adviser, Dr. Peter P. Sayeski, whose excellent guidance has been instrumental in my success as a graduate student. He has been avail able to answer all my questions, provide constructive criticism and has encouraged me to perform my best. Apart from being a mentor, Peter has been a wise friend whom I can rely on for advice. I would also like to thank the members of my supervisory commi ttee: Dr s. Jeff Harrison, Brad Fletcher and Satya Narayan. They have provided valuable scientific advice that has augmented my graduate work. In addition, I am grateful to Dr. David Ostrov, Dr. Gyrgy Keseru, and Andrew Magis for their assistance with in silico modeling applications. Without their help, our Jak2 inhibitor characterization would not have been possible. I appreciate the members of the Sayeski laboratory, past and present for their support : Tiffany Wallace, Michael Godeny, Issam McDoom, Rob ert Blair, Annet Kirabo, Anurima Majumder, Kavitha Gnanasambandan, and Nick Figueroa. They have provided an accommodating and encouraging environment. At times, they have also provided comic relief. In addition, I am grateful for meeting genuine and wo nderful friends in Gainesville I g reatly value the enjoyable time I shared with them throughout my graduate career. Fin ally, I would like to thank my mother, f ather and b rothers for their constant love and support I appreciate the fact that you encouraged me to pursue my interests and always believed that I can achieve my goals

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................................... 4 page LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 ABSTRACT ........................................................................................................................................ 11 CHAPTER 1 INTRODUCTION ....................................................................................................................... 13 History of Jak2 Tyrosine Kinase ................................................................................................ 13 Jak2 Structure .............................................................................................................................. 14 Jak2 Protein Domains .......................................................................................................... 14 Amino Acid Residues Critical for Jak2 Function .............................................................. 16 Jak2 Signal Transduction Pathway ............................................................................................ 19 Activation of Jak2 via Cytokine Receptors ........................................................................ 20 Activation of Jak2 via GPCR .............................................................................................. 20 Jak2 Regulatory Proteins ..................................................................................................... 21 SH2B family of proteins .............................................................................................. 21 SOCS family of proteins .............................................................................................. 22 Jak2 and Pathophysiology .......................................................................................................... 23 Role of Jak2 in Cardiovascular Disease ............................................................................. 23 Role of Jak2 in Hematological Disorders .......................................................................... 25 Jak2 chromosomal translocations and hematological malignancies ......................... 26 Jak2 somatic mutations and hematologic malignancies ............................................ 28 Jak2 V617F and myeloproliferative disorders ........................................................... 29 Other Jak2 mutations associated with myeloproliferative disorders ........................ 30 Inhibitors of Jak2 Tyrosine Kinase ............................................................................................ 31 Justification for Jak2 Inhibitors .......................................................................................... 31 Jak2 Selective Inhibitors ..................................................................................................... 32 Non Jak2 Selective Inhibitors ............................................................................................. 35 Purpose and Rationale of Studies ............................................................................................... 38 2 METHODS .................................................................................................................................. 43 Small Molecule Database ........................................................................................................... 43 In Silico Molecular Modeling of Jak2 ....................................................................................... 43 Mass Spectrometry ...................................................................................................................... 43 Cell Lines ..................................................................................................................................... 43 Cell Culture .................................................................................................................................. 44 Site Direc ted Mutagenesis .......................................................................................................... 44 BSC 40 Cell Transfection/Infection .......................................................................................... 45

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6 Transient Cell Transfections ....................................................................................................... 45 Cell Lysis and Immunoprecipitation .......................................................................................... 46 Western Blotting ......................................................................................................................... 47 c Src In Vitro Kinase Assay ....................................................................................................... 47 Luciferase Assay ......................................................................................................................... 47 Propidium Iodide Staining of Cells ............................................................................................ 48 Cell Proliferation A ssay .............................................................................................................. 48 Cell Cycle Analysis ..................................................................................................................... 48 Apoptosis Assays ........................................................................................................................ 48 Jak2 Mutation Analysis .............................................................................................................. 49 Colony Formation Assay ............................................................................................................ 49 Statistical Analysis ...................................................................................................................... 49 3 Z3, A NOVEL JAK2 TYROSINE KINASE SMALL -MOLECULE INHIBITOR THAT SUPPRESSES JAK2 -MEDIATED PATHOLOGIC CELL GROWTH ................................. 50 Summary ...................................................................................................................................... 50 In troduction ................................................................................................................................. 50 Results .......................................................................................................................................... 52 Z3 Inhibits Jak2 Tyrosine Autophosphorylation by Interacting with a Solvent Accessible Pocket Adjacent to the ATP Binding Pocket of Jak2 ................................. 52 Z3 Inhibits Jak2 Tyrosine Autophosphorylation in a Dose Dependent Manner that is Independent of Cellular Cytotoxicity.......................................................................... 54 Z3 is a Specific Inhibitor of Jak2 Tyrosine Kinase ........................................................... 56 Z3 Selectively Inhibits Jak2 -V617F Dependent Cell Proliferation and this Correlates with Suppression of Jak2 and STAT3 Tyrosine Phosphorylation .............. 57 Z3 Exerts its Effect on the Cell Cycle by Increasing the Percentage of HEL Cells in G1 Phase while Decreasing the Number of Cells in S phase .................................... 59 Z3 Reduces Hematopoietic Colony Formatio n Ex Vivo ................................................... 60 Discussion .................................................................................................................................... 61 4 CHA RACTERIZATION OF A SERIES OF NOVEL JAK2 INHIBITORS AND THEIR AFFECT ON JAK2 MEDIATED DISEASES ............................................................ 74 Summary ...................................................................................................................................... 74 Introduction ................................................................................................................................. 75 Results .......................................................................................................................................... 77 G6 Potently and Selectively Suppresses Jak2-V617F Dependent Aberrant Cell Growth .............................................................................................................................. 77 G6 Suppresses HEL Cell Proliferation by Inducing Cellular Apoptosis ......................... 79 G6 is a Specific Inhibitor of Jak2 Tyrosine Kinase Activity ............................................ 79 The G Compounds Effectively Block Jak2-Mediated Hematopoietic Colony Formation, Ex Vivo .......................................................................................................... 81 Discussion .................................................................................................................................... 82 5 TYROSINE 372 IS CRITICAL FOR JAK2 FUNCTION ....................................................... 89

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7 Summary ...................................................................................................................................... 89 Tyrosine 372 is Highly Conserved Among Species Expressing J ak2 and in Different Jak2 Kinase Family Members ......................................................................... 93 Loss of Tyrosine 372 and 373 Phosphorylation Reduce Jak2 Tyrosine Phosphorylation................................................................................................................ 93 Mutation of Jak2 at Tyrosine 372 Suppresses the Ability of Jak2 to Phosphorylate STAT1 .............................................................................................................................. 95 Loss of Tyrosine 372 Phosphorylation Reduces the Association of Jak2 with STAT1 .............................................................................................................................. 96 The Jak2 Y372F Mutation Abrogates LigandIndependent Gene Expression ................ 97 Loss of Tyrosine 372 Phosphorylation Hinders Interferon-Gamma -media ted Jak2 Activation ......................................................................................................................... 97 Loss of Tyrosine 372 Phosphorylation Impairs Epidermal Growth Factor -Mediated Jak2 Activation. ................................................................................................................ 98 Loss of Tyrosine 372 Phosphorylation Does Not Affect Hydrogen Peroxide Mediated Jak2 activation ................................................................................................. 99 Discussion .................................................................................................................................. 100 6 DISCUSSION ............................................................................................................................ 116 Overview .................................................................................................................................... 116 Characterization of the Jak2 Inhibitors, G6 and Z3 ................................................................ 117 Comparison of Z3 and G6 ........................................................................................................ 119 Additional Reflections Regarding Jak2 Inhibitors .................................................................. 120 The Role of Tyrosine 372 on Jak2 Function ........................................................................... 122 The Potential Role of Tyrosine 372 in Autoimmune Disorders ............................................. 123 The Potential Role of Tyrosine 372 Phosphorylation in Cell Growth ................................... 126 Conclusion ................................................................................................................................. 127 Reflections ................................................................................................................................. 128 Z3 and G6 ........................................................................................................................... 128 Tyrosine 372 ...................................................................................................................... 129 LIST OF REFERENCES ................................................................................................................. 132 BIOGRAPHICAL SKETCH ........................................................................................................... 146

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8 LIST OF TABLES Table page 1 1 A complete list of Jak2 gene aberrations repo rted in hematological disorders ................. 41 1 2 A complete list of class I and II Jak2 inhibitors identified since 1996. .............................. 42 3 1 Top six scoring Z compounds ............................................................................................... 65

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9 LIST OF FIGURES Figure page 1 1 Illustration of Jak2 structural domains. ................................................................................. 39 1 2 Jak/STAT signaling paradigm. .............................................................................................. 40 3 1 2 -methyl 1 -phenyl 4 pyridin 2 -yl 2 (2 pyridin 2 ylethyl)butan 1 -one (Z3) inhibits Jak2 V617F and Jak2WT tyrosine autophosphorylation. .................................................. 66 3 2 Z3 inhibits Jak2 tyrosine autophosphorylation in a dose dependent manner. .................... 67 3 3 Z3 selectively inhibits Jak2 tyrosine kinase activity ............................................................ 6 9 3 4 Z3 selectively inhibits Jak2 -V617F -dependent cell proliferation and this correlates with suppression of Jak2 and STAT3 tyrosine phosphorylation. ........................................ 70 3 5 Z3 induces cell cycle arrest in Jak2 -V617F transfor med human erythroleukemia cells ... 72 3 6 Z3 suppresses Jak2 -mediated hematopoietic colony formation ex vivo ............................ 73 4 1 G6 pot ently inhibits Jak2 -V617F -dependent cell growth. ................................................... 85 4 2 G6 reduces HEL cell numbers by increasing cellular apoptosis. ........................................ 86 4 3 G6 has no effect on Tyk2 or c -Src tyrosine kinase activity. ................................................ 87 4 4 The G compounds reduce hematopoietic colony formation ex vivo .................................. 88 5 1 Tyrosines 372 and 373 are Jak2 phosphorylation sites. ..................................................... 106 5 2 Tyrosine 372 is conserved in different Jak family members and among different s pecies ................................................................................................................................... 107 5 3 Loss of tyrosine 372 and 373 phosphorylation decrease the ability of Jak2 to autophosphorylate. ............................................................................................................... 108 5 4 Loss of Tyrosine 372 phosphorylation reduces J ak2 -medi ated STAT1 activation. ........ 109 5 5 The Jak2 Y372F mutant hinders the assoc iation between Jak2 and STAT1. .................. 110 5 6 Phosphorylation of Tyrosine 372 facilitates Jak2-dependent gene transcription ............. 112 5 7 Phosphorylation of tyrosine 372 is essential for interferon-gamma -dependent Jak2 activation .............................................................................................................................. 113 5 8 Tyrosine 372 is critical for epidermal growth fac tor -dependent Jak2 activation ............. 114

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10 5 9 Phosphorylation of tyrosine 372 does not affect hydrogen peroxide -dependent Jak2 activation. .............................................................................................................................. 115 6 1 N umber of reported Jak2 mutations and Jak2 inhibitors discovered by year. .................. 131

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11 Abstract of Dissertation Presented to the G raduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF NOVEL JAK2 TYROSINE KINASE INHIBITORS AND SITES OF JAK2 PHOSPHO REGULATION By Jacqueline Sayy ah May 2009 Chair: Peter P. Sayeski Major: Medical Sciences -Physiology and Pharmacology Jak2 is a nonreceptor tyrosine kinase that acts as a critical link in coupling ligand binding at the cell surface with gene transcription in the nucleus. Since it s discovery 17 years ago, Jak2 has emerged as an important protein in human health. In recent years, much attention has been devoted to understanding the role of the Jak2-V617F mutation and other gainof -function mutations of Jak2 in myeloproliferative d isorders and hematological malig nancies. T he constitutively active Jak2-V617F mutation has been found in a large percentage of myeloproliferative disorder patients and plays a critical role in the development of these disorders. Control of aberrant Jak2 kinase activity via Jak2 -specific inhibitors would therefore serve as a useful research tool and/or therapeutic agent. In this dissertation, we characterized two novel Jak2 inhibitors, termed Z3 and G6. We found that these compounds specifically inhibite d Jak2 kinase function with no effect on Tyk2 or c Src. In addition, Z3 and G6 selectively suppressed Jak2 -V617F -mediated human erythroleukemia cell growth in vitro Finally, these compounds reduced the ex vivo growth of hematopoietic progenitor cells de rived from myeloproliferative disorder patients carrying Jak2 mutations. Z3 and G6 could potentially serve as useful research tools or therapeutic agents against diseases related to elevated Jak2 tyrosine kinase activity.

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12 Since Jak2 has been linked to a number of disease states, it is also important to understand how the structure of Jak2 impacts its function. Here, we investigated structural elements in the FERM domain that influence Jak2 function. We found that tyrosines 372 and 373 are novel sites o f Jak2 phosphorylation in the FERM domain of Jak2. We demonstrated that loss of tyrosine 372 phosphorylation inhibited ligand -independen t Jak2 autophosphorylation. In contrast loss of tyrosine 373 phosphorylation resulted in more modest reductions in Ja k2 autophosphorylation levels. With specific focus on the more significant tyrosine 372, we also found that loss of tyrosine 372 phoshorylation hindered the ability of Jak2 to activate STAT1 and reduced Jak2dependent gene transcription. In addition, we found that loss of tyrosine 372 phosphorylation impaired interferon -gamma and epidermal growth factor -dependent Jak2 activation. Our results suggest that tyrosine 372 is important for Jak2 function.

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13 CHAPTER 1 INTRODUCTION History of Jak2 Tyrosine Kinase Over seventeen years ago, Wilks and colleagues cloned the Jak2 cDNA sequence and upon analysis found that the gene encoded a protein of roughly 130 kDa in mass (Harpur et al., 1992). The predicted amino acid sequence also revealed that Jak2 possessed a ki nase domain immediately adjacent to a pseudokinase domain and had seven conserved Jak homology domains Interestingly, the tandem manner by which the kinase and psuedokinase domains were linked was suggestive of Janus, the Roman God of two opposing faces, wh ich the Jaks were named after. Initially, Jak2 was characterized as a mediator of cytokine and growth factor signaling (Argetsinger et al., 1993; Silvennoinen et al., 1993; Witthuhn et al., 1993; Ihle et al., 1994). Later on, it was discovered that Jak 2 could transduce its signal through G -protein coupled receptors, such as the AT1 receptor, and be activated by reactive oxygen species thus revealing that Jak2 is a more versatile signaling mediator than previously thought (Marrero et al., 1995; Simon et al., 1998). Another important finding was that Jak2 is expressed in virtually every cell type and is necessary for animal survival as disruption of the Jak2 allele in mice resulted in embryonic lethality (Parganas et al., 1998; Neubauer et al., 1998). A c rucial event in the history of Jak2 tyrosine kinase was when a mutation in the psuedokinase domain of this protein, Jak2V617F, was linked to high percentage of myeloproliferative patients and shown to be the cause of this disease (Baxter et al., 2005; Jam es et al., 2005; Kralovics et al., 2005; Levine et al., 2005; Zhao et al., 2005). Given the above facts, it is not surprising that Jak2 tyrosine kinase has emerged as an important molecule in mammalian develo pment, physiology, and disease

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14 Jak2 Structure Jak2 Protein Domains Although the three -dimensional structure of the entire Jak2 protein has not been obtained, a portion of the Jak2 kinase domain has been resolved which gives us structural insight regarding the catalytic portion of JAK2 and the mechanis m by which it is regulated (Lucet et al., 2006). According to the Jak2 crystal structure, a loop structure situated between amino acids 1056 and 1078, termed the Jak2 kinase insertion loop, is a feature that is unique to the Jak family (Lucet et al., 200 6). This loop is relatively mobile and solvent accessible. In particular, the serine residue located at position 1056 on this loop is solvent exposed, suggesting that it could play a phosphorylation dependent regulatory role in Jak2 function. In ad ditio n, the Jak2 insertion loop is stabilized by the interaction of antiparallel -sheets with the base and the tip of the activation loop. Moreover, a number of lysine residues stabilize the conformation of phosphorylated tyrosine 1007, which is highly solven t exposed (Lucet et al., 2007). The exposed nature of phosphorylated tyrosine 1007 permits the binding of negative regulatory proteins such as SOCS 1 to this site (Giordanetto and Kroemer, 2003). Certain studies have contributed to the generation of a the oretical three -dimensional structure of the entire Jak2 protein through analysis of primary nucleotide and amino acid sequence as well as through application of homology modeling (Lindauer et al., 2001; Giordanetto and Kroemer, 2002). Jak2, like other Jak family members, contains seven highly conserved Jak homology (JH) domains termed JH1 through JH7 (Fig ure 1 1). The carboxyl terminus of Jak2 consists of the JH1 and JH2 domains. The JH1 region encodes the kinase domain, which contains the adenosine trip hosphate -binding region and the activation loop. This domain is a requisite for Jak2 tyrosine kinase function (Duhe and Farrar, 1995). Directly N terminal to the JH1 domain is the JH2 or pseudokinase domain. Although the JH2 psuedokinase

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15 domain of Jak2 shares conserved motifs with other Jak kinases, this region is catalytically inactive (Frank et al., 1995). It has been suggested that in the absence of ligand, the pseudokinase domain has a role in suppressing the function of the kinase domain by physica lly interacting with the JH1 domain. (Lindauer et al., 2001 ; Chen et al., 2000 ; Saharinen et al., 2003). The amino terminal region of Jak2 consists of domains JH3 through JH7. Although the Jak family members are thought to lack a canonical SH2 domain, i t was noted that in Tyk2, a portion of the JH4 domain along with the entire JH3 domain weakly resemble an SH2 domain (Bernards, 1991). Following cloning of the murine Jak2 cDNA, it was similarly observed that the sequence GLYVLRWS was weakly homologous to the core sequence element of SH2 domains (FLVRES) (Harpur et al., 1992). However, studies have reported conflicting finding as to whether a fully functional SH2 domain exists within Jak2 (Kampa and Burnside, 2000; G iordanetto and Kroemer, 2002). Directly N terminal to the putative SH2 domain of Jak2 resides the FERM domain, which spans from the middle of the JH4 domain through the JH7 domain. The FERM domain is involved in mediating stable interactions with other cellular proteins (Yonemura et al., 1998) In addition, the amino terminal end of Jak2, particularly, the JH6 and JH7 domains, has been shown to be crucial for Jak2 -cell surface receptor interactions (Frank et al ., 1994; Tanner et al., 1995). In brief, Jak2 is structurally composed of seven dist inct Jak homology domains. Each of these domains has a vital role in Jak2 function. The JH1 domain is essential for Jak2 kinase activation, while the JH2 domain is important for Jak2 autoregulation. Finally, JH3 JH7 domains are involved in interactions with substrates and membrane -bound re ceptors.

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16 Amino Acid Residues Critical for Jak2 Function Within the seven Jak2 homology domains, several amino acid residues have been identified as being important for regulating Jak2 function. These amino acids consis t of nontyrosine residues and phosphotyrosines. In the kinase domain, Trp 1020 and Glu 1024 are important for Jak2 catalytic activity as mutation of these amino acid residues together or individually render Jak2 catalytically inactive (Zhuang et al., 199 4; Vonderlinden et al., 2002). Furthermore, E1024 and R1113 form a hydrogen bond that is important for Jak2mediated angiotensin II signaling (Sandberg et al., 2004a). Likewise, the interaction between W1038 and E1046 has been demonstrated to be critical for preventi ng an inhibitory Jak2 phenotype in vivo (Frenzel et al., 2006). In contrast, there are certain non -phosphotyrosine amino acids within the pseudokinase autoinhibitory domain of Jak2 that when mutated result in constitutive Jak2 activation. The most well known is the Jak2 -V617F mutation, which is found in a high proportion of myeloproliferative disorder patients. Most likely, the V617 residue is important for the autoinhibitory interaction between the JH2 and JH1 domain. Current molecular mode ls of the psuedokinase domain suggest that this region interacts with the activation loop of the kinase domain (Lindauer et al., 2001; Giordanetto and Kroemer et al., 2002). Moreover, structure function studies have shown that amino acids located in the v icinity of V617 are critical for maintaining the inhibitory property of the pseudokinase dom ain (Saharinen et al., 2003). Jak2 tyrosine autophosphorylation is a significant process as it dictates the relay of signals from the cell surface to the nucleus. Murine Jak2 possesses 49 tyrosine residues and 10 of these are known to be phosphorylated with many more tyrosine residues awaiting characterization. Th ese characterized Jak2 tyrosine residues include: Y119, Y201, Y221, Y5 70, Y813, Y913, Y966, Y972, Y1007 and Y1008. The 11th and 12th phospotyrosine sites recently characterized in

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17 our lab are tyrosine 372 and 373 and they will be discussed later in detail. Many of these sites play important roles in Jak2 tyrosine kinase regulation. In the kinase domain, tyrosines 1007 and 1008 within the Jak2 activation loop were the first sites shown to be phosphorylated (Feng et al., 1997). While tyrosine 1007 is required for maximal Jak2 tyrosine kinase activity, the adjacent tyrosine at position 1008 seems to be dispensable for Jak2 tyrosine kinase function. The proposed mechanism by which phosphorylation of tyrosine 1007 results in maximal Jak2 activation is that the activation loop is moved away from the ATP binding site of the kinase domain upon phosphorylation, thus unblocking this region so that Jak2 can transfer the phosphate from ATP onto its substrates (Feng J et al., 1997). In addition, it has been demonstrated that phosphorylated tyrosine 1007 serves as a binding site for members of the Supperssors of Cytokine Signaling (SOCS) regulatory protein family in order to terminate Jak2 dependent signaling (Ungureanu et al., 2002). A recently characterized tyrosine residue in the kinase domain of Jak2 is tyrosine 972. In 2008, our laboratory demonstrated that tyrosine 972 is a novel Jak2 autophosphorylation site and found that this residue is important in Jak2 tyrosine kinase regulation (McDoom et al., 2008). Specifically, we found that phosphorylation of tyrosine 972 is critical for maximal Jak2 kinase functi on, in the context of a ligand -independent system. In addition, this site was shown to be essential for angiotensin II dependent Jak2 Y1007/Y1008 phosphorylation. In contrast, Funakoshi Tago and colleagues have found that phosphorylation of tyrosine 913, also situated in the kinase domain, negatively regulates Jak2 function by abrogating erythropoietindependent Jak2 activation (Funakoshi Tago et al., 2008). Finally, phosphorylation of tyrosine 966 has been shown to promote the binding of p70 to this res idue (Carpino et al., 2002). However, p70 does not have any known effect on any cytokines that employ Jak2. Therefore, the role of tyrosine 966 on Jak2-dependent function, if any, has yet

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18 to be defined. Collectively, phosphorylation of tyrosine residues in the kinase domain serves to upregulate or downregulate Jak2 tyrosine kinase activity. Certain tyrosine residues have also been characte rized in the JH2 domain that have opposing roles in of Jak2 function. For instance, tyrosine 570 has been shown to be phosphorylated in response t o growth hormone resulting in reduced Jak2 tyrosine kinase activity (Argetsinger et al., 2004). In contrast, when tyrosine 813 is phosphorylated, it serves as a binding site for the SH2B adaptor protein which is a potent act ivator of Jak2 (Kurzer et al., 2004). This in turn, enhances Jak2 tyrosine kinase activity. Although the JH2 domain is generally known to serve as a Jak2 autoinhibitory region, it is interesting that not all tyrosine residues within this domain contribut e to the suppression of Jak2 kinase activity. Therefore, it appears that the role of the pseudokinase domain in Jak2 regulatory function is quite complex, but may become more clear once the crystal str ucture of the JH2 domain is resolved. In addition, tyr osine residues within the Jak2 FERM domain are important for the association of Jak2 to a particular receptor. For example, our laboratory has shown that tyrosine 201 is phosphorylated and serves as a binding site for SHP 2 to recruit Jak2 to the AT1 rece ptor signaling complex. The consequence of this interaction is to promote downstream Jak2dependent signaling (Godeny et al., 2007). On the contrary, Y119 has a negative function with regard to the association of Jak2 to a particular receptor. Specifica lly, phosphorylation of Jak2 at tyrosine 119 results in the dissociation of Jak2 from the erythropoietin receptor (Funakoshi Tago et al., 2006). In summary, a number of Jak2 amino acids have been characterized that play diverse roles in regulating Jak2 fun ction. These amino acids consist of tyrosine as well as non-tyrosine residue s and they are situated on various Jak homology domains. Phosphorylation of these

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19 amino acid residues can result in either propagation or inhibition of Jak2 -dependent signaling. In addition, certain amino acid residues have been shown to be phosphorylated yet their role, if any, in the Jak2 signal transduction pathway is undefined. Although the structural elements of Jak2 play vital roles in regulating Jak2 function, a number of external factors also influence Jak2 so that the cell can bring forth the proper Jak/STAT response. In the subsequent section, we will discuss the external factors that affect the Jak2 signaling pathway. Jak2 Signal Transduction Pathway The significance of Jak2 in cell signaling was apparent when investigators showed that Jak2 is a critical mediator of cytokine dependent signaling (Argetsinger et al., 1993; Silvennoinen et al., 1993; Watling et al., 1993; Witthuhn et al., 1993; Narazaki et al., 1994; Rui et al., 1994). Subsequent work demonstrated a correlation between activation of Jak2 in the cytoplasm and amplified gene transcription in the nucleus. The Jak2 signaling paradigm was further elucidated when studies identified a new class of cytokine res ponsive transcription factors, named the Signal Transducers and Activators of Transcription (STAT) proteins which mediate gene transcription (Schindler et al., 1992; Shuai et al., 1992). Thus, within two years, it was defined that the primary cellular rol e of Jak2 is to relay signals from the cell surface to the nucleus. Aside from STATs, many different proteins participate in the Jak2 signaling pathway to permit a unique and specific signal to occur in the cell. These proteins consist of different cell s urface receptors including cytokine, growth factor and G -protein coupled receptors (GPCRs). Moreover, adaptor/regulatory proteins are also critical components of Jak2 -dependent signaling. Importantly, although Jak2 is expressed in numerous tissues and is activated by different types of cell surface receptors, specificity is maintained in this signaling system by the particular receptor and its associated STAT protein. In detail, the specificity of STAT activation by a

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20 certain receptor is established by th e particular SH2 domain situated within each STAT molecule and the specific phosphotyrosine -containing motif that is encoded on a given receptor (Heim et al., 1995; Stahl et al., 1995). Activation of Jak2 via Cytokine Receptors Jak2 activation through cytokine receptors is the most well understood Jak2 signaling model. An overview of the Jak/STAT signaling pathway is illustrated in Fig ure 1 2. In this paradigm, signaling is initiated by the binding of ligand to its cell surface receptor, resulting in rece ptor dimerization. Upon dimerization, the receptors undergo conformational changes that are thought to position the constitutively receptor -bound Jak2 molecules in close proximity to one another in order to facilitate Jak2 autophosphorylation (Brooks et a l., 2007). This liganddependent tyrosine phosphorylation occurs principally on Tyr 1007 (Feng et al., 1997). An activated Jak2 then phosphorylates specific tyrosine residues on the cytoplasmic tails of the receptors, creating docking sites for the SH2 domain containing STAT proteins. Once bound to the receptors, the STATs are themselves phosphorylated by Jak2 on tyrosine residues. The tyrosine -phosphorylated STATs then form active homodimers and heterodimers which translocate to the nucleus, where they bind to STAT recognition sequences and modulate gene transcription. Therefore, Jak2 is responsible for transducing a signal from the cell surface to the nucleus through a tyrosine phosphorylation signaling mechanism. Activation of Jak2 via GPCR In additi on to cytokine receptors, Jak2 is activated by G protein -coupled receptors, such as the AT1 receptor. GPCR are composed of a single polypeptide chain that spans the plasma membrane seven times and possess both intracellular and extracellu lar components (F ong, 1996). There are important differences in the mechanism by which GPCR and cytokine recep tors facilitate Jak2 activation, but the outcome is the same in that activation of Jak2 leads to STAT

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21 activation and subsequent modulation of gene transcription. The general differences in activation of Jak2 via the cytokine receptor versus the GPCR are illustrated in Fig ure 1 2. In the GPCR model, the Jak2 molecules are not constitutively bound to the GPCR in the absence of ligand. However, upon ligand binding Jak2 autophosphorylation is triggered and Jak2 is recruited from the cytosol to the GPCR (Ali et al., 1997; Sayeski et al., 2001). In contrast, in the cytokine receptor model, Jak2 is constitutively bound to the cytokine receptor. Another key differenc e is that while STATs directly associate with the cytoplasmic tail of the cytokine receptor, it is thought that Jak2 serves as a molecular bridge linking STATs t o the GPCR (Ali et al., 2000). Jak2 Regulatory Proteins As previously mentioned, in order for J ak2 to reach an initial state of activation, Tyr 1007 within the Jak2 activation loop must be phosphorylated in response to ligand stimulation. Furthermore, the catalytic state of Jak2 can be driven to even greater levels of activation through the associa tion of Jak2 with activator proteins such as SH2B When it is necessary for the cell to terminate Jak2 dependent signaling, there exist several Jak2 inhibitory proteins that exhibit their effect at different points of the signal transduction cascade to suppress Jak2 function. These Jak2 regulatory proteins will be discussed in detail below. SH2B family of proteins The SH2B family members include SH2B1, SH2B2 and SH2B3. These proteins all share a common domain structure that includes an SH2 domain, a PH domain domain, several proline rich regions and a dimerization domain (Maures et al., 2007). The SH2B1 or the simply stated SH2B transcript undergoes alternative splicing, yielding four protein products, SH2B and (Maures et al., 2007). The SH2B isoform has been shown to bind Jak2 at phosphorylated

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22 Y813 via its SH2 domain and enhance growth hormone mediated Jak2 activation (Kurzer et al., 2004). Two mechanism have been suggested to explain how SH2B augments Jak2 activity. One mechanism is that dimerization of SH2B leads to dimerization of the associated Jak2 proteins thereby resulting in enhanced Jak2 tyrosine kinase activity (Nishi et al., 2005). An alternative model proposes that SH2B increa ses the number of active Jak2 molecules by sustaining Jak2 in an active state (Kurzer et al., 2006). Nevertheless, both of the suggested mechanisms agree that the capacity of SH2B to increase Jak2 activity is directly due to SH2B function and not a consequence of recruitment of a Jak2 activator nor competition with a Jak2 inhibitor. SOCS family of proteins The suppressor of cytokine signaling (SOCS) family of proteins is a class of negative feedback regulators of cytokine receptor signaling. All SOCS family members share a central SH2 domain, a conserved C terminal motif called the SOCS box and a variable N terminal region (Ilangumaran and Rottapel, 2003). The SOCS proteins were first characterized as inhibitors of cytokine -dependent Jak2 signaling (S tarr et al., 1997). Specifically, SOCS1 has been shown to be a critical regulator of IFN -gamma as over -expression of SOCS1 inhibits Jak2 and blocks IFN gamma signaling (Song and Shuai, 1998; Sakamoto et al., 2000). SOCS expression is rapidly induced through transmission of the Jak/STAT signal. Subsequently, SOCS protein binds to Jak2 at phophorylated tyrosine 1007 on the activation loop resulting in ubiquitin -mediated degradation of Jak2 (Starr et al., 1997; Ungureanu et al., 2002; Ilangumaran S and R ott apel, 2003). Overall, the role of SOCS in physiology is important as it regulates the magnitude and duration of Jak2 dependent signaling to prevent excessive signaling and abnormal cellular

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23 activation that could result in oncogenic transformation. In fact aberrant methylation of SOCS genes have been associated with downregulation of SOCS proteins in solid tumors, leukemias and hematopoietic cells exhibiting constitutive Jak2 activity (Lee et al., 2006; Niwa et al., 2005; Jost et al., 2007). Jak2 and Path ophysiology Role of Jak2 in Cardiovascular Disease Jak2 plays an important role in cardiovascular disease as activation of this protein has been implicated in the molecular signaling events that lead to certain cardiovascular diseases such as cardiac hyper trophy, ischemia reperfusion and atherosclerosis. For example, Jak2 becomes activated in response cardiac hypertrophy-inducing stimuli. Specifically, it was demonstrated that acute pressure overload in rats increases Jak2 tyrosine phosphorylation levels by triggering autocrine/paracrine secretion of angiotensin II (Pa n et al., 1997). I nvestigators also showed that treatment of cardiomyocytes with the Jak2 inhibitor, AG490, reduced mechanical stretchinduced Jak2 phosphorylation levels thereby linking mec hanical stretch to Jak2 activation in cardiomyocytes (Pan J et al., 1998). In a different model, it was demonstrated that cardiotrophin1, an IL 6 related cytokine family member and potent inducer of cardiomyocyte hypertrophy, also stimulated Jak2 activat ion by increasing angiotensinogen mRNA (Pennica et al., 1995; Fukuzawa et al., 2000). Overall, evidence supports that diverse physical and chemical stimuli that generate cardiac hypertrophy also stimulate Jak2 activation. Jak2 signaling is also connected to cardiac injury resulting from ischemia reperfusion, a pathological condition characterized by impeded blood flow to an area of tissue followed by the reestablishment of circulation to the same area. Specifically, it was shown that treatment with the Ja k2 inhibitor, AG490 resulted in a reduction in cardiac infarct size and a reduction in apoptotic cell death in cardiomyocytes after ischemia reperfusion in a perfused rat heart

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24 (Mascareno et al., 2001). These results indicate that Jak2 activation is assoc iated with cardiac dysfunction during ischemia reperfusion. Approximately ten years ago, it was observed that intracellular reactive oxygen species or exogenous hydrogen peroxide activate the Jak2/STAT pathway in fibroblast cells, vascular smooth muscle cells as well as in human lymphocytes by behaving as intracellular signaling molecules (Simon et al., 1998; Carballo et al., 1999; Madamanchi et al., 2001). The activation of Jak2 by reactive oxygen species is important in the progression of atherosclerosis It has been shown that oxidative stress induced Jak2 activation plays a role in vascular smooth muscle cell proliferation, an important factor in the progression of atherosclerosis. Specifically, Madamanchi et al. demonstrated that hydrogen peroxide st imulates heat -shock protein 70 (HSP70) expression in vascular smooth muscle cells via activation of the Jak2/STAT signaling pathway (Madamanchi et al., 2001). The outcome of HSP70 activation in vascular smooth muscle cells could provide these cells a prol iferative advantage by protecting cells from undergoing apoptosis (Madamanchi N et al., 2001). Aside from vascular smooth muscle cell proliferation, apoptosis also plays an important part in the progression of atherosclerosis. It is thought that vascula r smooth muscle cell apoptosis in atherosclerotic plaques leads to plaque instability and eventual plaque rupturing (Hsieh et al., 2001). Several years ago, our laboratory defined a direct link between Jak2 and oxidative stress -induced apoptosis in vascul ar smooth muscle cells by demonstrating that inhibition of Jak2 results in an elimination of hydrogen peroxide -mediated apoptosis in these cells (Sandberg and Sayeski, 2004b). Collectively, there is evidence to support that Jak2 plays an important function in cardiovascular disease states such as cardiac hypertrophy and ischemia repurfusion. In addition, Jak2 plays a vital role in oxidative stress -mediated cell proliferation and apoptosis which are

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25 important components in atherosclerotic development. These findings suggest that Jak2 could be a potential therapeutic target in vascular diseases. Role of Jak2 in Hematological Disorders In addition to its critical role in a num ber of cardiovascular diseases, somatic Jak2 mutations have been linked to hematologi cal malignancies. These mutations include Jak2 amino acid substitutions, deletions, insertions, and chromosomal translocations that cause constitutive Jak2 kinase activity. For example, TEL Jak2 is a well characterized fusion protein resulting from a tra nslocation event between Jak2 on chromosome 9 and TEL on chromosome 12 that is found in some T -cell leukemia. Jak2 has also been linked to other chromosomal translocations. These include the REL Jak2, BCR Jak2, PCM1 Jak2 and Pax5 Jak2 fusions, which have been associated with myeloid or lymphoid leukemia. In addition, activating Jak2 mutations such as Jak2 T875N, Jak2-K607N, JakL611S and Jak2 IREED have been report ed in acute leukemia as well. In recent years, interest has focused on the gain -of -fun ction Jak2 -V617F mutation since it has been detected in almost all polycythemia vera patients and a substantial proportion of individuals with essential thrombocythemia and primary myelofibrosis (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2 005; Levine et al., 2005; Zhao et al., 2005). Moreover, the Jak2 V617F mutation has been shown to play a critical role in the pathogenesis of myeloproliferative disorders as expression of this mutation in murine hematopoietic cells leads to a polycythemia vera -like disease in mice (Lacout et al., 2006 ; Xing et al., 2008). As a consequence of the association of aberrant Jak2 activity with diverse hematological disorders, researchers are increasingly focused on identifying Jak2 inhibitors that suppress cons titutive Jak2 kinase activity. Some of these inhibitors may one day become therapeutically beneficial for

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26 individuals with Jak2 related hematologic malignancies or myeloproliferative disorders. Here, we will describe in detail the various Jak2 mutations that are associated with hematological disorders and assess some of the inhibitors of Jak2 that are either in the pre -clinical phase or in clinical trials. By examining these specific areas, we hope to have a better understanding regarding the role of Jak2 in hematological disorders and shed light on the utility of Jak2 inhibitors. Jak2 chromosomal translocations and hematological malignancies The first study that indicated that a mutant Jak kinase could result in a hematological malignancy was in 1995 when Luo et al. demonstrated that a glycine to glutamic acid substitution at position 341 in the Drosophila hopscotch gene caused a leukemia like hematopoietic defect (Luo et al., 1995). Two years later, studies linked Jak2 chromosomal translocations to human neoplastic growth. Specifically, a translocation event between the kinase domain of Jak2 and the helix-loop-helix domain of the ETS family transcription factor, Tel, was identified in a child with early B -precursor acute lymphoid leukemia and in an adult with atypical chronic myeloid leukemia (Lacronique et al., 1997; Peeters et al., 1997). The basis for the diverse phenotype detected in these two patients is due to two distinct translocation events within Jak2 and the Tel genes that consequently give ri se to distinct chimeras. Nevertheless, these TEL Jak2 fusion proteins caused increased oligomerization of the Jak2 proteins that lead to growth factor independent Jak2 activation and subsequent NF B signaling (Lacronique et al., 1997). Moreover, creation of TEL Jak2 transgenic mice revealed a causal relationship between the TEL Jak2 gene product and leukemogenesis, as over expression of this fusion protein resulted in the development of T cell leuk emia in these animals (Carron et al., 2000).

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27 Apart from TEL Jak2, studies have shown Jak2 to be implicated in other chromosomal translocations observed in various hematological malignancies. Myamoto et al. showed that the Jak2 inhibitor, AG490, reduced th e growth of human B precursor leukemic cells (Miyamoto et al., 2001). Specifically, they found that AG490 significantly downregulated Jak2 phosphorylation in these cells at a concentration that had little effect on normal hematopoiesis. Consequently, th is study correlated an l1q23 translocation or Philadelphia chromosome with constitutive Jak2 activation in human lymphoid leukemic cells. In addition, Joos and colleagues analyzed four Hodgkins lymphoma cell lines and identified chromosomal rearrangement s of the short arm of chromosome 2 involving REL, a transcription factor belonging to the NF B family of transcription factors (Joos et al., 2003). This resulted in a copy number increase of Jak2 (9p24) in three of the four cell lines. These results suggested that REL and Jak2 may play an important role in the pathogenesis of Hodgkins lymphoma Recent studies have demonstrated that human auto antigen pericentriolar material (PCM1) is a Jak2 translocation partner associated with chronic and acute leukemias including chronic eosinophilic leukemia, acute myeloid leukemia and acute lymphoblastic l eukemia (Murati et al., 2005; Reiter et al., 2005). In all cases, the PCM1 Jak2 fusion involved a t(8;9)(p22;p24) translocation event. The chimeric gene product was predicted to encode a protein that maintains several of the coiled -coil domains of PCM1 a nd the kinase domain of Jak2. The PCM1 coiled motifs possibly serve as a dimerization motif to bring about c onstitutive activation of Jak2. Furthermore, BCR Jak2 fusions have been identified in patients with typical and atypical chronic myeloid leukemia (Griesinger et al., 2005; Lane et al., 2008). In each case, in situ hybridization revealed a t(9;22)(p24;q11.2) translocation in these patients as opposed to the typical t(9;22)(q34;q11.2) translocation. Although the breakpoints were variable in each pat ient, the rearrangement resulted in a BCR Jak2 chimera

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28 rather than the classical BCR -ABL fusion protein. A common finding in these patients was that they exhibited relatively early blast crisis. Lastly, PAX5, a regulator of B -cell development, has been r ecently shown to form a fusion with the Jak2 gene in childhood acute lymphoblastic leukemia (Nebral et al., 2009) Jak2 somatic mutations and hematologic malignancies Activating Jak2 somatic mutations such as amino acid substitution mutations and deletions have also been identified in hematological malignancies. Mercher et al. identified a novel Jak2 T875N mutation in an acute megakaryoblastic leukemic cell line using a combination of mass spectrometry and growth inhibition assays via the use of a selective tyrosine kinase inhibitor (Mercher et al., 2006). The authors demonstrated that the Jak2 T875N was constitutively active in vitro and induced a myeloproliferative disease with characteristics of megakaryoblastic leukemia in a murine bone marrow transplan tation assay. Other novel mutations have been reported in the JH2 domain of Jak2, which confer constitutive activation of th e Jak -STAT signaling pathway. These include the Jak2 -K607N (Lee et al., 2006) and Jak2L611S (Kratz et al., 2006) mutations found in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. Finally, a deletion of amino acids 682 686 (Jak2IREED) has been observed in a patient with Down Syndrome and B cell precursor acute lymphoblastic leukemia (Malinge et al., 2007). In summary, the above studies indicate that the Jak2 locus is susceptible to chromosomal rearrangement, point mutations a nd deletions; all of which are associated with hematological malignancies. These Jak2 gene aberrations are summarized in Table 1 1. Jak2 translocation chimeras appear to increase Jak2 oligomerization and result in growth factor independent Jak2

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29 auto acti vation whereas Jak2 point mutations and deletions lead to hypersensitivity to growth factors through impaired Jak2 autoregulation. Nevertheless, the end result is that the aberrant Jak2 protein has constitutively active tyrosine kinase activity that resul ts in a neoplastic phenotype. Jak 2 V617F and myeloproliferative disorder s In 2005, five independent studies reported the identification of a Jak2 somatic mutation (Val 617 to Phe) in several myeloproliferative disorders at a high frequency (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2005; Levine et al., 2005; Zhao et al., 2005). Studies employing sensitive detection methodologies indicated that the Jak2 -V617F mutation on exon 14 can be detected in almost all polycythemia vera (PV) patients and approximately 50% of essential thrombocythemia (ET) and primary myelofibrosis patients (PMF) (Levine et al., 2006). These myeloproliferative disorders are characterized by the clonal over -production of normally differentiated hematopoietic lineages. F or instance, PV is characterized by the over -proliferation of erythrocytes while ET is defined as the unregulated expansion of megakaryocytes. The V617F substitution leads to constitutive activation of Jak2 and downstream effector signaling pathways inclu ding the STAT transcription pathway, PI3 kinase and ERK signaling networks, which in turn induce inappropriate cytokine independent proliferation of cells (Levine et al., 2005; Shannon and Van Etten, 2005). The nature of this gain-of -function mutation is that Val 617 lies in the JH2/pseudokinase auto inhibitory domain of Jak2. Current molecular models of the pseudokinase domain suggest that it interacts with the activation loop of the kinase domain (Giordanetto and Kroemer, 2002). Moreover, structure/function studies have shown that amino acids located between positions 619 to 970 are critical for maintaining the inhibitory property of the pseudokinase domain (Saharinen et al., 2003). Therefore, it is hypothesized that the V617F

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30 mutation impedes the pseudokinase domain from acting as an internal inhibitory regulator of the adjacent kinase domain, resulting in aberrant Jak2 tyrosine kinase activity. Other Jak2 mutations associated with myeloproliferative disorders Although the Jak2-V617F mutation is predom inantly associated with myeloproliferative disorders, it is evident that other activating alleles of Jak2 are also involved in these disorders. For example, Scott et al. identified a set of novel somatic Jak2 mutations on exon 12 in Jak2V617F negative polycythemia vera or idiopathic erythrocytosis patients (Scott et al., 2007). Specifically, these mutations mapped to amino acid residues 537543, which is a region that links the SH2 and JH2 domains of Jak2. Patients harboring these mutations displayed is olated erythrocytosis, reduced serum erythropoietin and factor -independent erythrocyte colony formation. Moreover, characterization of BaF3 cells expressing the erythropoietin receptor and carrying the exon 12 mutations revealed that these cells could proliferate in the absence of interleukin 3 and expressed increased Jak2 phosphorylation relative to cells transduced by w ild type Jak2 or Jak2-V617F. A separate group also identified Jak2 exon 12 mutations in 5 of 6 Jak2 -V617negative PV patients from a tota l of 220 PV cases (Pardanani et al., 2007). They found that approximately 80% of these patients harbored either the F537-K539delinsL or N542 E543del in the bone marrow and/or peripheral blood cells. In addition, they found that bone marrow derived from t he PV patients carrying the Jak2 exon 12 mutations displayed erythroid hyperplasia, megakaryocyte abnormalities, and reticulin fibrosis, similar to Jak2 -V617F harboring PV patients. Finally, Pietra and colleagues searched for Jak2 exon 12 mutations in 17 patients with Jak2 -V617F negative PV and found that 2 of the 8 mutations were novel, with the most frequent ones being N542E543del and E543-D544del (Pietra et al., 2008). In addition, they reported that most PV patients carrying an exon 12 mutation had i solated erythrocytosis at clinical onset. Collectively,

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31 the above reported findings imply that Jak2 exon 12 mutations are detected mainly in Jak2V617F negative PV cases and suggest that exon 12 could also be an active site for gain -of function mutations. O ther mutations have been identified in the JH2 ps e udokinase domain of Jak2. Schnittger et al. detected a novel Jak2 D620E mutation in a 56 year old myeloproliferative syndrome patient with leukocytosis (Schnittger et al., 2006). Simultaneously, another group identified the same Jak2 D620E mutation in a 27 year old PV patient who was also positive for the Jak2 V617F mutation (Grunebach et al., 2006). Their study revealed the Jak2D620E mutation was found in all peripheral blood subsets including B and T lymphocytes, suggesting that in this patient the D620E mutation arose at the multipotent hematopoietic stem cell level. Finally, Zhang et al. reported a novel Jak2 C616Y point mutati on in a PV patient who was Jak2 -V617F negative (Zhang et al., 2007). In summary, there is evidence to support that other somatic gain -of -function Jak2 mutations, aside from Jak2-V617F, could play an important role in myeloproliferative disorders. These mutations include deletions and insertions on exon 12 and substitutions i n the JH2 domain of Jak2. Inhibitors of Jak2 Tyrosine Kinase Justification for Jak2 Inhibitors The discovery of the Jak2-V617F mutation in nearly all polycythemia vera and a large subset of essential thrombocythemia and primary myelofibrosis patients has p rompted researchers to closely study the Jak2 gene and its role in myeloproliferative disorders. In addition, constitutive activation of Jak2 kinase activity by chromosomal translocations has been reported in various types of hematological malignancies (La cronique et al., 2007; Reiter et al., 2005). Currently however, no FDA approved Jak2 inhibitor therapies are available for use in the

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32 clinic, although a few are being examined for their efficacy and safety in Phase I/II clinical trials. Thus, the continu al identification of novel activating Jak2 mutations, and their correlation with hematological malignancies, highlights the requirement for the development of potent and therapeutically effective Jak2 inhibitors. Jak2-Selective Inhibitors The causal relati onship between constitutive Jak2 tyrosine kinase activity and neoplastic growth has prompted researchers to identify potent and selective Jak2 small molecule inhibitors. In 1995, Meydan et al. employed a high throughput screen of potential tyrosine kinase inhibitors and identified tyrphostin B42 (AG490) as the first Jak2 inhibitor (Meydan et al., 1996). Their important finding was that AG490 blocked the growth of leukemic cells derived from patients who expressed constitutive Jak2 tyrosine kinase activity The compound induced cellular apoptosis, without any deleterious effect on normal hematopoiesis. However, subsequent reports revealed that while AG490 is a potent inhibitor of Jak2, it suffered from a general lack of specificity (Gu et al., 2001). To c ircumvent this problem, researchers have utilized different approaches to identify novel Jak2 selective inhibitors. In 2004 for example, Flowers et al. developed a short peptide inhibitor of Jak2 termed, Tkip, that mimics the actions of the Jak2 inhibitor protein, SOCS 1. They reported that the inhibitor peptide mimicked SOCS 1 in that it specifically inhibited Jak2 tyrosine 1007 phosphorylation and suppressed IFN signaling (Flowers et al., 2004). In 2005, our group published a paper whereby we constructed a homology model of the Jak2 kinase domain and utilized a high -throughput program called DOCK to identify novel small molecule inhibitors of Jak2 tyrosine kinase (Sandberg et al., 2005). Specifically, we tested 6451 compounds of known chemical struct ure in silico for their ability to interact with a pocket

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33 positioned adjacent to the activation loop of Jak2. The top seven scoring compounds were obtained from National Cancer Institute and tested for their ability to inhibit Jak2 autophosphorylation in vitro We found that one compound, C7, directly inhibited Jak2 tyrosine kinase activity. Characterization of C7 revealed that this compound suppressed Jak2 tyrosine autophosphorylation in both a dose and time dependent manner. C7 significantly reduced growth hormone -dependent Jak2 autophosphorylation, but had no effect on epidermal growth factor receptor tyrosine phosphorylation. Moreover, C7 was not cytotoxic to cells at doses as high as 100 M idium iodide. All together, the results suggested that C7 may be a relatively specific Jak2 inhibitor and proposed that it may be useful for elucidating signaling mechanisms of Jak2. The discovery of the Jak2-V617F mutation in 2005 and its identification in a high percentage of myeloproliferative disorders has further spurred interest in the development of small -molecule inhibitors that selectively target Jak2. Moreover, the resolution of the crystal structures of portions of the kinase domains of Jak3 and Jak2 in 2005 and 2006, respectively, have provided a valuable tool for the design of potent and specific Jak2 small molecule inhibitors (Boggon et al., 2005; Lucet et al., 2006). Recently, a group developed several novel Jak2selective small molecule compounds (TG101209 and TG101348) while taking into consideration the crystal structures of the kinase domains of both Jak2 and Jak3 (Pardanani et al., 2007; Wernig et al., 2008). They showed that TG10120 9 and TG101348 potently inhibited Jak2 tyrosine kinase, with considerably less activity against other tyrosine kinases such as Jak3. These compounds suppress ed the proliferation of human erythroleukemia cells, which express the Jak2-V617F mutation. Fur thermore, they demonstrated that both compounds effectively treat ed Jak2 -V617F induced hematopoietic disease in mice and reduce d the growth of

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34 hemopoietic colonies from primary progenitor cells harboring Jak2 -V617F mutations. Currently, the TG101348 compo und has been assigned as a lead drug for clinical development for the potential treatment of Jak2 -V617F induced myeloproliferative disorders. Another Jak2-selective inhibitor, INCB18424, is presently under phase I/II clinical trial s at MD Anderson Cancer C enter for the treatment of primary myelofibrosis patients. While it has reduced splenomegaly, it unfortunately has not diminished the marrow fibros is (Verstovsek et al., 2008a). In 2008, Verstovsek et al. demonstrated that a novel analogue of AG490, WP1066, potently suppressed proliferation and induced apoptosis in erythroid human cells harboring the Jak2 V617F mutation (Verstovsek et al., 2008b). In addition, WP1066 inhibited the expansion of peripheral blood hematopoietic progenitors of PV patients who were positive for the Jak2V617F mutation. Interestingly, WP1066 was previously shown to inhibit phosphorylation of Jak2 in acute myelogenous leukemia cells, but unlike AG490, this compound also degraded the Jak2 protein (Ferrajoli et al., 2007). Collect ively, the data suggests that WP1066 is a potent Jak2 inhibitor in vitro and ex vivo and warrants further development for the treatment of myeloproliferative disorders and other hematological malignancies that are associated wi th constitutive Jak2 activity. Our laboratory has recently contributed to the continuing development of small molecule inhibitors that target aberrant Jak2 activity by using a rapid structure based approach combining molecular docking with cell -based functional testing. Like others, we took into consideration the crystal structure for portions of the Jak3 kinase domain in order to generate an atomic model of the kinase domain of murine Jak2 and then employed the DOCK program to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP -binding site.

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35 Consequently, we identified a Jak2 selective inhibitor termed Z3 (Sayyah et al., 2008). We found that it bound to Jak2 with a favorable energy score and inhibited Jak2 -V617F autophosphorylation in a dose -dependent manner, but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 as it had no effect on Tyk2 and c -Src kinase activity. Furthermore, Z3 significantly inhibited proliferation of the Jak2 V617F expressing HEL cells and this Z3 -mediated reduction in cell growth correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels, as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient carrying the Jak2-V617F mutation and a polycythemia vera patient harboring a Jak2 -F537I mutation. All together, our results suggest that Z3 is a specific inh ibitor of Jak2 tyrosine kina se. Non -Jak2 Selective Inhibitors In addition to the drugs that were targeted specifically for Jak2, there exists a group of drugs that were developed for the treatment of non-myeloproliferative disorders, but are now considered to have therapeutic potenti al in myeloproliferative because of their significant off target Jak2 inhibitory activity. Some of these drugs are even in phase I/II clinical trials. For example, MK 0457 (formerly, VX 680), a potent inhibitor of Aurora kinases effectively inhibits BCR -A BL, FLT3 and Jak2 (Pardanani, 2008). A phase I/II clinical trial of MK 0457 was initiated in patients with chronic myelogenous leukemia or Ph+ acute lymphoblastic leukemia who carried the T315I BCR -ABL resistance mutation, as well as in patients with refr actory Jak2 V617F positive myeloproliferative disease. This compound showed encouraging anti -neoplastic growth activity and a good safety profile (Wang and Serradell, 2007). Another off target Jak2 inhibitor is CEP 701 (Lestaurtinib), which was originally developed to suppress tropomyosin receptor kinase A activity for possible use in prostate cancer, but later discovered to exhibit

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36 FLT3 inhibitory activity as well. CEP 701 has been shown to inhibit Jak2 tyrosine kinase activity and inhibit the proliferat ion of progenitor cells obtained from myeloproliferative disorder patients (Hexner et al., 2008). Unfortunately, in Phase II clinical studies, CEP 701 has shown little to no activity in treating primary myelofibrosis. Finally, AT9283, another Aurora kina se as well as potent Jak2 inhibitor, is in phase I/II clinical trials for the treatment of acute leukemias, chronic myelogenous leukemia and primary myelofibrosis (Ravandi et al., 2007). There are other non Jak2 selective inhibitors that are still in pre -c linical testing for the treatment of Jak2 associated hematological disorders. One of these inhibitors is G6976, which is an inhibitor of the calcium -dependent isozymes of PKC and FLT3 tyrosine kinase. G6976 was subse quently found to be a potent inhibit or of Jak2 in vitro This compound also suppressed signaling, survival and proliferation of cells expressing either the TEL Jak2 fusion protein or the Jak2 V617F muta tion (Grandage et al., 2006). These data suggest that G6976 may be useful for the treat ment of myeloproliferative disorders or other Jak2associated hematological malignancies. In addition, Erlotinib (Tarceva) which is used for the treatment of patients with locally advanced or metastatic non -small cell lung cancer, inhibited the growth and expansion of Jak2 V617F -expressing polycythemia vera hematopoietic progenitor cells and human erythroleukemia HEL cells while having little effect on n ormal cells (Li et al., 2007). Another compound that possesses Jak2 inhibitory property is Atiprimod. A tiprimod is an orally bioavailable agent that has been investigated for its anti -inflammatory and anti -cancer properties. Faderl et al. reported that Atiprimod inhibits Jak2/STAT phosphorylation and blocks clonogenic growth of acute myelogenous leukemia c ell lines and patient -derived acute myelogenous leukemic marrow cells by inducing apoptosis (Faderl et al., 2007). Their data suggest that the anti proliferative and pro apoptotic activities of Atiprimod towards acute

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37 myelogenous leukemia cells might be a ttributed to the inhibition of the Jak -STAT signaling pathway. Interestingly, the inhibitory effect of this compound has not been evaluated on Jak2 V617F dependent pathologic cell growth. Thus, Atiprimod may warrant further evaluation as a drug candidate for the treatment of hematological disorders associated with constitutive Jak2 activation. Finally, CP 690,550, a selective JAK3 inhibitor, also exhibits Jak2 inhibitory properties. Mansh ouri et al. demonstrated that this compound exerts potent anti -prol iferative activity against cells expressing the Jak2 -V617F mutation (Manshouri et al., 2008). In fact, CP 690,550 suppressed Jak2-V617F -dependent cell growth in vitro (IC50 = 0.2 M) ten fold more potently than wild type Jak2 (IC50 = 2.1 M). It induced a marked pro apoptotic e ffect on cells harboring the Jak2-V6 17F mutation whereas a smaller e ffect was observed for cells carrying wild type Jak2. Furthermore, CP 690,550 selective ly inhibited the growth of Jak2 -V617F positive cells in ex-vivo expanded progenitors from polycythemia vera patients, which correlated with a decrease in Jak2 -V617F mutant allele frequency. Taken together, the data suggest that CP 690,550 is a putative inhibitor of Jak2-V617F both in vitro and ex vivo Collectively, work by many groups, including our own, have identified various small molecule inhibitors that suppress Jak2 tyrosine kinase activity. Some of these small molecule compounds may be classified as Jak2 -selective (class I) because they specifically target Jak2. Alternatively, a number of these compounds may be categorized as non Jak2 selective (class II) since they were initially developed for non-myeloproliferative disorders, but subsequently s hown to have considerable Jak2 inhibition. These inhibitors are summarized in Table 1 2.

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38 Purpose and Rationale of Studies Activating Jak2 mutations are found in almost all individuals with polycythemia vera and a substantial proportion of individuals wit h essential thrombocythemia and primary myelofibrosis. An increasing number of Jak2 aberrations such as substitution mutations, deletions, insertions and gene translocations are also being found in a number of hematopoietic malignancies. The expanding co mpendium of Jak2 aberrations found in hematological disorders justifies the need for quantitative Jak2 mutation testing in the clinic and validates their candidacy for targeted therapy. As previously mentioned, we have contributed to the identification of novel Jak2 small molecule inhibitors that target aberrant Jak2 tyrosine kinas e activity. In the subsequent C hapters 3 and 4 we will describe the characterization of our second and third generation inhibitors named Z3 and G6, respectively. Additionally, since Jak2 structure is intimately tied to its function, the identification and characterization of novel Jak2 phosphorylation sites will give us new insights regarding how this signal transduction pathway operates. In particular, we will define the role of tyrosine 372, a novel Jak2 phosphorylation site, on Jak2 tyrosine kinase function.

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39 N Terminal C TerminalJH7 JH6 JH5 JH4 JH3 JH2 JH1 AA 38 122 AA 144284 AA 288309 AA 322440 AA 451538 AA 543824 AA 8361123 JH1; Kinase domain JH2; Psuedokinase domain JH3 JH4; Putative SH2 domain JH4 JH7; FERM domain Figure 1 1 Illustration of Jak2 structural domains. The seven Jak homology (JH) domains and their relative positions within Jak2 are presented. The corresponding amino acid sequence of each domain is also shown. The JH1 domain represents the kinase activity site. The JH2 domain corresponds to the pseudokinase domain and exhibits an inhibitory effect on the kinase domain. The JH3 an d half of the JH4 domain encode a putative SH2 domain. The FERM domain extends from the second half of the JH4 to the JH7 domain and assists Jak2 association with the receptor

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40 Ligand ligand P P P P P Jak2 Jak2 Jak2 Jak2 STAT STAT P P STAT STAT Jak2 P P P STAT STAT P P STAT STAT P P SRE Cytokine Receptor G Protein Coupled Receptor Nucleus D B A C Figure 1 2 Jak/STAT signaling paradigm. Ja k2 signals through cytokine, tyrosine kinase or G protein -coupled receptors. A) With respect to cytokine receptors, Jak2 is noncovalently associated with the receptor. The binding of ligand to the extracellular surface of the cytokine receptor results in receptor dimerization and subsequent Jak2 activation. Activated Jak2 then phosphorylates the cytoplasmic tail of the receptor at particular tyrosine residues. The STAT proteins associate with phosphorylated tyrosine residues and are themselves phosphor ylated by Jak2. B) In the case of G protein -coupled receptors, Jak2 is not ubi quitously bound to the receptor, but becomes activated and associates with the cytoplasmic tail of the receptor after ligand binding. STAT proteins then bind to phosphorylated tyrosine residues on Jak2 and are themselves activated by Jak2. C) The phosphorylated STAT proteins dissociate from the receptor, form dimers and translocate into the nucleus. STAT proteins then bind specific STAT response elements (SRE) within gene pr omoters and initiate transcription.

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41 Table 1 1 A complete list of Jak2 gene aberrations reported in hematological disorders. These malignant mutations inc lude Jak2 amino acid substitutions deletions and chromosomal translocations that were identifie d from 1997 to 2008. Mutation Type Mutation Phenotype Year Identified Translocation TEL Jak2 ALL, aCML 1997 Translocation REL Jak2 aCML Hodgkins Lymp h. oma 2003 Translocation PCM1 Jak2 aCML, AML, ALL 2005 Translocation BCR Jak2 CML 2005 Translocation BCR Jak2 AML 2008 Substitution Jak2 V617F PV, ET, PMF 2005 Substitution Jak2T875N Megakaryoblastic Leuk emia 2006 Substitution Jak2K607N AML 2006 Substitution Jak2L611S ALL 2006 Substitution Jak2K539L PV, Idiopathic Erythrocytosis 2007 Deletion Jak2 IREED ALL 2007 Reprinted with permission from Current Medicine Group LLC Sayyah J and Sayeski PP. (2009) Jak2 inhibitors: Rationale and Role as Therapeutic Agents in Hematologic Malignancies. Curr Onc Rep 11:117124. Table 1, page 120.

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42 Table 1 2. A complete list of class I and II Jak2 inhibitors identified since 1996. The class I Jak2 inhibitors are considered as Jak2 selective compounds whereas the class II inhibitors are categorized as non Jak2 selective. The listed Jak2 small molecule inhibitors are either in pre -clinical or phase I/II clinical trials. Inhibitor Class Current Status Date Identified AG490 I Pre Clinical 1996 Tkip I Pre Clinical 2004 C7 I Pre Clinical 2005 TG101209 I Pre Clinical 2007 WP1066 I Pre Clinical 2008 Z3 I Pre Cl inical 2008 TG101348 I Phase I/II 2008 INCB018424 I Phase I/II 2008 Go6976 II Pre Clinical 2006 Erlotinib II Pre Clinical 2007 Atiprimod II Pre Clinical 2007 CP 690,550 II Pre Clinical 2008 AT9283 II Phase I/II 2006 CEP 701 II Phase II 2007 MK 045 7 II Phase I/II 2007 Reprinted with permission from Current Medicine Group LLC Sayyah J and Sayeski PP. (2009) Jak2 inhibitors: Rationale and Role as Therapeutic Agents in Hematologic Malignancies. Curr Onc Rep 11:117124. Table 2, page 122.

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43 CHAPT ER 2 METHODS Small Molecule Database The small molecules were obtained from the National Cancer Institute/Developmental Therapeutics Program (NCI/DTP), which maintains a repository of approximatel y 225,000 compounds. In Silico Molecular Modeling of Jak2 T he Swiss Model program was utilized to generate a homology model structure of murine Jak2 based on the human Jak2 crystal structure (PDB code: 2B7A). The Definition of Secondary Structure of Proteins (DSSP) program was used to calculate solvent accessible surface areas of tyrosine 372 (Kabsch and Sander, 1983). Mass Spectrometry Wild type Jak2 protein was expressed in BSC 40 cells using a vaccinia virus over expression system and the protein was subsequently purified as previously described (Ma X 2004). T he purified Jak2 protein was then subjected to SDS -PAGE, coommassie stained, excised from the gel and subjected to nano HPLC/ ESI ionization on an LTQ mass spectrometer as previously described (Godeny et al., 2007). Cell Lines The cell lines that were employed to carry out transient transfection experiments were COS 7, BSC 40, and MEF cells derived from Jak2 / mice. The COS 7 and BSC 40 are epithelial cell lines that originate from the monkey kidney. Since all these cells express very little to no endogenous Jak2 levels, they provide suitable conditions to carry out transient transfections. The BSC 40 cells are more permis sive to vaccinia virus infection, as such, they were preferentially used for virus -mediated over -expression assays. The mouse embryonic

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44 fibroblast cells (MEF) originate from Jak2 deficient mice and were a kind of Dr. Jim Ihle (Parganas et al., 1998). For our Jak2 inhibitor studies, HEL, Raji and CMK cells were employed. HEL cells are a human erythroleukemia cell line which expresses the V617F mutation on both Jak2 alleles (ATCC). Therefore, these cells provided a suitable background to determine the eff icacy of our novel Jak2 small molecule inhibitors. Raji cells are a human Burkitts lymphoma cell line (ATCC) whose mechanism of aberrant growth is due to a translocation event between the c -Myc gene and the heavy chain locus on chromosome 14 (Hamlyn and Rabbitts, 1983). Finally, CMK cells ( DSMZ, Braunschweig, Germany ) are a human megakaryoblastic leukemia cell line that expresses constitutively activated Jak3 (Verstovsek et al., 2008b). Thus, the Raji and CMK were appropriate control cell lines to use in our proliferation assays to determine the specificity of our Jak2 inhibitors. Cell Culture BSC 40 cells were maintained in high glucose (4.5 g/L) DMEM supplemented with 10% newborn calf serum. COS 7 and MEF cells were grown in high glucose DMEM supplem ented with 10% fetal bovine serum. HEL,Raji and CMK cells were maintained in RPMI 1640 medium (Mediatech, Herndon, VA) containing 10% fetal bovine serum and L -glutamine (2 mM final). All cells were cultured at 37oC in a 5% CO2 humidified atmosphere. Cell s treated with interferon -gamma, epidermal growth factor or hydrogen peroxide were growth arrested with serum -free DMEM for 18 hours prior to treatment. Site -Directed Mutagenesis The pRC -CMV Jak2 Y372F, pBOS Jak2 Y372F, pRC CMV Jak2 Y373F, and pRC CMV Jak 2 -V617F plasmids were generated using the Stratagene QuickChange Mutagenesis protocol. The sense primer for the Jak2Y372F plasmids is 5 -

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45 TTAATTGACGGGTTTTACAGACTAACT. The antisense primer sequence for these plasmids is 5 -AGTTAGTCTGTAAAACCCGTCAATTAA. Th e sense primer for the Jak2 Y373F plasmids is 5 -ATTGACGGGTATTTTAGACTAACTGCGDNA. The antisense primer for this plasmid is 5 -CGCAGTTAGTCTAAAATACCCGTCAAT. For the Jak2 -V617F plasmid, the sequence for the sense primer is 5 TATGGTGTCTGTTTCTGTGAAGAGGAG. Th e sequence for the antisense primer is 5 -CTCCTCTTCACAGAAACAGACACCATA. DNA sequencing was used to verify all mutations. BSC -40 Cell Transfection/Infection Jak2 autophosphorylation assays were performed in BSC 40 cells using the vaccinia virus transfection /infection procedure which results in high level Jak2 expression and subsequent tyrosine phosphorylation independent of exogenous ligand addition (Ma and Sayeski, 2004). Specifically, cells were transfected with 10 g of a plasmid encoding either the wild type murine Jak2 cDNA (pRC CMV Jak2 -WT), V617F mutant murine Ja k2 cDNA (pRC -CMV Jak2 V617F), Y372F mutant murine Jak2 cDNA (pRC CMV Jak2 Y372F) or Y373F mutant murine Jak2 cDNA (pRC CMV Jak2 Y373F) under the control of the bacteriophage T7 promoter, using Lipofectin per the manufacturers instructions (Invitrogen, Carlsbad, CA). After 4 hours of transfection, the cells were infected with the recombinant vaccinia virus, vTF7 3, at a multiplicity of infection (MOI) of 1.0 for 1 hour. The media containing Lipofectin/DNA/vTF7 3 was then removed from the cells, replaced with fresh serum -containing media and incubated overnight. Transient Cell Transfections For lipofectin -mediated transfections, plasmid DNA and lipofectin were incubated separately in 0.5 mL of serum -free DMEM at room temperature for 30 minutes. Plasmid DNA and lipofectin were then combined and incubated at room temperature for 10 minutes.

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46 Afterwards, an additional 2 mL of serum -free DMEM was added to DNA /Lipofectin complex and the 3 mL transfection mixture was added on to the plate of cells. The cells were incubated at 37oC for five hours. After five hours, the transfection mixtures were removed from the cells and replaced with 5 mL of serum -containing DMEM. The cells were allowed to recover for 48 hours. For Targefect -mediated transfections, plasmid DNA, Targefect (Targeting Systems, CA) and virofect enhancer (Targeting Systems) were combined in a total volume of 1 mL of serum free DMEM and incubated a t 37oC for 20 minutes. Afterwards, 1 mL of serum containing DMEM was added to the transfection complex and the 2 mL mixture was added onto a plate of cells. The cells were then incubated for 3 hours and after that, 3 mL of serum -containing media was adde d onto the cells. The cells were allowed to recover for 48 hours. Cell Lysis and Immunoprecipitation Cells were washed with two volumes of ice -cold PBS containing 1 mM Na3VO4. BSC 40, COS 7 and MEF cells were lysed in 0.8 mL of ice cold RIPA buffer (20 mM Tris pH 7.5, 10% glycerol, 1% Triton X 100, 1% deoxycholic acid, 0.1% SDS, 2.5 mM EDTA, 50 mM NaF, 10 mM Na4P2O7, 4 mM benzamidine, and 10 g/mL aprotinin) while HEL cells (1.0 x 107) were lysed in 0.8 mL of ice cold Gentle Lysis Buffer (25 mM Tris, pH 7. 4, 10% glycerol, 1% IGEPAL and 137 mM NaCl, 4 mM benzamidine, and 10 g/mL aprotinin). Cleared protein lysates were incubated with 2 g of antibody and 20 L of protein A/G plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 4oC. Protein complexes were washed three times with wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Triton X 100) and resuspended in SDS sample buffer. Bound proteins were boiled, separated by SDS -PAGE, and transferred onto nitrocellulose membranes.

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47 Wes tern Blotting Western blots were performed at room temperature. Nitrocellul ose membranes were blocked in 25 mL of either 5% BSA/TBST or 5% milk/TBST for 30 minutes. The membranes were then incubated in 25 mL of primary antibody solution for 2 hours and s ubsequently washed in TBST for 1 hour. Membranes were incubated in secondary antibody solution (1:4000, GE Healthcare) for 30 minutes and then washed in TBST for 30 minutes. Western blots were visualized using the enhanced chemiluminescence system (NEN L ife Science Products, Waltham, MA). Densitometry was performed using the automated digitizing software, Un-Scan It, Version 5.1 (Silk Scientific, Orem, Utah). All phosphorylation levels were normalized to total protein levels. c -Src In Vitro Kinase Assay Approximately 4 L (12 units) of catalytically active recombinant pp60c Src (Millipore) was incubated in 50 L of in vitro kinase reaction buffer as described (13). Reactions were terminated via the addition of SDS -containing sample buffer and subsequent ly Western blotted with either anti-ACTIVE c -Src or total c -Src antibody as described. Luciferase Assay Using either Lipofectin or Targefect, COS 7 cells were transiently transfected with the appropriate DNA plasmid and 2 g of a luciferase reporter plasmid consisting of four tandem copies of the interferon-gamma activating sequence (pLucGAS). Following 5 hours of transfection, approximately 7 x 105 cells were seeded onto six well culture dishes. The cells were allowed to recover in DMEM for 48 hours. The cells were then lysed in 1X Reporter Lysis buffer (Promega) for 5 hours. During lysis, the lysates were exposed to one freeze -thaw cycle

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48 between room temperature and 80oC. A 20 L sample of lysate was combined with 100 L of luciferase substrate and relative light units were assessed by a Monolight 3010 luninometer. Propidium Iodide Staining of Cells BSC 40 cells were seeded in 6 -well Lab Tek Chamber Slides (Nunc, Rochester, NY). After adherence, the cells were treated with either DMSO or Z3 at concentrations of 25, 100 or 250 M for 16 hours. Live cells were treated with 1 g/mL propidium iodide and then visualized using a Bio -Rad MRC 1024 confocal microscope. Same field image s were captured under phase contrast and fluorescent conditions. Cell Proliferation Assay HEL cells were plated on 96 -well dishes at 5 x 104 cells per well and treated with either DMSO or 25 M Z3 for the indicated times. Alternatively, 5 x 104 HEL, Raji or CMK cells were plated on 96 -well dishes and treated with either DMSO or G6 at the indicated concentrations for 48 hours. T he number of viable DMSO or inhibitor -treated cells was determined by trypan blue exclusion using a hemocytometer Cell Cycle Ana lysis The CycleTESTTM Plus DNA Reagent Kit (Becton Dickinson, San Jose, CA) was utilized to analyze nuclear DNA from HEL cell suspensions following the manufacturers instructions. Approximately 5 x 105 cells were treated with either DMSO or 25 M of Z3 for 16, 24, 48 and 72 hours. Cell suspensions were treated with the reagents stated in the protocol, filtered through a 50 m nylon mesh and analyzed on a FACSCalibur Flow Cytometer (Becton Dickinson). Apoptosis Assays HEL cells were treated wit h either DMSO or 25 M Z3 or G6 for 16, 24, 48 or 72 hours. Cells were subsequently measured for Annexin V staining via a FACSCalibur Flow Cytometer

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49 (Becton Dickinson). The percent of DMSO or inhibitor treated cells undergoing apoptosis was plotted as a function of time. Jak2 Mutation Analysis Residual bone marrow aspirates were obtained from patients diagnosed with ET or PV (WHO criteria) following approval by the University of Florida Institutional Review Board (approval # 4282006). Patient de -identif ied mononuclear cells were isolated by separation over Ficoll Paque (Pharmacia, Peapack, NJ). Genomic DNA was isolated from 106 cells using the Easy DNA kit (Invitrogen) following the manufacturers instructions. Exons 12 and 14 were amplified via PCR us ing previously published exon specific primers and annealing conditions (Scott et al., 2007). Each amplified exon was then subjected to direct DNA sequence analysis. Colony Formation Assay Marrow derived mononuclear cells were washed three times in IMDM media and plated at 4 x 105 cells/mL in 1 mL methylcellulose media (0.9% methylcellulose, 30% heat inactivated FCS, 0.1 mM 2 -mercaptoethanol, 0.9% BSA, 0.05% NaHCO3, 2 mM/L glutamine, penicillin, streptomycin, 50 ng/mL SCF, and 20 ng/mL IL 3 (Stem Cell Te chnologies, Vancouver, BC). Z3 /G6 (25 M) and TPO (50 ng/mL) or EPO (1 U/mL) were either added or omitted as described in the legend. The cultures were then incubated at 37oC and 5% CO2 until assessment of colony formation at day 14. Results were expres sed as the average number of colonies from duplicate cultures per 4 x 105 cells. Statistical Analysis For IC50 determination, data was analyzed using one -way analysis of variance (ANOVA). For proliferation assays and cell cycle analysis, statistical significance between each group was analyzed using a two -way ANOVA. For all other experiments, data were analyzed via Students t -test. Significance was set at p<0.05.

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50 CHAPTER 3 Z3, A NOVEL JAK2 TYROSINE KINASE SMALL -MOLECULE INHIBITOR THAT S UPPRESSES JAK2 -M EDIATED PATHOLOGIC CELL GROWTH Summary Jak2 tyrosine kinase is essential for animal development and hyper kinetic Jak2 function has been linked to a host of human diseases. Control of this pathway using Jak2 specific inhibitors would therefore potentially serve as a useful research tool and/or therapeutic agent. Here, we used a high throughput program called DOCK, to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP binding site of murine Jak2. One sma ll molecule, 2 -methyl 1 -phenyl 4 -pyridin 2 -yl 2 (2 pyridin 2 -ylethyl)butan 1 -one (herein designated as Z3) bound to Jak2 with a favorable energy score. Z3 inhibited Jak2-V617F and Jak2 WT autophosphorylation in both a dose and time -dependent manner, but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 k inase function with no effect on Tyk2 or c Src kinase function. Z3 significantly inhibited proliferation of the Jak2-V617F -expressing, human erythroleukemia cell line, HEL 92.1.7. The Z3 -mediated reduction in cell proliferation correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cel ls isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2 F537I mutation. Collectively, the data suggest that Z3 is a novel specific inhib itor of Jak2 tyrosin e kinase. Introduction Aside from its essential role in embryonic development (Parganas et al., 1998), Jak2 plays an important role in pathophysiology. Jak2 has been linked to several hematological malignancies including acute lymphoid leukemia and chroni c myeloid leukemia (Lacronique et

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51 al, 1997, Peeters et al., 1997; Shuai K et al., 1996). Recently, a Jak2 -V617F somatic mutation has been identified in approximately 98% of patients with polycythemia vera (PV) and in a substantial proportion (50%) of pati ents with essential thrombocythemia ( ET) and primary myelofibrosis (PMF) (Levine et al., 2005). This valine to phenylalanine substitution (V617F) occurs in the JH2 pseudokinase domain of Jak2. These myeloproliferative disorders are characterized by the c lonal overproduction of normally differentiated hematopoietic lineages. The V617F substitution leads to constitutive activation of Jak2 and downstream effector signaling pathways including the STAT transcription pathway and the phosphatidylinositol 3 kina se and extracellular signal -regulated kinase signaling networks, which in turn induce inappropriate cytokine independent proliferation of cells (Levine et al., 2005; Shannon and Van Etten, 2005). In addition, the Jak2-V617F mutation contributes to myelofi brosis by constitutively phosphorylating STAT3 and diminishing myeloid c ell apoptosis (Mesa, 2007). Collectively, these results implicate dysregulated Jak2 signaling in various hematological and myeloproliferative disorders. As components of the Jak-STAT signaling pathway are hyper activated in some leukemias and myeloproliferative disorders, control of this pathway using pharmacological inhibition is highly desirable. As a research tool, AG490 is by far the most extensively used commercially available Jak2 inhibitor. Although it has been valuable in identifying Jak2 as a therapeutic target, it suffers from a general lack of specificity. For example, AG490 inhibits calf -serum inducible cell growth and DNA synthesis (Kleinberger Doro n et al., 1998) and is a partial blocker of c -Src activity (Oda et al, 1999). Most importantly, AG490 inhibits epidermal growth factor receptor autophosphorylation more potently than it inhibits Jak2 activity (Osherov et al.,

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52 1993, Gu et al., 2001). Thus, identification and c haracterization of novel Jak2 specific inhibitors may serve as useful research tools and therapeutic agents. Here, we utilized a rapid structure -based approach combining molecular docking with cell based functional testing in order to identify a Jak2 -selective inhibitor. One small molecule, 2 methyl 1 -phenyl 4 -pyridin 2 -yl 2 (2 pyridin 2 -ylethyl)butan 1 -one (herein designated as Z3) bound to a pocket that was adjacent to the ATP binding site of Jak2 with a favorable energy score. Our functional testing d ata suggest that Z3 is a novel Jak2 tyrosine kinase specific, small molecule inhibitor. Results Z3 Inhibits Jak2 Tyrosine Autophosphorylation by Interacting with a Solvent Accessible Pocket Adjacent to the ATP Binding Pocket of Jak2 Taking the crystal str ucture for portions of the Jak3 kinase domain (pdb code 1YVJ) into consideration, a comparative structural modeling approach was employed to generate an atomic model of the kinase domain of murine Jak2 using ProModII as described previously (Sandberg et al ., 2005). The program SPHGEN, which identifies potential ligand binding sites based on chemical and shape characteristics, was employed to select the target pocket on Jak2 for small molecule docking. To prepare the site for docking, all water molecules we re removed. Protonation of receptor residues was performed with the program Sybyl. The sphere set utilized for molecular docking was based on the position of a Jak3 inhibitor in the crystal structure of the kinase domain of Jak3 (Figure 3 1A). The posit ion of a pan Jak2 inhibitor that was subsequently crystallized in the kinase domain of h uman Jak2 is also shown (Fig ure .3 1A). The site selected for molecular docking was adjacent to the ATP binding site of murine Jak2 and the number of spheres in the sit e (50 100) was in the ideal range for binding to small molecules.

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53 Utilizing the DOCK program, we tested 20,000 compounds of known chemical structure, in silico, for their ability to interact with a structural pocket adjacent to the ATP binding site of the murine Jak2 kinase domain. The top six scoring compounds (Table 1) were obtained from the NCI/DTP and tested for their ability to inhibit Jak2 -WT and Ja k2-V617F autophosphorylation. A Jak2 over expression system w as used to first determine the e ffect of each inhibitor on Jak2 autophosphorylation. Specifically, BSC 40 cells were transfected with an expression vector encoding either empty vector control (no Jak2), the mutant murine Jak2 cDNA (Jak2-V617F) or the wild type Jak2 cDNA (Jak2-WT) under the contr ol of T7 RNA polymerase. This resulted in high -level Jak2 expression and subsequent tyrosine phosphorylation independent of exogenous ligand addition. The six inhibitor compounds (Z1 Z6) were each added at a concentration of 100 M and incubated overnight. Sixteen hours following addition of the compounds, cellular lysates were immunoprecipitated with an anti Jak2 polyclonal antibody and then immunoblotted with an anti -phosphotyrosine monoclonal antibody to detect tyros ine phosphorylated Jak2 (Fig ure 3 1B ). The results show that compound Z3 (methyl 1 phenyl 4 -pyridin 2 -yl 2 (2 pyridin 2 -ylethyl)butan 1 one) most effectively inhibited both Jak2-V617F and Jak2 WT tyrosine autophosphorylation. Clearly however, the ability of Z3 to inhibit Jak2 WT autophosphorylation was greater than when compared to Jak2 -V617F autophosphorylation. The membrane was then stripped and reprobed with an anti Jak2 polyclonal antibody to demonstrat e equal protein loading (Figure 3 1C ). The structure of th e Z3 compound is shown in Figure 3 1D Based on the data in Figure 3 1B, we returned to our molecular model of Jak2 and utilized the DOCK program to determine the position of Z3 on the Jak2 structure. Based on contact points and energy scores, the Z3

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54 inh ibitor was predicted to bind to the solvent accessible pocket adjacent to the ATP binding s ite as we had intended (Figure 3 1E ). Collectively, the data in Figure 3 1 indicat e that Z3 inhibits both Jak2WT and Jak2 -V617F tyrosine autophosphorylation (ie, kinase func tion) via an interaction with a critical solvent accessible pocket adjacent to the ATP binding site. Z3 Inhibits Jak2 Tyrosine Autophosphorylation in a Dose -Dependent Manner that is Independent of Cellular Cytotoxicity In order to obtain a bett er understanding of the inhibitory properties of Z3, we examined whether it could inhibit Jak2-WT and Jak2 -V617F tyrosine autophosphorylation in a dose dependent manner. Specifically, we sought to determine whether Z3 inhibits phosphorylation of the criti cally important tyrosine 1007 residue, as phosphorylation of this residue within the activation loop of Jak2 is necessary for maximal Jak2 activation (Feng et al., 1997). To meet this end, BSC 40 cells were transfected/infected with either a Jak2 -WT or a Jak2 -V617F plasmid and then treated for 16 hours with Z3 at the indicated concentrations. For the Jak2WT expressing cells, cell lysates were first immunoprecipitated with a Jak2 polyclonal antibody and then Western blotted with a phospho -specific (pY1007/pY1008) Jak2 antibody to detect phosphorylated Jak2 at this residue (Figure 3 2A ). The results indicate that Z3 inhibited phosphorylation of the critically important tyrosine 1007 residue on Jak2WT, in a dose dependent manner. The membrane was subseque ntly stripped and re -probed with an anti Jak2 polyclonal antibody to determine Jak2 expression levels amongst all samples (Figure 3 2B ). In order to quantitate Jak2 WT tyrosine 1007 phosphorylation levels in the presence of increasing amounts of Z3, densit ometry analysis on four representative w estern blots was performed (Figure 3 2C ). We found that Jak2 -WT tyrosine autophosphorylation was reduced by approximately 60 percent between the doses of 10 to 30 M of Z3. A 100 M dose of Z3 maximally suppressed Jak2 phosphorylation by about 92 percent. The estimated IC50 of Z3 for

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55 Jak2 WT was 15 M. For the Jak2 -V617F expressing samples, cell lysates were immunoprecipitated with a Jak2 antibody and then immunoblotted with the phospho-specific (pY1007/pY1008), a s we did previously (Figure 3 2D ). Similar to the Jak2 -WT expressing cells, Z3 inhibited Jak2 -V617F tyrosine 1007 phosphorylation in a dose -dependent fashion. The membrane was then stripped and reprobed with an anti Jak2 antibody in order to determine re lative Jak2 expression levels across all samples (Figure 3 2E ). Densitometry analysis on four representative Western blots showed that Jak2 -V617F tyrosine 1007 phosphorylation was inhibited by approximately 60 percent between the concentrations of 10 to 1 00 M of Z3 (Figure 3 2F ). The IC50 of Z3 for Jak2 -V617F was approximately 28 M. To rule out the possibility that the affects of Z3 were due to non-specific cellular toxicity rather than specific Jak2 inhibition, we treated these same BSC 40 cells with either DMSO or with 25, 100 or 250 M Z3 for 16 hours. The live cells were then stained with propidium iodide. Propidium iodide selectively stains necrotic cells and fluoresces red, but is excluded from the plasma membrane of intact cells. The results s how that cells treated with 25 or 100 M of Z3, showed propidium iodide staining similar to that of DMSO treated cells (Figure 3 2G ). However, cells treated with 250 M Z3 displayed a marked increase in propidium iodide staining, indicating that this dose of Z3 is cytotoxic. The same propidium iodide fields were also visualized by phase contrast microscopy to determine total cell numbers and o ve rall cellular morphology. Overall, the data indicate that the range of Z3 that inhibits Jak2 tyrosine kinase au tophosphorylation by 50% 100% (25100 M), does so in a manner that is independent of cellular cytotoxicity. Taken together, our results indicate that the Z3 compound blocks Jak2 autophosphorylation in a dose -dependent manner, but is not cytotoxic to cells at concentrations

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56 that inhibit Jak2 tyrosine kinase activity. Moreover, the ability of Z3 to inhibit Jak2 WT (IC50 = ~15 M) is greater when compared to Jak2 -V617F (IC50 = ~28 M). Z3 is a Specific Inhibitor of Jak2 Tyrosine Kinase To determine whether Z3 is selective for suppressing Jak2 autophosphorylation when compared to other Jak family members, we employed an autophosphorylation assay to allow for robust Jak family kinase activation independent of ligand treatment (Ma and Sayeski, 2004). Here, COS 7 cells were transiently transfected with an expression vector encoding the wild type human Tyk2 cDNA. The following day, the cells were treated with either DMSO or 25 M of Z3 for 16 hours. The cells were then lysed and Tyk2 was immunoprecipitated from the harvested lysates by the addition of a Tyk2 specific antibody. The immunoprecipitated protein was then immunoblotted with an anti -phosphotyrosine antibody to evalua te the level of Tyk2 tyrosine autophosphorylation (ie, catalytic activity). We found that Z3 did not inhibit Tyk2 tyrosine kinase autophosphorylation when compared to DMSO control (Figure 3 3 A ), bearing in mind that this same 25 M dose reduced Jak2 WT ty rosine kinase autophosphorylation by approximately 70% (Figure 3 2C ). The membrane was subsequently stripped and reprobed with anti Tyk2 antibody to demonstrate equal Tyk2 expre ssion for both samples (Figure 3 3 B). We next investigated the effect of Z3 on the ubiquitously expressed nonreceptor tyrosine kinase, c Src. Similar to Jak2, c -Src has been shown to activate several effectors that are also involved in aberrant cell growth. Here, catalytically active recombinant c Src protein was incubated in kina se reaction buffer, either in the presence of DMSO, 25 M of Z3, or 25 M of the Src kinase inhibitor, PP2. The reactions were incubated for 20 minutes at room temperature and then terminated by the addition of SDS containing sample buffer. The samples w ere separated by SDS -PAGE and subsequently Western blotted with an anti active c Src (pY418)

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57 antibody to determine c Src catalytic activity (Figure 3 3 C). We found that Z3 had no effect on c Src tyrosine kinase activity when compared to DMSO control. How ever, in the presence of the known c Src inhibitor, PP2, c -Src tyrosine kinase activity was completely abolished. To show equal c -Src protein content among all conditions, the membrane was stripped and reprobed wi th anti -c -Src antibody (Figure 3 3 D ). Coll ectively, the results demonstrate that while Z3 suppresses Jak2 tyrosine kinase activity, it does not inhibit Tyk2 and c Src. This suggests that Z3 is therefore reasonably specific for Jak2. Z3 Selectively Inhibits Jak2 V617F Dependent Cell Proliferation and this Correlates with Suppression of Jak2 and STAT3 Tyrosine Phosphorylation It is known that the human erythroleukemia cell line, termed HEL, is homozygous for the Jak2 V617F mutation (Quentmeir et al., 2006). The single point mutation leading to a V617F substitution in the JH2 domain of Jak2 has been associated with its proliferative phenotype (Quentmeir et al., 2006). In addition, it has been shown that expression of the constitutively activated Jak2 -V617F mutation is required for transformation of H EL cells. Mechanistically, the Jak2 V617F mutation promotes G1/S phase transition in HEL cells and subsequent increases in cellular proliferation (Walz et al., 2006). Due to the presence of the constitutively activated Jak2-V617F mutation in HEL cells, we wanted to determine whether Z3 could suppress Jak2-V617F dependent cell proliferation in this cell line. Here, 5 x 104 HEL cells were treated with either DMSO or 25 M of Z3 for 0, 16, 24, 48 and 72 hours. At the end of each time point, the number of vi able DMSO or Z3 treated cells was determined by trypan blue exclusion using a hemocytometer (Fig ure 3 -4 A). The results show that in the presence of DMSO, HEL cells were in rapid growth. However, treatment with Z3 reduced cell numbers when compared to DM SO. Analysis of variance indicated that the Z3 growth curve was significantly different from that of DMSO (p= 1.06 x 1014).

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58 To determine whether Z3 specifically suppresses Jak2 -V61F dependent cell growth as opposed to non-specific inhibition of cell pr oliferation we examined the inhibitory effect of Z3 on Raji cells. The molecular mechanism responsible for the aberrant growth of Raji cells is a translocation event between the c -Myc gene on chromosome 8 and the heavy chain locus on chromosome 14 (Hamly n and Rabbitts, 1983). Here 5 x 104 HEL or Raji cells were treated for 72 hours with either DMSO or Z3 at the indicated concentrations The number of viable cells was then determined (Figure 3 4 B) The results indicate that HEL cells were more sensitiv e to the inhibitory effects of Z3, over a wide range of doses, when compared to Raji cells (p= 5.28 x 1011). We next examined whether Z3 -dependent inhibition of cell proliferation correlated with suppression of Jak2 tyrosine autophosph orylation. Specifically, HEL cells were treated with either DMSO or 25 M Z3 for the indicated times. Protein lysates were first immunoprecipitated with anti Jak2 antibody and then Western blotted with an anti phosphotyrosine antibody to detect total tyrosi ne phosphorylated Jak2 (Figure 3 4 C). We found that Z3 dramatically reduced total Jak2 tyrosine autophosphorylation by 48 hours when compared to DMSO control and this correlated with inhibition of HEL cell prol iferation at this time point. The membrane was then stripped and reprobed with anti Jak2 antibody to demonstrate total Jak2 expression amongst all samples (Figure 3 4 D ). We next sought to determine whether Z3 could inhibit Jak2 phosphorylation at Tyr 1007 in these same cells. After treatment of the cells with DMSO or 25 M Z3 for the indicated times, whole cell protein lysates were Western blotted with the phospho -specific (pY1007/pY1008) Jak2 antibody to detect phosphorylate d Jak2 at this residue (Figure 3 4 E ). The results show that Z3 inhibited Jak2 phosphorylation at tyrosine 1007 in a time -dependent manner when compared

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59 to DMSO control. The membrane was subsequently stripped and reprobed with anti Jak2 antibody to demonstrate total Jak2 protein expressi on amongst all samples (Figure 3 4 F ). STAT3 is a known substrate of Jak2 and previous studies have shown that STAT3 is constitutively phosphorylated in HEL cells (Spiekermann et al., 2001; Faderl et al., 2007). We therefore examined whether Z3 -mediated inhibition of Jak2-dependent cell proliferation in HEL cells also c orrelates with reduced STAT3 phosphorylation. Specifically, protein cell lysates were immunoprecipitated with a STAT3 antibody and then immunoblotted with a phospho-specific (pY705) STAT3 antibody to asse ss active STAT3 levels (Figure 3 4 G ). We found tha t Z3 inhibited STAT3 tyrosine phosphorylation in HEL cells when compared to DMSO control, thus demonstrating that reduced phospho -STAT3 levels also correlate with reduced cell numbers. The membrane was stripped and reprobed with a STAT3 antibody to demons trate equal STAT3 conte nt amongst all samples (Fig ure 3 4 H ). Overall, the data in Figure 3 4 demonstrate that Z3 selectively blocks Jak2 -V617F pathologic cell growth and this corresponds with reduced levels of activated Jak2 and STAT3. Z3 Exerts its Effect on the Cell Cycle by Increasing the Percentage of HEL Cells in G1 Phase while Decreasing the Number of Cells in S phase To determine the mechanism of Z3 -mediated inhibition in cell growth, we first examined whether treatment with Z3 corresponded to an in crease in apoptosis in the HEL cells. Specifically, HEL cells were treated with either DMSO or 25 M of Z3 for 16, 24, 48 or 72 hours and cells were measured for Annexin V staining via flow cytometry. Treatment of cells with Z3 did not result in a signif icant increase in the percentage of Annexin V -positive -stained cells when compared to DMSO -treated cells (data not shown), indicating that Z3 did not induce apoptosis in this cell type. In addition, HEL cells treated with 25 M of Z3 stained negative for

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60 an early marker of apoptosis, APO2.7, confirming that the mechanism by which Z3 suppresses HEL cell proliferation is independent of apoptosis (data not shown). We next examined cell cycle distribution as a function of Z3 treatment using the CycleTestTM PLU S DNA Kit. HEL cells were treated with either DMSO or 25 M of Z3 for 16, 24, 48 or 72 hours and cell cycle variables were determined by fluorescence activated cell sorting analysis. We found that Z3 significantly increased the percentage of cells in the G1 phase (Fig ure 3 5 A) and significantly decreased the pe rcentage of cells in S phase (Figure 3 5 B) when compared to DMSO control treated cells. A representative experiment of the cell cycle profile for DMSO and Z3 treated H EL cells is also shown (Figure 3 5 C and 3 5 D, respectively). All together, the result s in Fi gure 3 5 reveal that the mechanism by which Z3 reduces cell numbers is by blocking G1S transition. Z3 Reduces Hematopoietic Colony Formatio n Ex Vivo Somatic Jak2 mutations have been described in myeloproliferative disorders (Gruneback et al., 2006; Malinge et al, 2007; Scott et al., 2007). The most common Jak2 mutation, Jak2V617F, h as been shown to play an essential role in the pathogenesis of several myeloproliferative diseases including polycythemia vera, essential thrombocythemia and primary my elofibrosis. We have already shown that Z3 suppresses Jak2 -dependent cell growt h in the HEL cell line (Fig ure 3 4 A). We next turned our attention to determining whether Z3 could inhibit the growth of hematopoietic progenitor cells isolated from the bone marrow of confirmed myeloproliferative disorder patients. Here, residual bone marrow aspirates were obtained from an essential thrombocythemia patient who was Jak2 -V617F positive (Figure 3 6 A) and from a polycythemia vera patient who w as Jak2 -F537I positi ve (Figure 3 6 B). Their hematopoietic progenitor cells were cultured in a semisolid colony assay medium in the presence

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61 of DMSO or 25 M of Z3. In addition, since hematopoietic progenitors taken from patients with myeloproliferative disorders are known to be hypersensitive to cytokine stimulation (Axelrad et al., 2000), the cells taken from the ET patient were cultured in both the prese nce and absence of human thrombopoietin, whereas the cells taken from the polycythemia vera patient were cultured in both the presence and absence of human erythropoietin. The results show that, as expected, treatment of the essential thrombocythemia pati ents hematopoietic progenitor cells with thrombopoietin markedly increased megakary ocyte colony formation (Figure 3 6 A). However, this cytokine -dependent increase in cell growth was significantly blunted when the cells were cultured in the presence of Z3. Similarly, treatment of the polycythemia vera patients progenitor cells with erythropoietin significantly increased erythrocyte colony formation. However, treatment of the cell with Z3 significantly reduced both cytokine independent as well as cytokin e -dependent erythrocyte colony formation (Figure 3 6B). Colle ctively, the results in Figure 6 show that Z3 greatly reduces Jak2 -V617F as well as Jak2F537I -mediated, human pathologic cell growth ex vivo Discussion Hyperkinetic Jak2 tyrosine kinase activit y has been linked to a variety of human diseases including cardiovascular disease, diabetes and cancer (Neubauer H et al., 1998, Wang X et al., 2002, Real PJ et al., 2002). In recent years, the Jak2-V617F gain-of -function mutation in myeloproliferative di sorders has also been well described (Van Etten and Shannon, 2004; Levine RL et al, 2005; Shannon and Van Etten, 2005; Mesa, 2007; Wernig et al., 2006). Mutations in Exon 12 of Jak2 which cause dysregulated Jak-STAT signaling have been identified in Jak2 V617F negative myeloproliferative disorders as well (Pardanani et al., 2007a; Scott et al., 2007;

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62 Pietra et al., 2008). Thus, the continued identification of novel agents that can inhibit aberrant Jak2 tyrosine kinase function will be of great value. Here, we have used in silico homology modeling and high throughput molecular docking to identify a novel small molecule inhibitor of Jak2. Our results are significant for a number of reasons. First, Z3 inhibits Jak2 WT and Jak2 -V617F autophosphorylation, but is not cytotoxic to cells at concentrations that inhibit Jak2. Second, Z3 inhibits phosphorylation of tyrosine 1007, a residue whose phosphorylation is concomitant with hyper kinetic Jak2 function. Third, Z3 selectively inhibits Jak2 tyrosine kinase act ivity as it has no effect of Tyk2 and c -Src. Fourth, Z3 preferentially suppresses proliferation of HEL cells that express the Jak2 -V617F mutation by inducing cell cycle arrest. This arrest in cell growth directly correlates with reduced levels of active Jak2 and STAT3 proteins. Fifth, Z3 significantly blocks the growth of hematopoietic progenitor cells isolated from the bone marrow of myeloproliferative disorder patients carrying Jak2 mutations. The Z3 compound is yet another step in the continuing devel opment of Jak2 inhibitors. High throughput screening of potential tyrosine kinase inhibitors identified tyrphostin B42 (AG490) as the first Jak2 inhibitor. Initially, AG490 was regarded as a specific inhibitor of Jak2 because it concomitantly inhibited J ak2 tyrosine phosphorylation levels and suppressed acute lymphoblastic leukemia by inducing cellular apoptosis (Meydan et al., 1996). However, other studies suggested that it suffered from a general lack of specificity (Osherov et al., 1993; Oda et al., 1 999; Gu et al., 2001). Ensuing work by multiple groups, including our own, has identified various small molecules or in some cases, a protein mimetic, that block Jak2 kinase function (Sandberg et al., 2005; Flowers et al., 2004; Duan et al, 2007, Pardanani et al., 2007). Despite the fact that these agents all inhibit Jak2 kinase function in some manner, differences in both the

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63 structure as well the mechanism of action of the inhibitors is striking. For example, AG490 inhibits Jak2 and promotes cellular a poptosis (Meydan et al., 1996) whereas our Z3 compound inhibits Jak2 and promotes cell cycle arrest. Interestingly, the TG101209 compound described by Pardanani et al inhibits Jak2 and causes both increased apoptosis and cell cycle arrest (Pardanani et a l., 2007). Finally, work by Flowers and colleagues characterized a peptide inhibitor of Jak2 that mimics the Jak2 inhibitory protein, SOCS 1 (Flowers et al., 2004). The peptide mimetic was designed to recognize this autophosphorylation site of Jak2 simil ar to SOCS 1. They found that the peptide mimicked SOCS 1 in that it suppressed Jak2 tyrosine autophosphorylation and subsequent IFN dependent signaling independent of marked changes in apoptosis or cell cycle progression. Collectively, these works suggest that the relationship between blocking Jak2 kinase function and the eventual fate of the cell is complex and warrants further exa mination. Although the Jak2-V617F mutation on exon 14 is the predominant disease associated allele in myeloproliferative disorders, several other Jak2 exon 14 mutations have been identified in Jak2 -V617F -negative, myeloproliferative disorder patients. For example, unique C616Y and D620E substitution mutations have been identified in V617F -negative, myeloproliferative disorder individuals (Schnittger et al., 2006; Zhang et al., 2007). Additionally, a number of mutations have been identified in Jak2 exon 12, including an F537 deletion in one individual and F537 duplication in another (Scott et al., 2007). Furthermore, chromosomal translocations between the Jak2 allele and other alleles, including TEL, REL, PCM1, and BCR, have all been linked to a number of hematological malignancies (Peeters et al., 1997; Joos et al., 2003; Griesinger et al., 2005; Reiter et al., 2005). Although each specific translocation gives rise to a unique chimeric protein, they all share one common feature in that they all exhibit hyperkinetic

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64 Jak2 kinase activity and subsequent malignant hyperplasia. Therefore, given the growing number of known Jak2 somatic cell mutations and chromosomal translocations, as well as the diverse human diseases that hyperkinetic Jak2 kinase activity has been associated with, identifying inhibitors that can block multiple Jak2 mutations, such as our Z3 compound, will be of great value. In summary, our results suggest that Z3 specifically inhibits Jak2 tyrosine kinase function. It suppresses Jak2 dependent pathologic cell growth in vitro via a Jak2/STAT3 dependent mechanism that results in cell cycle arrest. Additionally, it blocks ex vivo hematopoietic progenitor cell growth from an essential thrombocythemia patient who harbors Jak2 -V617F and a polycythe mia vera patient who carries a novel Jak2 -F537I mutation. As such, this compound may have practical applications in Jak2 related research.

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65 Table 3 1. Top six scoring Z compounds Compound # NSC # Formula Name mol wt Z1 302088 C8H6O5 5 hydroxybenzene 1,3 dicarboxylic acid 182 Z2 302311 C8H5FO4 5 fluorobenzene 1,3 dicarboxylic acid 184 Z3 42834 C23H24N2O 2 methyl 1 phenyl 4 pyridin 2 yl 2 (2 pyridin 2 ylethyl)butan 1 one 344 Z4 43744 C9H10O4 3,5 dimethoxybenzoic acid 182 Z5 70313 C8H9NO4S 4 methanesulfonamidobenzoic acid 215 Z6 113790 C19H24N2O3 3 hydroxy 4 [(4 methylpiperazin 1 yl)methyl] 7,8,9,10tetrahydrobenzo[c]chromen 6 one 328

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66 Z1 DMSO Z1 Z2 Z3 Z4 Z5 DMSO Z6No JaksZ3 Z2 Z4 Z5 Z6111 kDa 111 kDa Jak2(P ) Jak2 IP: anti Jak2pAb IB: anti Tyr(p) mAb IP: anti Jak2pAb IP: anti Jak2pAb Jak2V617F Jak2WT O N N A B E D C Figure 3 1 2 methyl 1 -phenyl 4 pyridin 2 yl 2 -(2 -pyridin 2 -ylethyl)butan -1 one (Z3) inhibits Jak2 V617F and Jak2WT tyrosine autophosphorylation. A) The sphere set used for molecular docking was based on the position of a solvent accessible pocket adjacent to the Jak2 activation loop. For reference, th e positions of the resolved structures for portions of both the Jak2 and Jak3 kinase domains are indicated. Also shown is the position of the IZA pan Jak2 inhibitor that was crystallized within the Jak2 kinase domain. B) BSC 40 cells were transfected with empty vector control, Jak2-V617F, or Jak2 -WT expression plasmids and then infected with vaccinia virus to drive high level expression and subsequent Jak2 tyrosine autophosphorylation. The six highest scoring compounds identified by DOCK were incubated wi th the cells at a concentration of 100 M each, for 16 hours. Cell lysates were immunoprecipitated with antiJak2 antibody and immunoblotted with anti -phosphotyrosine antibody to detect Jak2 tyrosine p hosphorylation levels C) The membrane was stripped and reprobed with anti Jak2 antibody to demonstrate equal Jak2 expression among all samples Shown is one of tw o independent experiments. D ) Shown is the molecular st ructure of the Z3 compound. E ) Based on contact points and energy scores, Z3 docks into a structural pocket on the Jak2 kinase domain that is adjacent to the ATP binding site.

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67 10 M 0 M Jak2WTA D 1 M 3 M 30 M 100 MIP: anti Jak2 pAb IB: anti Jak2 pY1007/Y1008pAb0 M 1 M 3 M 10 M 30 M 100 MIP: anti Jak2 pAb IB: anti Jak2 pY1007/Y1008pAb Jak2V617F Jak2 Jak2 Jak2(P) Jak2(P) IP: anti Jak2pAb IP: anti Jak2pAb IB: anti Jak2 pAb IB: anti Jak2 pAb 111 kDa 111 kDa 111 kDa 111 kDaC0 1 3 10 30 100 MFJak2 Phos :Percent Control Jak2 Phos :Percent Control0 1 3 10 30 100 M20 40 60 80 100 120 140 0 0 20 40 60 80 100 120 140 Jak2WT Jak2V617F[Z3] [Z3] DMSO Z3 (25 Z3 (100 M ) Z3 (250 M ) Phase Contrast Propidium IodideG B E Figure 3 2. Z3 inhibits Jak2 tyrosine autophosphoryla tion in a dose -dependent manner. A) BSC 40 cells were transfected/infected as described above. Jak2-WT -expressing ce lls were incubated with either vehicle control (DMSO) or with Z3 at the indicated doses. Protein cell lysates were then immunoprecipitated with anti Jak2 antibody and immunoblotted with a phospho-specific (pY1007/pY1008) Jak2 antibody to detect phosphoryl ated Jak 2 at this specific residue B) The membrane was stripped and reprobed with anti Jak2 antibody to demonstrate equal Jak2 expression among all samples C ) Densitometrical analysis was performed on four representative Western blots to quantify Z3 -me diated inhibition of WT Jak2 phosphorylation at the tyrosine 1007 residue. Data are represented as the ratio of phosphorylated Jak2 to total Jak2

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68 and presented as the mean +/ SEM. The IC50 of Z3 for Jak2WT was approximately 15 M. Statistical signific ance between the vehicle control and Z3 treated cells was determined by a one -way analysis of variance (ANOVA) (p<0.001). D ) For Jak2 V617F expression, cells were first immunoprecipitated with an anti Jak2 polyclonal antibody and then Western blotted with a phospho-specific (pY 1007/pY1008) Jak2 antibody. E) The membrane was then stripped and reprobed with a nti Jak2 antibody F ) Densitometry analysis of four representative Western blots showing Z3 -mediated inhibition of Jak2-V617F phosphorylation at the t yrosine 1007 residue. Data are represented as the ratio of phosphorylated Jak2 to total Jak2 and presented as the mean +/ SEM. The IC50 of Z3 for Jak2 -V617F was approximately 28 M. There was a statistically significant difference between the vehicle c ontrol and Z3 treated cells (p<0.001). G ) BSC 40 cells were treated for 16 hours with either DMSO or with 25, 100, or 250 M of Z3. Live cells were then stained with 1 g/mL propidium iodide to determine whether Z3 was cytotoxic. The cells were visualiz ed using confocal mi croscopy under fluorescent and phase contrast conditions. Shown is one of two representative results.

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69 IB: anti c Src pY418pAb IB: anti c Src pAb c Src(P ) c Src 49 49 DMSO Z3, 25 M PP2, 25 MC A IP: anti Tyk2 pAb IB: anti Tyr(p) mAb IB: anti Tyk2 pAb Tyk2 Tyk2(P) 111 79 111 79 172 172 DMSO Z3, 25 MB D Figure 3 3. Z3 selectively inhibits Jak2 tyrosine kinase activity A) COS 7 cells were transientl y transfected with an expression vector encoding wild type human Tyk2 cDNA. Cells were subsequently treated with either DMSO or 25 M of Z3 for 16 hours. Protein lysates were immunoprecipitated with anti Tyk2 antibody and then Western blotted with antiphosphotyrosine antibodies to detec t Tyk2 autophosphorylation. B) The membrane was stripped and reprobed with anti Tyk2 antibody to show equal Tyk2 expression among all samples C ) Catalytically active c -Src protein was incubated in kinase reaction buffer containing either DMSO, 25 M of Z3, or 25 M of PP2. The samples were subsequently immunoblotted with anti phospho c -Src (pY418) antibody to determine relative c -Sr c tyrosine kinase activity D) The membrane was then stripped and reprobed with c -Src antibody to asse ss c -Src protein levels Shown is one of three representative experiments for each.

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70 Jak2(P) Jak2 IP: anti Jak2pAb IB: anti Tyr(p) mAb IP: anti Jak2pAb IB: anti Jak2 pAb 111 kDa 111 kDaDMSO Z3 DMSO Z3 DMSO Z3 Z3 DMSO 16 24 48 72 hrs.B A STAT3(P) STAT3 IB: anti STAT3 pY705mAb IP: STAT3 pAb IP: STAT3 pAb IB: STAT3 pAb 80 kDa 80 kDa DMSO Z3 DMSO Z3 DMSO Z3 Z3 DMSO 16 24 48 72 hrs.G Raji HEL 0 0.1 0.3 1 3 10 30 20 40 60 80 100 120 MViable Cells : Percent Control0 20 40 60 80 100x 103200x 103300x 103400x 103500x 103600x 103700x 103800x 1030Viable Cells Time (hours) DMSO Z3 [Z3]C Jak2(P) Jak2 IB: anti Jak2 pY1007/Y1008pAb IB: anti Jak2 pAb 111 kDa 111 kDaZ3 DMSO Z3 DMSO DMSO Z3 DMSO Z3 72 48 24 16 hrs.E D F H Figure 3 4. Z3 selectively inhibits Jak2-V617F -dependent cell proliferation and this correlates with suppression of Jak2 and STAT3 tyrosine phosphorylation A) Approximatlely 50,000 HEL cells were treated with either DMSO or 25 M of Z3 for 0, 16, 24, 48, or 72 hours. The numbers of viable DMSO or Z3 treated cells were assessed by trypan blue exclusion. All data points were measured in triplicate. Statistical significance between each group was analyzed using a two-way analysi s of variance (ANOVA) by comparing the entire DMSO curve with the entire Z3 curve. The two conditions were found to be significantly different (p=1.06 X 1014). B) Approximately 50,000 HEL and Raji cells were treated for 72 hours with either DMSO or with 0.1, 0.3, 1, 3, 10,

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71 or 30 M of Z After 72 hours, the numbers of viable DMSO or Z3 treated cells were assessed as described above. The graph shown is a compilation of three independent experiments. Statistical significance between each group was analyzed using a two-way analy sis of variance (ANOVA) by comaring the entire HEL curve with the entire Raji Curve The two conditions were found to be significantly different (p=5.28 X 1011). C) Approximately 50,000 HEL cells were treated with either DMSO or 25 M of Z3 for 16, 24, 48, or 72 hours. Protein lysates were immunoprecipitated with anti Jak2 antibody and then immunblotted with anti phosphotyrosine antibody to determine total J ak2 phosphorylation levels D) The membrane was stripped and reprobed with Jak2 antibody to demonstrate Jak2 expression among all samples E ) Approximately 50,000 HEL cells were treated as indicated. Jak2 Y1007 phosphoryalation levels were determined by immunoblotting with anti active Jak2 antibody F) The membrane was stripped and reprobed with J ak2 to show equal Jak2 expre ssion among all samples G ) Approximately 50,000 HEL cells were treated as indicated. STAT3 phosphorylation levels were determined by first immunoprecipitating with STAT3 antibody and then immunobloting with a phospho-speci fic (pY705) STAT3 antibody. H) The membrane was stripped and reprobed with STAT3 to show total STAT3 protein content among all samples Shown is one of three (A, B E F) or two (C, D, G, H ) representative experiments

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72 CDMSO, 72 hrsDZ3, 72 hrs DMSO Z3 DMSO Z3 10 20 30 40 50 60 70 80Time (hours)Percent G1Phase10 20 30 40 50 60 0A10 20 30 40 50 60 70 80Percent S Phase10 20 30 40 50 60 0BTime (hours) Figu re 3 5. Z3 induces cell cycle arrest in Jak2 -V617F transfor med human erythroleukemia cells. Aprroximately 50,000 HEL cells were treated with either DMSO or 25 M of Z3 for 16, 24, 48, or 72 hours and cell -cycle effects were determined. FACSCalibur flow cytometer along with Modfit software (Verity Software) was used to analyze DNA contents. The mean and standard deviation of samples were determined. Statistical significance between each group was analyzed using a two -way an alysis of variance (ANOVA) by c omaring the entire DMSO curve with the entire Z3 curve A) Percentage of HEL cells in G1 phase following treatment with either DMSO or Z3 for the indicated times. The two conditions were considered to be significantl y different (p=0.000407). B) Percenta ge of HEL cells in S phase following treatment with either DMSO or Z3 for the indicated times. The two conditions were considered to be significantly different (p=0.0256). Shown is one of three independent experiments (A and B). Representative cell cycl e analysis profile from one of those three experiments after 72 hours treatment with either DMSO (C) or Z3 (D).

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73 0 5 10 15 20 25 30 + +# CFU -MegsTPODMSO Z3 *A 0 10 20 30 40 50 + +# CFU -EDMSO Z3 ** BEPO Figure 3 6. Z3 suppresses Jak2-mediated hematopoietic colony formation ex vivo A) Marrow derived mononucle ar cells from an essential thrombocythemia patient who was Jak2 V617F positive were cultured in methylcellulose media containing either DMSO (0.25%, vol/vol) or 25 M Z3. The number of megakaryocyte colonies was assessed 14 days later. Results are expres sed as the average number of colonies from duplicate cultures per 4 x 105 cells. Statistical significance between each group was analyzed using Students t test. p = 0.017. B) Marrow derived mononuclear cells from a polycythemia vera patient who was J ak2 F537I positive were cultured in methylcellulose media containing either DMSO (0.25%, vol/vol) or 25 M Z3. The number of erythrocyte colonies was assessed 14 days later. Results are expressed as the average number of colonies from duplicate cultures per 4 x 105 cells. Statistical significance between each group was analyzed using Students t test. p = 0.0245, ** p = 0.0195.

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74 CHAPTER 4 CHARACTERIZATION OF A SERIES OF NOVEL JAK2 INHIBITORS AND THEIR AFFECT ON JAK2 MEDIATED DISEASES Summary Jak2 tyrosine kinase is important in both physiology and pathophysiology as it plays significant roles in embryo nic development, cell signaling, as well as heart disease, diabetes and cancer. In addition, a novel Jak2 gain-of -function, somatic mutation (Jak2 -V617F) has been linked to several myeloproliferative disorders including polycythemia vera, essential thromb ocythemia and primary myelofibrosis. Control of aberrant Jak2 kinase function through the use of a novel Jak2 selective small molecule inhibitor would therefore potentially serve as a useful research tool and/or therapeutic agent. Recent work from our la b has been aimed at identifying novel Jak2 tyrosine kinase inhibitor molecules. Here, we refined our previous Jak2 molecular model using information obtained from the crystal structures of the Jak2 and Jak3 kinase domains. A total of 223,481 compounds wi thin the NIH small molecule database were then screened in silico using FlexX 1.13.2 in order to identify compounds that specifically bind and inhibit Jak2. In what is now our third generation of Jak2 inhibitors, analysis of the highest scoring compounds identified a set of structurally diverse molecules that potently inhibited Jak2 autophosphorylation n ot only at Tyr 1007, but at other tyrosine residues as well These compounds significantly inhibited proliferation of the human erythroleukemia (HEL) cell s, which express the Jak2-V617F mutation on both alleles. One compound in particular, herein designated as G6, was further examined in greater detail. The mechanism by which G6 inhibits Jak2 V617F dependent cell growth is via a marked increase in cellula r apoptosis. We found that G6 does not inhibit c Src or Tyk2 autophosphorylation at doses that completely inhibit Jak2, therefore sugge sting a degree of specificity. In addition, G6 selectively suppressed Jak2 -V617F mediated pathologic cell growth. Fina lly, w e found that the G6 and G13 compounds

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75 significantly reduced ex vivo hematopoietic colony formation of cells derived from an ET patient harboring the Jak2-V617F mutation and a PV patient c arrying a Jak2 -F537I mutation. Collectively, our data demonstr ate that these small molecule compounds inhibit Jak2 function in vitro and ex vivo As such, they may have therapeutic value in treating diseases that are caused by aberrant Jak2 kinase function. Introduction Myeloproliferative neoplasms which include po lycythemia vera, essential thrombocythemia and primary myelofibrosis entered the spotlight in 2005 when a somatic acquired Jak2 -V617F mutation was described in these disorders. To date, the Jak2-V617F mutation is harbored by virtually all polycythemia ver a patients and by more than 50% of essential thrombocythemia and primary myelofibrosis patients (James et al., 2005; Baxter et al., 2005; Kralovics et al., 2005; Levine et al., 2005; Zhao et al., 2005). Subsequent reports have identified Jak2 exon mutations in polycythemia vera patients who do not carry the Jak2-V617F mutation (Scott et al, 2007, Pardanani et al., 2007a, Sayyah et al., 2008). Specifically, several deletions and insertions were noted in the case of exon 12 mutations (Scott et al., 2007; Butcher et al., 2008). A common feature between the exon 12 mutations and the V617F mutation on exon 14 is that they both result in constitutive Jak2 tyrosine kinase activity. The driving force for the identification of Jak2 inhibitors has been the discover y of the Jak2 V617F mutation in myeloproliferative disorders. However, the continued observation of novel Jak2 somatic cell mutations and chromosomal translocations in hematologic disorders also validates the search for specific inhibitors that target abe rrant Jak2 activity (Schnittger et al., 2006; Scott et al., 2007; Zhang et al, 2007; Kearney et al., 2008; Nebral et al., 2009). In fact, several Jak2 inhibitors have been developed. One such molecule, TG101209, was able to inhibit phosphorylation of Jak 2 -V617F, STAT5 and STAT3 in a Jak2-V617F -expressing acute myeloid

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76 leukemia cell line (Pardanani et al., 2007). In addition, TG101209 suppressed the proliferation of both Jak2-V617F and TpoR W515L/K -expressing hematopoietic cells (Pardanani et al., 2007a) Another Jak2-specific inhibitor, TG101348, was effective in significantly reducing hematocrit levels in a Jak2 -V617F induced myeloproliferative disorder mouse model and inhibiting the growth of primary hematopoietic cells derived from myeloproliferative disorder patients with Jak2-V617F, MPLW515K and Jak2 exon 12 mutations (Lasho et al., 2008; Wernig et al, 2008). Moreover, this compound inhibited the engraftment of Jak2-V617F positive hematopoietic stem cells and myeloid progenitors in a bioluminescent xenogeneic mouse transplantation model (Geron et al., 2008). Importantly, TG101348 reduced GATA1 phosphorylation, as phosphorylation of this transcription factor can be associated with erythroid skewing of Jak2-V617F -positive progenitor differentiation. All together, characterization of the TG compounds reveal that not only do they potently inhibit aberrant Jak2 activity, but they also suppress pathologic cell growth due mutation in the thrombopoietin receptor. The most extensive clinical trial of a Ja k2 inhibitor for the treatment of myeloproliferative disorders has been with ICNB018424. ICNB018424 inhibits Jak1 and Jak2, but not Jak3 or TYK2 at clinically achievable concentrations (Verstovek et al., 2008a). Primary myelofibrosis patients treated wit h this compound displayed reduced splenomegaly and showed marked improvements in disease associated symptoms. To date, there have only been modest reductions in Jak2 -V617F allele burden. Collectively, a growing number of Jak2 inhibitors are being identif ied that selectively target aberrant Jak2 tyrosine kinase activity in vitro ex vivo and in vivo In addition, some of these inhibitors have already entered or are expected to go into clinical trials for the treatment of myeloproliferative disorders.

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77 Our laboratory has contributed to the identification of novel Jak2 -selective inhibitors that could one day have therapeutic value for the treatment of myeloproliferative disorders or other Jak2 related hematologic malignancies. By using a structure based appr oach of combining molecular docking with cell -based functional testing, we have identified a series of novel Jak2 inhibitors that potently inhibit Jak2 tyrosine kinase function. In particular, one of these compounds, designated as G6, potently and selecti vely inhibits Jak2-mediated aberrant cell growth by causing an increase in cellular apoptosis. Furthermore, G6 blunts the growth of hematopoietic progenitor cells derived from myeloproliferative disorder patients displaying Jak2 mutations. All together, our results suggest that G6 is a novel, selective and potent inhibitor of Jak2 tyrosine kinase activity. Results G6 Potently and Selectively Suppresses Jak2 V617F -Dependent Aberrant Cell Growth An aim of our laboratory in recent years has been directed to wards the identification of novel Jak2 inhibitors (Sandberg et al. 2005; Sayyah et al., 2008). Here, we refined our earlier Jak2 molecular model using information from the crystal structures of the Jak2 and Jak3 kinase domain. We subsequently screened in silico a total of 223, 281 compounds of known structure using FlexX in order to identify compounds that specifically bind to Jak2. The top scoring G compounds were then examined for their ability to inhibit proliferation of a pathologically relevant cell line which expresses the Jak2 -V617F mutation. For this, we employed HEL cells which are human erythroleukemia cells that require the constitutively activated Jak2-V617F mutation for their transformation and proliferation (Quentmeir et al., 2006; Walz et al., 2006). To ascertain if the G compounds could block Jak2-V617F -dependent cell proliferation in this cell line, 5 x 104 HEL cells were treated with either DMSO or 25 M of each of the G compounds for 0, 16, 24, 48, and 72 hours. Our previous work has established that Z3

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78 suppresses HEL cell proliferation (Say yah et al., 2008). To compare the HEL cell growth inhibitory potential of G6 with that of Z3, HEL cells were al so treated with 25 M Z3 for the indicated times. At the end of each time point, the number of viable DMSO, Z3, or G compoundtreated cells was determined by trypan blue exclusion via the use of a hemacytometer (Figure 4 1A). Relative to DMSO control, th e G13, G11 and Z3 compounds moderately reduced HEL cell growth by approximately 28%, 30%, and 42%, respectively. However, the G6 compound was more successful in inhibiting cell growth in comparison. By 72 hours, G6 inhibited HEL cell proliferation by ove r 90% relative to DMSO control. ANOVA indicated that the G6, G13, G11 and Z3 growth curves were statistically significant from that of DMSO. Although G6 exhibited potent anti -cell growth ability, it was important to determine whether this compound was sel ectively suppressing Jak2-V61 7 F dependent cell growth or inhibiting cell proliferation in a non -specific manner. To answer this question, we examined the growth inhibitory effect of G6 on several cell lines such as, Raji, CMK and BSC 40 that proliferate i ndependent of Jak2. The mechanism responsible for the abnormal growth of Raji cells is a translocation event involving the c -myc gene while the abnormal growth of CMK cells is a result of an activating A572V mutation in the Jak3 pseudokinase domain (Hamly n and Rabbitts, 1983; Walters et al, 2006). BSC 40 cells are monkey kidney epithelial cells transformed with SV40 T antigen. Here, 5 x 104 HEL, Raji, CMK, or BSC -40 cells were treated with G6 at the indicated concentrations. The number of viable cells w as subsequently determined (Figure 4 1B). The results reveal that HEL cells were more susceptible to the growth suppressive effects of G6, over a wide range of doses, when compared with Raji, CMK, or BSC 40 cells. This indicates that G6 inhibits Jak2 -V61 7F dependent cell growth in a relatively

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79 specific manner. All together, the results in Figure 4 1 show that G6 is a potent and selective inhibitor of Jak2 -V617F -dependent pathologic cell growth. G6 Suppresses HEL Cell Proliferation by Inducing Cellular A poptosis We then wanted to uncover the mechanism by which G6 blocks Jak2-V617F dependent HEL cell growth by examining whether treatment with G6 corresponds to an increase in apoptosis in HEL cells. Specifically, HEL cells were treated with either DMSO or 25 M G6 for 16, 24, or 48 hours and cells were analyzed by flow cytometry for Annexin V staining (Fig ure 4 2). The simultaneous application of propidium iodide allowed for the distinction between necrotic and apoptotic cells since propidium iodide will s tain the DNA of only leaky necrotic cells. A representative experiment at 48 hours treatment with G6 showed an increase in the percentage of Annexin V -stained HEL cells (Fig ure 4 2A) relative to DMSO control (Fig ure 4 2B), indicating that G6 induced apopt osis in this cell type. The percent of DMSO or G6 treated cells were then plo tted as a function of time (Fig ure 4 2C). The results show that G6 increased the percentage of HEL cells undergoing apoptosis over time. By 48 hours treatment cells with G6, t here was approximately a 45% increase in cellular apoptosis. Collectively, the results in Figure 4 2 show that the mechanism by which G6 reduces Jak2 -V617F dependent pathologic cell growth is by promoting apoptosis. G6 is a Specific Inhibitor of Jak2 Tyro sine Kinase Activity While our results showed that G6 selectively inhibits Jak2 -V617F -dependent HEL cell growth, another way to establish the specificity of G6 for Jak2 would be to examine the effect of this compound on the phosphorylation of other Jak fam ily tyrosine kinases, such as Tyk2. To achieve this goal, we employed an autophosphorylation assay to allow for strong Jak family kinase activation independent of ligand treatment, as we had done previously (Ma and Sayeski, 2004; Sayyah et al., 2008). He re, COS 7 cells were transiently transfected with an expression

PAGE 80

80 vector encoding the wild -type human Tyk2 cDNA. The cells were subsequently treate d with either DMSO, 100 M Z3, or 100 M G6 for 16 hours. The cells were then lysed and Tyk2 was immunoprecipitated from the lysates through the use of an anti Tyk2 antibody. The immunoprecipitated protein was then immunoblotted with an anti phosphotyrosine antibody to assess t he level of Tyk2 tyrosine kinase autophosphorylation (catalytic activity). The results show that G6, similar to Z3, had little to no effect on inhibiting Tyk2 tyrosine kinase autophosphorylation whe n compared to DMSO control (Figure 4 3A ). The membrane was then stripped and reprobed with an anti Tyk2 antibody to demonstrate equal Tyk 2 expression among samples (Figure 4 3B ) To further confirm the specificity of G6, we investigated the effect of this compound on c Src tyrosine kinase activity. Here, we in cubated catalytically active recombinant cSrc protein either in the presence of DMSO, 25 M Z3, 25 M G6, or 25 M of the Src kinase inhibitor, PP2 and incubated the reactions for 20 minutes at room temperature. The reactions were then terminated by the addition of SDS -containing sample buffer, separated by SDS PAGE and subsequently Western blotted with an anti active c Src (pY418) antibody to assess c Src tyrosine kinase activity (Fig ure 4 3C ). We observed that G6 similar to Z3, had no significant eff ect on c Src catalytic activity when compared to DMSO control, but in the presence of PP2, c -Src tyrosine kinase activity was completely eliminated. Equal c Src protein content among all samples was verified by stripping and reprobing the membrane with anti -c -Src antibody (Figure 4 3D ). All together, the results confirm that G6 is a relatively specific inhibitor of Jak2 tyrosine kinase activity as demonstrated by the fact that this compound has little to no activity against Tyk2 or c Src.

PAGE 81

81 The G C ompounds Effectively Block Jak2-Mediated Hematopoietic Colony F ormation Ex V ivo Our previous report shows that Z3 suppresses the ex vivo growth of Jak2 -mediated hematopoietic colonies (Sayyah et al., 2008). We have already demonstrated that G6 potently and specif ically inhibits Jak2 -dependent pathologic cell growth in vitro We next wanted to determine if G6 could block the ex vivo growth of hematopoietic progenitor cells isolated from the bone marrow of confirmed myeloprolilferative disorder patients. We also w anted to compare the ex vivo cell growth inhibitory potential of G6 with that of Z3. Specifically, residual bone marrow aspirates were obtained from an essential thrombocythemia patient w ho was Jak2 V617F positive (Figure 4 4A) or from a polycythemia vera patient who displayed a Jak2F537I exon 12 mutation (Fig ure 4 4B). The hematopoietic progenitor cells isolated from these patients were subsequently cultured in a semisolid growth medium in the presence or absence of the appropriate cytokine and in the presence of DMSO, 25 M Z3 or 25 M G6. As expected, treatment of the essential thrombocythemia patients hematopoietic progenitor cells with thrombopoietin dramatically increased megakaryocyte colony formation (Fig ure 4 4A). However, G13 and Z3 significantly reduced this thrombopoietin-dependent increase in cell growth. In addition, G6 completely blocked cytokine -independent megakaryocyte colony formation (Figure 4 4A). In a similar manner, erythropoietin treatment of progenitor cells obtained from the polycythemia vera pa tient considerably increased erythrocyte cell growth, but G6 and Z3 significantly suppressed cytokine -dependent as well as -independent erythrocyte colony formation (Fig ure 4 4B). However, the ability of G6 to block cytokine dependent erythrocyte colony f ormation was greater relative to Z3 (p= 0 .049). All together, the results in Figure 4 4 show that the G -co mpounds significantly reduce J ak2 -mediated hematopoietic cell

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82 growth ex vivo with G6 blunting Jak2 -F537I -mediated, human pathologic cell growth more effectively. Discussion Myeloproliferative disorders, which include polycythemia vera, essential thrombocythemia, and primary myelofibrosis are disorders of hematopoietic stem cells, where myeloid progenitors become hypersensitive and/or independent of cyt okines for survival and proliferation (Dameshek, 1951). The discovery of the Jak2-V617F mutation in almost all individuals with polycythemia vera and about 50% of patients with essential thrombocythemia and primary myelofibrosis suggested that this Jak2 pseudokinase domain mutation plays a crucial role in these disorders. Confirmation on the role of Jak2-V617F in the pathogenesis of myeloproliferative disorders came from animal models. In particular, retroviral transduction of Jak2 V617F in murine hematopoietic stem cells followed by transplantation into lethally irradiated mice resulted in the development of polycythemia vera phenotype (Bumm et al., 2006; Lacout et al., 2006; Wernig et al, 2006). Later on, several Jak2 exon 12 mutations were detected in Jak2 -V617F -negative polycythemia vera patients, which also led to constitutive activation of Jak STAT signaling pathway. In order to identify therapeutically effective compounds that target aberrant Jak2 kinase function, we revised our previous Jak2 model yet again using information obtained from the crystal structures of the Jak3 and Jak2 kinase domain. In addition, we screened a significantly larger number of compounds in silico 223,481 to be exact, with the intention of identifying small molecules tha t specifically bind and inhibit Jak2 more potently than Z3. In what is now our third generation of Jak2 inhibitors, our studies showed that a set of structurally diverse molecules potently suppressed Jak2 autophosphorylation not onl y at tyrosine 1007, but at other tyrosine residues as well. These compounds also significantly inhibited the growth of human

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83 erythroleukemia cells that express the Jak2 -V617F mutation on both alleles. One particular compound, G6, was further characterized since it was more eff ective in inhibiting Jak2 -V617F dependent cell proliferation when compared to the other G compounds. We found that G6 selectively blocked Jak2 -V617F pathologic cell growth by causing an increase in cellular apoptosis. In addition, G6 had no effect on Tyk2 or c -Src tyrosine kinase activity at concentrations that completely inhibited Jak2, thus confirming the relative specificity of this compound. Finally, we demonstrated that the G6 and G13 compounds significantly reduced the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2 -V617F mutation and a polycythemia vera patient carrying a Jak 2 F537I exon 12 mutation. Upon analysis, we found that there is a difference between the G co mpounds and Z3 in terms of their efficacy. First, G6 reduced Jak2-V617F dependent HEL cell growth approximately 43% more than Z3. Another direct comparison between the efficacy of the G compounds and Z3 is demonstrated with our ex vivo data. Here, we sh owed that G6 suppressed hematopoietic colony formation of cells derived from a polycythemia vera patient carrying a novel Jak2 F537I mutation more effectively than Z3 (p=0.049). Collectively, our r esults show that the G6 is more effective than Z3 in suppr essing Jak2 -dependent pathologic cell growth in vitro and ex vivo In summary, our laboratory has invested effort toward the identification and characterization of novel Jak2 -specific inhibitors. Our ability to identify novel Jak2 inhibitors has evolved over the years. Specifically, with every generation of Jak2 inhibitor identified by our lab, our Jak2 homology model has become a more accurate structural representation of the Jak2 kinase domain to be utilized for high throughput molecular docking. In addition, due to an

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84 increase in computational ability, we have screened more compounds for their ability to bind and inhibit Jak2. As a result, with each succeeding generation of Jak2 inhibitor indentified, we have observed an increase in Jak2 -mediated ce ll growth inhibition. In what is now our third generation Jak2 inhibitor, we have demonstrated that G6 is an effective inhibitor of Jak2 dependent pathologic cell growth in vitro and ex vivo As such, we believe that G6 could be considered as a lead ther apeutic agent.

PAGE 85

85 Time (hours) 0 20 40 60 80 0 200x103400x103600x103800x103 DMSO Z3 G6 G11 G13 0 20 40 60 80 100 120 BSC40 Raji HEL CMK Viable Cells: Percent of Control[G6] MA B G6 G11 G13 Z3 DMSO 109 108107106 105104Number of Viable Cells Figure 4 1. G6 potently inhibits Jak2 -V 617F dependent cell growth. A) Approximately 50,000 HEL cells were treated with either DMSO or 25 M of the G compounds for 0, 16, 24, 48, and 72 hours. At the end of each time point, the number of viable DMSO or G compound treated cells was determined by trypan blue exclusion. All data points were measured in triplicate. Statistical significance between each group was analyzed using a two-way statistical analysis of variance (ANOVA). Values of p=0.025 (G13), p=7.52 x 109 (G11), p=2.98 x 1025 (G6) were considered to be significant whe n compared to DMSO control. B) Approximately 50,000 HEL, Raji, CMK, and BSC 40 cells were treated with either DMSO or with G6 at the indicated concentrations. After 48 hours, the numbers of viable cells were assessed as described above. The graph shown is a compilation of three independent experiments. Statistical significance between each group was determined using a two-way ANOVA. Values of p=2.09 x 1025 (Raji), p=1.95 x 1025 (CMK), p=6.4 x 1023 (BSC 40) were regarded as significant relative to DMSO control

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86 DMSO (0.25%, 48 hrs) G6 (25 M, 48 hrs) 10 15 20 25 30 35 40 45 50 0 10 20 30 40 50 Time (hours)Percent Apoptosis DMSO G6 C A B Figure 4 2. G6 reduces HEL cell numbers by increasing cellular apoptosis. Approximate ly 50,000 HEL cells were treated with either DMSO or 25 M G6 for 16, 24 or 48 hours and cells were measured for Annexin V staining via flow cytometry. A representative experiment at 48 hours shows the percentage of Annexin V and propidium iodide stained cells treated with either 0.25% DMSO ( A) or 25 M G6 B). C). The percent of DMSO or G6 treated cells undergoing apoptosis is plotted as a function of time. Shown is one of two representative experiments.

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87 DMSO Z3, 100 G6, 100 DMSO Z3, 25 PP2, 25 G6, 25 A CIP: antiTyk2pAb IB: antiTyr(P)mAb IB: antiTyk2pAb Tyk2(P) Tyk2 111 111 IB: antic Src pY418pAb IB: antic Src pAb c Src(P ) c Src 61 61 49 49B D Figure 4 3. G6 has no effect on Tyk2 or c Sr c tyrosine kinase activity. A) COS 7 cells were transiently transfected with an expression vector encoding wild type human Tyk2 cDNA. Cells were subsequen tly treated with either DMSO, 100 M Z3 or 100 M G6 for 16 hours. Pr otein lysates were immunoprecipitated with anti Tyk2 antibody and then Western blotted with anti -phosphotyrosine antibodies to detec t Tyk2 autophosphorylation. B) The membrane was stripped and reprobed with anti Tyk2 antibody to demonstrate equal Tyk2 exp ress ion among samples C ) A catalytically active recombinant c Src prot ein was incubated in kinase buff er containing either DMSO, 25 M Z3, 25 M PP2 or 25 M G6. The samples were subsequently immunoblotted with anti phospho c Src (pY418) antibody to assess relative c Sr c tyrosine kinase activity. D) The membrane was then stripped and reprobed with c -Src antibody to determine tot al c -Src protein levels Shown is one of three representative experiments for each.

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88 Figure 4 4. The G compounds reduce hematopoietic colony formation ex vivo A) Marrow derived mononuclear cells from an essential thrombocythemia patient who was Jak2 V617F positive were cultured in methylcellulose media containing DMSO (0.25%, vol/vol), 25 M Z3, 25 M G6 or 25 M G13. The number of megakaryocyte colonies was assessed 14 days later. Results are expressed as the average number of colonies of duplicate cultures per 4 x 105 cells. Statistical significance between each group was analyzed usi ng Students t test, *p=0.011, **p=0.017, ***p=0.005. B) Marrow derived mononuclear cells from a polycythemia vera patient was who was Jak2 F537I positive were cultured in methylcellulose media containing either DMSO (0.25%, vol/vol), 25 M Z3 or 25 M G6 Results are expressed as the average number of colonies from duplicate cultures per 4 x 105 cells. Statistical significance between each group was determined using Students t test, *p=0.01, **p=0.0195, ***p=0.01, ****p=0.002, *****p=0.049.

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89 CHAPTER 5 T YROSINE 372 IS CRITICAL FOR JAK2 FUNCTION Summary Jak2 is a non receptor tyrosine kinase whose key function is to transduce gene transcription signals from the cell surface to the nucleus through a tyrosine phosphorylation signaling mechanism. Jak2 consis ts of 49 tyrosine residues and already a handful of these are known to be phosphorylated and play important roles in regulating Jak2 tyrosine kinase activity. Here, we demonstrated by electrospray mass spectrometry that tyrosines 372 and 373 are novel sit es of Jak2 phosphorylation. We introduced tyrosine to phenylalanine point mutations at positions 372 and 373 in plasmids encoding wildtype Jak2 protein and expressed these plasmids in cells to determine the effect of loss of tyrosines 372 and 373 phospho rylation on Jak2 tyrosine kinase function. Using a Jak2 autophosphorylation assay, we found that loss of tyrosine 372 phosphorylation not only inhibited Jak2 tyrosine 1007 phosphorylation, but also suppressed phosphorylation of other Jak2 tyrosine residue s, indicating that tyrosine 372 is important for Jak2 catalytic activity in the context of a ligand independent signaling system. Conversion of tyrosine 373 to phenylalanine on the other hand, resulted in more modest reductions in total Jak2 phosphorylat i on levels and phosphorylation of tyrosine 1007. With particular focus on the more significant tyrosine 372, we also found that loss of phosphorylation at tyrosine 372 suppressed the ability of Jak2 to phosphorylate STAT1 via a mechanism involving reduced Jak2 and STAT1 co association. Finally, we found that loss of tyrosine 372 phosphorylation blocked interferongamma and epidermal growth factor -dependent Jak2 activation relative to wild type Jak2, but had no effect on hydrogen peroxide -mediated Jak2 acti vation. All together, our results demonstrate that tyrosine 372 phosphorylation plays an important role in Jak2 tyrosine

PAGE 90

90 autophosphorylation. In addition, tyrosine 372 has a significant and differential role in Jak2 activation in response to ligand. In troduction Jak2 is a nonreceptor tyrosine kinase belonging to the Janus family of tyrosine kinases that also includes Jak1, Jak3 and Tyk2. A key cellular role of Jak2 is to phosphorylate and hence activate members of the Signal Transducers and Activators of Transcription (STAT) family of latent cytoplasmic transcription factors. Once activated, the dimerized STAT proteins translocate to the nucleus, bind DNA promoter elements and modulate gene expression. On an extrinsic level, Jak2 is activated by a var iety of cytokine and growth factor receptors as well as by oxidative stress in the form of hydrogen peroxide, resulting in signaling cascades that are involved in the regulation of cell growth, proliferation and death. In addition, Jak2 is intrinsically r egulated via specific autophosphorylation of a handful of its tyrosine residues. The Jak kinases are structurally composed of seven Jak homology (JH) domains. The JH1 domain, located at the C terminus of Jak kinases, corresponds to the catalytically activ e tyrosine kinase domain (Duhe and Farrar 1995). The JH2 domain which exhibits sequence similarity with the JH1 domain, but lacks catalytic activity, is termed the pseudokinase domain. The pseudokinase domain has been proposed to negatively regulate Jak kinase activity (Lindauer et al., 2001; Chen et al., 2000; Saharinen et al., 2003). The JH3 JH4 regions of Jak kinases represent the SH2 like domain whose function is unknown to date. At the amino terminus, the JH4 JH7 regions of Jaks comprise the FERM domain. The FERM domain has been shown to be the region that is involved in receptor -Jak association (Girault et al., 1998; Hilkens et al., 2001; Zhou YJ et al., 2001). The significance of the FERM domain in Jak function was established by the observation of three naturally occurring point

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91 mutations in Jak3 in severe combined immunodeficiency (SCID) patients which resulted in loss of Jak3 kinase activity (Cacalano et al., 1999; Zhou et al., 2001). Specifically it was shown that these mutations reduced the interaction of Jak3 with the common -chain of the IL 2 subfamily of cytokine receptors and in parallel inhibited the ability of Jak3 to be activated in response to ligand binding (Cacalano et al., 1999; Zhou et al., 2001). Therefore, it appears that structural changes in the FERM domain br ought about by point mutation can alter the activity of Jak kinases. Of the 49 Jak2 tyrosine residues encoded in murine Jak2, a number have been shown to be phosphorylated and play important roles in overall Jak2 tyrosine kinase regulation. Interestingly, many of these characterized tyrosine residues are situated at the C terminus of Jak2, where the pseudokinase and kinase domains reside. For example, in the activation loop of the kinase domain, phosphorylation of tyrosine 1007 is required for maximal Jak2 activation while phosphorylation of tyrosine 1008 has no effect on Jak2 kinase activity (Feng et al., 1997). Recently, Funakoshi Tago et al., showed that autophosphorylation at tyrosine 913 in the kinase domain negatively regulates Jak2 by suppressing e rythropoietin induced Jak2 activation (Funakoshi Tago et al., 2008). In addition, our laboratory recently identified tyrosine 972 as a novel site of Jak2 functional regulation. We showed that phosphorylation of tyrosine 972 is necessary for Jak2 kinase a ctivity in response to angiotensin II (McDoom et al., 2008). Finally, phosphorylation of tyrosine 570 situated in the pseudokinase domain of Jak2 was shown to modulate Jak2 kinase function by suppressing Jak2 tyrosine kinase activity (Argetsinger et al., 2004; Feener et al., 2004). Collectively, the data suggest that the activation or inhibition of Jak2 tyrosine kinase is dependent upon the phosphorylation of its numerous tyrosine residues.

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92 Fewer phosphorylated tyrosine residues have been characterized in the N terminal region of Jak2 which comprise the FERM domain. The characterized phosphorylated tyrosine residues in the FERM domain have different consequences for Jak2 tyrosine kinase regulation based on the presence or absence of ligand activation and the type of ligand receptor system involved. For instance, Argetsinger et al. have shown that phosphorylation of tyrosine 221 increases ligandindependent Jak2 tyrosine kinase activity while Feener et al have demonstrated that phosphorylation of tyrosine 221 has no effect on Jak2 dependent signaling in the presence of an erythropoietinleptin receptor chimera (Argetsinger et al., 2004; Feener et al., 2004). In addition, Funakoshi Tago et al. have reported that phosphorylation of tyrosine 119 in response t o erythropoietin down regulates Jak2 kinase activity by promoting dissociation of activated Jak2 from the erythropoietin receptor while phosphorylation of this residue has no effect on Jak2 regulation in the presence of the interferon -gamma receptor (Funa koshi Tago et al., 2006). Given the limited knowledge of how the FERM domain regulates Jak2 function, the identification of novel Jak2 tyrosine phosphorylation sites, within this region, will be important for our understanding of Jak2-dependent signaling and its regulation. In this study, we have identified tyrosine 372 as a novel Jak2 phosphorylation site in the FERM domain of Jak2. We have found that phosphorylation of tyrosine 372 is critical for the maintenance of maximal Jak2 phosphorylation, STAT1 a ctivation and Jak2 -dependent gene transcription in the context of a ligand independent system. In addition, tyrosine 372 phosphorylation has an important and differential role on Jak2 -dependent signal transduction in response to ligand. In particular, ph osphorylation of tyrosine 372 facilitates interferon -gamma and epidermal growth factor -medated Jak2 activation but has no affect on hydrogen peroxide -

PAGE 93

93 mediated Jak2 activation. As such, this work demonstrates the importance of tyrosine 372 in the regulatio n of the Jak2 signaling pathway. Results Tyrosine 372 and 373 are Sites of Jak2 Autophosphorylation Jak2 protein was expressed at a high level via a vaccinia virus -mediated overexpression system and then purified to homogeneity as previously described (Ma and Sayeski, 2004). The purified Jak2 protein was then subjected to a combination of na no HPLC/ ESI ioniz ation on a LTQ mass spectrometer The spectra corresponding to the peptide fragment containing tyrosine 372 and 373 was identified and tyrosines 372 and 373 were found to be phosphorylated (Fig ure 5 1). Tyrosine 372 is H ighly Conserved A mong S pecies Expressing Jak2 and in Different Jak2 Kinase Family Members To further characterize the significance of tyrosine 372 and 373, we determined whether these amino acid residues were conserved throughout the evolutionary history (Fig ure 5 2). Compariso n of the amino acid sequence of Jak2 from diverse species revealed that tyrosines 372 and 373 are conserved. However, evaluation of the amino acid sequence of the different Jak family members revealed that while tyrosine 372 is well conserved, tyrosine 373 is not. The higher conserved nature of tyrosine 372 relative to tyrosine 373 suggests that tyrosine 372 could play a more critical role in Jak2 function. Loss of Tyrosine 372 and 373 Phosphorylation Reduce Jak2 Tyrosine Phosphorylation Published reports have demonstrated that a number of the tyrosine residues of Jak2 are phosphorylated and play important roles in regulating Jak2 tyrosine kinase activity (Ihle et al., 1994; Feng et al., 1997; Ungureanu et al., 2002; Kurzer et al. 2004; Argetsinger et al., 2004; Funakoshi Tago et al., 2006; Brooks et al., 2007; Godeny et al., 2007; McDoom et al., 2008).

PAGE 94

94 While our mass spectrometry data revealed that tyrosine 372 and 373 are phosphorylated, the next critical step was to determine if these tyrosines are impo rtant for Jak 2 tyrosine autophosphorylation. To determine how tyrosine 372 and 373 influence Jak2 function the affect of tyrosine to phenylalanine substitution mutations on total Jak2 tyrosine phosphorylation was assessed. For this, BSC 40 cells were tr ansfected to overexpress either empty vector, wild -type Jak2, Jak2Y372F, or Jak2-Y373F. Cell lysates were prepared, Jak2 protein was isolated by immunoprecipitating with a Jak2 antibody and total tyrosine phosphorylation was assessed by immunoblotting wi th anti phosphotyrosine antibody (Fig ure 5 3A ). Subsequently, total Jak2 phosphorylation levels were quantified and averaged (Fig ure 5 3D ). The results show that the loss of phosphorylation at tyrosines 372 or 373, individually, reduced total Jak2 tyrosi ne phosphorylation levels relative to wild type Jak2 in a stati stically significant manner. However, mutation at tyrosine 372 suppressed overall Jak2 phosphorylation significantly more than mutation at tyrosine 373. Specifically, the tyrosine 372 mutant reduced total Jak2 phosphorylation by roughly 70% whereas the tyrosine 373 mutant reduced phosphorylation by approximately 40%, when compared to wild -type Jak2 protein. Since phosphorylation of tyrosine 1007 in Jak2 has been shown to be essential for maxim al Jak2 tyrosine kinase activity (Feng et al., 1997), we also investigated whether individually mutating tyrosines 372 and 373 would affect the phosphorylation of this critical residue. For this, the same blot was probed with an anti active Jak2 antibody to detect tyrosine 1 007 phosphorylation levels (Figure 5 3B ). Jak2 tyrosine 1007 phosphorylation levels were then quantified and averaged (Figure 5 3E ). Similarly, we found that the loss of phosphorylation at tyrosine 372 or 373 significantly decreased Ja k2 tyrosine 1007 phosphorylation, when compared to wild type Jak2 protein. However the tyrosine 372 mutant completely inhibited Jak2 tyrosine

PAGE 95

95 1007 phosphorylation while the tyrosine 373 mutant reduced phoshporylation by approximately 70%. To verify equa l Jak2 expression among samples, the membrane was stripped and re blotted with anti -Jak2 polyclonal antibody (Fig ure 5 3C ). Collectively, the results indicate that phosphorylation of Jak2 at tyrosines 372 and 373 is important for the regulation of the kin ase activity of Jak2 in a ligand independent system. Additionally, the data suggest that phosphorylation at tyrosine 372 is more important for the catalytic activity of Jak2 when compared to tyrosine 373, since mutation of tyrosine 372 reduced Jak2 tyrosi ne 1007 phosphorylation as well as total Jak2 tyrosine phosphorylation more effectively than mutation at tyrosine 373. As such, for the remainder of the study, we focused on investigating the specific role of tyrosine 372 on Jak2 function. Mutation of Jak2 at Tyrosine 372 Suppresses the Ability of Jak2 to Phosphorylate STAT1 To determine the outcome of the Jak2Y372F mutant on the ability of Jak2 to activate substrates, we specifically examined the effect of the Y372F mutant on STAT1 phosphorylation. STAT1 is a signaling protein that is phosphorylated on tyrosine 701 in the presence of active Jak2 (Darnell et al., 1994). Here, BSC 40 cells that endogenously express STAT1 were transiently transfected with either empty vector control, wild type Jak2, or Jak2Y372F expressing plasmids. Whole cell lysates were prepared and samples were blotted with an anti active STAT1 antibody to detect STAT1 phosphorylation at tyrosine 701 (Figure 5 4A ). We found that loss of tyrosine 372 phosphorylation significantly reduc ed the ability of Jak2 to phosphorylate STAT1 at tyrosine 701 by approximately 65%, when compared to wild-type Jak2 (p=0.004) (Fig ure 5 4D ). The membrane was stripped and re -probed with a STAT1 po lyclonal antibody to confirm equ al STAT1 protein expression among samples (Fig ure 5 4B ). To confirm equal Jak2 expression levels, the membrane was stripped again and re-blotted with a Jak2

PAGE 96

96 polyclonal antibody (Fig ure 5 4C ). These results suggest that phosphorylation of Jak2 at tyrosine 372 facilitates Jak2-dependent STAT1 activation. Loss of Tyrosine 372 Phosphorylation Reduces the Association of Jak2 with STAT1 In the Jak STAT signaling paradigm, Jak2 associates with receptor bound STAT proteins and subsequently tyrosine phosphorylates them. Here, we examined th e effect of loss tyrosine 372 phosphorylation on Jak2-STAT1 co association. BSC 40 cells were transiently transfected with empty vector, wild -type Jak2 or Jak2Y372F plasmid. The following day, protein lysates were then immunoprecipitated with anti -STAT1 polyclonal antibody and immunoblotted with anti Jak2 polyclonal antibody to detect STAT1/Jak2 coassociation (Figure 5 5A ). We found that elimination of tyrosine 372 phosphorylation hindered the ability of Jak2 to associate with STAT1. In particular, th e Jak2 Y372F mutation reduced STAT/Jak2 coprecipitation by approximately 80 % (*p=3.87 x 106) (Figure 5 5D ). To demonstrate equal STAT1 precipitation across all samples, the membrane was stripped and re blotted with anti -STAT1 polyc lonal antibody (Figur e 5 5B ). In addition, whole cell lysates were blotted with anti Jak2 polyclonal antibody to determ ine Jak2 expression levels (Figure 5 5C ). To verify that the Jak2 Y372F mutation hinders Jak2/STAT1 coassociation, the inverse experiment was performed wh ereby protein lysates were first immunoprecipitated with anti Jak2 antibody and then immunoblotte d with anti STAT1 antibody (Figure 5 -E ). Similar to the experiment above, mutation at tyrosine 372 decreased Jak2/STAT1 coassociation by approximately 85% (*p=1.35 x 108) (Fig ure 5 5I ). To demonstrate equal Jak2 precipitation among samples, the membrane was stripped and re -probed with an anti Jak2 polyclonal antibody (Fig ure 5 5F ). To assess STAT1 protein levels, whole cell lysates from these same samples w ere blotted with an anti -STAT1 polyclonal antibody (Fig ure 5 5G ). All together, the results in Figure 5 5 suggest that loss of tyrosine 372 phosphorylation reduces the ability of Jak2 to

PAGE 97

97 associate with STAT1 and thereby provides a mechanistic explanation as to why this Jak2 mutation disrupts STAT1 activation. The Jak2 -Y372F Mutation Abrogates Ligand -Independent Gene Expression It is known that Jak2 is capable of driving a basal level of gene expression even in the absence of ligand stimulation (Chatti et al., 2004; Wallace et al., 2006). This ligandindependent gene expression corresponds to Jak2s intrinsic functional activity. Here, we sought to determine whether loss of phosphorylation of tyrosine 372 affects the intrinsic functional capacity of Jak2 to induce ligand independent gene expression by performing a luciferase gene reporter assay. Specifically, COS 7 cells were transiently transfected with a plasmid encoding four tandem repeats of the activating sequence upstream of the firefly luciferase cDNA. In addition, these cells were co transfected with plasmid encoding either empty vector control, wild type Jak2, or Jak2 Y372F protein. Two days later, luciferase activity was determined and p lotted as a function of Jak2 expression status (Fig ure 5 6A). We found that the Jak2 Y372F mutation significantly reduced the ability of Jak2 to drive luciferase gene expression when compared to wild-type Jak2 (p= 0.00036). To confirm equal expression lev els of wild type Jak2 and Jak2Y372F protein, a portion of the transfected protein lysates were immunoblotted with a Jak2 polyclonal antibody (Figure 5 6B). Loss of Tyrosine 372 Phosphorylation Hinders Interferon Gamma -mediated Jak2 Activation Having sho wn the importance of tyrosine 372 on ligand-independent Jak2 function, we then examined the functional significance of tyrosine 372 phosphorylation in the context of ligand -dependent Jak2 signaling. Interferongamma is a cytokine that has been well characterized to activate the receptor associated Jak2 tyrosine kinase (Pestka et al., 1997, Parganas et al., 1998). Given the critical role of Jak2 in interferon-gamma receptor signaling, we

PAGE 98

98 sought to determine the effect of tyrosine 372 phosphorylation on int erferon -gamma -mediated Jak2 activation. Here, mouse embryonic fibroblast cells (MEF) that endogenously express the interferon -gamma receptor, but that lack Jak2, were transiently transfected with empty -vector, wild -type Jak2 or Jak2Y372F expressing plasm ids. These cells were treated with interferon gamma for 0 or 10 minutes and then lysed. Jak2 protein was immunoprecipitated from the lysate and Jak2 tyrosine 1007 phosphorylation levels were measured by immunoblotting with an anti Jak2 pY1007/pY1008 anti body. In agreement with published reports, we found that by 10 minutes of interferon -gamma treatment, wild typ e Jak2 was activated (Parganas et al., 1 998, Funakoshi Tago et al., 2006). However, loss of tyrosine 372 phosphorylation blocked the interferon -gamma -mediated increase in Jak2 tyrosine 1007 phosphorylation (Fig ure 5 7 A ). The membrane was subsequently stripped and equal Jak2 protein expression was verified via Western blot with an anti Jak2 antibody (Fig ure 5 7 B). The results in Figure 5 7 sugges t that phosphorylation of tyrosine 372 is critical for interferon-gamma -dependent Jak2 activation. Loss of Tyrosine 372 Phosphorylation Impairs Epidermal Growth Factor -Mediated Jak2 Activation. Jak2 also becomes activated in response to growth factors, lik e epidermal growth factor (Shuai et al., 1993; Andl et al., 2004). However, the relevance of Jak2 tyrosine phosphorylation in growth -factor signaling has not been well characterized. Thus, we sought to determine the effect of Jak2 tyrosine 372 phosphoryl ation on the ability of Jak2 to respond to epidermal growth factor. Mouse embryonic fibroblasts that were derived from Jak2 / mice were transiently transfected with empty vector, wildtype Jak2 or Jak2-Y372F expressing plasmids. The cells were then treated with epidermal growth factor for 0 or 10 minutes. To detect Jak2 tyrosine 1007 phosphorylation, protein extracts were immunoprecipitated with anti Jak2 antibody and then immunoblotted with anti Jak2 pY1007/pY1008 antibody. By ten minutes of epiderma l

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99 growth factor treatment, wild type Jak2 was increasingly activated, which is in agreement with previously published reports (Shuai et al., 1993; Andl et al., 2004). On the other hand, loss of tyrosine 372 phosphorylation inhibited the epidermal growth factor -mediated increase in Jak2 tyrosine 1007 phosphorylation (Figure 5 8 A ). The membrane was stripped and re -probed with an anti Jak2 antibody to verify e qual protein loading (Figure 5 8 B). The results in Figure 5 8 suggest that Jak2 tyrosine 372 phosp horylation is important for epidermal growth factor mediated Jak2 activation. Loss of Tyrosine 372 Phosphorylation Does Not Affect H ydrogen Peroxide -Mediated Jak2 A ctivation Although Jak2 is tradionally considered a mediator of cytokine signaling, other st imuli can activate this pathway suc h as, oxidative stress (Simon et al., 1998; Madamanchi et al., 2001, Sandberg et al., 2004B). Jak2 can be potently activated in a number of cell types by oxidative stress in the form of hydrogen peroxide (Simon et al., 1 998; Madamanchi et al., 2001, Sandberg et al., 2004B). We have already shown that tyrosine 372 plays an important role in cytokine and growth factor -dependent Jak2 activation. We next wanted to determine the impact of tyrosine 372 phosphorylation on Jak2 activation in response to a nontraditional ligand such as, hydrogen peroxide. For this, mouse embryonic fibroblasts that lack Jak2 were transiently transfected with empty vector, wildtype Jak2 or Jak2Y372F expressing plasmids. These cells were treated with hydrogen peroxide for 0 or 10 minutes. Cellular lysates were immunoprecipitated with anti Jak2 antibody and then immunoblotted with anti Jak2 pY1007/pY1008 antibody to detect Jak2 tyrosine 1007 phosphorylation. Consistent with previous reports, wi ld type Jak2 was potently activated by 10 minutes of treatment w ith hydrogen peroxide (Simon et al., 1998; Madamanchi et al., 2001; Sandberg et al., 2004B). However, loss of tyrosine 372 phosphorylation had no effect on hydrogen peroxide -mediated Jak2 tyr osine 1007 phosphorylation (Fig ure 5 9 A ). To

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100 demonstrate equal Jak2 expression among samples, the membrane was stripped and re -blott ed with antiJak2 antibody (Figure 5 9 B). These results suggest that tyrosine 372 phosphorylation is dispensable for hydro gen peroxide -dependent Jak2 activation. Discussion In this study, we identified tyrosines 372 and 373 as sites of Jak2 autophosphorylation via mass spectrometry analysis. Interestingly, comparison of the amino acid sequence of the different Janus kinase family members, and the amino acid sequence of Jak2 from various species, indicated that tyrosine 372 is a highly conserved residue through evolution. As such, tyrosine 372 could play a critical role in Jak2 kinase function. To determine the importance of tyrosine 372 in the Jak2 signaling pathway, we utilized site directed mutagenesis to mutate tyrosine 372 to phenylalanine. We found that loss of tyrosine 372 phosphorylation eliminated the potential of Jak2 to become maximally activated as demonstrated by the loss of tyrosine 1007 phosphorylation. The Jak2 Y372F mutation also suppressed the phosphorylation of other Jak2 tyrosine residues. These results show that phosphorylation of tyrosine 372 is important for Jak2 catalytic activity in the context of a ligand independent signaling system. Loss of tyrosine 372 phosphorylation also affected the capacity of Jak2 to activate its downstream substrate, STAT1, and hindered the intrinsic ability of Jak2 to drive gene transcription. Moreover, we believe that the Jak2Y372F -mediated disruption in STAT1 phosphorylation could be due to the impaired ability of Jak2 to co associate with STAT1. Tyrosine 372 was also investigated within a signaling context. We found that loss of tyrosine 372 phosphorylation blocke d interferon and epidermal growth factor -dependent Jak2 activation relative to wild type Jak2, but had no effect on hydrogen peroxide -mediated Jak2 activation, suggesting that tyrosine 372 plays a significant and differential role in Jak2 activation

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101 in res ponse to ligand. All together, our results indicate that tyrosine 372 is a novel site of Jak2 functional regulation. An important finding of this study is that while our results suggest that tyrosine 372 is critical for ligand -independent Jak2 catalytic activity, this residue may not be essential for the catalytic activity of Jak2 in the context of a ligand -dependent system, as demonstrated by the fact that loss of tyrosine tyrosine 372 phosphorylation has no affect on hydrogen peroxide -dependent Jak2 ac tivation. A possible explanation for this disparity is that phosphorylation of tyrosine 372 may facilitate Jak2 dimerization and thus enhance ligand independent Jak2 catalytic activity, whereas in response to nontraditional ligands like hydrogen peroxide tyrosine 372 may not play a role in Jak2 dimer formation and therefore have no direct affect on the catlayitic activity of Jak2. Since hydr ogen peroxide activates Jak2 by inhibiting phosphatases via their oxidation, it is possible that phosphorylation o f Jak2 at sites other than tyrosine 372 could block the association of Jak2 with a phosphatase such as SHP 2, thus maintaining Jak2 in the active state. The FERM domain of Jak kinases has been shown to be important for the interaction with cytokine recepto rs (Frank et al., 1995; Chen et al., 2000; Kohlhuber et al., 1997; Zhou et al., 2001). We hypothesize that tyrosine 372, situated in the FERM domain of Jak2, could possibly mediate Jak2 association with various receptors. Another possible mechanistic ex planation for the Jak2Y372F effect on Jak2 function is that loss of tyrosine 372 phosphorlation may promote the dissociation of Jak2 tyrosine kinase from receptor complexes thereby downregulating Jak2 depedent signaling. Therefore, it is possible that ph osphorylation of tyrosine 372 causes a conformational change in Jak2 that could stabilize the FERM domain of Jak2 in a more active conformation when bound to receptor, maintaining Jak2 in the active state. Future studies should

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102 be aimed at verifying wheth er the Jak2 Y372F mutation disrupts the interaction between Jak2 and its associated receptor in response to ligand treatment. Of the 49 Jak2 tyrosine residues, a number are known to be phosphorylated and play important roles in Jak2 tyrosine kinase function. Interestingly, many of these characterized Jak2 tyrosine phosphorylation sites are situated either in the pseudokinase or kinase domains of Jak2. Less is known regarding the consequences of phosphorylation of tyrosines in the N terminus of Jak2, where the FERM domain resides. Argetsinger et al. have shown via mass spectrometry and two dimensional peptide mapping that tyrosine 221 in the FERM domain of Jak2 is phosphorylated. When tyrosine 221 was mutated to phenylalanine, Jak2 tyrosine autophosphoryl ation was reduced suggesting that tyrosine 221 could serve to regulate Jak2 tyrosine kinase activity (Argetsinger et al., 2004). In contrast, Feener et al. reported that when the Jak2Y221F mutant is overexpressed with an erythropoietinleptin receptor ch imera in HEK 293 cells, Jak2-dependent signaling was unaffected (Feener et al., 2004). The apparent discrepancy in the findings regarding the role of tyrosine 221 could be due to the fact that Feener et al. employed a receptor chimera in their system whil e there was an absence of coexpressed cytokine receptor in that of the Argetsinger group. These results imply that Jak2 tyrosine 221 phosphorylation could regulate the activity of Jak2 in the absence of cytokine receptor association, or conceivably in ass ociation with other cytokine receptors that dont include the erythropoietin or leptin receptors. The role of the FERM domain in regulating kinase activity has also been suggested by Jak2 tyrosine 119 phosphorylation (Funakoshi Tago et al., 2006). Using a Jak2 Y119F mutant, Funakoshi Tago et al. demonstrated this mutation corresponded to a more stable association of Jak2 with the erythropoietin receptor, resulting in prolonged of Jak2 activation (Funakoshi Tago

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103 et al., 2006). Thus they concluded that phos phorylation of tyrosine 119 within the Jak2 FERM domain downregulates Jak2 tyrosine kinase activity by promoting the dissociation of activated Jak2 from the erythropoietin receptor. Interestingly, by using a phosphorylation mimic Jak2Y119E mutation, this group further showed that this mutation reduced the interaction of Jak2 with the erythropoietin receptor, while there was no consequence of this mutation on the ability of Jak2 to associate with the interferon-gamma receptor. These results suggest that t here are underlying differences in the way Jak2 interacts with erythropoietin receptor and the interferon gamma receptor. In addition, in 2006, our laboratory verified another Jak2 phosphorylation site in the FERM domain of Jak2. We showed that phosphor ylation of tyrosine 201 served as a binding site for the SHP 2 regulatory protein. This Jak2/SHP 2 interaction allowed the recruitment of Jak2 to the angiotensin II type 1 receptor signaling complex and in turn promoted downstream Jak2dependent signaling (Godeny et al., 2006). Finally, Funakoshi Tago and colleagues have recently investigated the effect of several conserved Jak2 tyrosine residues on Jak2 function, including tyrosine 372. Although they have not provided evidence that tyrosine 372 is phosph orylated, they have shown that a Jak2 Y372F mutant has no effect on Jak2 -mediated erythroid progenitor col ony formation (Funakoshi Tago et al., 2008). Their results suggest that phosphorylation of tyrosine 372 is not important for the regulation of Jak2 kinase activity. In contrast, we have shown that phosphorylation of tyrosine 372 is critical for the regulation of Jak2, as loss of tyrosine 372 phosphorylation suppresses ligand -independent Jak2 tyrosine autophosphorylation as well as interferon-gamma and epidermal growth factor -mediated Jak2 activation. The discrepancy between our findings and those of Funakoshi Tago et al. could be due to a few factors. First, we have examined how

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104 phosphorylation of tyrosine 372 affects the inherent kinase activity of Jak2 in a ligand independent system, whereas Funakoshi Tago et al. have observed the consequence of loss of tyrosine 372 phosphorylation on Jak2dependent cell growth in response to erythropoietin stimulation. Second, while Funakoshi Tago et al. have exam ined the effect of the Jak2Y372F mutation on Jak2 regulation in the context of the erythropoietin signaling pathway, we have studied the effect of the Jak2 Y372F mutant on interferon-gamma and epidermal growth factor signaling. The Jak2 Y372F mutation co uld differentially influence the manner in which Jak2 interacts with the interferon -gamma/epidmeral growth factor receptors and the erythropoeitin receptor leading to differences in Jak2 activity. Collectively, studies show that phosphorylation of tyrosine resides in the FERM domain can have differential effects on Jak2 tyrosine kinase activity based on the presence of ligand and the type of ligandreceptor system involved. Undeniably, regulation of Jak2 through the phosphorylation of tyrosines in the FERM domain is quite complex and therefore further insight into the exact mechanism by which ligand binding to a particular receptor activates Jak2 is required. In conclusion, our laboratory has contributed to the understanding of Jak2 tyrosine kinase regulation by the FERM domain. We have identified tyrosine 372 as a novel site of Jak2 autophosphorylation in the FERM domain and subsequently demonstrated the importance of the phosphorylation of this amino acid residue on Jak2 function. Specifically, we showed that phosphorylation of tyrosine 372 is important for the catalytic activity of Jak2, for the ability of Jak2 to associate with STAT1 leading to STAT1 activation and for Jak2 dependent gene transcription. Additionally, we showed that tyrosine 372 is crit ical for interferon -gamma and epidermal growth factor -dependent Jak2 activation. Future studies should seek to elucidate the

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105 mechanism by which tyrosine 372 regulates Jak2 function.

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106 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z 0 100 E A L S F V S L I D G pY pY R130 201 314 401 548 647 734 847 960 1075 1132 1375 1618 1792 1792 1663 1592 1479 1392 1245 1146 1059 946 833 718 661 418 175byb3 b4 y2 b5 b6 y4 y6 y7 y8 y9 y10 y11 b12 b10 y5 h2o Figure 5 1. Tyrosines 372 and 373 are Jak2 phosphorylation sites. Purified Jak2 protein was subjected to mass spectrometry. Analysis revealed that tyrosines 372 and 373 are phosphorylated The data in Figure 5 1 was generated by Dr. Jennifer Busby.

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107 In different species:Mouse Jak2 361E A L S F V S L I D G Y Y R L T A D A H H Y L C K E V A P P390 Rat Jak2 E A L S F V S L I D G Y Y R L T A D A H H Y L C K E V A P P Pig Jak2 E A L S F V S L I D G Y Y R L T A D A H H Y L C K E V A P P Human Jak2 E A L S F V S L I D G Y Y R L T A D A H H Y L C K E V A P P Puffer fish Jak2 E A L S F V S L I D G Y Y R L T T D A H H Y L C K E V A P P Zebra fish Jak2 E A L S F V S L I D G Y Y R L T T D A H H Y L C K E V A P P Chicken Jak2 E A L S F V S L I D G Y Y R L T A D A H H Y L C K E V A P PIn different JAK family members:Mouse Jak2 E A L S F V S L I D G Y Y R L T A D A H H Y L C K E V A P P Mouse Jak1 E G H FG K V E LC R Y D P E G D N T G E Q V A V K S L K P Mouse Jak3 E A L S F V S L V D G Y F R L T A D A H H Y L C T D V A P P Mouse Tyk2 E A L S F V A L V D G Y F R L T A D S S H Y L C H E V A P P Figure 5 2. Tyrosine 372 is conserved in different Jak family members and among different species. Comparison of the amino acid sequence of Jak2 from different species indicates that tyrosines 372 and 373 are conserved amino acid residues. In addition, tyrosin e 372 is highly conserved among different Janus kinase family members. F igure 5 2 was generated by Sushama Kamarjugadda.

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108 Empty Vector Jak2 WT Jak2 Y372F Jak2 Y373F IP: anti -Jak2 -pAb IB: anti -Jak2 pY1007/Y1008-pAb IB: anti -Jak2 -pAb Jak2(P) Jak2 IB: anti -Tyr(p)-mAb Jak2(P) Empty Vector WT Y372F Y373F Empty Vector WT Y372F Y373FJak2 Y1007 Phosphorylation : Percent of Control Total Jak2 Phosphorylation : Percent of Control120 0 0 20 20 40 40 60 60 80 80 100 100 120 20 20 ** ** 111 111 111A D E B C Figure 5 3. Loss of tyrosine 372 and 373 phosphorylation decrease the ability of Jak2 to autophosphorylate. A) BSC 40 cells were transfected with 10 g of the indicated plasmid DNA and the constructs were overexpressed via a vaccinia virus -mediated expression system. Proteins were immunoprecipitated using a Jak2 polyclonal antibody and resolved by SDS -PAGE. The immunoprecipitated Jak2 was immunoblott ed with an anti -phosphotyrosine monoclonal antibody B) The membrane was then strip ped and re -probed with an anti -p Y1007/ p Y1008 polyclonal antibody. C) To verify equal Jak2 expression among all samples, the membrane was stripped again and re blotted with an anti Jak2 polyclona l antibody. D ) Densitometry analysis of total Jak2 phosphorylation levels of three representative Western blots. Statistical significance was determined via Students ttest. Values of p=0.001469 (Jak2 Y372F) and p=0.0454 (Jak2Y3 73F) were considered signif icant relative to Jak2 WT. E ) Densitometrical analysis of three Western blots quantitating Jak2 tyrosine 1007 phosphorylation levels. Values of p=2.86 x 10 12 (Jak2Y372F) and p=.0.0032 (Jak2Y373F) The data in Figure s 5 3A C were generated by Dr. Peter Sayeski.

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109 IB: anti -STAT1 -pAb IB: anti -phospho STAT1 -pAb IB: anti -Jak2 -pAb STAT1 STAT1(P) Jak2Empty Vector Jak2 WT Jak2 Y372FSTAT1 Phosphorylation : Percent of Control 0 20 40 60 80 100 120 20 Empty Vector Jak2WT Jak2Y372F 80 80 111A D B C Figure 5 4. Loss of Tyrosine 372 phosphorylation reduces Jak2 mediated STAT1 activation. BSC 40 cells were transiently transfected with 10 g empty vector, Jak2 wild t ype or Jak2 Y372F plasmid. A) STAT1 phosphorylation at tyrosine 701 was determined via western blot using an anti a ctive STAT1 antibody. B) Equal STAT1 protein levels were verified by stripping membrane and immunoblotting with a STAT1 po lyclonal antibody. C)To show Jak2 expression levels among all samples, the membrane was stripped again and probed with a Jak2 polyclona l antibody D ) Densitometry analysis of STAT1 phosphorylation levels of three representative Western blots. Statistical significance (p=0.004) was determined using Students t test.

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110 Empty Vector Jak2 WT Jak2 Y372FIB: anti Jak2 pAb IB: anti STAT1 pAb IP: antiSTAT1 pAb 0 20 40 60 80 100 120Jak2 STAT1 Co Association: Percent of ControlEmpty Vector Jak2 WT Jak2 Y372F STAT1 Jak2 WCL IB: anti Jak2 pAb Jak2 Empty Vector Jak2 WT Jak2 Y372FIP: antiJak2 pAb IB: anti STAT1 pAb IB: anti Jak2 pAb WCL IB: anti STAT1 pAb Jak2 STAT1 STAT1 Empty Vector Jak2 WT Jak2 Y372FJak2 STAT1 Co Association: Percent of Control0 20 40 60 80 100 120 WCL IB: anti Jak2 pAb Jak2 20 20 *A D E I 111 111 80 80 111 111 80B C F G H Figure 5 5. The Jak2Y372F mutant hinders the association between Jak2 and STAT1. BSC 40 cells were transiently transfected with 10 g of empty vector, Jak2 wild t ype or Jak2 Y372F plasmid. A) Lysates were immunoprecipitated with anti -STAT1 polyclonal antibody and then Western blotted with anti Jak2 antibody to determine STAT1/Jak2

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111 co association B) The membrane was stripped and reprobed with STAT1 polyclonal antibody to demonstrate equal STAT1 precipitat ion among samples C) Whole cell lysates were probed with anti Jak2 polyclonal antibody to assess J ak2 protein levels D ) Densitometry analysis of STAT1/Jak2 association levels of three representative experiments. Statistical significance (*p=3.87 x 106) was determined using Students t -test. E) Lysates were immunoprecipitated with anti Jak2 antibody and then immunoblotted with anti STAT1 polyclonal antibody to determine Jak2/ STA T1 co association F) The membrane was stripped and re -blotted with Jak2 antibody to demonstrate equal Jak2 precipitat ion among samples G) Whole cell lysates were immunoblotted with anti STAT1 antibody to assess STAT1 prot ein levels H) Whole cell lysa tes were also immunoblotted with anti Jak2 antibody to assess Jak2 protein levels. I ) Densitometry analysis of Jak2/STAT1 association levels. Statistical significance (*p=1.35 x 108) was assessed using Students t test.

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112 20 40 60 80 100 120 0RLU: Percent of Jak2 WTJak2 WT Empty Vector *Jak2 Y372F No Jak2 Jak2 WT Jak2 Y372FIB: antiJak2 pAbA B Figure 5 6. Phosphorylation of Tyrosine 372 facilitates Jak2-dep endent gene transcription. A) COS 7 cells were transiently transfected with 2 g of luciferase plasmid and 5 g of empty vector, wildtype Jak2 or Jak2Y372F plasmids. Two days later, rela tive light units (RLU) were read by a luminometer and were taken as a measure of l uciferase gene expression. B) Equal Jak2 expression was ascertained via Western blot analysis with an anti Jak2 polyclonal antibody. Statistical significance (p= 0.00036) wa s determined using a Students t test.

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113 0 10 0 10 0 10 IFN minutes Empty Vector Jak2 WT Jak2 Y372F IP: antiJak2 pAb IP: antiJak2 pAb IB: antiJak2 pAb Phospho Jak2 Jak2 IB: antiJak2 pY1007/Y1008 pAb111 111 A B Figure 5 7 Phosphorylation of tyrosine 372 is essential for interferon-gamma dependent Jak2 activation. MEF cells were transiently transfected with 10 g empty vector plasmid, Jak2 wildtype plasmid or Jak2 Y372F plasmid. Following transfection, the cells were treated with 1 g/mL interferon -gamma for 0 or 10 minutes. A) The cells were subsequently lysed and Jak2 protein was immunoprecipitated from the lysates. Jak2 tyrosine 1007 phosphorylation was determined via Western blot analysis with an anti J ak2 pY1007/pY1008 antibody. B) The membrane was stripped and re -blotted with anti Jak2 antibody to verify equal Jak2 e xpression among samples Shown is one of three independent results.

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114 0 10 0 10 0 10 minutes EGF Empty Vector Jak2 WT Jak2 Y372F IP: antiJak2 pAb IB: antiJak2 pY1007/Y1008 pAb IP: antiJak2 pAb IB: antiJak2 pAb Phospho Jak2 Jak2 111 111 A B Figure 5 8 Tyrosine 372 is critical for epidermal growth factor dependent Jak2 activation. MEF cells were transiently transfected with 10 g empty vector, Jak2 wild type or Jak2 Y372F plasmids. After transfection, these cells were treated with 200 ng/mL epidermal growth factor for 0 or 10 minutes. A) Lysates were immunoprecipitated with antiJak2 antibody and Western blotted with anti Jak2 pY1007/pY1008 antibody to detect Jak2 tyr osine 1007 phosphorylation B) The membrane was subsequently stripped and re -probed with anti Jak2 antibody to confirm total Jak2 protein levels Shown is one of two independent results.

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115 0 10 0 10 0 10 Jak2 WT Jak2 Y372F Empty Vector IP: antiJak2 pAb IB: antiJak2 pY1007/Y1008 pAb IP: antiJak2 pAb IB: antiJak2 pAb H2O2Phospho Jak2 Jak2 111 111 A B Figure 5 9 Phosphorylation of tyrosine 372 does not affect hydrogen peroxide dependent Jak2 activation. MEF cells were transiently transfected with 10 g empty vector plasmid, Jak2 wildtype plasmid, or Jak2 Y372F plasmid. A) Following transfection, cells were treated with 0.5 mM hydrogen peroxide for 0 or 10 minutes and Jak2 protein was immunoprecipitated from the lysates. Jak2 tyrosine 1007 phosphory lation was then assessed by immunoblotting with anti J ak2 pY1008/pY1008 antibody B) The membrane was stripped and re -probed with an anti Jak2 antibody to confir m equal protein loading. Shown is one of two independent results.

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116 CHAPTER 6 DISCUSSION Overview Since the discovery of Jak2 seventeen years ago, a wealth of evidence has linked this protein to diverse pathophysiological processes. Specifically, Jak2 has been implicated in cardiovascular disease, autoimmune disorders and cancer. Over the past f ew years, much attention has been focused on the role of Jak2 in myeloproliferative disorders, as a gain-of function Jak2 -V617F mutation has been found in almost all polycythemia vera patients and a substantial proportion of essential thrombocythemia and p rimary myelofibrosis patients. The fact that the constitutively active Jak2 -V617F mutation is an important contributor to the pathogenesis of myeloproliferative disorders has made this mutation an attractive target for inhibition via small molecule inhibi tors. Our laboratory has invested effort toward the identification and characterization of Jak2 inhib itors that target aberrant Jak2 kinase activity. In this process, we identified two novel Jak2 tyrosine kinase small molecule inhibitors, termed Z3 and G 6. Z3 and G6 are valuable additions to the growing number of Jak2 inhibitors for a number of reasons. First, both these compounds inhibited Jak2-V617F mediated pathologic cell growth in vitro Second, Z3 and G6 suppressed the ex vivo growth of hematopoi etic progenitor cells isolated from the bone marrow of a myeloproliferative disorder p atient carrying a Jak2 gain -of -function -mutation Third, Z3 and G6 had no effect on Tyk2 or c -Src tyrosine kinase activity at concentrations that inhibited Jak2, thus su ggesting the relative specificity of these compounds for Jak2. Finally, both compounds inhibited the proliferation of Jak2V617F expressing cells more effectively than cells that dont rely on Jak2 for their aberrant growth, confirming the specificity of these compounds for inhibiting Jak2 -dependent aberrant cell

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117 growth. Overall, our results suggest that Z3 and G6 effectively inhibit Jak2 tyrosine kinase function in vitro and ex vivo In addition, the association of Jak2 to a number of different disease s tates emphasizes the need to fully understand Jak2 function. However, while much progress has been made in trying to comprehend the factors that control Jak2 function, much work still needs to be accomplished. Eight of the ten known tyrosine phosphorylat ion sites within Jak2 have been linked to regulatory mechanisms, but many more Jak2 phosphorylation sites remain to be identified. In this dissertation we characterized a novel phosphorylation site in the FERM domain of Jak2 and found that Jak2 tyrosine 372 phosphorylation has important consequences for Jak2 function. Mainly, we found that phosphorylation of tyrosine 372 is important for ligand independent Jak2 tyrosine autophosphorylation, STAT1 activation and Jak2-dependent gene expression. In addition, we showed that tyrosine 372 phosphorylation is critical for interferongamma and epidermal growth factor -mediated Jak2 activation. In Chapter 6 we will discuss the impact of the characterization of Z3 and G6 on Jak2mediated disease. Moreover, we wi ll discuss the effect of tyrosine 372 on Jak2 function and its potential role in autoimmune disorders and cell growth. Characterization of the Jak2 Inhibitors, G6 and Z3 A driving force for the identification of Jak2-specific small molecules can be contrib uted to the identification of the constitutively active Jak2 -V617F m utation that has been observed in almost all polycythemia vera patients and a substantial proportion of essential thrombocythemia and primary myelofibrosis cases. Our laboratory has contr ibuted to the growing number of Jak2 small molecules by identifying two novel Jak2-specific small molecules termed, Z3 and G6. Both Z3 and G6 effectively inhibited Jak2-dependent aberrant cell growth.

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118 Although we have clearly shown that Z3 and G6 inhibit Jak2 -mediated pathologic cell growth in vitro and ex vivo the characterization of these compounds in vivo would give us important information about the therapeutic efficacy of these compounds. Work by others in our laboratory has preliminarily shown the efficacy of G6 in a Jak2 -dependent erythroleukemia murine model, in vivo By intravenously injecting SCID NOD mice with HEL cells expressing the Jak2-V617F mutation, we found the pathologic appearance of blast cells in the peripheral blood and this was si gnificantly reduced with G6 treatment. In addition, we established that HEL cell injection alone resulted in an increased spleen weight to body weight ratio and decreased the myeloid to erythroid ratio due to a marked increase in erythroid cells. However we discovered that treatment with G6 corrected these pathologic effects. Collectively, these results preliminarily show that G6 suppresses Jak2-V617F mediated pathologic cell growth, in vivo If Z3 and G6 have the potential to one day treat myeloprolife rative disorders in patients, it is imperative that the efficacy of these compounds be tested in a myeloproliferative disorder mouse model. A myeloproliferative mouse model could be generated by reconstituting the bone marrow of mice with hematopoietic st em cells that are retrovirally transduced with the Jak2 V617F mutation. We would anticipate that the Jak2-V617F mutation will induce erythrocytosis and splenomagaly in these mice. We hypothesize that both Z3 and G6 will abr ogate these pathologic effects. In addition, since we have demonstrated these compounds to be Jak2selective, we predict that the reduction in erythrocytosis and spleen size will correlate with suppression in Jak2 and STAT activation in these animals. We have shown that Z3 inhibits the growth of Jak2-V617F -expressing human erythroleukemia cells by inducing cell cycle arrest while we have demonstrated that G6 inhibits

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119 the growth of these cells by promoting apoptosis. To enhance our understanding regarding the mechanisms by which Z3 and G6 inhibit aberrant Jak2 activity, it would be useful to know what cell survival associated proteins are downregulated by G6 and Z3 and what apoptotic related proteins are upregulated by G6 in Jak2 -V617F expressing human erythroleukemia cells. To achieve this goal, human erythroleukemia cells that are homozygous for the Jak2V617F mutation can be treated with DMSO, G6 or Z3 and then protein lysates can be separated by 2 dimensional gel electrophoresis to compare the protein expression profile between DMSO and Jak2 inhibitor -treated cells. Subsequently, the differentially expressed proteins would be isolated and subjected to mass spectrometry for their identification. This information could potentially inform us what key proteins are involved in the Jak2-V6 17F signaling pathway that leads to aberrant cell growth. Comparison of Z3 and G6 In this dissertation, we characterized two novel Jak2 tyrosine kinase inhibitors, termed Z3 and G6. Upon analysis, we found that Z3 and G6 shared general similarities, but also displayed differences in their inhibitory properties. The similarities and differences between Z3 and G6 could impact their potential to inhibit Jak2 dependent aberrant cell growth. A main difference between Z3 and G6 is that G6 was more effective th an Z3 in suppressing Jak2-V617F -mediated pathologic cell growth In addition, G6 was more effective than Z3 in blocking the ex vivo growth of hematopoietic progenitor cells isolated from the bone marrow of a myeloproliferative disorder patient. These resu lts suggest that the structure of G6 could be more favorable for binding to the ATP pocket of Jak2 resulting in a greater inhibition of Jak2 V617F tyrosine kinase activity when compared to Z3. Interestingly, the mechanism by which Z3 and G6 inhibited Jak 2 -V617F mediated pathologic cell growth was also different. Z3 inhibited the proliferation of Jak2 V617F

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120 expressing cells by inducing cell cycle arrest while G6 suppressed the growth of these cells by promoting apoptosis. Perhaps we could take advantage of the fact that Z3 and G6 inhibit Jak2V617F function via different mechanism s by treating Jak2-V617F expressing cells with both Z3 and G6 to determine if the combination of both drugs inhibits Jak2 -V617F mediated pathologic cell growth more effectively t han either drug alone. Despite the differences between Z3 and G6, they nevertheless share similarities. Both Z3 and G6 had no effect on Tyk2 or c -Src tyrosine autophosphorylation at concentrations that completely inhibit Jak2. In addition, HEL cells expr essing the Jak2 -V617F mutation were more sensitive to the inhibitory effects of Z3 and G6, when compared to Raji cells that rely on a c -Myc translocation for their aberrant growth. Overall, these results suggest that Z3 and G6 are relatively selective inh ibitors of Jak2 tyrosine kinase. Additionally, the fact that Z3 and G6 have no effect on Tyk2 kinase function in vitro is encouraging given that Tyk2 deficiency has been linked to immune functional defects and increased tumor susceptibility (Nakamura et a l., 2008; Minegishi et al., 2006; Stoiber et al., 2004). Additional Reflections Regarding Jak2 Inhibitors In response to the growing numbers of Jak2 mutations connected with hematological malignancies, there has been an increase in the identification of Jak2 inhibitors (Figure 6 1). A possible weakness facing the current state of Jak2 inhibitors is that although these compounds are suppressing mutant Jak2 tyrosine kinase activity, they are also inhibiting wildtype Jak2 function. For example, Pardanani et al. demonstrated that a 500 nM dose of TG101209 completely inhibits wild type Jak2 tyrosine kinase activity (Pardanani et al., 2007). Moreover, our laboratory show ed that the Z3 compound inhibited Jak2 WT tyrosine autophosphorylation (IC50 = ~15 M) mo re effectively relative to Jak2 -V617F (IC50 = ~28 M) (Sayyah et al., 2008).

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121 Given that normal Jak2 function is critical for hematopoiesis and for the transmission of the growth hormone signal, one wonders about the possible deleterious effects of blocking wild type Jak2 function. The lack of structural information regarding full length Jak2 may currently be an impediment for the design of inhibitors that selectively target aberrant Jak2 kinase activity. In order to overcome this obstacle, the crystal s tructure of the entire Jak2 protein needs to be resolved so that we may have a better understanding regarding the structural differences between mutant and wild type Jak2 protein and therefore develop inhibitors that block only mutant Jak2 kinase activity. Once our structural knowledge regarding the entire Jak2 protein increases, we may evolve to develop Jak2 designer drugs based on specific mutations and par ticular hematological malignancies Another concern is that there is evidence suggesting that addit ional mutations could occur before the acquisition of the Jak2-V617F mutation in some myeloproliferative disorder patients. For example, Nussenzveig et al., found homozygous wild-type Jak2 erythropoietinindependent colonies together with Jak2-V617F -posit ive erythroid -independent colonies in Jak2 -V617F positive polycythemia vera patients (Nussenzveig et al., 2007). These results suggests that an undefined molecular lesion, preceding the Jak2-V617F mutation, may be responsible for clonal hematopoiesis in p olycythemia vera. Therefore, this raises doubts that inhibiting the Jak2 V617F protein, which may not be the founding pathogenic event in myeloproliferative disorders, will be useful In summary, activating Jak2 mutations are found in almost all individua ls with polycythemia vera and a substantial proportion of individuals with essential thrombocythemia and primary myelofibrosis. Interestingly, since the discovery of the Jak2 -V617F mutation in

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122 2005, there has been a steady increase in the number of report ed Jak2 gene aberrations in hematological disorders as well as the number of class I and II small molecule compounds that effectively target constitutive Jak2 kinase activity (Figure 6 1). The question that arises is whether inhibitors that inhibit Jak2 -V 617F tyrosine kinase could also block other Jak2 mutations associated with hematological malignancies. In addition, will a compounds absolute mutant over wild type selectivity be essential for the effective treatment of hematological malignancies? Or, w ill we evolve to develop Jak2 designer drugs based on specific mutations and particular disease? Furthermore, will a Jak2 -V617F -selective inhibitor be useful in light of the fact that a genetic lesion, preceding the Jak2 -V617F mutation, could be responsib le for the pathogenesis of myeloproliferative disorders? Hopefully in the coming years we will have the answers to these questions as our knowledge regarding the role of Jak2 mutations in hematological malignancies increases and we acquire more information regarding the efficacy and safety of Jak2 inhibitors that ar e currently in clinical trials. The Role of Tyrosine 372 on Jak2 Function Tyrosine autophosphorylation is a fundamental process in Jak2 -dependent signaling. In Chapter 5, we investigated the rol e of tyrosine 372 on Jak2 function. We found via electrospray mass spectrometry that tyrosine 372 is a novel site of Jak2 autophosphorylation. We demonstrated that tyrosine 372 phosphorylation is important for maximal tyrosine autophosphorylation, STAT1 activation and Jak2 -dependent gene expression. Within the context of ligand -dependent Jak2 signaling, we found that phosphorylation of tyrosine 372 is important for interferon gamma and epidermal growth factor -mediated maximal Jak2 activation. Based o n t hese results, we suggest that tyrosine 372 is critical for Jak2 function.

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123 Since tyrosine 372 plays a significant role in ligandindependent and dependent Jak2 signaling, we hypothesize that tyrosine 372 could have an impact on Jak2 -mediated pathologies and Jak2 dependent cell growth. The Potential Role of Tyrosine 372 in Autoimmune Disorders Interferon -gamma is a strong activator of inflammatory response and plays a pivotal role in host defense by increasing antigen presentation of macrophages and promot ing natural killer cell activity (Schroder et al., 2004). In addition, its excessive release has been associated with the pathogenesis of chronic inflammatory and autoimmune disease. For example, transgenic mice harboring the interferon -gamma gene linked to the human insulin promoter lead to a deficiency in insulin producing beta cells and develop insulin -dependent type I diabetes (Sarvetnick et al., 1988; Sarvetnick, 2000). In addition, interferongamma overexpression in the epidermis or liver of transg enic mice causes lupus nephritis or chronic active hepatitis, respectively (Toyonaga et al., 1994; Seery et al., 1997). Moreover, hyperesponsiveness to interferon-gamma activates the Jak2 signaling pathway and results in sustained STAT1 activation in huma n inflammatory diseases and autoimmune disorders (Bach et al., 1995; Sampath et al., 1999; Yasukawa et al., 2000; Kuhbacher et al., 2001; Mazzarella et al., 2003; Dong et al., 2007). Interestingly, there is a lack of information regarding the molecular me chanisms leading to interferon gamma -mediated STAT1 activation in inflammatory diseases and autoimmune disorders. Therefore, understanding how Jak2 tyrosine autophosphorylation regulates the Jak2 signaling pathway in inflammatory disease and autoimmune di sorders could increase our knowledge regarding these pathologies. We have shown in C hapter 5 that loss of tyrosine 372 phosphorylation reduces Jak2 Y1007 phosphorylation, total Jak2 tyrosine phosphorylation and STAT1 activation thereby revealing the impo rtance of tyrosine 372 in the upregulation of Jak2dependent signaling. Importantly, we

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124 have also shown that phosphorylation of tyrosine 372 is important for interferon -gamma mediated Jak2 activation in mouse embryonic fibroblasts. Since the pathologic ef fect of interferon -gamma is evident in autoimmune disorders like type 1 diabetes, and is mediated through the Jak/STAT signaling pathway, it would be important to determine whether tyrosine 372 phosphorylation is important for interferon -gamma -mediated Jak 2 and STAT1 phosphorylation in immortalized insulin producing pancreatic beta cells. In addition, it would be worthy to examine if interferon -gamma dependent tyrosine 372 phosphorylation leads to beta cell dysfunction. To carry out the above e xperiments, immortalized pancreatic beta cells that display a normal glucose -dependent insulin secretion could be stably overexpressed with wildtype Jak2 or Jak2Y372F and then treated with interferon-gamma to examine the consequence of tyrosine 372 phosphorylation on Jak2 and STAT1 activation in these cells (Efrat et al., 1995; Cottet et al., 2001). If interferon-gamma mediated Jak2 tyrosine 372 phosphorylation is critical for Jak2 signaling in insulin secreting beta cells, then we would anticipate the Jak2 Y372F m utation to suppress Jak2 and STAT1 phosphorylation levels in these cells relative to wild type Jak2. These results would therefore suggest that tyrosine 372 is important for interferon -gamma mediated Jak2 signaling in physiologically relevant insulin prod ucing cell system. Subsequently, it would be important to demonstrate that the requirement of tyrosine 372 phosphorylation for Jak2 signaling in response to interferon-gamma accompanies pathologic events in insulin producing pancreatic beta cells. It has been reported that interferon -gamma diminishes insulin gene expression, insulin cellular content and glucose -stimulated insulin secretion in beta cells and that these effects could be mediated by activation of the Jak/STAT pathway (Baldeon et al., 1998; Cottet et al., 2001). In particular, Cottet et al. have shown that when SOCS 1, a Jak2 negative regulatory protein, is stably expressed in beta cells, it can

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125 effectively block the suppressive effects of interferongamma on insulin expression and secretion (Cottet et al., 2001). Therefore, their results suggest that the Jak2 signaling pathway is involved in the interferon gamma induced defect in insulin expression and secretion. Examining the effect of Jak2 tyrosine 372 phosphorylation on interferongamma d ependent insulin expression and glucose -stimulated insulin secretion in beta cells would give us more insight on how the Jak/STAT pathway mediates this pathologic process. Specifically, insulin mRNA and protein expression levels could be determined in int erferon -gamma treated beta cells stably expressing wild -type Jak2 or Jak2Y372F. In addition, the amount of glucose -stimulated insulin secretion could be determined in these interferon -gamma stimulated cells that are treated with glucose. We hypothesize that loss of tyrosine 372 phosphorylation would increase insulin expression and glucose -stimulated insulin levels in beta cells relative to wild -type. These results would suggest that Jak2 tyrosine 372 phosphorylation mediates the downregulation of insuli n and glucose induced insulin secretion in beta cells in response to interferon gamma which could lead to beta cell dysfunction. If interferon -gamma signaling in insulin -secreting beta cells requires Jak2 tyrosine 372 phosphorylation to mediate the defect in insulin expression and secretion that occurs in these cells, then perhaps this pathologic effect can be blocked by developing a pharmacological inhibitor that is targeted toward tyrosine 372. We foresee that a tyrosine 372 specific inhibitor would inhi bit interferon -gamma mediated Jak2 activation in beta cells leading to suppression in STAT1 phosphorylation. Furthermore, by pharmacologically blocking Jak2 tyrosine 372 phosphorylation and thus inhibiting interferon gamma dependent Jak2 signaling, we cou ld perhaps prevent deficits in insulin expression and excretion which lead to beta cell dysfunction in type I diabetes.

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126 The Potential Role of Tyrosine 372 Phosphorylation in Cell Growth Activation of epidermal growth factor receptor (EGFR) is implicated in diverse cell functions, including regulation of cell proliferation, differentiation and cell survival. In addition EGFR activation is involved in processes that are critical for cancer progression such as metastasis and angiogenesis. Epidermal growth fa ctor receptor signaling has been shown to be mediated by Jak2 tyrosine kinase and result in the activation of STAT3 (Andl et al., 2004; Colomiere et al., 2009). In turn, STAT3 activation has been shown to increase cell proliferation and survival in vitro and promote tumor growth rates in vivo Interestingly, not much is known regarding which of the 49 Jak2 tyrosine residues could be critical for epidermal growth factor mediated activation of Jak2 and STAT3. In addition, there is no information about whic h of the phosphorylated Jak2 tyrosine residues could have an impact on cell growth and survival in response to epidermal growth factor stimulation. The more we understand about how Jak2 structure influences its cellular function, the more insight we will gain into its essential role in physiology and pathophysiology. We have shown in C hapter 5 that loss of tyrosine 372 phosphorylation suppresses epidermal growth factor -mediated Jak2 tyrosine 1007 phosphorylation. These results suggest that phosphorylation of tyrosine 372 is critical for epidermal -growth factor -dependent Jak2 activation. Since it is established that activated Jak2 phosphorylates its substrate, STAT3 and activated STAT3 increases cell proliferation and survival, it would be pertinent to exa mine if Jak2 tyrosine 372 phosphorylation is required for epidermal growth factor -mediated STAT3 activation. Furthermore, it would be important to determine it tyrosine 372 phosphorylation has functional consequences for cells such as, increasing cell pro liferation and survival. To answer the above questions, it would be important to utilize fibroblast cells that stably express wild type Jak2 or the Jak2Y372F mutant to ensure longterm Jak2 gene expression in

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127 cells. To determine whether tyrosine 372 is critical for Jak2 -dependent -STAT3 activation in response to epidermal growth factor, we would analyze STAT3 phosphorylation levels in these cells in response to epidermal growth factor. We hypothesize that cells stably expressing the Jak2 Y372F mutation would suppress STAT3 activation relative to Jak2-WT expressing cells. These results would imply that Jak2 tyrosine 372 phosphorylation is critical for Jak2-dependent STAT3 phosphorylation in response to epidermal growth factor. Subsequently, it would be important to investigate if tyrosine 372 is essential for cell growth and survival. To determine the role of tyrosine 372 phosphorylation on cell growth, the number viable stably expressing wild type Jak2 or Jak2-Y372F cells that have been treated with e pidermal growth factor could be compared. In addition, the role of tyrosine 372 in cell survival could be addressed by serum starving cells to introduce cell stress and then determining the number of viable cells. We anticipate that loss of tyrosine 372 phosphorylation could result in a lower number of viable cells relative to wild -type expressing cells. These results would link Jak2 tyrosine 372 phosphorylation to upregulation of cell growth and survival. Conclusion In C hapters 3 and 4 of this dissertat ion, we have identified and characterized two novel Jak2 specific small molecule inhibitors termed, Z3 and G6, which effectively inhibit Jak2 mediated pathologic cell growth in vitro and ex vivo Due to the selective nature by which Z3 and G6 inhibit Jak2 these compounds could serve as useful research tools to further understand the Jak2 signaling pathway in the context of physiology and pathophysiology. In addition G6 could potentially serve as a therapeutic agent for the treatment of myeloproliferative disorders based on the encouraging fact that this compound potently inhibits Jak2 -mediated aberrant cell growth in vitro ex vivo and preliminarily, in vivo

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128 We have also advanced our understanding of how structural changes in the FERM domain affect Jak2 function by identifying a novel site of Jak2 tyrosine phosphorylation. In C hapter 5, we have shown that phosphorylation of tyrosine 372 is important for the upregulation Jak2 tyrosine autophosphorylation in the context of a ligand -independent system. In addition, we have found that phosphosphorylation of tyrosine 372 has a significant role in ligand-dependent Jak2 function by facilitating interferongamma and epidermal growth factor -mediated Jak2 activation. Due to the critical role of tyrosine 372 in Ja k2 function, future analysis of this phosphorylation site could give us insight on how to regulate abnormal Jak2 activation in disease states. Reflections The identification and characterization of novel Jak2 tyrosine kinase small molecule inhibitors and the elucidation of the role of tyrosine 372 in Jak2 function are important ongoing research components in our laboratory. Characterization of Z3 and G6 contribute to the body of knowledge pertaining to Jak2 inhibition, whereas characterization of tyrosine 372 enhances our understanding regarding the relationship between Jak2 structure and function. Upon reflection, we realize that our investigations could be enhanced and rendered more complete if certain elements or important experiments are considered i n the future. Z3 and G6 Since Jak3 plays a significant role in immune function and is structurally similar to Jak2, it would be important to investigate the specificity of Z3 and G6 for inhibiting the Jak2 -V617F mutant protein versus Jak3. A direct appr oach would be to obtain a recombinant Jak2 -V617F along with a recombinant Jak3 protein and perform an in vitro kinase assay in the presence or absence of ATP and in the presence of DMSO or Z3/G6. The inhibition of Jak2V617F and Jak3 tyrosine kinase activities by the Jak2 inhibitors could then be compared. In addition to Jak3,

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129 Tyk2 and c -Src, it would be important to determine if Z3 and G6 directly inhibit other tyrosine kinases so that we may have a broader knowledge regarding the specificity of these co mpounds for inhibiting Jak2. We have shown that Z3 and G6 effectively block the ex vivo growth of hematopoietic progenitor cells isolated from an essential thrombocythemia patient carrying the Jak2 -V617F mutation and from a polycythemia vera patient harbor ing the Jak2-F537I mutation. Our studies would have more significance if we examine the efficacy of Z3 and G6 for inhibiting the ex vivo growth of hematopoietic progenitor cells obtained from a greater number of myeloproliferative disorder patients carryi ng Jak2 mutations. Importantly, it would be informative to genotype colonies in our ex vivo colony formation assays to compare the percentage of Jak2 -V617F postive myeloid and erythroid colonies in the absence or presence of Z3 or G6. These results would reveal whether selective suppression of Jak2-V617F harboring colonies derived from myeloproliferative disorder patients occurs in the presence of Z3 or G6. Tyrosine 372 While mass spectrometry analysis revealed that tyrosine 372 is a site of Jak2 phosphor ylation, acquisition of an antibody that specifically recognizes phosphorylated tyrosine 372 on Jak2 would confirm that tyrosine 372 is a Jak2 phosphorylation site. In addition, the characterization of tyrosine 372 would be augmented by the creation of construct in which tyrosine 372 is mutated to glutamic acid (Jak2 Y372E). Since the Jak2Y372E mutation would be used to mimic the consequence of phosphorylation, we anticipate that expression of the Jak2Y372E construct in cells would enhance Jak2 and STAT 1 phosphorylation levels, validating the importance of tyrosine 372 in Jak2 functional regulation. Furthermore, given the fact that tyrosine 372 is conserved in different Jak family members, it would be informative to determine whether phosphorylation of tyrosine 372 is important for Tyk2, Jak1 or Jak3 tyrosine kinase

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130 function. Finally, the investigation of tyrosine 372 lacks a mechanistic explanation for the tyrosine 372 mediated effects on Jak2 function. Since tyrosine 372 is situated in the FERM domai n of Jak2, we hypothesized that tyrosine 372 phosphorylation may be important for Jak2 association with the cytokine receptor. Our study would greatly benefit from an evaluation of t he role of tyrosine 372 in Jak2/ receptor co association.

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131 Figure 6 1 T he number of reported Jak2 mutations and Jak2 inhibitors discovered by year. The reported Jak2 gene aberrations include Jak2 amino acid substitutions, deletions, insertions and chromosomal translocations that were identified since 1997. The Jak2 inhi bitors consist of Jak2 and nonJak2 selective compounds that are either in pre clinical or clinical trials Reprinted with permissi on from Current Medince Group LLC Sayyah J and Sayeski PP. (2009) Jak2 inhibitors: Rationale and Role as Therapeutic Agen ts in Hematologic Malignancies. Curr Onc Rep 11:117124. Figure 1, page 124.

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146 BIOGRAPHICAL SKETCH Jacqueline Sayyah grew up in Santa Monica, California and attended the University of California Los Angeles where she received a Bachelor of Science degree in biochemistry. While attending UCLA, her interest in science was sparked by taking an introductory cancer biology class. As an undergraduate student, her first exposure to research was in t he laboratory of Dr. Gayle Baldwin, where she investigated the effects of tobacco, marijuana and cocaine on human pulmonary immune cell function. Jacqueline subsequently moved to Washington D.C. to earn her M.S. degree in biochemistry and was then awarded a Cancer Research Training Award from NIH to study the role of AKT activation in lung tumorigenesis. In 2004, Jacqueline began pursuing her Ph.D. in biomedical sciences at the University of Florida, under the mentorship of Dr. Peter P. Sayeski in the D ep artment of Physiology and Functional Genomics She received her Ph.D. from the University of Florida in the spring of 2009.