Negative Regulation of Cytokine Signalling in the Myeloid Lineage

NEGATIVE REGULATION OF CYTOKINE SIGNALLING IN THE MYELOID LINEAGE

Investigating the Role of CBL and SH2B1

Mojib Javadi Javed

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Medical Biophysics University of Toronto

NEGATIVE REGULATION OF CYTOKINE SIGNALLING IN THE MYELOID LINEAGE

Investigating the Role of CBL and SH2B1

Mojib Javadi Javed

Doctor of Philosophy

Graduate Department of Medical Biophysics University of Toronto

2012

ABSTRACT

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of

EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R

Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of

EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation.

Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling.

Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-

C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases.

Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.

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ACKNOWLEDGMENTS

The past number of years have had their fill of challenges and moments of excitement. I was very fortunate to have had the support of colleagues, family and friends, throughout this process.

I would like to thank Dr. Dwayne Barber for all his support and mentorship. You have always challenged me to ask the right scientific questions, and have been patient and receptive as I worked towards the answers. I greatly appreciate the exceptional training I have received under your supervision.

I would also like to thank my committee members, Dr. Jane McGlade and Dr. Vuk Stambolic for their guidance. Your critique, input and advice have been an integral part of my advancement as a scientist.

To my lab mates in the Barber lab, past and present, thank you for your sense of humor and encouragement. Thank you for listening to all my rants. Sharing this time with all of you has been an amazing experience. I look forward to our continued friendship and camaraderie.

To my family and friends, thank you for your relentless support. Especially to my parents, thank you for instilling me with the passion to learn, and the drive to achieve anything I set my mind to.

Finally to Sara Zokaei, thank you for helping me stay positive, motivated and inspired. Your support has made the tough “all my experiments failed” moments bearable and the moments of success even sweeter.

TABLE OF CONTENTS

ACKNOWLEDGMENTS...... iv

TABLE OF CONTENTS ...... v

LIST OF TABLES...... viii

LIST OF FIGURES ...... ix

ABBREVIATIONS ...... xii

1 INTRODUCTION...... 1 1.1 HEMATOPOIESIS ...... 2 1.1.1 Erythropoiesis ...... 4 1.2 ERYTHROPOIETIN SIGNALLING ...... 6 1.2.1 Activation of the Erythropoietin Receptor...... 6 1.2.2 Downstream Signalling Pathways...... 7 1.2.3 Attenuation of EPO Signalling...... 13 1.3 GM-CSF SIGNALLING...... 15 1.3.1 GM-CSF Receptor Complex ...... 15 1.3.2 Downstream Signalling ...... 16 1.3.3 Attenuation of GM-CSF Signalling ...... 23 1.4 SH2B Family of Adaptor Proteins ...... 25 1.4.1 SH2B Family...... 25 1.4.2 SH2B1 Regulation of Signalling...... 25 1.4.3 Role of SH2B family in EPO signalling ...... 29 1.5 Casitas B-lineage Lymphoma (Cbl)...... 30 1.5.1 Structural Organization of Cbl...... 30 1.5.2 Cbl as an E3 Ligase...... 31 1.5.3 Adaptor Protein Functions of Cbl...... 36 1.5.4 CBL Associated Oncogenesis ...... 38 1.6 RATIONAL AND HYPOTHESIS...... 42 1.7 THESIS OBJECTIVES ...... 44

2 THE SH2B1 ADAPTOR PROTEIN ASSOCIATES WITH A PROXIMAL REGION OF THE ERYTHROPOIETIN RECEPTOR ...... 46 2.1 ABSTRACT ...... 47 2.2 INTRODUCTION...... 48 2.3 EXPERIMENTAL PROCEDURES:...... 50 2.4 RESULTS ...... 55 2.4.1 COLT Screening Identifies SH2B1β Interacting with EPO-R Y343...... 55 2.4.2 SH2B1 Associates with the EPO-R in Hematopoietic Cell Lines...... 55 2.4.3 SH2B1 Binds Specifically to pY343 and pY401 of the EPO-R upon EPO Stimulation ...... 57 2.4.4 SH2B1 Associates with Unphosphorylated EPO-R ...... 61 2.4.5 SH2B1 is Phosphorylated in Response to EPO Stimulation...... 65 2.4.6 SH2B1 Associates with the EPO-R in Primary Splenic Erythroblasts ...... 68 2.4.7 SH2B1 is a Negative Regulator of Downstream EPO Signaling...... 71 2.5 DISCUSSION...... 73

3 CBL linker region and RING finger mutations lead to enhanced GM-CSF signalling via elevated levels of JAK2 and LYN...... 78 3.1 ABSTRACT ...... 79 3.2 INTRODUCTION...... 80 3.3 MATERIALS AND METHODS ...... 83 3.4 RESULTS ...... 87 3.4.1 Enhanced and Prolonged Phosphorylation of the GM-CSFR βc in CBL Mutant Expressing Cells...... 87 3.4.2 Elevated levels of JAK2 kinase in CBL-Y371H and CBL-C384R expressing TF-1 cells ...... 89 3.4.3 Increased expression of LYN kinase in CBL mutant cells...... 91 3.4.4 Enhanced phosphorylation of SHP2 in CBL mutant cells ...... 93 3.4.5 Expression of CBL mutants result in elevated S6 phosphorylation and enhanced factor-free survival...... 96

3.4.6 Constitutive and enhanced phosphorylation of CBL mutants is dependent on a SRC family kinase...... 96 3.4.7 TG101348 mediated inhibition of enhanced phosphorylation of GM- CSFR βc in CBL mutant expressing cells...... 102 3.5 DISCUSSION...... 104

4 DISCUSSION AND FUTURE DIRECTIONS ...... 111

5 CONCLUDING REMARKS...... 125

REFERENCES ...... 127

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LIST OF TABLES

TABLE 1.1. Cbl Binding Proteins ...... 38

TABLE 1.2 Frequency of CBL Mutations in Myeloid Mallignancies ...... 39

TABLE 3.1. Primers utilized in the QuickChange mutagenesis of CBL constructs ...... 82

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LIST OF FIGURES

FIGURE 1.1. Hematopoiesis ...... 3

FIGURE 1.2. Erythropoiesis ...... 5

FIGURE 1.3. EPO Mediated Signalling ...... 8

FIGURE 1.4. Formation of GM-CSF/GM-CSFRα/GM-CSFR βc Dodecamer Complex...... 17

FIGURE 1.5 GM-CSF Mediated Signalling ...... 19

FIGURE1.6. GM-CSFR Signalling Pathway Mutations associated with Juvenile Myelomonocytic Leukemia (JMML) ...... 22

FIGURE 1.7. SH2B Family of Adaptor Proteins ...... 26

FIGURE 1.8. Cbl Family of E3 Ligases ...... 32

FIGURE 1.9. The Ubiquitin Cascade ……...... 34

FIGURE 1.10. Phosphorylation of Tyr371 activates CBL E3 Ligase ...... 37

FIGURE 1.11. Linker and RING Finger are Hot Spots for CBL Mutations ...... 41

FIGURE 2.1. Specificity of SH2B1 for pY343, pY401 and pY429 ...... 56

FIGURE 2.2.A,B. SH2B1 co-immunoprecipitates with phosphorylated EPO-R in hematopoietic cell lines, via its SH2 domain ...... 58-59

FIGURE 2.3.A-C. Co-immunoprecipitation with EPO-R truncation mutants confirms SH2B1 binds specifically to pY343, and pY401 of the EPO-R ...... 60

FIGURE 2.3.D,E. Co-immunoprecipitation with EPO-R truncation mutants confirms SH2B1 binds specifically to pY343, and pY401 of the EPO-R ...... 62

FIGURE 2.4. The PH domain and amino acids 1-266 of SH2B1 mediate a constitutive association with the membrane proximal region of the EPO-R ...... 63-64

FIGURE 2.5. The association of SH2B1 with EPO-R is independent of Jak2 ...... 66

FIGURE 2.6. SH2B1 and Jak2 do not co-immunoprecipitate in cell lines expressing EPO-R ...... 67

FIGURE 2.7. EPO stimulation induces SH2B1 phosphorylation in a time and dose dependent manner ...... 69

FIGURE 2.8. SH2B1 associates with the EPO-R in primary erythroblasts ...... 70

FIGURE 2.9. Knock down of SH2-B results in enhanced EPO mediated signaling ...... 72

FIGURE 3.1. Enhanced phosphorylation of GM-CSF receptor β upon expression of CBL mutants ...... 88

FIGURE 3.2. TF-1 cells expressing CBL mutants have lower expression levels of GM-CSFR βc ...... 90

FIGURE 3.3. Expression of CBL-Y371H and CBL-C384R mutants results in elevated levels of JAK2 kinase ...... 92

FIGURE 3.4. Increased expression of LYN kinase in CBL-Y371H and CBL-C384R mutant TF-1 cells ...... 94

FIGURE 3.5. Mutant forms of CBL elevate GM-CSF induced phosphorylation of SHP2 ...... 95

FIGURE 3.6. Expression of CBL mutants results in constitutive phosphorylation of S6 ...... 97

FIGURE 3.7. Expression of JMML associated CBL mutants results in enhanced survival of TF-1 cells in the abscence of GM-CSF ...... 98

FIGURE 3.8. CBL-Y371H and CBL-C384R mutants show enhanced phosphorylation in response to GM-CSF stimulation ...... 100

FIGURE 3.9. Dasatinib treatment abolishes the enhanced phosphorylation of CBL-Y371H and CBL-C384R ...... 101

FIGURE 3.10. Treatment with TG101348 abolishes the enhanced phosphorylation of GM-CSFR β in CBL mutant expressing cells ...... 103

FIGURE 3.11. CBL linker and RING finger mutants lead to elevated GM-CSF signalling and enhanced survival ...... 108

ABBREVIATIONS

°C degree(s) Celsius µg microgram µl microliter µM micromolar A alanine APS Adaptor molecule containing PH and SH2 domains ATP adenosine triphosphate Bcl-2 B cell lymphoma 2 Bcl-XL B cell lymphoma X large BFU-E burst forming unit-erythroid BSA bovine serum albumin C cysteine Cbl c-Cbl, Casitas B-lineage lymphoma CD71 transferrin receptor CD116 granulocyte-macrophage colony-stimulating factor receptor α CD131 IL-3/IL-5/GM-CSFR βc CFU-E colony forming unit-erythroid CFU-GEMM colony forming unit – granulocyte, erythrocyte, monocyte, megakaryocyte c-Kit CD117/stem cell factor receptor CRK CT10 regulator of kinase CRKL Crk-like CSF colony stimulating factor C3G Crk SH3 domain-binding guanine nucleotide-releasing factor DNA deoxyribonucleic acid DTT dithiothreitol E1 ubiquitin activating enzyme E2 ubiquitin conjugating enzyme E3 ubiquitin ligase ECL enhanced chemiluminescence EDTA ethylene diamine tetra acetic acid EGF epidermal growth factor EGFR epidermal growth factor receptor EPO erythropoietin EPO-R erythropoietin recetpor ERK extracellular signal related kinase FBS fetal bovine serum FCS fetal calf serum FOXO forkhead family of transcription factor g gram G glycine

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GAS Interferon γ Activated Sequence G-CSF granulocyte colony-stimulating factor GM-CSF granulocyte-macrophage colony-stimulating factor GMP granulocyte/macrophage progenitor GRB growth-factor receptor binding protein GSK3 glycogen synthase kinase 3 GST glutathione S-transferase H histidine HA hemagglutinin HCl hydrochloric acid HEPES N-(2-hydroxyethyl) piperazine-Nʼ-2(-ethanesulfonic acid) HRP horseradish peroxidase HSC hematopoietic stem cell IL interleukin IL-3R interleukin 3 receptor IP immunoprecipiration IRS insulin receptor substrate JAK janus kinase JH janus homology JNK c-Jun N-terminal kinase K lysine kDa kilodalton L liter Lyn lck-yes-related novel tyrosine kinase M molar MAPK mitogen-activated protein kinase MDS myelodysplastic syndromes MEK MAP kinase/Erk-activated kinase MEP megakaryocyte/erythroid progenitor mM milimolar MPD myeloproliferative disease MPN myeloproliferative neoplasm mRNA messenger RNA ng nanogram Myc myelocytomatosis viral oncogene p85 85 kDA regulatory subunit of PI-3K p110 110 kDa catalytic subunit of PI-3K PBS phosphate buffered saline PDGFR platelet derived growth-factor receptor PHZ phenylhydrazine PI phosphatidylinositide PIP3 phosphatidylinositide-3,4,5-triphosphate PI-3K phosphatidylinositol 3-kinase PMS phenazine methosulfate PMSF phenylmethylsulfonyl fluoride AKT PKB, protein kinase B xiii

PTK protein tyrosine kinase pTyr phosphotyrosine PV polycythemia vera PVDF polyvinylidene fluoride pY phosphotyrosine Q glutamine R argenine RBC red blood cell RF RING finger RING really interesting new thing RNA ribonucleic acid RTK receptor tyrosine kinase SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SHC Src homology and collagen homology SHIP SH2-domain-containing 5ʼ inositol phosphatase SHP1 SH2 domain containing phosphatase 1 SHP2 SH2 domain containing phosphatase 2 SOCS suppressor of cytokine signalling Sos son of sevenless Src Rous sarcoma virus STAT signal transducer and activator of transcription TBS Tris-buffered saline TBST Tris-buffered saline containing Tween-20 TKB tyrosine kinase binding Tris tris (hydroxymetyl) aminomethase Triton X-100 t-octylphenoxypoly-ethoxyethanol Tween 20 polyoxyethylene sorbitan monolaurate UbcH7 ubiquitin conjugating enzyme H7 UPD uniparental disomy WT wild type XTT 3,3ʼ-{1-[(phenylamino)carbonyl]-3,4-tetrazolium)bis(4- methoxy-6-nitro)benzene sulfonic acid hydrate Y tyrosine Zn zinc

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1 INTRODUCTION

1.1 HEMATOPOIESIS

Hematopoiesis is the developmental process that gives rise to the entire blood lineage, whose functions ranges from coagulation, immune responses, to oxygen delivery (Figure 1.1). Central to hematopoiesis is the hematopoietic stem cell (HSC). One of the unique properties of HSCs is their ability to undergo symmetric self-renew, whereby the daughter cells maintain HSC multipotency. HSCs can be classified as long-term or short-term HSCs based on their capacity to self-renew, and their ability to provide hematopoietic engraftment in mouse models. HSCs also differentiate to various progenitors, which will go on to form all of the terminally differentiated blood cells. As these hematopoietic progenitors differentiate, they lose their ability to regenerate and become committed to specific lineages. HSCs differentiate to give rise to the common lymphoid progenitor (CLP), and the common myeloid progenitor (CMP). CLPs ultimately give rise to adaptive immune cells; B- and T-cells, as well as natural killer innate immune cells. CMPs differentiate into granulocyte-monocyte progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP). GMPs produce various granulocytes: neutrophils, eosinophils, basophils and mast cells, as well as monocytes and macrophages. MEPs give rise to red blood cells and platelet producing megakaryocytes. The differentiation process of hematopoietic progenitors is regulated by an array of hormones, growth factors and cytokines, as well as the hematopoietic niche in the bone marrow. The expression, availability and levels of various cytokines direct progenitors towards specific lineages. Cytokine signalling is important not only for mediating differentiation, but also regulating the survival, and proliferation of various progenitors in the differentiation process.

FIGURE 1.1. Hematopoiesis. Hematopoiesis is the developmental process that gives rise to all blood cells. HSC - hematopoietic stem cell, LT - long term, ST - short term, MPP - multipotent progenitor, CMP - common myeloid progenitor, CLP - common lymphoid progenitor, GMP - granulocyte monocyte progenitor, MEP - megakaryocyte erythroid progenitor. Modified from (Doulatov et al., 2012).

1.1.1 Erythropoiesis

Erythropoiesis is the developmental process that gives rise to circulating red blood cells. In the developing fetus, primitive erythropoiesis begins in the yolk sac (YS) at embryonic day (E) 7.25. The YS provides erythroid cells for the developing fetus, during which time definitive erythropoiesis becomes established in the fetal liver (FL). HSC from the FL seed the hematopoietic tissue found in the bone marrow, which is responsible for adult hematopoiesis (McGrath et al., 2005). The bone marrow is the only site of adult erythropoiesis in humans. However, murine erythropoiesis can occur in the bone marrow as well as the spleen. Under anemic conditions an erythropoietic stress response is mounted which results in the spleen expanding up to 10-fold to compensate for the absence of effective bone marrow erythropoiesis (Krystal, 1983). Differentiation of committed erythroid progenitors to mature enucleated erythrocytes requires the cytokine, erythropoietin (EPO). EPO is a 34 kDa glycoprotein, produced by the kidney in response to hypoxia (Krantz, 1991; Zhu et al., 2002). EPO responsiveness is determined by expression of its cognate receptor, the erythropoietin receptor (EPO-R), which is first expressed by the Burst Forming Unit- Erythroid (BFU-E) (Figure 1.2). BFU-E progenitors do not require EPO or the EPO-R for their differentiation or survival (Lin et al., 1996; Wu et al., 1995), but these cells are dependent on other factors including IL-3 and GM-CSF (Emerson et al., 1988). EPO dependent growth, survival and differentiation begins at the Colony Forming Unit- Erythroid (CFU-E) progenitor stage, and persists until the end of the erythroblast stage (Figure 1.2, represented by red italics). Both EPO and EPO-R are essential for definitive erythropoiesis, as demonstrated by knock-out mouse models. Deletion of EPO or EPO-R inhibits erythroid development at the CFU-E stage, and results in embryonic lethality at E13 due to severe anemia (Lin et al., 1996; Wu et al., 1995).

FIGURE 1.2. Erythropoiesis.

Development of red blood cells is dependent on the cytokine erythropoietin (EPO). EPO dependent growth and survival begins at the CFU-E stage and lasts to the erythroblast stage (red italics). CFU-GEMM – colony forming unit – granulocyte, erythrocyte, monocyte, megakaryocyte, BFU-E - burst forming unit-erythroid, CFU-E - colony forming unit-erythroid (Richmond et al., 2005)

1.2 ERYTHROPOIETIN SIGNALLING

1.2.1 Activation of the Erythropoietin Receptor

The 507 amino acid erythropoietin receptor is classified as a type I cytokine receptor. The EPO-R has an N-terminal extracellular domain, composed of two fibronectin type II domains (D1 and D2), which contain the specific binding site for EPO (Richmond et al., 2005; Wilson et al., 1999). The EPO-R also contains a transmembrane domain, the sequence and orientation of which are critical in the activation of downstream signalling pathways (Seubert et al., 2003). The cytoplasmic domain of EPO-R does not contain intrinsic kinase activity; therefore, EPO-R depends on Janus Kinase 2 (JAK2) to mediate EPO induced tyrosine phosphorylation (Ihle et al., 1995). The N-terminal JH3-JH7 (Jak Homology) domains of JAK2 comprise the FERM domain, which via binding to the juxtamembrane Box 1 domain of EPO-R mediates the constitutive binding of JAK2 to EPO-R (Jiang et al., 1996; Zhuang et al., 1994). The majority of newly transcribed EPO-R is found in the endoplasmic reticulum (ER). Only about 20% of EPO-Rs successfully undergo proper post-translational modifications, including glycosylation, to give rise to mature Endo H resistant glycosylated EPO-R (Neumann et al., 1993; Yoshimura et al., 1990). Of these mature EPO-R, only a fraction (5% of the total EPO-R pool) are presented at the cell surface (Hilton et al., 1995). JAK2 associates with EPO-R in the ER, mediating the proper modification and transportation of EPO-R through the Golgi Apparatus to the plasma membrane (Huang et al., 2001). Considering the functional consequences of the EPO-R/JAK2 association, it is not surprising that JAK2 is essential for erythropoiesis and EPO-dependent signalling. JAK2 deficient mice are embryonic lethal due to severe anemia. JAK2 knock-out mice lack both BFU-E and CFU-E erythroid progenitors, exhibiting a more pronounced erythroid phenotype than EPO and EPO-R deficient mice (Neubauer et al., 1998; Parganas et al., 1998). This is indicative of the essential role of JAK2 in several other cytokine signalling pathways, including Thrombopoietin, IL-3 and GM-CSF.

The unliganded EPO-R is present at the plasma membrane as a preformed homodimer. Crystallographic studies have shown that in its unliganded dimeric form, the transmembrane domains and the cytoplasmic regions are too distal (73 angstroms) to allow activation of the constitutively bound JAK2 kinases. Upon EPO binding, there is a conformational change in the D1 and D2 extracellular domains of EPO-R bringing the transmembrane domains and cytoplasmic regions of each receptor closer (39 angstroms) permitting JAK2 transphosphorylation and activation (Livnah et al., 1999a; Remy et al., 1999). The activated JAK2 kinases go on to phosphorylate EPO-R tyrosine residues, of which four of eight lie in YXX[L/I/V] motifs preferentially recognized by JAK2 (Argetsinger et al., 2004). Phosphorylated EPO-R tyrosines recruit a variety of effectors, which leads to the activation of downstream signalling pathways (Figure 1.3), which will be further discussed in the next sections.

1.2.2 Downstream Signalling Pathways

Of the eight tyrosines residues in the cytoplasmic domain of EPO-R, seven have been shown to become phosphorylated and recruit various effectors leading to activation of the JAK/STAT, PI-3K and Ras/MAPK signalling pathways (reviewed in (Richmond et al., 2005)). There has been considerable effort to determine which of the EPO-R tyrosine residues are essential for EPO signalling and erythroid development. These investigations showed EPO-R Tyr343 plays an important, although not essential, role in stress erythropoiesis and erythroblast survival and growth (Wojchowski et al., 2006).

1.2.2.1 EPO-R Y343 is Critical to EPO Mediated Signalling

Utilizing chimeric EPO-R, initial investigations into the role of EPO-R Y343 showed that expression of a truncated receptor containing only Y343 allowed for normal survival, expansion, whereas mutating Y343 to a phenylalanine severely attenuated the functionality of the chimeric receptor (Miller et al., 2002; Socolovsky et al., 1997b). Since

FIGURE 1.3. EPO Mediated Signalling Binding of EPO to the EPO-R leads to the activation of Jak/Stat, PI-3K, and MAPK pathways.

Signal Transducer and Activator of Transcription (STAT) 5 is the main interactor of Y343 (Klingmuller et al., 1996b), functional deficiencies observed upon mutation of Y343 were attributed to disruption of STAT5 activation (Li et al., 2003b; Miller et al., 2002). The Ihle group created two knock-in mouse models to study the importance of EPO-R Y343 in an animal model. The EPO-R H strain expressed a truncation of the distal portion of the cytoplasmic domain of the EPO-R containing Y343, but no other cytoplasmic tyrosines. The EPO-R HM strain expressed the identical truncation, except for a Y343F mutation (Zang et al., 2001b). Both EPO-R H and EPO-R HM mice are viable. EPO-R H mice show a decreased fetal liver BFU-Es, although normal hematocrit levels are observed throughout gestation and adult life. EPO-R HM mice also have lower fetal liver BFU-Es, however, in contrast to EPO-R H mice, the HM mice have consistently lower hematocrit levels (Zang et al., 2001b). At low EPO concentrations (close to physiological relevant concentration of 0.5 U/ ml (Socolovsky et al., 1999a)) erythroid progenitor cells from EPO-R HM mice display decreased proliferation, increased apoptosis and disrupted differentiation (Li et al., 2003a). Interestingly, steady- state renal EPO transcript levels are higher in EPO-R HM mice when compared to wild type littermate controls, indicating a potential compensatory mechanism (Menon et al., 2006a). In bone marrow-derived erythroid progenitors, Akt activation is significantly decreased in both strains of mice, whereas Extracellular-Regulated Kinase 1/2 (Erk1/2) is hyperactivated in EPO-R HM animals, relative to EPO-R H or wild-type mice. These observations suggest that EPO-R HM can mediate erythroid progenitor survival, growth and differentiation, via a tyrosine-independent, but JAK2 dependent pathway(s), leading to Erk1/2 activation (Menon et al., 2006a). Aspects of adult erythropoiesis can be dissected by exposing mice to stress agents such as 5-fluorouracil (5-FU) and phenylhydrazine (PHZ). 5-FU is a uracil analogue; it leads to cytotoxicity via misincorporation of fluoronucleotides into RNA and DNA and by inhibiting the nucleotide synthetic enzyme thymidylate synthase. 5-FU treatment leads to depletion of rapidly proliferating progenitor cells, thereby inducing anemia (Longley et al., 2003). Administration of phenylhydrazine (PHZ) causes hemolytic anemia, the destruction of mature erythrocytes (Itano et al., 1975; Krystal,

1983). The rate and level of recovery of the erythroid compartment can be monitored after exposure to 5-FU or PHZ. When EPO-R HM mice are challenged with either 5-FU or PHZ, their recovery is significantly diminished in comparison to EPO-R H and WT mice (Menon et al., 2006a; Menon et al., 2006b). This lack of response to erythropoietic stress is due to the absence of downstream activation of STAT5 response genes in EPO-R HM mice (Menon et al., 2006b).

1.2.2.2 JAK/STAT Pathway

Signal transducer and activator of transcription (STAT) proteins are found in their monomeric form in the cytoplasm. STATs are recruited to phosphorylated tyrosines of cytokine receptors via their SH2 domain, where they are tyrosine phosphorylated by Janus kinases. Upon phosphorylation, STATs are released from the receptor and homo- or hetro-dimerize, translocating to the nucleus. Once in the nucleus, STATs regulate transcription by binding to Interferon γ Activated Sequence (GAS) sites within the promoters of the target genes (Ihle, 1996). EPO stimulates the activation of three STATs; STAT1, STAT3 and STAT5a/b. STAT1 is activated directly downstream of JAK2 and does not require an EPO-R phospho-tyrosine docking site (Haq et al., 2002). Stat1-/- mice have decreased number of bone marrow CFU-Es, which is compensated by an increase in splenic BFU-E and CFU-Es. Stat1 deficient erythroblasts also show a block in differentiation, and an elevated level of apoptosis (Halupa et al., 2005a). Stat3 is recruited to pY431 of the EPO-R (Kirito et al., 2002). Stat3 knock-out mice are embryonic lethal (Takeda et al., 1997), and although Stat3fl/fl animals have been generated, they have not been utilized in erythropoietic specific studies by crossing with Epo-R-Cre mice (Heinrich et al., 2004). Two STAT5 genes (STAT5a and STAT5b) encode proteins that are approximately 95% identical in amino acid sequence (Liu et al., 1995). STAT5 is the main effector downstream of EPO-R Y343; therefore there has been numerous studies to determine whether it plays a critical role in erythropoiesis. The initial Stat5a/b knock

11 out mouse model was produced by deleting the first exon of both Stat5 genes (Stat5a/bΔN/ΔN) (Teglund et al., 1998b). Analysis of Stat5a/bΔN/ΔN revealed no significant difference in erythrocyte levels in adult mice. Adult Stat5a/bΔN/ΔN mice were found to have a slower rate of recovery following PHZ induced erythropoietic stress (Socolovsky et al., 2001). Investigation of fetal erythropoiesis showed that Stat5a/b deficient E13.5 embryos are anemic (Socolovsky et al., 1999a). Fetal liver derived progenitors were found to have differentiation block at an early erythroblast stage. Increased apoptosis of Stat5a/bΔN/ΔN fetal liver-derived erythroid progenitors was reported. These embryonic erythroid phenotypes were attributed to the inability of Stat5a/bΔN/ΔN mice to induce the anti-apoptotic protein Bcl-XL, which is an immediate early gene proposed to be activated by Stat5 in response to EPO (Socolovsky et al., 1999a; Socolovsky et al., 2001). The Stat5a/bΔN/ΔN alleles were found to be hypomorphic, producing a truncated form of Stat5a/b (Hoelbl et al., 2006; Yao et al., 2006), which may explain the mild erythroid phenotype observed. However, complete deletion of Stat5a/b has been shown to result in anemia due to increase in apoptosis of erythroid progenitors. Furthermore, Stat5a/b deficient erythroid progenitors were demonstrated to have lower expression of the transferrin receptor-1 (TfR-1) and iron regulatory protein 2 (Irp-2). Both of these genes are transcriptional targets of Stat5 and are involved in the iron uptake and accumulation of erythroid cells (Kerenyi et al., 2008; Zhu et al., 2008).

1.2.2.3 MAPK Pathway

EPO stimulates the Mitogen-Activated Protein Kinase (MAPK) pathway. Engagement of the guanine nucleotide exchange factor, Son of Sevenless (Sos) to the receptor leads to an elevation of activated Ras-GTP levels, which in turn leads to the downstream activation of various MAPKs. Sos is recruited to the receptor by the adaptor protein, Growth-factor Receptor Bound protein-2 (Grb2), which interacts with Sos in an SH3 dependent manner (Rozakis-Adcock et al., 1993). Grb2 can be recruited directly to the EPO-R via SH2 domain mediated binding to EPO-R pY464 (Barber et al., 1997), or

12 indirectly by binding to SH2 domain-containing 5ʼ Inositol Phosphatase-1 (Ship1) (Mason et al., 2000), Src and Collagen Homology (Shc) (Damen et al., 1993; He et al., 1995), or SH2-containing Phosphatase (Shp2) (Tauchi et al., 1996). Shp2 and Ship1 are recruited to pTyr401 (reviewed in (Richmond et al., 2005)). Erk 1/2 are one set of the MAPKs activated downstream of the EPO-R, and play a role in EPO mediated mitogenesis. SAP kinase (SAPK)/ Jun kinase (JNK) and p38 are other MAPKs activated by EPO (Haq et al., 2002; Jacobs-Helber et al., 2000). p38α has been shown to stabilize EPO mRNA; therefore, p38α deficient mice die of a fatal fetal anemia (Tamura et al., 2000).

1.2.2.4 PI-3K Pathway

The activation of the phosphatidylinositol 3 kinase (PI-3K) pathway contributes to EPO mediated survival and growth. Upon EPO stimulation, PI-3K can be recruited to the EPO-R either directly via binding to EPO-R pY479 (Damen et al., 1995a), or indirectly via interacting with Ship-1, Grb2-associated binder 1 (Gab1), insulin receptor substrate 2 (Irs2) (Bouscary et al., 2003), Shp-1 (Cuevas et al., 1999) and Vav (Shigematsu et al., 1997). Recruitment of PI-3K to the receptor results in the phosphorylation of phosphatidylinositols (PI), ultimately forming phosphatidylinositol-3,4,5-triphosphate

(PIP3), which in turn attracts 3-phosphatidylinositide dependent protein kinase 1 (Pdpk1) via interaction with its pleckstrin homology (PH) domain. Pdpk1 phosphorylates and activates Akt, which phosphorylates various downstream targets leading to survival and proliferative signals (reviewed in (Manning et al., 2007)). The importance of PI-3K signalling in erythroid development and function has been demonstrated via examination of knock out mouse models. p85α deficient mice have reduced number of BFU-E and CFU-E colonies (Huddleston et al., 2003). Also, expression of Akt in JAK2-/- erythroid progenitors rescues deficiencies in erythroid maturation (Ghaffari et al., 2006).

1.2.3 Attenuation of EPO Signalling

1.2.3.1 Phosphatases

As EPO mediates survival and proliferative signals, it is essential that those signals be temporally regulated to prevent aberrant growth, which can lead to myeloproliferative diseases. Protein and inositol phosphatases have been demonstrated to play an important role in the attenuation of EPO signalling. There are three major protein phosphatases that regulate EPO signalling; SH2 domain-containing phosphatase 1 (Shp1), protein tyrosine phosphatase 1B (Ptp-1b) and cluster designation 45 (Cd45). Shp1 binds via its SH2 domain to EPO-R pY429 and pY431. Disruption of Shp1 binding to EPO-R results in EPO hypersensitivity due to prolonged Jak2 phosphorylation, indicating Shp1 is responsible for dephosphorylating and inactivating Jak2 downstream of EPO-R (Klingmuller et al., 1995). Erythroid progenitors of motheaten mice, which have a germline Shp1 mutation, have been shown to be hypersensitive to EPO (reviewed in (Neel et al., 2003)). Cd45 and Ptp-1b are also JAK2 phosphatases and therefore negative regulators of EPO mediated signalling. Over-expression of wild type Ptp-1b leads to decreased EPO-R phosphorylation, where as the expression of an inactivating ʻsubstrate-trappingʼ mutant of PTP-1B results in significantly enhanced EPO-R phosphorylation (Cohen et al., 2004). Cd45 deficient mice have decreased bone marrow derived BFU-E numbers (Irie- Sasaki et al., 2001). It is unclear whether this is a direct effect or a secondary cytokine effect, potentially through altered cytokine secretion from the Cd45-deficient bone marrow (Hesslein et al., 2006). The main inositol phosphatase which has been identified to be functional downstream of the EPO-R is Ship1. Ship1 binds via its SH2 domain to EPO-R pY401 (Mason et al., 2000). Elevated numbers of BFU-E and CFU-E progenitors have been reported in Ship1-deficient mice (Helgason et al., 1998).

1.2.3.2 Suppressor of Cytokine Signalling

The transcription of three members of the Suppressor of Cytokine Signalling (Socs) family of proteins, Socs1, Socs3 and Cis, are induced by EPO stimulation (Matsumoto et al., 1997; Sarna et al., 2003). Cis and Socs3 binding to EPO-R pY401 compete with STAT5 binding, which can potentially restrict Stat5-mediated signalling. Socs1 has been shown to interact with Jak2 pY1007 (in the Jak2 activation loop), leading to the ubiquitination and proteasomal degradation of the kinase (Ungureanu et al., 2002). Socs1-/- mice were found to have decreased hematocrit levels (Metcalf et al., 1999), but fetal liver-derived erythroid progenitors were hypersensitive to EPO (Sarna et al., 2003). Socs3 is recruited to EPO-R pY401, pY429 and pY431 via its SH2 domain (Hortner et al., 2002; Sasaki et al., 1999). Socs3-deficient mice are embryonic lethal at day 12-16, possibly due to an observed erythrocytosis. Interestingly, Socs3-/- fetal liver cells were able to reconstitute hematopoiesis in lethally irradiated recipients, indicating that Socs3 may be dispensable for adult erythropoiesis (Marine et al., 1999).

1.2.3.3 EPO-R Ubiquitination and Internalization

β-transducin repeat-containing protein (β-Trcp) mediates the ubiquitination of phosphorylated targets by forming a complex with Skp1 and Cullin1. Upon EPO stimulation, this complex is recruited to the receptor and mediates the ubiquitination of the receptor leading to its proteasomal degradation (Meyer et al., 2007). Receptor internalization is another mechanism by which EPO-R signalling can be attenuated. EPO-R has been demonstrated to undergo clathrin-mediated endocytosis, via a mechanism that is dependent on p85 recruitment, but independent of PI-3 kinase activity. It has been postulated that binding of p85 to the receptor recruits effectors in the endocytic pathway (Sulahian et al., 2009). EPO-R ubiquitination also plays an important role in receptor endocytosis. Bulut et al. has shown that ubiquitination on Lys256 mediates EPO-R endocytosis, whereas Lys428 ubiquitination marks the receptor for lysosomal degradation (Bulut et al., 2011).

1.3 GM-CSF SIGNALLING

Granulocyte-monocyte colony-stimulating factor (GM-CSF) is a cytokine produced by a variety of cells including B-cells, eosinophils, neutrophils, basophils and macrophages. GM-CSF has variable and pleiotropic effects on hematopoietic cells (reviewed in (Barreda et al., 2004)); one of its major functions is to induce the proliferation and maturation of granulocytes and macrophages. The functional effects of GM-CSF are dependent on several variables such as the target cell type, presence of other cytokines and particularly the concentration of GM-CSF. At low concentrations of GM-CSF, mouse GMPs differentiate to macrophages, whereas at high doses, GM-CSF gives rise to granulocyte colonies (Metcalf, 1980). Furthermore, low GM-CSF doses enhances cell survival, via inhibition of apoptosis (Williams et al., 1990). Beyond driving proliferation and maturation, GM-CSF can also modulate monocyte functions. GM-CSF treatment has been shown to enhance the cytotoxic activity of monocytes against various leukemic cell lines (Williams et al., 1997). The pleiotropic functionality of GM- CSF has led to its use in various clinical settings.

1.3.1 GM-CSF Receptor Complex

GM-CSF mediates its functions by binding to the GM-CSF receptor (GM-CSFR). The GM-CSF receptor is composed of GM-CSFRα (CD116) and GM-CSFR βc (CD131) subunits. The GM-CSFα sub-unit of the receptor binds specifically to GM-CSF, albeit with a low affinity (Gearing et al., 1989). GM-CSFR βc subunit is the major signalling component of the receptor complex, and it is shared by closely related IL-3 and IL-5 cytokine receptors (Hayashida et al., 1990). GM-CSF engagement of its receptor is a multi-step process. GM-CSF initially binds to GM-CSFRα. Two GM-CSF/GM-CSFRα binary complexes then engage a preformed GM-CSFR βc homodimer, forming a hexameric complex (Carr et al., 2001). In this conformation, the GM-CSFR βc- associated JAK2 kinases are too distant to allow for transphosphorylation. In order for optimal GM-CSF signalling to be initiated, two GM-CSF/GM-CSFR α/βc hexameric

16 complexes must interact. This effectively forms a dodecamer complex, which brings the βc associated JAK2 kinases in close proximity, allowing for transphosphorylation of the kinases (Figure 1.4) (Hansen et al., 2008).

1.3.2 Downstream Signalling

1.3.2.1 JAK/STAT Pathway

As a type I cytokine receptor, GM-CSFR does not have its own intrinsic kinase activity and is dependent on GM-CSFR βc associated JAK2 kinase for downstream signalling (Quelle et al., 1994). Binding of GM-CSF to its receptor activates JAK2, which phosphorylates the tyrosine residues in the cytoplasmic tail of the βc sub-unit (Itoh et al., 1998; van Dijk et al., 1997). One of the major canonical signalling pathways activated in cytokine signalling is the JAK/STAT pathway. Although GM-CSF has been shown to activate Stat1 (Brizzi et al., 1996; Welte et al., 1997) and Stat3 (Al-Shami et al., 1998), the main Stat downstream of the GM-CSF receptor is Stat5 (Al-Shami et al., 1998; Mui et al., 1995; Welte et al., 1997). Macrophage, monocyte and granulocyte numbers and maturation in Stat1 deficient mice are comparable to their wild type counterparts, indicating that Stat1 is dispensable for GM-CSF mediated differentiation of GMP progenitors (Durbin et al., 1996). Upon βc phosphorylation, Stat5 is recruited to Tyr612, Tyr695, or Tyr750 (Itoh et al., 1998; van Dijk et al., 1997) via its SH2 domain, and becomes phosphorylated, allowing for its dimerization and activation as a transcription factor. Binding of Stat5 to the βc subunit plays an important role in GM-CSF induced proliferation (Matsuguchi et al., 1997; Quelle et al., 1994). Studies utilizing Stat5a-/- macrophages have demonstrated that in the absence of Stat5a there is an inhibition of growth in response to GM-CSF. Furthermore, GM-CSF stimulation of Stat5a-/- BM derived macrophages results in decreased expression of Cis and bcl-2 like A1 gene, which contribute to cellular differentiation, growth and survival (Feldman et al., 1997). The hematopoietic

FIGURE 1.4. Formation of GM-CSF/GM-CSFRα/GM-CSFR βc Dodecamer Complex. GM-CSF binding to GM-CSFRα leads to the formation of a hexameric complex with GM-CSFR βc. In order for downstream signalling to be induced, two hexamers must interact to form a dodecameric complex (Hansen, Hercus et al. 2008).

18 deletion of Stat5a and Stat5b in Stat5a/bfl/fl Mx-Cre mice has shown that in the absence of Stat5a/b, GM-CSF induced survival of neutrophils and differentiation and maturation of GMPs to granulocytes is significantly compromised (Kimura et al., 2009). These mouse models demonstrate the critical role of Stat5 in GM-CSF signalling.

1.3.2.2 PI-3K Pathway

The GM-CSFR βc does not have a YXXM consensus motif to allow for direct recruitment of PI-3 kinase, and therefore requires adaptor protein dependent mechanisms to couple to this signalling pathway. Guthridge et al. have demonstrated that the mechanism of PI-3K activation is dependent on the concentration of GM-CSF used to stimulate signalling (Guthridge et al., 2006). Stimulation with low doses of GM- CSF leads to protein kinase A phosphorylation of GM-CSFR βc Ser585 (Guthridge et al., 2000), which binds adaptor protein 14-3-3ζ (Stomski et al., 1999). 14-3-3 recruits the p85 subunit of PI-3 Kinase to GM-CSFR βc (Guthridge et al., 2000) (Figure 1.5). There is a unidirectional switch from Ser585 phosphorylation, to Tyr577 phosphorylation at higher doses of GM-CSF (Guthridge et al., 2006). GM-CSFR βc pTyr577 binds the adaptor protein Shc (Pratt et al., 1996). Stimulation with high doses of GM-CSF also results in 14-3-3ζ phosphorylation on Tyr179, mediating binding to the Shc SH2 domain (Barry et al., 2009). Formation of GM-CSFR βc-pTyr577/Shc/14-3-3 complex couples GM-CSF stimulation to the activation of the PI-3K pathway (Figure 1.5). Activation of the PI-3K pathway at low doses of GM-CSF result in enhanced cell survival, whereas activation of the pathway in response to higher concentrations of GM-CSF leads to elevated cell growth as well (Guthridge et al., 2006). There is also evidence for the activation of the PI-3K pathway via LYN downstream of the GM-CSF receptor. GM-CSF stimulation can lead to formation of a complex containing p85 subunit of PI-3K, Lyn and GM-CSFR βc (Jucker et al., 1995), potentially allowing Lyn to phosphorylate p85 (al-Shami et al., 1997), culminating in the activation of the PI-3 Kinase pathway downstream of GM-CSFR (Corey et al., 1993).

FIGURE 1.5. GM-CSF Mediated Signalling Binding of GM-CSF to the GM-CSF receptor complex leads to the activation of Jak/Stat, PI-3K, and MAPK pathways.

Furthermore, Lyn binds to GM-CSFRα, and upon stimulation, induces a Jak2- independent survival signal. This GM-CSFRα/Lyn survival signal requires the activation of the PI-3K pathway (Perugini et al., 2010), potentially via direct binding of p85 to GM- CSFRα (Dhar-Mascareno et al., 2003). The GM-CSF stimulated survival signal is mediated by a PI-3K dependent inhibition of apoptosis. Upon cytokine deprivation, there is transcriptional up-regulation of Bim and Puma that activate pro-apoptotic Bcl-2 family members, Bax and Bak, which in turn induce apoptosis (Ekert et al., 2006). In order to inhibit apoptosis, GM-CSF stimulation of the PI-3K pathway activates Akt, which phosphorylates Foxo3a, inhibiting the transcription factor from translocating to the nucleus and inducing Bim and Puma transcription (You et al., 2006). Alternatively, GM-CSF stimulation maintains the expression of the anti-apoptotic Bcl-2 family member, Mcl-1 (Steimer et al., 2009). Glycogen synthase kinase-3 (GSK-3) phosphorylates Mcl-1, targeting it for proteasomal degradation. However, GM-CSF activated Akt phosphorylates GSK3, thereby inactivating GSK3, and preventing Mcl-1 degradation (Maurer et al., 2006).

1.3.2.3 Ras/MAPK Pathway

GM-CSF stimulation leads to the activation of various MAPK pathway effectors including Erk1/2 (Coffer et al., 1998; Okuda et al., 1992), p38 (Nahas et al., 1996), and Sapk/Jnk (Foltz et al., 1997; Terada et al., 1997). Activation of Ras signalling plays a critical role in GM-CSF mediated growth and proliferation (Okuda et al., 1994; Satoh et al., 1992). Tyrosine phosphorylation of GM-CSFR βc is required for the activation of Ras/MAPK pathway, in particular Tyr577, Tyr612 and Tyr695 (Sato et al., 1993). The Shc adaptor protein binds to pTyr577 (Itoh et al., 1996; Pratt et al., 1996) and becomes phosphorylated, this in turn recruits Grb2 (Lanfrancone et al., 1995). Grb2 itself can directly bind to GM-CSFR βc pTyr577 and pTyr612. Engagement of Grb2 by GM- CSFR βc leads to Ras activation by the Grb2 associated guanine nucleotide exchange factor, Sos (Lanfrancone et al., 1995). The Shp2 phosphatase, whose activation plays a positive regulatory role in Ras signalling, binds to βc pTyr577 and pTyr612 (Itoh et al.,

1996). Shp2 can also recruit Grb2-Sos, leading to Erk activation (Pazdrak et al., 1997) (Figure 1.5). Mutations that affect the regulation of the Ras signalling pathway downstream of GM-CSFR have been shown to lead to GM-CSF hypersensitivity (Figure 1.6), which is a hallmark of Juvenile Myelomonocytic Leukemia (JMML) (Loh, 2010). Similar mutations have been identified in Noonan syndrome patients, which have the propensity to develop JMML. Mouse modeling of the various mutations identified in JMML have provided a great deal of understanding of how deregulation of the Ras signalling pathway can lead to the pathogenesis of myeloid malignancies. About 20-25% of JMML patients have mutations in Ras (Flotho et al., 1999; Kalra et al., 1994; Miyauchi et al., 1994). Hematopoietic-restricted expression of the K-Ras G12D mutant results in a fatal myeloproliferative disease. Bone marrow colony assays show enhanced colony formation in the presence of GM-CSF (Braun et al., 2004; Chan et al., 2004). NF-1 is a GTPase activating protein (GAP), and negatively regulates Ras signalling by enhancing the hydrolysis of Ras-GTP to Ras-GDP. NF-1 is mutated in 10- 15% of JMML patients (Side et al., 1997; Side et al., 1998). Deletion of Nf-1 in the hematopoietic linage in Nf-1fl/fl Mx-Cre mice also results in a MPD. Similar to the K-Ras G12D mice, bone marrow cells deficient in Nf1 show enhanced colony formation in response to GM-CSF (Le et al., 2004). Thirty-five per cent of JMML patients have been found to harbor activating gain- of-function mutations in SHP2 (PTPN11) phosphatase (Kratz et al., 2005; Loh et al., 2004; Tartaglia et al., 2003). Transformation of murine fetal liver or bone marrow cells with Shp2-D61Y or Shp2-E76K mutants results in enhanced colony formation in response to GM-CSF, as well as enhanced Erk and Akt phosphorylation in bone marrow derived macrophages and mast cells, indicating GM-CSF hypersensitivity (Chan et al., 2005; Mohi et al., 2005; Schubbert et al., 2005). Mice transplanted with mutant Shp2 transduced bone marrow succumb to a fatal and invasive JMML-like myeloproliferative disorder (MPD) (Mohi et al., 2005). Similarly Shp2-D61Y knock-in mice develop a fatal MPD after a long latency (Chan et al., 2009a). A potential mechanism to explain how

FIGURE1.6. GM-CSFR Signalling Pathway Mutations associated with Juvenile Myelomonocytic Leukemia (JMML). The GM-CSF signal transduction pathway is illustrated. Mutations in genes, which lead to JMML and/or Noonan Syndrome are indicated. Modified from (Loh, 2011)

Shp2 activating mutations lead to enhanced Ras/MAPK signalling and GM-CSF hypersensitivity has been investigated by Huang et al. They have shown that Shp2 dephosphorylation of interferon response factor 8 (Irf8) is critical for the role of Irf8 in transcriptional activation of Nf1. Therefore, activating mutations of Shp2 leads to enhanced dephosphorylation of Irf8 and decreased expression of Nf1, resulting in elevated Ras activity (Huang et al., 2006).

1.3.3 Attenuation of GM-CSF Signalling

1.3.3.1 Phosphatases

Phosphatases play a critical role in the negative regulation of cytokine signalling by targeting kinases, downstream effectors as well as cytokine receptors for dephosphorylation. The two main phosphatases engaged in response to GM-CSF are Shp1 and Shp2. As discussed above Shp2 plays a positive regulatory role in GM-CSF signalling. However, Shp1 negatively regulates GM-CSF signalling. Shp1 is recruited via its SH2 domain to pTyr612 of IL-3/IL-5/GM-CSFR βc in response to IL-3 stimulation (Bone et al., 1997; Yi et al., 1993). Shp1 dephosphorylates βc, leading to attenuation of GM-CSF signalling. Shp1 binds and dephosphorylates activated Janus kinases (Jiao et al., 1996; Klingmuller et al., 1995). Therefore, an alternative mechanism by which Shp1 may negatively regulate GM-CSF signalling is through the direct dephosphorylation of Jak2 associated with GM-CSFR βc. Macrophages isolated from the motheaten Shp1 loss-of-function mutant mice display enhanced GM-CSF induced proliferation, providing in vivo evidence for the negative regulatory role of Shp1 in GM-CSF mitogenic signalling (Jiao et al., 1997). SH2-containing inositol 5-phosphatase-1 (Ship1) plays an important role in the negative regulation of GM-CSF signalling via modulation of the PI-3K pathway. Ship1 has been shown to bind to IL-3/IL-5/GM-CSFR βc in response to IL-3 stimulation (Liu et al., 1996a). Investigations utilizing Ship1 deficient mice have clarified the critical role of SHIP-1 in GM-CSF signalling. Ship1-/- mice succumb to a myeloid hyperplasia, which

24 leads to splenomegaly and myeloid infiltration of vital organs (Helgason et al., 1998; Liu et al., 1996a). SHIP-1-/- bone marrow derived progenitors show dose-dependent hypersensitivity to GM-CSF in colony forming assays (Helgason et al., 1998), as well as elevated and prolonged activation of Akt and accumulation of PIP3 in response to GM- CSF stimulation (Liu et al., 1996a). Furthermore, over-expression of SHIP-1 in CD34+ cells collected from JMML patients harboring either SHP2 or KRAS mutations significantly reduces their enhanced proliferation and colony formation, effectively abolishing GM-CSF hypersensitivity (Metzner et al., 2007). Interestingly, the authors also demonstrated that expression of the enzymatically inactive SHIP-1 D672A mutant, does not inhibit GM-CSF hypersensitivity in JMML patient progenitor cells.

1.3.3.2 Receptor Modulation Post-Stimulation

Binding of GM-CSF to the receptor initiates events that lead to endocytosis of the receptor from the cell surface (Nicola et al., 1988). Events leading up to GM-CSFR endocytosis have also been shown to play an important role in attenuation of signalling. Studies investigating IL-5 induced receptor endocytosis can be used to gain insight into how other βc sharing cytokine receptors are regulated. Martinez-Moczygemba et al. have shown that the cytoplasmic portion of the βc becomes ubiquitinated in response to stimulation (Martinez-Moczygemba et al., 2001). This JAK2-dependent ubiquitination event occurs at the cell surface and is maintained after endocytosis (Lei et al., 2011; Martinez-Moczygemba et al., 2007). Ubiquitination marks the cytoplasmic region of the βc receptor for proteasomal degradation, which occurs once the receptor has been endocytosed. Proteasomal degradation of the cytoplasmic domain of the βc contributes to attenuation of downstream signalling. The remaining cytokine/receptor complex is then trafficked to the lysosome for degradation (Lei et al., 2008; Martinez-Moczygemba et al., 2001). It is likely that the GM-CSF/GM-CSFRα/GM-CSFR βc complex is similarly regulated, providing an important role of receptor ubiquitination in the negative regulation of GM-CSF signalling.

1.4 SH2B Family of Adaptor Proteins

1.4.1 SH2B Family

Adaptor proteins are a class of proteins, which are devoid of intrinsic enzymatic activity, but composed of several modular protein-protein interaction domains. Adaptor proteins are responsible for the formation of protein complexes, which lead to the engagement of various effectors and regulators downstream of activated receptors. The SH2B family of adaptor proteins, has three members, SH2B1 (SH2-B, PSM), SH2B2 (APS (Adaptor molecule containing PH and SH2 domains)) and SH2B3 (Lnk) (Maures et al., 2007). All three members contain an amino-terminal pleckstrin homology (PH) and dimerization domains, a carboxy terminal Src Homology 2 (SH2) domain and several proline rich regions. Sequence similarity between each domain is very high (~ 60-80%) among the family members (Figure 1.7) (Maures et al., 2007; O'Brien et al., 2002). There are four isoforms of SH2B1 (α, β, γ, δ). These isoforms are a result of mRNA splicing, and differ only in the length of their carboxyl terminus. SH2B1 is ubiquitously expressed, although SH2B1β is the prominent isoform observed in hematopoietic cells. SH2B family proteins have been shown to be involved in signalling downstream of several receptor tyrosine kinases as well as receptors engaging Janus kinase family members.

1.4.2 SH2B1 Regulation of Signalling

1.4.2.1 SH2B1 Modulation of JAK2

Jak2 was one of the first identified binding partners of SH2B1. Rui et al. demonstrated that SH2B1 not only interacts with Jak2 but is also a substrate of Jak2 and becomes phosphorylated upon Growth Hormone (GH) stimulation (Rui et al., 1997a). SH2B1 has a more complex role in its interaction with Jak2, than just an adaptor protein. It can also act as an activator of Jak2 (Kurzer et al., 2004; Kurzer et al., 2006; Nishi et al., 2005; O'Brien et al., 2002; Rui et al., 2000b).

FIGURE 1.7. SH2B Family of Adaptor Proteins. DD – dimerization domain, SH2 – Src Homology 2, PH – Pleckstrin Homology, P – proline rich region. Adapted from (Maures, Kurzer et al. 2007).

Tyrosine 813 of Jak2, and SH2 domain of SH2B1 are both required for the positive regulatory activity of SH2B1 (Kurzer et al., 2004; O'Brien et al., 2002). There are at least two models of how SH2B1 may be regulating Jak2 activity, the first model hypothesizes that dimerization of SH2B1 leads to dimerization of its associated Jaks, which in turn enhances Jak2 activity (Nishi et al., 2005). The other model suggests that binding of SH2B1, causes a conformational change in Jak2 which allows Jak2 to remain in an active state (Kurzer et al., 2006). SH2B1 has been shown to also associate with inactive Jak2. Structural- functional studies of SH2B1 have shown that a region of SH2B1 consisting of aa 269- 555 (which includes the PH domain) allows SH2B1 to interact with inactive Jak2, with relatively low affinity (Rui et al., 2000b). There are several models for the functional consequences of this interaction. The first is that binding of SH2B1 to inactive Jak2 prevents spontaneous and abnormal activation of Jak2. The second functional consequence would be that association of SH2B1 with inactive JAK2 increases the local concentration of SH2B1 around Jak2 and once JAK2 is activated and becomes tyrosyl phosphorylated, the SH2 domain of SH2B1 is able to bind more rapidly and with high affinity to Jak2, leading to enhancement of Jak2 activity (Rui et al., 2000b).

1.4.2.2 SH2B1 and Associated Signalling Pathways

SH2B1 has been studied in context of a number of different receptor tyrosine kinases, including receptors for Insulin, Insulin-like growth Factor I (IGF-I), nerve growth factor (NGF), and platelet-derived growth factor (PDGF). SH2B1 acts as a positive regulator of signalling downstream of these receptors and leads to enhanced activation of the PI- 3K pathway (Deng et al., 2007; Riedel et al., 2000; Wang et al., 2004). SH2B1 is recruited, in a SH2 dependent manner, to the NGF receptor, TrkA, post-stimulation (Choi et al., 2001). The Ginty group has shown that in NGF-responsive PC12 cells, SH2B1 exists in a homo-dimeric form and even in a heteromeric complex with APS. SH2B1 is able to multimerize via an N-terminal dimerization domain (aa 1- 237), which also contains a proline-rich region. This multimerization domain is critical

28 for NGF-induced signalling pathways mediated by SH2B1, allowing morphological differentiation of neural PC12 cells (Qian et al., 2001a). Murine SH2B1 also has a large number of phosphorylation sites, including 9 tyrosines, 82 serines and 29 threonines (Rui et al., 1998). The Carter-Su group has shown that SH2B1 can be differentially phosphorylated on these residues, and that the phosphorylation pattern is dependent on the stimuli. For example, GH or PDGF stimulate JAK2-dependent SH2B1 tyrosine phosphorylation (Rui et al., 1998; Rui et al., 2000b). In contrast, NGF stimulates Ser/Thr phosphorylation of SH2B1 (Rui et al., 1999b). Differential serine/threonine phosphorylation of SH2B1 has also been observed in response to PDGF and epidermal growth factor (EGF) (Rui et al., 1998). SH2B1 undergoes shuttling between the cytoplasm and the nucleus in response to NGF (Chen et al., 2004). The amino terminus of SH2B1 contains a nuclear export sequence (NES) as well as a polybasic nuclear localization sequence (NLS), which regulate the nucleocytoplamic shuttling of SH2B1. NES deactivating mutations lead to decreased NGF-induced transcription of genes required for neural differentiation (Maures et al., 2009). NGF induced serine phosphorylation of SH2B1 has been shown to play an important role in the nuclear shuttling of the adaptor protein. The polybasic NLS of SH2B1 interacts with the negatively charged plasma membrane, and SH2B1 dimerization further stabilizes this interaction. Upon stimulation, serine residues in the polybasic region surrounding the NLS become phosphorylated. These phosphorylated serine residues electrostatically disrupt SH2B1 binding to the plasma membrane and SH2B1 dimerization, effectively releasing SH2B1 from the plasma membrane and allowing for localization to the nucleus (Maures et al., 2011).

1.4.2.3 Role of SH2B1 in Energy Homeostasis and Body Weight

Utilizing SH2B1-/- mice, the Rui group has demonstrated that deficiency in SH2B1 results in severe metabolic defects including hyperglycemia, hyperinsulinemia, hyperleptinemia, hyperlipidemia, glucose intolerance, and insulin resistance, all leading to development of type 2 diabetes and obesity (Duan et al., 2004b; Ren et al., 2005).

Deficiency in SH2B1 leads to global impairment of insulin signalling, including decreased insulin receptor (IR) auto-phosphorylation, decreased IR kinase activity and decreased activation of downstream targets Erk 1/2, Irs1 and Irs2, whose activation is required for insulin induced activation of PI-3K (Duan et al., 2004b; Morris et al., 2009). Binding of leptin to its receptor results in the activation of Jak2, which mediates downstream signalling. As SH2B1 is a positive regulator of Jak2, deficiency in SH2B1 results in impairment in leptin-mediated signalling. SH2B1 has also been shown to bind to Irs1 and Irs2, thereby recruiting these adaptor proteins to the Leptin Receptor, leading to the enhancement of PI-3Kinase activity. Furthermore, SH2B1 binding protects Irs proteins from dephosphorylation (Li et al., 2007b). Interestingly, neuron- specific reconstitution of SH2B1 in SH2B1-/- restores leptin and insulin sensitivity and reverses obesity, and neuron-specific over-expression of SH2B1 protects against high fat diet induced obesity (Ren et al., 2007). The importance of SH2B1 in regulating energy homeostasis and body weight has also been demonstrated in humans. Genome-wide association studies have identified single nucleotide polymorphisms within the SH2B1 gene to be linked to obesity (Jamshidi et al., 2007; Speliotes et al., 2010; Thorleifsson et al., 2009; Willer et al., 2009).

1.4.3 Role of SH2B family in EPO signalling

1.4.3.1 SH2B3 (Lnk): Negative Regulator of JAK2 and EPO Signalling

Murine knock out models showed that deficiency in Lnk results in an expansion of hematopoietic progenitors and that Lnk plays a role in HSC quiescence and self- renewal (Bersenev et al., 2008). Lnk deficient hematopoietic progenitors are also found to be hypersensitive to stem cell factor (SCF) and interleukin -3 (IL-3) (Takaki et al., 2002; Velazquez et al., 2002). Lnk-/- animals have increased numbers of erythroid progenitors, show faster recovery from erythropoietic stress, and enhanced EPO-

30 induced signalling. Molecular investigations revealed that Lnk acts as a negative regulator of EPO signalling by attenuating Jak2 activation (Tong et al., 2005). The role of Lnk as a negative regulator of Jak2 signalling was confirmed in TEL- JAK2 and JAK2 V617F mediated murine MPN models, whereby deficiency in Lnk resulted in enhancement of disease (Bersenev et al., 2010; Gery et al., 2009). Furthermore, mutations in Lnk have been identified in patients with JAK2-V617F positive and negative MPN (Baran-Marszak et al., 2010; Hurtado et al., 2011; Oh et al., 2010).

1.4.3.2 SH2B2 (APS) Negatively Regulates EPO Signalling

The Yoshimura group demonstrated that upon EPO stimulation, Aps binds to tyrosine 343 of the EPO-R. Furthermore, they showed that Aps becomes phosphorylated on a carboxy-terminal tyrosine upon EPO stimulation and this phosphorylated tyrosine acts as a recruitment site for Cbl. The Aps/Cbl complex was found to negatively regulate EPO induced activation of STAT5 (Wakioka et al., 1999a). However the physiological applicability of these studies is under question, as APS is not highly expressed in hematopoietic cells. Furthermore, these studies were performed in a 293T over- expression system, with exogenous over-expression of EPO-R, Aps and Cbl. Aps-/- mice are viable and fertile (Iseki et al., 2004; Li et al., 2006). Aps deficient mice were found to have normal steady state erythropoiesis, although a detailed investigation of the role of SH2B2 in erythropoiesis would require induction of erythropoietic stress.

1.5 Casitas B-lineage Lymphoma (Cbl)

1.5.1 Structural Organization of Cbl v-Cbl was originally isolated as the transforming oncogene from the murine Cas-Br-M ecotropic retrovirus, causing pre-B and pro-B lymphomas (Langdon et al., 1989a), and cellular Cbl or c-Cbl (herein referred to as Cbl) was the first mammalian homolog to be

31 identified (Langdon et al., 1989b). Other members of the Cbl family include Cbl-b and Cbl-3 (Cbl-c) (Figure 1.8). There are several conserved domains among the three Cbl family members (reviewed in (Thien et al., 2005b)). The amino terminus of Cbl proteins contains a highly conserved Tyrosine Kinase Binding (TKB) domain. The Cbl TKB domain consists of three sub-domains; a four-helix bundle, a calcium binding EF hand and an SH2 domain variant. This multi-subunit TKB domain is unique to Cbl proteins. All sub- domains are required for functional binding of Cbl to phosphotyrosine residues (Meng et al., 1999). The TKB domain is followed by a highly conserved alpha helix linker region (Please see section 1.5.2.3 for more details). Cbl and Cbl-b also contain a RING finger domain, which is essential for E3 ligase activity. The carboxy terminus of Cbl and Cbl-b is composed of a proline-rich region, serine and tyrosine phosphorylation sites, as well as an ubiquitin associated domain (UBA). The UBA domain has been shown to allow for homo- and heterodimerization of Cbl and Cbl-b (Peschard et al., 2007). Cbl-b UBA domain is capable of binding to ubiquitin, whereas the Cbl UBA domain shows decreased affinity to ubiquitin (Davies et al., 2004).

1.5.2 Cbl as an E3 Ligase

1.5.2.1 The Ubiquitin Cascade

Ubiquitination is classified as a post-translational modification, which culminates in the formation of a covalent bond between an ubiquitin moiety and a target protein. Ubiquitin (Ub) is a highly conserved, 76 amino acid protein, which is ubiquitously expressed. Ubiquitination is a multi-step process and begins with activation of Ub via an ATP dependent formation of a thiol-ester bond between Ub and the active-site cysteine of the ubiquitin-activating enzyme (E1). The activated Ub is transferred to the active-site cysteine of the ubiquitin-conjugating enzyme (E2), again via formation of a thiol-ester bond. The E2-Ub then interacts with an E3 ligase, which mediates the transfer of the Ub

FIGURE 1.8. Cbl Family of E3 Ligases. TKB – Tyrosine Kinase Binding domain, 4H – Four helix bundle, EF – EF hand, SH2 – atypical SH2 domain, L – linker, RF – RING finger, UBA – Ubiquitin associated domain.

33 to a lysine residue of the target protein (Figure 1.9). The specificity of ubiquitination is determined by the interaction between the substrate and E3 ligase. A substrate can be modified by the addition of a single Ub (monoubiquitination), or by monoubiquitination at multiple sites (multiubiquitination). Lysine residues within Ub can also be ubiquitinated to give rise to ubiquitin chains (polyubiquitination) (reviewed in (Deshaies et al., 2009). The different forms of ubiquitination can have varying biological effects. Monoubiquitination and multiubiquitination have been shown to play a role in endocytosis and endosomal sorting (Hicke et al., 1996). All seven lysine residues of ubiquitin can be utilized to form polyubiquitin chains, of which, Lys48 and Lys63 are the best characterized. Lys48 chains target the modified substrate for proteasomal degradation (Chau et al., 1989), whereas Lys63 chains are involved in modulation of non-proteolytic signalling (Deng et al., 2000; Spence et al., 2000; Spence et al., 1995).

1.5.2.2 Cbl as an E3 Ubiquitin Ligase

Extensive genetic and biochemical studies have confirmed the role of Cbl as an E3 ubiquitin ligase in down regulation of receptor tyrosine kinases, as well as a number of non-receptor protein tyrosine kinases. Upon activation, Cbl is recruited via its TKB domain to phosphotyrosine residues of the RTK or PTK. Lysine residue(s) of the associated RTK/PTK is then ubiquitinated, which can signal for receptor internalization or target the substrate for proteasomal degradation. The role of Cbl has been most extensively studied in context of epidermal growth factor receptor (EGFR) ubiquitination and degradation. Stimulation induced phosphorylation of EGFR leads to the recruitment of Cbl, either directly by binding to EGFR pY1045 (Levkowitz et al., 1999), or indirectly via associating with Grb2 (Batzer et al., 1994; Huang et al., 2005; Waterman et al., 2002). Cbl then interacts with the UbcH7 E2 to ubiquitinate the EGFR. Cbl mediated ubiquitination is sustained throughout endocytosis and within the endosome, suggesting that Cbl plays an important role in the maintaining the receptor in the endosome to ensure proper endosomal sorting (Longva et al., 2002; Schmidt et al., 2005). Interestingly, evidence suggests that Cbl and its RING finger are necessary for

FIGURE 1.9. The Ubiquitin Cascade. Ub – ubiquitin, E1 – Ub activating enzyme, E2 – Ub conjugating enzyme, E3 – E3 ligase.

EGFR internalization (Huang et al., 2005), however, EGFR ubiquitination is not required for initial internalization but essential for receptor degradation (Huang et al., 2007). It has been hypothesized that deubiquitination of the receptor may promote sorting to recycling endosomes, returning the receptor to the cell surface instead of undergoing lysosomal degradation (reviewed in (Schmidt et al., 2005)). Cbl has also been shown to regulate the down-regulation of a variety of RTKs including: platelet derived growth factor receptor (PDGFR), colony stimulating factor-1 receptor (CSF-1R), and c-Kit. Non-receptor protein tyrosine kinases that are regulated by Cbl include, but are not limited to members of the Src family kinase (Fyn, Lck, Fgr, Hck, and Lyn), as well as Syk, c-Abl (reviewed in (Swaminathan et al., 2006)), and Jak2 (Nagao et al., 2011).

1.5.2.3 Importance of the Linker Region and RING Finger Domains in E3 Ligase Activity

The RING finger plays a critical role in the E3 ligase activity of Cbl, as it is responsible for mediating the interaction with the E2 ubiquitin conjucating enzyme. The RING finger is a zinc finger, and the consensus sequence of Cbl family RING fingers is -C-X2-C- X11-C-X-H-X2-C-X2-C-X(11-13)-C-X2-C-, with the highly conserved cysteine (C) and histidine (H) residues coordinating zinc ions (Lipkowitz et al., 2011). The mutation or deletion of the first cystine residue, CBL-C381, or the conserved histidine, CBL-H398, has been shown to result in inhibition of E3 ligase activity (Thien et al., 2001). Mouse knock-in models that express the murine Cbl-C379A (corresponding to human CBL- C381A) RING finger mutants have shown that the RING finger and E3 ligase activity of Cbl are required for T-cell receptor signalling and thymic development (Thien et al., 2005a). Furthermore, Cbl-C379A mice have increased myeloid lineage cells, and aberrant FLT3 signalling leading to development of myeloid leukemia (Rathinam et al., 2010). The residues between the sixth and seventh cystines form a groove, which interacts with two highly conserved loops within the E2. Mutagenesis experiments have

36 found that tryptophan 408 in this region is critical to the interaction between E2 and E3 (Joazeiro et al., 1999). Mutagenesis experiments have also been utilized to elucidate the importance of Cbl linker region in E3 ligase activity. Studies utilizing the Cbl-70Z mutant were the first to clearly indicate the critical role of the Cbl linker region. A splice site mutation leading to deletion of 17 amino acids (366-382), which encompasses most of the carboxy terminus of the linker region and the first conserved cystiene residue in the RING finger, gives rise the the Cbl-70Z mutant. Expression of the Cbl-70Z mutant led to transformation of the transfected cells as well as enhanced phosphorylation of Cbl (Andoniou et al., 1994). Further investigations into the linker region reveal that Tyr368 and Tyr371 may be a critical to E3 ligase functionality (Thien et al., 2001). An interesting dichotomy was discovered, as Y371F mutations abolished E3 ligase activity (Levkowitz et al., 1999), where as Y371E mutations led to a constitutively active E3 ligase (Kassenbrock et al., 2004), suggesting that the polarity and structural consequences of the residue at 371 plays an important role in regulating E3 ligase activity. Duo et al. have provided a mechanistic explanation for the role of CBL-Y371. Prior to activation, CBL is in an autoinhibitory conformation, whereby the E2 binding region of the RING finger contacts the TKB domain, preventing E2 binding. Phosphorylation of CBL-Y371 leads to a conformational change of the linker region and flips the RING finger providing an enhanced E2 binding module and bringing the E2 in close proximity to the substrate that is to be ubiquitinated. Furthermore CBL-pY371 has been shown to stabilize the E2 binding pocket of the RING finger (Figure 1.10) (Dou et al., 2012).

1.5.3 Adaptor Protein Functions of Cbl

As outlined previously, Cbl contains multiple protein-protein interaction motifs and domains, such as the TKB domain, a proline-rich region, phospho-serine and tyrosine sites, which allow for many binding partners. In fact, to date, over 150 Cbl interacting proteins have been identified (Schmidt et al., 2005), of which just a few are outlined in

FIGURE 1.10. Phosphorylation of Tyr371 activates CBL E3 Ligase. Prior to activation Cbl is in an autoinhibitory conformation. Phosphorylation of Tyr371 causes a conformation change, flipping the RING finger to bring the E2 and the ubiquitination target into close vicinity (Dou, Buetow et al. 2012).

Table 1.1. These interactions allow Cbl to play an important role in modulating signalling, independent of its E3 ligase activity. The role of Cbl in EGFR endocytosis is an excellent example of Cbl as an adaptor protein. Upon EGFR activation, Cbl is recruited to the phosphorylated receptor. Cbl contributes to the formation of clathrin- coated pits via an interaction between the Cbl proline rich region and SH3 domains of Cbl-interacting protein of 85K (CIN85). The Cbl-CIN85 interaction leads to the recruitment of various members of the endocytic machinery, including endothelins and AP2 (Soubeyran et al., 2002). Furthermore, Cbl can link the endocytosing pre-vesicle to the cytoskeleton via an interaction with CD2AP/cortactin/actin related protein 2/3 (ARP2/3) (Lynch et al., 2003). Cbl mediated activation of PI-3K activity is another example of the role of Cbl as an adaptor protein. Cbl couples to PI-3K via two possible interactions; a) binding via the SH3 domain of p85 subunit of PI-3K (Dombrosky-Ferlan et al., 1997) and b) via p85 SH2 domain dependent recruitment to pY731 of Cbl (Liu et al., 1997a). These interactions can recruit PI-3K to activated receptors, contributing to activation of PI-3K signalling.

1.5.4 CBL Associated Oncogenesis

Since Cbl has been shown to play a negative regulatory role in such a wide array of signalling pathways, it is not surprising that mutations in Cbl lead to malignancies. Since 2007, there have been numerous studies that have identified Cbl mutations in myeloid malignancies. Interestingly, the majority of Cbl mutations have been identified in patients with various myelodysplastic syndrome – myeloproliferative neoplasms (MDS/MPN) as well as secondary acute myeloid leukemia (sAML) (Kales et al., 2010) (Table 1.2).

TABLE 1.1. Cbl Binding Proteins

Identified Cbl Site(s) of Protein References interaction

Adaptor proteins

Slap TKB (but not pTyr dependent) (Swaminathan et al., 2007)

Crk pTyr (Buday et al., 1996)

Grb2 Proline rich region (Donovan et al., 1994; Meisner et al., 1995; Odai et al., 1995)

14-3-3 pSer (Liu et al., 1996b; Liu et al., 1997b)

CIN85 Proline rich region (Borinstein et al., 2000; Take et al., 2000)

Receptor Tyrosine Kinases

EGFR TKB domain (Bowtell et al., 1995; Galisteo et al., 1995; Levkowitz et al., 1999)

PDGFR TKB domain (Bonita et al., 1997; Miyake et al., 1998)

Non-receptor Protein Tyrosine Kinases

Syk TKB domain, Proline rich region (Deckert et al., 1998; Lupher et al., 1998; Marcilla et al., 1995)

Src TKB, Proline rich region, pTyr (Ojaniemi et al., 1997; Sanjay et al., 2001; Tanaka et al., 1995)

Fyn Proline rich region (Fukazawa et al., 1995; Reedquist et al., 1994)

Lyn Proline rich region, pTyr (Dombrosky-Ferlan et al., 1997; Tezuka et al., 1996)

Abl pTyr (Bhat et al., 1997)

Other p85 Proline rich region, pTyr (Fukazawa et al., 1995; Hartley et al., 1995; Meisner et al., 1995)

Vav pTyr (Marengere et al., 1997)

TABLE 1.2 – Frequency of CBL Mutations in Myeloid Mallignancies

Myeloid Malignancy Frequency of CBL mutations

JMML 15%

CMML 13%

aCML 8%

sAML 10%

JMML – Juvenile Myelomonocytic Leukemia, CMML – Chronic Myelomonocytic Leukemia, aCML – atypical Chronic Myeloid Leukemia, sAML – secondary Acute Myeloid Leukemia.

The linker region and RING finger have been found to be the hotspots for oncogenic CBL mutations (Figure 1.11) (Naramura et al., 2011b). As discussed, these regions play a critical role in the E3 ligase function, therefore, it is likely that CBL mutants identified in myeloid malignancies are E3 ligase inactive. Knock-in mice expressing CBL-C379A (corresponding to human C381A) RING finger mutations have been shown to develop a MPD that progresses to myeloid leukemia, confirming the role of CBL RING finger mutations in oncogenesis (Rathinam et al., 2010). Furthermore, copy number analyses have shown that many of the CBL mutations identified in patients are associated with a copy-neutral uniparental disomy (UPD). This indicates that the presence of single copy of a CBL mutant is not sufficient for disease development. This is confirmed by Sanada et al. who have shown that expression of wild type allele of Cbl rescues Cbl mediated transformation (Sanada et al., 2009).

FIGURE 1.11. Linker and RING Finger are Hot Spots for CBL Mutations. The domain structure of CBL is illustrated. The location of the mutations is marked with disease-associated asterisks. (Naramura, Nadeau et al. 2011).

1.6 RATIONALE AND HYPOTHESES

Hematopoiesis is a tightly regulated process, the deregulation of which leads to the development of various leukemias and lymphomas. Cytokines play an important role in the pathogenesis of various hematopoietic malignancies; therefore, understanding the signalling pathways mediated by various cytokines throughout hematopoiesis holds the promise of potential treatment options. We present our investigation of the negative regulation of two cytokines associated with the myeloid lineage. Adaptor proteins play a critical role in positive and negative regulation of cytokine signalling. We identified SH2B1 as a novel erythropoietin responsive adaptor protein and assessed its role in EPO mediated signalling (Chapter 2). We also investigated the mechanism by which CBL mutations inactivating E3 ligase activity lead to dysregulation of GM-CSF signalling, contributing to the GM-CSF hypersensitivity observed in patients with JMML (Chapter 3).

Chapter 2

EPO-R Tyr343 has been shown to be critical for EPO mediated signalling, especially in response to erythroid stress. STAT5 is the main effector downstream of EPO-R Tyr343. However, when the first STAT5 knockout mice (STAT5ΔN/ΔN) did not present with a prominent erythroid phenotype, we hypothesized that other SH2 domain containing proteins may be playing an important regulatory role downstream of EPO-R Tyr343. The SH2B1 adaptor protein was identified as such an interactor. Since SH2B1 had been demonstrated to be a positive regulator of Jak2 (downstream of GH and leptin receptors) as well as several receptor tyrosine kinases, we hypothesized SH2B1 to also be a positive regulator of EPO signalling. Interestingly the data presented in this thesis shows otherwise and establishes SH2B1 as a novel negative regulator of EPO signalling.

Chapter 3

Mutations in the linker region and RING finger of CBL have been shown to be present in 15% of JMML patients. Although a number of studies have demonstrated that mutations in the linker region and RING finger of CBL lead to aberrant signalling, none of them have investigated the role of these mutations specifically in context of GM-CSF signalling. We focused our investigation on the two most common CBL mutations found in JMML, CBL-Y371H and CBL-C384R. We hypothesized that since these mutations result in a loss of CBL E3 ligase activity, they will lead to changes in the stability of the receptor or downstream kinases, leading to GM-CSF hypersensitivity.

Ultimately, the aim of these studies is to provide greater insight into various mechanisms negatively regulating cytokine signalling. We also provide further evidence for the importance of adaptor protein functionality in these mechanisms. Disrupting the function of these negative regulators leads to enhanced cytokine signalling, which has significant impact on disease development

1.7 THESIS OBJECTIVES

Chapter 2

Hypothesis: SH2B1 binds to the EPO-R and acts as a regulator of EPO mediated signalling.

Objectives: 1. Confirm the binding of SH2B1 with EPO-R in hematopoietic cell lines and in primary murine erythroid progenitors 2. Determine whether binding of SH2B1 to EPO-R is dependent on Jak2 3. Characterize the interaction between SH2B1 and EPO-R: a. Determine which EPO-R pTyr residue(s) act as binding site(s) for the SH2 domain of SH2B1 b. Establish which regions of SH2B1 and EPO-R are responsible for constitutive association 4. Determine whether SH2B1 is responsive to EPO stimulation, via assessment of EPO induced SH2B1 phosphorylation 5. Determine the functional consequences of the SH2B1-EPO-R interaction on EPO signalling

Chapter 3

Hypothesis: CBL linker region and RING finger mutations lead to GM-CSF hypersensitivity by stabilizing the receptor and/or downstream kinases.

Objectives: 1. Determine the effects of CBL-Y371H and CBL-C384R expression on GM-CSF induced phosphorylation of GM-CSFR βc. 2. Assess whether the expression of CBL mutants result in changes in the expression of GM-CSFR βc, JAK2 and LYN.

3. Compare and evaluate signalling downstream of the GM-CSFR between WT- CBL and CBL mutant expressing cells. 4. Determine whether expression of CBL mutants effects cell survival. 5. Assess whether treatment with dasatinib or TG101348 can inhibit enhanced signalling observed in CBL mutant expressing cells.

2 THE SH2B1 ADAPTOR PROTEIN ASSOCIATES WITH A PROXIMAL REGION OF THE ERYTHROPOIETIN RECEPTOR

A modified form of this chapter has been published in the Journal of Biological Chemistry (Javadi, M., Tschirch, E., Stickle, N., Beattie, B.K., Jaster, R., Carter-Su, C., and Barber, D.L. (2012). The SH2B1 adaptor protein associates with a proximal region of the Erythropoietin receptor. J Biol Chem 287, 26223-26234  the American Society for Biochemistry and Molecular Biology). Attributions: The COLT screen was completed by Bryan Beattie and Natalie Stickle. Edda Hoffstatter completed figures 2A, 3B-C and 6.

2.1 ABSTRACT

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R) and associated Janus tyrosine kinase, Jak2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Y343 is important in EPO-mediated mitogenesis. While Stat5 binds to EPO-R phosphoY343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional SH2 domain- containing effectors may bind to EPO-R Y343 and couple to downstream signaling pathways. We have utilized Cloning of Ligand Target (COLT) screening to demonstrate that EPO-R pY343 and pY401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pY-X-X-L. Previous studies have shown that SH2B1 binds directly to Jak2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-pY343/pY401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPOR, indicating that SH2B1 is a negative regulator of EPO signaling.

2.2 INTRODUCTION

Erythropoietin (EPO) is the primary cytokine required for the development of red blood cells, specifically driving definitive erythropoiesis (Wu et al., 1995). The EPO Receptor (EPO-R) (D'Andrea et al., 1989), a 66 kDa transmembrane protein, belongs to the cytokine receptor superfamily. The EPO-R is dependent on a constitutively associated Janus tyrosine kinase, Jak2, to mediate its downstream signaling. Upon EPO binding, the pre-formed EPO-R homodimer undergoes a conformational change leading to activation of Jak2 (Livnah et al., 1999b). The activated Jak2 phosphorylates tyrosine residues of the EPO-R, generating docking sites for SH2 effector proteins (reviewed in (Richmond et al., 2005)). Gene targeting studies have elucidated the critical role of EPO (Wu et al., 1995), EPO-R (Kieran et al., 1996; Lin et al., 1996; Wu et al., 1995) and Jak2 (Neubauer et al., 1998; Parganas et al., 1998) in erythropoiesis as loss of any of these genes leads to embryonic lethality due to defective definitive erythropoiesis. These findings suggest that EPO-R and/or Jak2 deliver signals crucial to EPO-dependent proliferation, differentiation and cell survival. Initial structural-functional studies identified the membrane proximal region of the EPO-R, specifically Tyr343, to play an important role in EPO signaling and Colony Forming Unit-Erythroid (CFU-E) formation (Miller et al., 2002; Socolovsky et al., 1997a). The role of EPO-R cytoplasmic tyrosines was examined in greater detail through the generation of knock-in mice. Although mice expressing EPO-R H (truncated EPO-R possessing only Tyr-343) and EPO-R HM (truncated EPO-R with a Y343F mutation) are both viable (Zang et al., 2001a), the ability of the EPO-R HM mice to respond to the stress agent phenylhydrazine is impaired (Li et al., 2003a). EPO-R HM mice also have elevated serum EPO (Menon et al., 2006a), decreased reticulocyte production (Menon et al., 2006b), defective erythroid repopulation (Menon et al., 2006b), and increased apoptosis of erythroid progenitors (Li et al., 2003a). These studies illustrate that while EPO-R Tyr343 may not be required for viability, it plays an essential role in signal amplification contributing to erythroid survival and mitogenesis, especially in response to erythroid stress.

The Jak-Stat pathway is one of the major pathways activated downstream of the EPO-R. Stat5a and Stat5b, which are largely redundant, are recruited to pTyr343 of the EPOR via their SH2 domains (Barber et al., 2001; Damen et al., 1995b; Klingmuller et al., 1996a). Studies utilizing Stat5 deficient EPOR-H erythroid progenitors indicated that the phenotypes observed in EPO-R HM mice may be due to the inability of EPO-R HM to activate Stat5a/b (Zang et al., 2001a). Interestingly, the embryos from the initial Stat5a/b deficient mice (Stat5a/bΔN/ΔN) were anemic (Socolovsky et al., 1999b; Zang et al., 2001a), but in the adult mice there was only a subtle decrease in erythroid survival (Socolovsky et al., 2001; Teglund et al., 1998a). However, these data must be interpreted with caution, as it is now known that Stat5a/b ΔN alleles are hypomorphic (Hoelbl et al., 2006; Yao et al., 2006). Conditional targeting of Stat5a/b in the erythroid lineage has demonstrated that Stat5a/b plays a critical role in the regulation of several erythroid genes including Transferrin Receptor-1 and Iron Regulatory Protein-2 (Kerenyi et al., 2008; Zhu et al., 2008). We hypothesized that there are additional SH2 effectors that bind to EPO-R Tyr-343 and play a role in earlier steps of erythroid development. We performed an expression screen utilizing a tyrosine phosphorylated EPO-R Y343 peptide to address this question, and identified the adaptor protein SH2B1 (SH2- B/PSM) as an EPO-R Y343 binding partner. SH2B1 is a member of the SH2B family of adaptor proteins, whose other members include SH2B2 (APS), and SH2B3 (Lnk). All three members contain amino-terminal pleckstrin homology (PH) and dimerization domains, a carboxy terminal Src Homology 2 (SH2) domain and several proline rich regions (Maures et al., 2007). SH2B2 and SH2B3 are capable of modulating EPO signaling (Tong et al., 2005; Wakioka et al., 1999b), and SH2B1 has been shown to regulate Jak2 activity in the context of growth hormone and leptin signaling (Duan et al., 2004a; Rui et al., 1997b). In the present study, we characterize the interaction between SH2B1 and EPO-R. We show that SH2B1 becomes phosphorylated in response to EPO, and acts as a negative regulator of signaling downstream of the EPO-R.

2.3 EXPERIMENTAL PROCEDURES:

Cloning of Ligand Target (COLT) Expression Screen

A Day 16 murine embryo phage expression library (Novagen, Madison, WI, USA) was diluted at 4 x 104 pfu/plate and protein expression was induced as per manufacturerʼs instructions. The nitrocellulose filters containing immobilized proteins were washed in 20 mM Tris HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20 (TBST) and blocked overnight in TBST containing 2% (w/v) BSA, 1 mM dithiothreitol (DTT) (Buffer A). Biotinylated EPO- R peptides were briefly incubated with Streptavidin-Alkaline Phosphatase, conjugated and mixed with Buffer A. Blocked filters were incubated in blocking buffer at room temperature for 3 hours with biotinylated peptides (25 pmol/ml). Filters were washed five times with TBST and detection performed with a colorimetric nitro-blue tetrazolium/5- bromo-4-chloro-3ʼindolyphophate p-toluidine (NBT-BCIP, ThermoScientific, Rockford, IL) reaction. Plaques interacting with EPO-R pY343 were back-screened with a non- phosphorylated EPO-R Y343 peptide. Plaques that specifically interacted with EPO-R pY343 were purified and plasmid DNA was isolated from the phage according to the manufacturerʼs directions and inserts were sequenced.

Cell Lines and Culture

Ba/F3 cells stably expressing wild-type or mutant EPO-R were maintained in RPMI 1640 medium, 10% (v/v) fetal calf serum, 100 units of penicillin per ml, 100 µg of streptomycin per ml, and 50 µM β-mercaptoethanol (RPMI 1640 complete medium) supplemented with 0.1 ng per ml recombinant mouse IL-3 in the presence of 1 mg/ml G418 (Mason et al., 2000; Miller et al., 1999). DA-3-EPO-R cells were maintained in RPMI 1640 complete medium and 0.5 units/ml human recombinant EPO (Mason et al., 2000). HCD-57 cells were cultured in Iscoveʼs modified Dulbeccosʼs medium supplemented with 20% fetal calf serum, 50 µM β-mercaptoethanol, and 0.5 units/ml human recombinant EPO (Mason et al., 2000). Various EPO-R constructs were electroporated into Ba/F3 cells (Mason et al., 2000; Miller et al., 1999). EPO-R deletion mutants were generated as previously

51 reported (Mason et al., 2000; Miller et al., 1999). Individual G418-resistant subclones were isolated by limiting dilution. The expression of the EPO-R was confirmed by Western blotting, and the EPO-dependent growth characteristics of each subclone were examined by performing an XTT assay as described (Barber et al., 1994). 293T cells were maintained in Dulbeccoʼs Modified Eagleʼs Medium (DMEM) H- 21, 10% FBS. 293T cells were transiently transfected with EPO-R and SH2B1 constructs using Lipofectamine 2000 (Invitrogen) as per manufacturerʼs instructions. The SH2B1 constructs were previously described (Rui et al., 2000a). siRNA Mediated Knock-down of SH2B1

SH2B1 specific ON-TARGET plus pooled siRNA was purchased from Dharmacon RNAi Technologies (Lafayette, CO). ON-TARGETplus siCONTROL Non-targeting Pool was used as controls in the knock-down experiments. Ba/F3-EPO-R cells were transfected with siRNA via electroporation (Walters et al., 2005). Ba/F3-EPO-R cells were maintained in their log phase of growth prior to transfection. The cells were washed once with RPMI-1640 and re-suspended at 107 cells/200 µl. The cells were incubated with 1 µg of siRNA in a 0.4-cm electroporation cuvette for 10 minutes at room temperature. The cells were pulsed once at 300 V, 450 µF using the Bio-Rad Gene Pulser II electroporator. The cells were then re-suspended in 5 ml of RPMI-1640- IL3/G418.

Cytokine Deprivation and Stimulation

Ba/F3, DA-3 and HCD-57 cells were washed three times in 10 mM HEPES (pH 7.4), Hanksʼ balanced salts, incubated in RPMI 1640 medium supplemented with 10% fetal calf serum and 50 µM β-mercaptoethanol for 4 h at 37°C, and then stimulated with 10 ng/ml murine recombinant IL-3, or 50 units/ml murine recombinant IL-2 or human recombinant EPO, or vehicle for 10 min at 37°C. 293T cells were washed three times with PBS and starved in DME H-21 supplemented with 2.5 mg/ml of BSA overnight at 37°C. The cells were stimulated with 5 units/ml of human recombinant EPO or vehicle for 10 min at 37°C.

The cells were washed once in 10 mM HEPES (pH 7.4), Hanksʼ balanced salts containing 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM EDTA, and 1 mM sodium orthovanadate, and lysed in ice-cold lysis buffer containing 1% Triton X- 100, 50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 2 mg/ml leupeptin, and 1 mg/ml pepstatin A. After 5 min on ice, the lysates were centrifuged at 10,000 x g for 5 min at 4°C.

Antibodies

The polyclonal SH2B1 antibody that recognizes amino acids 527 to 670 of SH2B1β was produced by Antibodies Inc. (Davis, CA). Purified GST-SH2B1 fusion protein (corresponding to amino acids 527-670) was used to immunize rabbits. Protein-A- Sepharose columns were used to purify anti-SH2B1 antibodies from the sera of the immunized rabbits. Purified anti-SH2B1 antibodies were used in immunoprecipitation experiments. The polyclonal anti-pY343-EPO-R (Halupa et al., 2005b) and the antibody targeting the N-terminus of the EPO-R were produced in-house. The anti- phosphotyrosine monoclonal antibody, 4G10, and the total Jak2 antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The anti-pY479 EPO-R, anti- EPO-R, monoclonal anti-GST and phospho-Erk1/2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SH2B1 specific antibodies used for immunoblotting were graciously provided by Dr. Liangyou Rui (University of Michigan).

Immunoprecipitations

Antibodies along with a 50 µl volume of protein A-Sepharose 4B beads (Amersham Pharmacia Biotech) were added to 2 mg of lysates for an overnight incubation. The beads were washed three times in ice-cold lysis buffer. The immune complexes were eluted by boiling in Laemmli sample buffer containing 100 mM DTT. Samples were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane for Western blotting.

GST Fusion Protein Binding Experiments

Two mg of cell lysate were incubated for 1 hour at 4°C with GST fusion proteins expressing the SH2 domain of SH2B1β immobilized on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). The beads were washed three times in ice-cold lysis buffer, and precipitated complexes were eluted by boiling in Laemmli sample buffer with 100 mM DTT. Samples were resolved by SDS-PAGE and analyzed by Western Blotting.

Western Blotting

Following the electrophoretic transfer of proteins to PVDF membrane (NEN Life Science Products), the membranes were blocked at room temperature with 2.5% BSA in Tris- buffered saline (50 mM Tris (pH 8.0) and 150 mM NaCl) for 1 h. Membranes were then incubated with an optimal concentration of the primary antibody in TBST for 1 hour at room temperature or overnight at 4°C, washed four times in TBST, and incubated with the relevant HRP-conjugated secondary antibody for 30 - 60 min. Membranes were washed four times in TBST and visualized by enhanced chemiluminescence with autoradiographic film (ECL, Amersham Pharmacia Biotech). For reprobing, membranes were stripped in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 0.1 M β-mercaptoethanol for 30 min at 50°C; rinsed twice in TBST; and blocked in 2.5% BSA in Tris-buffered saline prior to primary antibody incubation. Western blots were scanned using film exposures in the linear range and quantified using ImageJ software.

CIAP Treatment of SH2B1 Immunoprecipitations

Following SH2B1 immunoprecipitation, the sepharose beads were washed twice in dephosphorylation buffer (50 mM HEPES, 1 mM MgCl2, pH 7.5). The immunoprecipitates were re-suspended in dephosphorylation buffer and 30 units of calf intestinal alkaline phosphatase was added to the beads and incubated at 30 °C. To terminate the reaction, SDS-PAGE sample buffer was added to the beads and the sample boiled to elute off the bound protein.

Preparation of Primary Erythroblasts

C57/Bl6 mice (8-12 weeks old) were injected intraperitonealy on day 1 and 2 with a sterile solution of phenylhydrazine (6mg/ml) in PBS solution (final dose: 60 mg/kg) (Barber et al., 2001). After the mice were sacrificed on day 5, a single spleen cell suspension was prepared. Cells were washed once in 10mM HEPES (pH 7.4) and Hanksʼ balanced salts and starved in Iscoves-Media containing 2% fetal calf serum and 50 µM β-mercaptoethanol for 4 hours at 37°C. Cells were then incubated with either no factor or 50 units/ml of human recombinant EPO for 10 min at 37°C. Cells were washed once in 10 mM HEPES (pH 7.4) and Hanksʼ balanced salts containing 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM EDTA, and 1 mM sodium orthovanadate. Lysates were prepared as described above. Studies were approved by the Animal Care Committee at the Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.

2.4 RESULTS

2.4.1 COLT Screening Identifies SH2B1β Interacting with EPO-R Y343

We hypothesized that in addition to Stat5, EPO-R Y343 may recruit other SH2 signaling effectors. SH2 effectors that bind to EPO-R Y343 were identified in an unbiased fashion via Cloning of Ligand Target (COLT) screening (Liu et al., 1998; Pirozzi et al., 1997). A biotinylated EPO-R pY343 phosphopeptide was utilized to screen a murine Day 16 embryonic library. Positive clones were back-screened with an identical EPO-R Y343 non-phosphorylated peptide. Clones that met this specificity selection were sequenced. All were found to contain a SH2 domain. One of the clones isolated corresponded to the SH2 adaptor protein, SH2B1β (Rui et al., 1997b). To examine the specificity of the EPO-R–SH2B1β interaction, isolated SH2B1β plaques were incubated with specific biotinylated EPO-R phosphopeptides corresponding to the sequences of the eight EPO- R cytoplasmic tyrosines (Figure 2.1). In addition to EPO-R pY343, EPO-R pY401 and EPO-R pY429 were also found to interact with SH2B1β, although to a lower extent. All three EPO-R sequences possess a common pYXXL motif which is known to be recognized by several SH2 domains (Songyang et al., 1993; Songyang et al., 1994) including the SH2 domain of SH2B1 (Kurzer et al., 2004). Considering that SH2B1 was characterized as a Jak2 binding partner (Rui et al., 1997b), we were interested in determining the role of SH2B1 in EPO-mediated signaling pathways.

2.4.2 SH2B1 Associates with the EPO-R in Hematopoietic Cell Lines

To investigate the role of SH2B1 in EPO signaling, we tested Ba/F3 and DA-3 hematopoietic cells, transfected to express EPO-R, as well as HCD-57 cells expressing endogenous EPO-R. These cells were depleted of cytokine and then incubated with no factor or with IL-3 or EPO. Immunoprecipitation experiments were performed using a polyclonal SH2B1 antibody, followed by Western blotting with the anti-phosphotyrosine antibody 4G10. Upon EPO stimulation, SH2B1 co-immunoprecipitated a

FIGURE 2.1. Specificity of SH2B1 for pY343, pY401 and pY429. Phage expressing SH2B1 were incubated with BL21 E. coli cells and plated on 2xYT plates. Plaques were transferred onto nitrocellulose membranes and then cut into eight sections. Each section was incubated with a biotinylated phosphopeptide corresponding to one of the eight cytoplasmic tyrosines of EPO-R (as labeled). Positives were detected with colorimetric detection solution.

57 phosphoprotein with a molecular weight corresponding to EPO-R in all cell lines (Figure 2.2A, lanes 3, 6 and 8). To show that the phosphoprotein that co-immunoprecipitates with SH2B1 is EPO-R, a pY343-EPO-R specific antibody was also used for immunoblotting. Figure 2.2B confirms that the phosphoprotein that co-IPs with SH2B1 (Figure 2.2B, lanes 6 and 8) is indeed phosphorylated EPO-R (Figure 2.2B, lanes 2, 4). COLT screening suggested that SH2B1 bound to EPO-R in an SH2 domain- dependent manner. In vitro mixing experiments were performed utilizing the following fusion proteins: GST-SH2B1 SH2, GST-SH2B1 SH2 R555K (an inactive SH2 domain mutant), and GST-Ship SH2 as a positive control (Mason et al., 2000). Bound proteins from Ba/F3-EPO-R lysates were detected using an anti-phosphotyrosine antibody (Figure 2.2C, lanes 1-8), and the pY343-EPO-R specific antibody (Figure 2.2C, lanes 11-16). The GST-SH2B1 SH2 fusion protein associated with phosphorylated EPO-R upon EPO stimulation (Figure 2.2C, lane 6 and 14), and the SH2 inactivating R555K mutation (Figure 2.2C, lanes 8 and 16) abolished this interaction. These results indicate that SH2B1 interacts with tyrosine phosphorylated EPO-R via its SH2 domain.

2.4.3 SH2B1 Binds Specifically to pY343 and pY401 of the EPO-R upon EPO Stimulation

To confirm the COLT screen results that SH2B1 binds specifically to pY343 and/or pY401 of the EPO-R, we examined the interaction of SH2B1 with a panel of EPO-R deletion mutants. Ba/F3 cells expressing wild-type EPO-R, EPO-RΔ43 (containing Y343, Y401, Y429 and Y431), EPO-RΔ69 (containing Y343 and Y401), EPO-RΔ99 (containing Y343) and EPO-RΔ99,F1 (containing a Y343F mutation) (Figure 2.3A) were depleted of cytokine and stimulated with EPO or left unstimulated. SH2B1 was immunoprecipitated and Western blotting using an anti-phosphotyrosine antibody was performed (Figure 2.3B). EPO stimulation resulted in co-immunoprecipitation of SH2B1 with the tyrosine phosphorylated EPO-R in Ba/F3-cells expressing full-length EPO-R (lane 2), EPO-RΔ43 (lane 4) and EPO-RΔ69 (lane 6), indicating that SH2B1β is specifically recruited to pY343 and/or pY401 of the EPO-R. To examine whether the

FIGURE 2.2. SH2B1 co-immunoprecipitates with phosphorylated EPO-R in hematopoietic cell lines, via its SH2 domain. A, Ba/F3-EPO-R (lanes 1-3), DA-3-EPO-R (lanes 4-6), and HCD-57 (lanes 7 and 8) cells were depleted of cytokine for 4 h and incubated with no factor, 10 ng/ml murine IL- 3 or 50 units/ml human recombinant EPO for 10 min at 37°C. Immunoprecipitation (IP) was performed with an anti-SH2B1 antibody, and the immunoblot was probed with the anti-phosphotyrosine antibody 4G10. Molecular mass standards are indicated in kilodaltons. B, Ba/F3-EPO-R and DA-3-EPO-R cells were processed as in A, and the immunoblot was probed with pY343-EPO-R antibody (upper panel). The immunoblot was stripped and reprobed for SH2B1. Lanes 11-14 (SH2B1 IB) show a lighter exposure than lanes 1-10.

FIGURE 2.2. SH2B1 co-immunoprecipitates with phosphorylated EPO-R in hematopoietic cell lines, via its SH2 domain. C, Ba/F3-EPO-R cells were cytokine-depleted then either left unstimulated, or stimulated with EPO. Lysates were incubated with GST, GST-SH2B1 SH2, or GST- SH2B1 SH2-R555K. The immunoblots were probed with the anti-phosphotyrosine 4G10 or pY343-EPO-R antibody and then stripped and reprobed for GST.

FIGURE 2.3. Co-immunoprecipitation with EPO-R truncation mutants confirms SH2B1 binds specifically to pY343, and pY401 of the EPO-R. A, Panel of EPO-R truncation mutants used. Deletion mutant numbers correspond to the number of amino acids deleted from the cytoplasmic tail of the EPO-R. B, SH2B1 IPs were performed on lysates from EPO stimulated and unstimulated Ba/F3 cells expressing the various EPO-R mutants, followed by phospho-tyrosine immunoblotting (IB). The membrane was reprobed with anti-SH2B1. C, GST-SH2B1 SH2 pull downs were performed on lysates collected from EPO stimulated and unstimulated Ba/F3 cells expressing the EPO-R truncation mutant panel. Isolated complexes were analyzed via phospho-tyrosine IB. Lysates were probed with anti-GST.

61 truncated EPO-R mutants associate with the SH2 domain of SH2B1, in vitro mixing experiments were performed (Figure 2.3C). Lysates were incubated with GST-SH2B1 SH2, and tyrosine-phosphorylated proteins were detected via anti-phosphotyrosine western blotting. Upon EPO stimulation, association of the full-length EPO-R (lane 2), EPO-RΔ43 (lane 4) and EPO-RΔ69 (lane 6) with the SH2B1 SH2 domain was detected. To determine whether the binding of SH2B1 to phosphorylated EPO-R was dependent on pY343 or pY401 or both EPO-R tyrosines, we tested several mutant EPO-R (Figure 2.3D). SH2B1 co-immunoprecipitated EPO-R-Y7, F1 lacking Y343 (lane 10) and EPO-R-Y7, F2 lacking Y401 (lane 12), but the interaction was abolished upon the expression of EPO-R-Y6, F1F2 lacking both Y343 and Y401 (lane 8). Tyrosine phosphorylation of the EPO-R was also examined in this panel of EPO-R receptor mutants (Figure 2.3E). EPO-R (lane 4), EPO-R Y6, F1F2 (lane 8), EPO-R Y7,F1 (lane 10) and EPO-R Y7,F2 are tyrosine phosphorylated in response to EPO-R. The lack of SH2B1 binding to EPO-R Y6,F1F2 is not due to lack of phosphorylation. These results suggest that upon EPO stimulation SH2B1, via its SH2 domain, specifically binds to phospho-Tyr343 and phospho-Tyr401 of the EPO-R.

2.4.4 SH2B1 Associates with Unphosphorylated EPO-R

Stripping SH2B1 immunoprecipitation blots and reprobing for total EPO-R revealed that SH2B1 co-immunoprecipitated with unphosphorylated EPO-R in unstimulated samples (Figure 2.4A, lane 11). This indicates that SH2B1 may associate with the EPO-R prior to EPO stimulation, and upon EPO-R phosphorylation binds to pY343/pY401 of the EPO-R via its SH2 domain. To determine which region of SH2B1 was responsible for associating with unphosphorylated EPO-R, the EPO-R was co-expressed with a panel of myc-tagged SH2B1 truncations mutants (Figure 2.4B, C) (Rui et al., 2000a). The cytokine-deprived cells were stimulated with EPO or left unstimulated, myc-SH2B1 was immunoprecipitated using a myc antibody. Probing with an EPO-R antibody revealed that SH2B1 full length, C410, C266 and to a lesser extent N269 and PH truncation mutants associate with unphosphorylated EPO-R (Figure 2.4E). However,

FIGURE 2.3. Co-immunoprecipitation with EPO-R truncation mutants confirms SH2B1 binds specifically to pY343, and pY401 of the EPO-R. D, 293T cells were transfected to express Myc-tagged SH2B1 and the indicated EPO-R tyrosine mutants. Transfected cells were depleted of cytokines and either left unstimulated or stimulated with 5 U/ml EPO for 10 min. Immunoprecipitation was performed with an anti-Myc antibody, and the membranes were probed with anti-pTyr, and reprobed with anti-EPO-R and anti-myc (to detect myc-SH2B1β). E, From identical lysates as 3D, immunoprecipitations were performed with an EPO-R antibody, followed by phosphotyrosine immunoblotting and reprobing with anti-EPO-R and anti-Myc.

FIGURE 2.4. The PH domain and amino acids 1-266 of SH2B1 mediate a constitutive association with the membrane proximal region of the EPO-R. A. Lysates from Ba/F3 and Ba/F3-EPO-R cells stimulated or unstimulated with EPO were subjected to immunoprecipitation using anti-EPO-R, non-reactive serum (NRS), and anti-SH2B1 antibodies. Immunoblotting was performed using anti-phospho-tyrosine antibody and anti-EPO-R antibody. B, Panel of Myc-tagged SH2B1 truncation mutants used. 293T cells were transfected with the indicated expression vectors. Lysates collect from the cells were subjected to anti-EPO-R (C) or anti-SH2B1 (D) immunoblotting.

FIGURE 2.4. The PH domain and amino acids 1-266 of SH2B1 mediate a constitutive association with the membrane proximal region of the EPO-R. E, 293T cells were transfected to express WT EPO-R and SH2B1 truncation mutants. The cells were cytokine-deprived over-night, then stimulated with 10 U/ml EPO for 10 min. Myc-SH2B1 was immunoprecipitated using an anti-Myc antibody. IB was performed with anti-phosphotyrosine and anti-EPO-R antibodies. F, 293T cells were transfected to express WT SH2B1 and EPO-R truncation mutants. The cells were cytokine-deprived overnight, then stimulated with 10 U/ml EPO for 10 min. EPO-R IPs were followed by anti-pTyr and anti-Myc immunoblotting analysis.

65 phosphotyrosine immunoblotting indicated that only full length SH2B1 was capable of associating with both unphosphorylated and phosphorylated EPO-R. These results indicate that both the PH domain and the region encompassing amino acids 1 – 266 are required for maximal association of SH2B1 with unphosphorylated EPO-R. We were also interested to determine which region of the EPO-R was involved in the constitutive association with SH2B1. Therefore, EPO-R truncation mutants (Figure 2.3A, Figure 2.4D) were co-expressed with myc-tagged wild-type SH2B1. SH2B1 co- immunoprecipitated with all EPO-R mutants, indicating that the membrane proximal region of the EPO-R mediates constitutive association with SH2B1 (Figure 2.4F). Since SH2B1 also associates with both inactive and active Jak2 (Li et al., 2007b; Rui et al., 2000a), we wanted to confirm that the constitutive association between SH2B1 and the EPO-R was independent of Jak2. We investigated whether SH2B1 could constitutively bind to EPO-R carrying the W282R point mutation, which abolishes EPO-R binding to Jak2 (Huang et al., 2001; Miura et al., 1994). SH2B1 co- immunoprecipitated to a similar extent with wild type EPO-R and EPO-R W282R (Figure 2.5). Since EPO-R W282R is unable to bind Jak2, it is also not phosphorylated in response to EPO stimulation. Therefore, as anticipated, only the co-precipitating wild type EPO-R and not the co-precipitating EPO-R W282R was detected using pY343 EPO-R antibody. SH2B1 also failed to co-immunoprecipitate with Jak2 in co- immunoprecipitation experiments (Figure 2.6). These data demonstrate that the constitutive association between SH2B1 and EPO-R is not dependent on Jak2.

2.4.5 SH2B1 is Phosphorylated in Response to EPO Stimulation

We observed that upon EPO stimulation, SH2B1 migrates as a broader, slower migrating band, consistent with SH2B1 phosphorylation (Figure 2.2B, lower panel). To test whether this EPO-dependent upward mobility shift is due to increased phosphorylation of SH2B1, we treated immunoprecipitated SH2B1 with calf intestinal alkaline phosphatase (CIAP), a non-specific phosphatase. CIAP treatment results in the collapse of the broader SH2B1 band in the stimulated samples, generating a single

FIGURE 2.5. The association of SH2B1 with EPO-R is independent of Jak2. SH2B1 immunoprecipitations were performed on lysates from EPO stimulated and unstimulated Ba/F3 cells expressing the wild type EPO-R or the EPO-R W282R mutant. Phospho-tyrosine immunoblotting (IB), was followed by a re-probe for total EPO-R.

FIGURE 2.6. SH2B1 and Jak2 do not co-immunoprecipitate in cell lines expressing EPO-R. A) Lysates were collected from cytokine depleted Ba/F3-EPO-R and DA-3-EPO-R cells, which were either unstimulated or stimulated with EPO. Immunoprecipitations were performed using anti-SH2B1 antibody or an N-terminal specific Jak2 antibody (R80) (Barber et al. Mol Cell Biol 14: 6506, 1994). Blots were probed with 4G10 phosphotyrosine antibody, then stripped and reprobed for SH2B1. B) Lysates were collected from unstimulated and EPO stimulated Ba/F3-EPO-R cells. Immunoprecipitation was performed using anti-SH2B1 antibody and anti-Jak2 (DE12) antibody (Cell Signaling Technology). Blots were probed with anti-phosphotyrosine, then stripped and reprobed for either SH2B1 or Jak2.

68 dense band similar to that precipitated from the unstimulated samples (Figure 2.7A). SH2B1 has been reported to undergo both tyrosine (Rui et al., 1997b; Yao et al., 2006) and serine/threonine (Rui et al., 1999c) phosphorylation. Since no phosphotyrosine signal was observed at the molecular weight corresponding to SH2B1 (Fig 2A, B), it is likely that SH2B1 is becoming serine/threonine phosphorylated in response to EPO. Although it is possible that the 4G10 phosphotyrosine antibody may not recognize the EPO specific phosphorylated tyrosines in SH2B1, Rui et al. have shown the 4G10 antibody recognizes tyrosine phosphorylated SH2B1 (Rui et al., 1998; Rui et al., 1999c; Rui et al., 1997b). A time course study (Figure 2.7B) indicated that SH2B1 exhibits increased phosphorylation after one minute of EPO stimulation and that phosphorylation returns to its basal state within 60 minutes post-EPO stimulation. SH2B1 phosphorylation was observed at the physiological dose of 0.5 U/ml of EPO, and was further enhanced with increasing EPO dosage (Figure 2.7C). These results indicate that SH2B1 becomes rapidly and transiently phosphorylated, most likely on serine and threonine residues, in response to physiological doses of EPO.

2.4.6 SH2B1 Associates with the EPO-R in Primary Splenic Erythroblasts

Phenylhydrazine priming of mice results in the generation of EPO-responsive splenic erythroblasts (Halupa et al., 2005a), by inducing hemolytic anemia. The physiological response to the induced low hematocrit levels is an increase in hematopoiesis, whereby four days after the phenylhydrazine treatments, the spleen and bone marrow consist of greater than 90% primary erythroblasts. To test whether SH2B1 binds to EPO-R in primary splenocytes, C57BL/6J mice were treated with phenylhydrazine and after four days the splenocytes were harvested. The cells were depleted of cytokine for four hours and stimulated with EPO or vehicle. SH2B1 and EPO-R immunoprecipitations were performed. Anti-phosphotyrosine Western blotting demonstrated that SH2B1 co-immunoprecipitated with tyrosine phosphorylated EPO-R in primary splenic erythroblasts (Figure 2.8A).

FIGURE 2.7. EPO stimulation induces SH2B1 phosphorylation in a time and dose dependent manner. A, Immunoprecipitations were preformed on unstimulated and EPO stimulated Ba/F3- EPO-R lysates. The IPs were treated with 30 units of Calf Intestinal Alkaline Phosphatase (CIAP), and immunoblotted with SH2B1 specific antibodies. Cytokine- deprived Ba/F3-EPO-R cells were stimulated with 50 U/ml of EPO for the indicated times (B), or stimulated with indicated doses of EPO for 10 min (C). Cell lysates were immunoprecipitated and immunoblotted with a SH2B1 antibody.

FIGURE 2.8. SH2B1 associates with the EPO-R in primary erythroblasts. Splenocytes were collected from phenylhydrazine-treated C57/Bl6 mice and depleted of cytokine for 4 hours. Cells were unstimulated or stimulated with 50 U/ml EPO for 10 min. A, Following cell lysis, immunoprecipitations were performed with anti-SH2B1 or anti-EPO-R antibodies, followed by immunoblotting with an anti-phosphotyrosie antibody. B, The same lysates used in (A) were incubated with GST, GST-SH2B1 SH2, or GST-SH2B1 SH2 R555K. Bound proteins were identified by immunoblotting with pTyr antibody.

To determine whether the SH2 domain is required for the interaction of SH2B1 with activated EPO-R in the primary splenic erythroblasts as in the cultured cell systems, an in vitro mixing experiment was performed (Figure 2.8B). Lysates were incubated with GST-SH2B1 SH2 and mutant GST-SH2B1 SH2 R555K fusion proteins, followed by anti-phosphotyrosine Western blotting. As shown previously for Ba/F3-EPO- R cells (Figures 2.2C), in primary erythroblasts SH2B1 also associates with phosphorylated EPO-R in an SH2-dependent manner (Figure 2.8B).

2.4.7 SH2B1 is a Negative Regulator of Downstream EPO Signaling

To determine the functional signaling consequences of the association of SH2B1 with EPO-R, siRNA was used to knock-down SH2B1 in Ba/F3-EPO-R cells (Figure 2.9A). Lysates from unstimulated or EPO stimulated knock-down cells were probed for signaling effectors downstream of the EPO-R. When compared to mock and control siRNA transfected cells, EPO stimulation of SH2B1 knockdown cells resulted in an increase in tyrosine phosphorylation of the EPO-R (detected by pY343 EPO-R (Figure 2.9B) and pY479 EPO-R (data not shown) Western blotting), Jak2 (assessed by blotting with an antibody to the activatory pTyr-1007/pTyr1008 of Jak2) and Erk1/2 phosphorylation (detected by an antibody to the activatory pTyr-204 of Erk) (Figure 2.9B). These results indicate that SH2B1 is a negative regulator of EPO signaling.

FIGURE 2.9. Knock down of SH2B1 results in enhanced EPO mediated signaling. A, The indicated siRNAs were electroporated into Ba/F3-EPO-R cells (Mock, no siRNA). The resulting knock down was assessed via SH2B1 immunoblotting. Experiments were performed in duplicates. Quantification of the knock down is presented as a ratio of the density of the SH2B1 band to tubulin. B, Ba/F3-EPO-R cells were electroporated with the indicated siRNAs. The lysates from unstimulated or EPO stimulated cells were analyzed via phospho-Jak2, pY343-EPO-R, and phospho-Erk1/2 western blotting. The blots were stripped and reprobed for amounts of total EPO-R, Jak2 and Erks 1/2. Quantification of signaling blots is presented as the ratio of the density of the phospho band to the total band + SD (n=4).

2.5 DISCUSSION

Several structural-functional studies and the EPO-R H and EPO-R HM knock-in mice have demonstrated that tyrosine-343 of the EPO-R plays an important role in erythropoiesis, especially in response to erythropoietic stress (Li et al., 2003a; Menon et al., 2006a; Menon et al., 2006b; Miller et al., 2002; Socolovsky et al., 1997a; Zang et al., 2001a). The phenotypes observed in EPO-R HM mice were attributed to the inability of the mutant receptor to activate Stat5a/b downstream of pY343 (Zang et al., 2001a). Stat5a/b is not essential for erythroid differentiation (Kerenyi et al., 2008), however, these transcription factors play a critical role in iron metabolism in erythroid progenitors as demonstrated by floxed alleles of Stat5a/b deleted in the erythroid lineage (Kerenyi et al., 2008; Zhu et al., 2008). This raises the question of whether there are other effectors downstream of pY343 of the EPO-R, which may be playing an important role in earlier stages of erythroid development and erythroblast differentiation. We utilized a COLT screen to identify additional SH2 effectors that bind to EPO-R pY343 to address this question in an unbiased fashion. We identified the adaptor protein SH2B1 as a novel EPO-R binding protein. Since SH2B1 was originally identified as a Jak2 interacting protein, we were interested in examining the recruitment and role of SH2B1 in EPO-mediated signaling pathways in hematopoietic cells. The major findings of this study include: i) SH2B1 binding to tyrosine phosphorylated EPO-R in an SH2 dependent manner in hematopoietic cell lines and primary erythroblasts, ii) tyrosine-dependent binding is mediated via a membrane- proximal region of the EPO-R containing pY343 and pY401, iii) SH2B1 and EPO-R also constitutively associate, iv) SH2B1 preferentially associates with EPO-R rather than Jak2, v) EPO enhances phosphorylation of SH2B1 and vi) SH2B1 negatively regulates EPO signalling. We have shown that SH2B1 interacts with EPO-R in several hematopoietic cell lines as well as in primary murine splenic erythroblasts. Detailed investigation of the SH2B1/EPO-R interaction revealed that SH2B1 binds pY343 and/or pY401 of the EPO- R in an SH2-dependent manner. SH2B1 has been reported to interact with Jak2 (Maures et al., 2007), and tyrosine-813 of Jak2 is the primary binding site of the SH2B1

SH2 domain (Kurzer et al., 2004). SH2B1 binds to inactive Jak2 via a region encompassing amino acids 410 – 555 (Kurzer et al., 2006; Li et al., 2007b). Like Jak2 Y813, EPO-R Y343 and Y401 are in an YXXL motif. We have also shown that SH2B1 binds to unphosphorylated EPO-R. However, the constitutive association of SH2B1 with EPO-R appears to be primarily mediated by the N-terminal region (amino acids 1-266) of SH2B1, binding to the membrane proximal region of the EPO-R (amino acids 248- 384). This N-terminal segment of SH2B1 contains the polybasic nuclear localization sequence (NLS), which is required for the recruitment of SH2B1 to the plasma membrane (Maures et al., 2011). These data raise the possibility that the localization of SH2B1 to the plasma membrane is required for the constitutive association with EPO-R. It is important to note that SH2B1 mutants (SH2B1-N269, SH2B1-N397) containing the SH2 domain but with the EPO-R constitutive association region deleted, are unable to co-immunoprecipitate phosphorylated EPO-R (Figure 2.4). This indicates that optimal constitutive association between SH2B1 and inactive EPO-R at the plasma membrane is required for the binding of the SH2 domain of SH2B1 to pY343/pY401 of EPO-R upon stimulation. Although there are a number of similarities between the SH2B1/Jak2 and SH2B1/EPO-R interactions, the constitutive association of SH2B1 with EPO-R appears to be independent of Jak2 binding to the erythropoietin receptor. This leads us to conclude that in context of signaling downstream of the EPO-R, SH2B1 preferentially binds to the EPO receptor, and not to Jak2. This is a novel interaction for SH2B1, as this adaptor protein has been shown to preferentially bind to Jak2 downstream of both leptin (Li et al., 2007b) and growth hormone receptors (Rui et al., 1997b), instead of the cognate receptors. It is possible that the dynamic interaction observed between SH2B1 and EPO-R serves a similar function as the interaction between SH2B1 and Jak2, whereby constitutive association of SH2B1 with the EPO-R increases the local concentration of SH2B1 around the receptor, and upon EPO stimulation allows for rapid binding of the SH2 domain of SH2B1 with pY343 and/or pY401 of EPO-R. This would allow for robust recruitment of effectors downstream of SH2B1. STAT5 is recruited to pY343 and pY401 of the EPO-R (Klingmuller et al., 1996a). We have shown that SH2B1 also binds to these phospho-tyrosine residues via its SH2

75 domain, which raises the question of what effects binding of SH2B1 to EPO-R pY343 and pY401 may have on STAT5 activation in response to EPO. Potentially, SH2B1 may compete with STAT5 for binding to pY343 and pY401 of the EPO-R, which could lead to decreased activation of STAT5 in response to EPO stimulation. However we did not observe consistent increase in STAT5 phosphorylation upon knock-down of SH2B1. This may have been due to residual presence of SH2B1 in our knock-down system. SH2B1 has 9 tyrosine, 82 serine and 29 threonine residues, and is capable of becoming phosphorylated at multiple sites. Specifically NGF and PDGF have been shown to induce serine/threonine phosphorylation of SH2B1 (Rui et al., 1998; Rui et al., 1999c). In this study, we have shown that SH2B1 is responsive to EPO and upon EPO stimulation becomes phosphorylated. We believe that phosphorylation is most likely on serines/threonines because tyrosyl phosphorylation is not detected using 4G10 antibody, which recognizes the tyrosines in SH2B1β that are phosphorylated in response to growth hormone (Rui et al., 1997b), NGF (Rui et al., 1999c) and PDGF (Rui et al., 1998). This EPO-induced phosphorylation of SH2B1 is both dose- and time- dependent. SH2B1 becomes serine/threonine phosphorylated by MEK or a downstream kinase in response to NGF stimulation (Rui et al., 1999c). Considering that EPO activates the MAP kinase pathway (Haq et al., 2002), and a number of SH2B1 serine/threonine residues lie in the Erk1/2 consensus phosphorylation sites (Rui et al., 1998; Rui et al., 1999c), it is likely that in response to EPO, SH2B1 becomes serine/threonine phosphorylated by Erk 1/2. EPO also activates Protein kinase C (von Lindern et al., 2000) and could lead to phosphorylation of SH2B1 Ser-161 and Ser-165, as these sites are phosphorylated by Protein kinase C. EPO-stimulated phosphorylation of SH2B1 could have very important functional consequences. SH2B1 phospho-sites can either recruit phosphoserine binding proteins, or cause conformational changes, which may lead to enhanced accessibility of proline-rich regions and recruitment of SH3-containing proteins, in both scenarios leading to recruitment of downstream effectors. Furthermore, phosphorylation of serine/threonine residues in the vicinity of the polybasic NLS region of SH2B1 causes the release of SH2B1 from the plasma membrane (Maures et al., 2011) and facilitates entry into the nucleus. Therefore, EPO

76 mediated phosphorylation of SH2B1 may be releasing SH2B1 from the plasma membrane to allow binding to phosphorylated EPO-R via the SH2 domain. siRNA depletion of SH2B1 results in enhanced EPO-stimulated phosphorylation of EPO-R, Jak2, Stat5 and Erk1/2, indicating that SH2B1 is a negative regulator of EPO signaling. Increased phosphorylation of EPO-R pY343 and pY479 was observed. Whether altered expression of SH2B1 affects tyrosine phosphorylation of other EPO-R tyrosines remains to be resolved. SH2B1-dependent regulation of Jak2 has been well documented (Maures et al., 2007). In the absence of SH2B1, there is not only hyperactivation of Jak2, but also increased phosphorylation of the EPO-R and enhanced activation of several downstream pathways. These results suggest that SH2B1 mediates its inhibitory effect on EPO signaling by modulating Jak2 activity. This result was somewhat surprising, as SH2B1 has been shown to be a positive regulator of Jak2 activity downstream of GH and leptin receptors (Li et al., 2007b; Rui et al., 1999a) and to enhance the kinase activity of the insulin receptor (Zhang et al., 2008) and NGF receptor, TrkA (Qian et al., 2001b). However, both of the other members of the SH2B family of adaptor proteins, SH2B2 (Aps) and SH2B3 (Lnk), have been shown to be negative regulators of EPO signaling. The Yoshimura group has shown that SH2B2/Aps negatively regulates EPO signaling by recruiting Cbl to the EPO-R/Jak2 complex (Wakioka et al., 1999b). Studies investigating the erythroid phenotype of SH2B3 (Lnk-/-) deficient mice have shown that SH2B3/Lnk is also a negative regulator of erythropoiesis and EPO signaling (Tong et al., 2005). Interestingly, the erythroid compartment of Lnk-/- mice is normal at steady state (Tong et al., 2005), indicative of a potential compensatory mechanism, which may be fulfilled by SH2B1. One potential mechanism by which SH2B1 may be inhibiting EPO signaling is by recruiting negative regulators via its proline-rich regions or its EPO stimulated phospho-serine sites, and targeting them to Jak2. SH2B1 has been shown to be capable of heterodimerizing with SH2B2 (Nishi et al., 2005). Therefore, another possible mechanism may involve heterodimerization with either SH2B2 or SH2B3 to form an inhibitory complex, which can then modulate the kinase activity of Jak2 downstream of the EPO-R.

In conclusion, our data suggest that SH2B1 binds constitutively to the EPO-R. Upon EPO stimulation, SH2B1 is phosphorylated, most likely on serine/threonines, releasing it from the plasma membrane and allowing SH2B1 to bind to EPO-R pY343 and pY401. This binding can cause conformational changes, which could lead to the recruitment of SH2B1 binding partners to EPO-R. We have also demonstrated that SH2B1 preferentially binds to the EPO-R and not Jak2. Similar to its other family members, SH2B1 is a negative regulator of EPO signaling. Assessment of the definitive role of SH2B1 in erythroid development will be best addressed through analysis of SH2B1 and compound SH2B family gene targeted animals tested under conditions of erythroid stress.

3 CBL linker region and RING finger mutations lead to enhanced GM-CSF signalling via elevated levels of JAK2 and LYN.

A modified version of this chapter is in preparation for submission. Attributions: Dr. Kai Huang generated the CBL expression constructs utilized in this study.

3.1 ABSTRACT

Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, NRAS and KRAS have been found to mutated in JMML patients, leading to aberrant regulation of the Ras signalling pathway. A subset of JMML patients have been shown to harbour CBL mutations associated with 11q acquired uniparental disomy. Mutations in CBL have also been identified in a number of other myeloid malignancies. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated how the CBL linker region mutant, CBL-Y371H, and the CBL RING finger mutant, CBL-C384R lead to enhanced GM-CSF signalling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Stimulation induced phosphorylation of GM-CSF receptor βc subunit was elevated in TF-1 cells expressing CBL mutants, although total levels of GM- CSFR βc were lower. This suggested enhanced kinase activity downstream of GM- CSFR. JAK2 and LYN kinase levels were found to be elevated in CBL-Y371H and CBL- C384R mutant expressing cells. The increased expression of these kinases resulted in enhanced phosphorylation of CBL, SHP2 and S6 in response to GM-CSF stimulation in CBL mutant samples. JAK2 inhibition via treatment with the JAK kinase inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR βc in CBL mutant expressing cells, where-as treatment with SRC family kinase inhibitor, dasatinib, resulted in equalization of GM-CSFR βc phosphorylation signal between CBL wild type and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signalling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

3.2 INTRODUCTION

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that regulates the differentiation, survival, proliferation and functional activation of granulocytes and monocytes in the myeloid lineage (Hercus et al., 2012). In order to mediate its functional activity, GM-CSF binds to its receptor the GM-CSF receptor (GM- CSFR), consisting of GM-CSR α, and GM-CSFR β common (βc) sub-units. The βc subunit of the receptor is shared by the IL-3 and IL-5 receptors. βc is the major signalling subunit, and is tyrosine phosphorylated in response to cytokine stimulation, which recruits various effectors leading to activation of downstream signalling (Barreda et al., 2004; Hayashida et al., 1990; Park et al., 1992). GM-CSFR α is the major binding subunit, which dictates the binding specificity of the receptor complex for GM-CSF (Barreda et al., 2004; Gearing et al., 1989; Park et al., 1992). The engagement of GM-CSFR by GM-CSF results in the formation of a dodecomeric complex of GM-CSF/GM-CSFR α/GM-CSFR βc, which leads to the trans-phosphorylation of GM-CSFR βc associated JAK2 kinases (Hansen et al., 2008). The activated JAK2 kinases phosphorylate GM- CSFR βc tyrosine residues, allowing for the recruitment of various effectors and the activation of downstream signalling. GM-CSF stimulation results in the activation of the JAK-STAT, PI-3Kinase, and the RAS/MAPK pathways, contributing to GM-CSF mediated differentiation, proliferation and survival (reviewed in (Barreda et al., 2004; Hercus et al., 2012). GM-CSF hypersensitivity is one of the defining characteristics of Juvenile Myelomonocytic Leukemia (JMML). This disease is classified by the World Health Organization as a mixed myelodysplastic syndrome/myeloproliferative neoplasm (MPD/MPN) (Emanuel, 2008). JMML patients must show persistent monocytosis in the absence of the BCR/ABL oncogene (Chan et al., 2009b). Genetic lesions deregulating the Ras signalling pathway have been identified to lead to JMML pathogenesis. Ten to fifteen percent of JMML patients harbor mutations in neurofibromin protein (NF1) (Side et al., 1997; Side et al., 1998), a GTPase activating protein (GAP) which negatively regulates RAS by enhancing the hydrolysis of the active GTP bound conformation of RAS to the inactive GDP bound form (Xu et al., 1990). The largest proportion of

81 patients (35%) have mutations in SHP2 (Kratz et al., 2005; Loh et al., 2004; Tartaglia et al., 2003), a protein tyrosine phosphatase, which positively regulates the Ras signalling pathway (Noguchi et al., 1994; Shi et al., 2000). Ras activating mutations account for another 20-25% of JMML associated mutations (Flotho et al., 1999; Kalra et al., 1994; Miyauchi et al., 1994). The presence of NF1, SHP2 and RAS mutations in JMML patients are mutually exclusive. Several groups determined that a proportion of JMML patients have 11q uniparental disomy (UPD). CBL mutations were identified upon further analysis of the 11q UPD samples (Loh et al., 2009; Makishima et al., 2009; Muramatsu et al., 2010). Similar to NF1, SHP2 and RAS mutations, the occurrence of CBL mutations is mutually exclusive to the presence of the other identified JMML mutations. CBL mutations have been identified in numerous other myeloid malignancies, including Acute Myeloid Leukemia, MDS, MPN, and mixed MDS/MPN disease (Dunbar et al., 2008; Fernandes et al., 2010; Grand et al., 2009; Reindl et al., 2009; Sanada et al., 2009; Sargin et al., 2007). The majority of these mutations localize to the linker region or the RING finger of CBL. CBL is an ubiquitin E3 ligase, which specifies target proteins for ubiquitination. The linker region and RING finger of CBL play very important roles in its E3 ligase functionality. The linker region contains two conserved tyrosine residues, Y368 and Y371, whose phosphorylation activates and positively regulates E3 ligase activity of CBL (Dou et al., 2012; Kassenbrock et al., 2004; Levkowitz et al., 1999). The CBL RING finger is responsible for recruiting active E2s carrying a ubiquitin moiety, allowing for the transfer of ubiquitin to the target substrate (Joazeiro et al., 1999; Levkowitz et al., 1999). Therefore, it is not surprising that mutations in the linker region and RING finger of CBL identified in myeloid malignancies result in a loss of E3 ligase activity. Expression of CBL linker region and RING finger mutants inhibit stimulation- induced ubiquitination of the EGF-R (Levkowitz et al., 1999; Sanada et al., 2009), FLT3, c-KIT, and JAK2 (downstream of EPO-R) (Sanada et al., 2009). Investigating the signalling consequences of CBL mutations associated with myeloid malignancies, Niemeyer et al. has shown that expression of linker region mutants in Ba/F3-EPO-R cells results in an enhanced phosphorylation of AKT, ERK 1/2, and S6 (Niemeyer et al.,

2010). In Ba/F3 cells expressing the receptor tyrosine kinase, FLT3, expression of CBL-Y371 mutants has been shown to lead to elevated phosphorylation of the receptor itself, as well as AKT and STAT5 (Fernandes et al., 2010). Although these studies indicate that CBL linker region and RING finger mutations lead to elevated downstream signalling, there is a lack of empirical evidence that specifically shows the role of CBL downstream of the GM-CSF receptor, and how CBL JMML associated mutations may affect GM-CSF signalling. CBL is known to become phosphorylated downstream of the βc in response to stimulation by IL-3 (Anderson et al., 1997; Barber et al., 1997) and GM-CSF (Naccache et al., 1997; Odai et al., 1995). Furthermore, CBL has been observed to associate with the βc sub-unit upon IL-5 stimulation (Martinez-Moczygemba et al., 2001). However, these studies do not directly address the role of CBL in GM-CSF signalling. The objective of this study is to investigate how JMML-associated CBL mutations modulate GM-CSF signaling and lead to GM-CSF hypersensitivity. We expressed wild type CBL, CBL-Y371H and CBL-C384R in the TF-1 hematopoietic cell line, which expresses the endogenous GM-CSF receptor (Kitamura et al., 1989). Phosphorylation of the GM-CSFR βc was elevated in TF-1 cells expressing CBL mutants, despite lower expression of GM-CSFR βc in CBL mutant cells. Increased expression and phosphorylation of JAK2 and LYN was observed in cells expressing CBL-Y371H and CBL-C384R. Elevated SHP2 and S6 phosphorylation was also observed in CBL mutant TF-1 cell lines. In addition, enhanced survival of TF-1 cells expressing CBL-Y371H and CBL-C384R was documented. The increased phosphorylation of GM-CSFR βc observed in TF-1 cells expressing CBL mutants was blocked with treatment with the JAK2-selective inhibitor, TG101348. In summation, our data show that CBL JMML mutants result in enhanced GM-CSF signaling via modulation of JAK2 and LYN tyrosine kinases.

3.3 MATERIALS AND METHODS

Constructs pMSCV-HA-Cbl retro-viral vectors were used to create JMML associated Cbl mutants. HA-tagged Cbl-Y371H and Cbl-C384R were constructed using QuickChange XL Site- Directed Mutagenesis Kits (Stratagene) according to the manufacturerʼs protocol. Primers utilized for mutagenesis are listed in Table 3.1.

TABLE 3.1. Primers utilized in the QuickChange mutagenesis of CBL constructs

Mutant Primer Sequence

CBL-Y371H Forward 5ʼ-CCAGGAACAATATGAATTACACTGTGAGATGGGCTCC-3ʼ

Reverse 5ʼ-GGAGCCCATCTCACAGTGTAATTCATATTGTTCCTGG-3ʼ

CBL-C384R Forward 5ʼ-GGCTCCACATTCCAACTATGTAAAATACGTGCTGAAAATGATAA-3ʼ

Reverse 5ʼ-CTTATCATTTTCAGGACGTATTTTACATAGTTGGAATGTGGAGCC-3ʼ

Retro-virus production

HEK 293T were plated onto 10 cm dishes (Sarstedt) and grown to around 75% confluence. The 293T cells transiently transfected with pSV (gag and pol proteins) and pVSV-G (envelope protein) and pMSCV-GFP retroviral vectors expressing HA-CBL-WT, HA-CBL-Y371H or HA-CBL-C384R using Lipofectamine 2000 (Invitrogen) as directed in the manufacturers protocol. Briefly, 4.5 µg pSV, 1.5 µg VSV-G and 4.5 µg pMSCV- IRES-GFP-HA-Cbl constructs were mixed with Lipofectamine 2000 in serum and antibiotics free DMEM H-21 for 20 minutes at room temperature. 293T cells were washed with serum-free DMEM H-21, and 4 ml of serum and antibiotics free DMEM H- 21 was added to the cells. DNA-Lipofectamine 2000 mixture was added to the plated

84 cells and incubated for 6 hours at 37 °C. Transfection media was removed from the cells and 10 ml RPMI 1640 medium with 10% (v/v) fetal calf serum was added. Transfected 293T supernatant containing VSV-g psuedotyped virus carrying pMSCV- HA-CBL was collected at 48 and 72 hours post transfection.

Cell lines and culture

TF-1 cells were maintained in RPMI 1640 medium, 10% (v/v) fetal calf serum, 100 units of penicillin per ml, 100 µg of streptomycin per ml, and 2 ng/ml recombinant human GM- CSF. In order to stably express wild type and mutant CBL, TF-1 cells were infected with VSV-G pseudotyped retro-virus carrying WT-CBL, CBL-Y371H and CBL-C384R constructs. Briefly, TF-1 cells were suspended in retroviral supernatant supplemented with 8 ng/ml polybrene and centrifuged in 50 ml tubes at 12,000 rpm for 75 minutes. TF-1 cells were then re-suspended in fresh retroviral-polybrene solution and incubated overnight in 6-well dishes at 37 °C. A final round of spinoculation with fresh retroviral supernatant was performed on day 2. TF-1 cells were allowed to expand for 48 hours. GFP positive infected TF-1 cells expressing HA-CBL constructs were purified using fluorescence-activated cell sorting (FACS).

XTT Assay

TF-1 cells were cytokine-depleted overnight, as described below, prior to plating for XTT assay. Two thousand cells were plated per well of a 96-well plate in a final volume of 100 µl of RPMI-10% FCS with indicated concentrations of GM-CSF. Plates were incubated at 37 °C for 48 hours prior to the addition of 3 µM phenazine methosulfate (PMS) (Sigma) and 2 mg/ml sodium 3,3ʼ-[1-[(phenylamino)carbonyl]-3,4- tetrazolium)bis(4-mehoxy-6-nitro)benzene sulfonic acid hydrate] (XTT) (Diagnostics Chemicals). The cells were incubated with the XTT/PMS solution for 6 hours at 37 °C. The absorption of the reduction product at 450 nm was measured using a spectrophotometer plate reader.

Cytokine Deprivation, Stimulation, and Lysis

TF-1 cells were washed three times in 10 mM HEPES (pH 7.4), Hanksʼ balanced salts, depleted in RPMI 1640 medium supplemented with 10% fetal calf serum overnight at 37°C, and then stimulated with 0.5 ng/ml recombinant human GM-CSF. Proteasomal and lysosomal degradation was inhibited by incubation of cells with 30 µM MG132 (Calbiochem/Millipore) and 100 µM chloroquine (Sigma), respectively, for 2 hours prior to stimulation. JAK2 and LYN kinase activity was inhibited by treatment with 2 M of TG101348 (generously provided by Dr. R. Levine) for four hours, or with 0.1 M of dasatinib (gift from Dr. D. Hedley) for two hours, respectively. TF-1 cells were stimulated with 0.5 ng/ml rhGM-CSF for the indicated times at 37 °C. The cells were washed once in 10 mM HEPES (pH 7.4), Hanksʼ balanced salts containing 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM EDTA, and 1 mM sodium orthovanadate and lysed in ice-cold lysis buffer containing 1% Triton X-100, 50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and supplemented with “cOmplete” protease inhibitor cocktail tables (Roche). After 5 min on ice, the lysates were centrifuged at 10,000 x g for 5 min at 4°C.

Antibodies

The 4G10 phosphotyrosine specific monoclonal antibody and anti-Erk1/2 were purchased from Upstate Biotechnology/Millipore. Anti-IL-3/IL-5/GM-CSFR βc (N-20 – used for immunoblotting) and (K-17 – used for immunoprecipitations), anti-Shp2, and anti-phospho-Erk 1/2 antibodies were purchased from Santa Cruz biotechnology. Antibodies specific for Phospho-Jak2 (Tyr1007/1008), Jak2, phospho-Src family (Tyr416), and Lynp56 were acquired from Cell Signaling Technologies. Anti-phospho- STAT5a/b antibodies were purchased from Zymed/Invitrogen. Anti-STAT5 antibody was purchased from BD Biosciences. The monoclonal 12CA5 anti-HA antibody was acquired from Roche.

Immunoprecipitations

Antibodies along with a 50 µl volume of protein A or protein G-Sepharose 4B beads (Amersham Pharmacia Biotech) were added to 2 mg of lysates for an overnight incubation at 4°C. The beads were washed three times in ice-cold lysis buffer. The immune complexes were eluted by boiling in Laemmli sample buffer containing 100 mM DTT. Samples were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane for Western blotting.

Western Blotting

Following the electrophoretic transfer of proteins to PVDF membrane (NEN Life Science Products), the membranes were blocked at room temperature with 2.5% BSA (W/V) or 5% non-fat dry milk (W/V) in Tris-buffered saline (50 mM Tris (pH 8.0) and 150 mM NaCl) with Tween 20 for 1 h. Membranes were then incubated with an optimal concentration of the primary antibody in Tris-buffered saline containing Tween 20 (TBST) for 1 hour at room temperature or overnight at 4°C. Membranes were washed four times in TBST and incubated with the relevant HRP-conjugated secondary antibody for 30 - 60 min. Membranes were washed four times in TBST and visualized by enhanced chemiluminescence with autoradiographic film (ECL, Amersham Pharmacia Biotech). For reprobing, membranes were stripped in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 0.1 M β-mercaptoethanol for 30 min at 50°C; rinsed twice in TBST; and blocked in 2.5% BSA in Tris-buffered saline prior to primary antibody incubation. Western blots were scanned and quantified using ImageJ software.

3.4 RESULTS

3.4.1 Enhanced and Prolonged Phosphorylation of the GM-CSFR βc in CBL Mutant Expressing Cells.

GM-CSF hypersensitivity is one of the hallmark features of JMML (Emanuel et al., 1991). To investigate the role of JMML associated Cbl mutations in vitro, we utilized the TF-1 hematopoietic cell line. TF-1 cells express the GM-CSF receptor α (GM-CSFRα) and the IL-3/IL-5/GM-CSF receptor beta common chain (GM-CSFR βc), and are responsive to GM-CSF. TF-1 cells also have a low endogenous expression of CBL (Figure 3.1A). This is especially advantageous since CBL mutations identified in JMML are associated with acquired UPD, and studies have shown that expression of wild type CBL can rescue or mask the effects of CBL mutants (Niemeyer et al., 2010; Sanada et al., 2009). Retroviral expression vectors were used to stably express HA-tagged wild type CBL, as well as CBL-Y371H and CBL-C384R mutants (Figure 3.1A). Binding of GM-CSF to the GM-CSF receptor complex, activates JAK2, which in turn leads to the tyrosine phosphorylation of GM-CSFR βc. In order to investigate the effects of CBL-Y371H and CBL-C384R mutants on GM-CSF signalling, we examined the phosphorylation of the GM-CSFR βc in response to stimulation. TF-1 cells expressing CBL-WT, -Y371H and -C384R were cytokine depleted and stimulated with GM-CSF. GM-CSFR βc was immunoprecipitated from lysates, and immunoblotted with the anti-phosphotyrosine antibody, 4G10. Upon GM-CSF stimulation cells expressing CBL-Y371H and CBL-C384R mutants (Figure 3.1B – lanes 6-8, 10-12) show enhanced phosphorylation of GM-CSFR βc compared to the wild type CBL controls (lanes 2-4). Furthermore in CBL mutant samples, prolonged phosphorylation of GM-CSFR βc was observed. In TF-1 cells expressing WT-CBL, GM-CSFR βc phosphorylation is significantly reduced 120 minutes post GM-CSF stimulation (Figure 3.1B – lane 4). However, in CBL-Y371H and CBL-C384R cells, this decrease in GM-CSFR βc phosphorylation was not as pronounced (lane 8 & 12).

FIGURE 3.1. Enhanced phosphorylation of GM-CSF receptor βc upon expression of CBL mutants. A, Lysates from parental TF-1 cells, as well as TF-1 cells expressing wild type CBL, CBL-Y371H and CBL-C384R were probed for CBL expression. B, GM-CSFR βc immunoprecipitations were preformed on lysates collected from TF-1 cells expressing CBL wild type, CBL-Y371H and CBL-C384R mutants. The cells were stimulated with 0.5 ng/ml GM-CSF for indicated amount of time. Blots were probed with pTyr and GM- CSFR βc specific antibodies.

CBL plays an important role in stimulation induced endocytosis, trafficking and degradation of numerous tyrosine kinase receptors (Joazeiro et al., 1999; Levkowitz et al., 1999) and cytokine receptors (Saur et al., 2010; Tanaka et al., 2008). To determine whether expression of CBL-Y371H and CBL-C384R mutants have an effect on the expression of the GM-CSFR βc, as well as its GM-CSF induced degradation, lysates collected from GM-CSF stimulated TF-1 cells were probed for GM-CSFR βc. Since it is likely that CBL-Y371H (Dou et al., 2012; Sanada et al., 2009) and CBL-C384R (Thien et al., 2001) mutations result in a loss of CBL E3 ligase activity, GM-CSFR βc levels were expected to be higher in mutant CBL expressing cells. Surprisingly, expression of CBL- Y371H and CBL-C384R (Figure 3.2A – lanes 5-12) results in a decrease in GM-CSFR βc compared to WT-CBL (lanes 1-4) expressing cells at all time points tested (Figure 3.2B). However, it is important to note that GM-CSFR βc expression decreased post GM-CSF stimulation in wild type and mutant CBL expressing cells at comparable rates. These results indicate that although expression of CBL-Y371H and CBL-C384R result in an overall decreased expression of GM-CSFR βc, the degradation of the receptor post stimulation is not disrupted.

3.4.2 Elevated levels of JAK2 kinase in CBL-Y371H and CBL-C384R expressing TF-1 cells

Upon binding of GM-CSF to GM-CSFRα, a GM-CSFR βc homodimer is recruited (Hansen et al., 2008) bringing the βc associated JAK2 kinase (Brizzi et al., 1994; Quelle et al., 1994) in close proximity allowing their transphosphorylation and activation. Activation of JAK2 is essential for downstream GM-CSF signalling (Parganas et al., 1998; Watanabe et al., 1996). The enhanced and prolonged phosphorylation of GM- CSFR βc, along with a decrease in the over all expression of the receptor in CBL mutant expressing cells may be indicative of elevated kinase activity downstream of GM-CSFR βc. Since JAK2 is the main kinase downstream of GM-CSFR, the GM-CSF induced activation of JAK2 was assessed. Upon GM-CSF stimulation, TF-1 cells expressing

FIGURE 3.2. TF-1 cells expressing CBL mutants have lower expression levels of GM-CSFR βc. Lysates were collected as described in figure 3.1. A, Immunoblotting was performed with GM-CSFR βc specific antibody. Blots were reprobed for tubulin as a loading control. B, Blots were quantified using ImageJ software. Values provided are ratios of GM-CSFR βc band to the tubulin loading control, normalized to wild type values at time point zero. (n=8)

CBL-Y371H and CBL-C384R displayed elevated levels of phosphorylated JAK2 (Figure 3.3A – top panel – lanes 6-8, 10-12), compared to the wild type CBL controls (lanes 2- 4). Re-probing the blots for total JAK2, revealed that the observed enhancement of the phospho-JAK2 signal may be due to an increase in the expression of JAK2 in CBL mutant expressing cells (Figure 3.3A – middle panel – lanes 5-12, Figure 3.3B). Linker and RING finger mutations disrupt the E3 ligase activity of CBL (Joazeiro et al., 1999; Kassenbrock et al., 2004; Levkowitz et al., 1999). Such loss of E3 ligase activity has been shown to result in a significant decrease in the EPO-induced ubiquitination of JAK2 (Sanada et al., 2009). In order to determine whether the increase in the expression of JAK2 in CBL mutant expressing cell lines was due to compromised ubiquitination and degradation of the kinase, TF-1 cells expressing wild type CBL, CBL- Y371H and CBL-C384 were treated with proteasomal (MG-132) and lysosomal (chloroquine) inhibitors prior to stimulation. Inhibition of proteasomal and lysosomal degradation stabilized JAK2 to comparable levels in all three cell lines (Figure 3.3C – middle panel). These results indicate that the expression of CBL-Y371H and CBL- C384R mutants results in decreased degradation and therefore elevated levels of JAK2 in TF-1 cells, which in turn contribute to increased JAK2 phosphorylation in response to GM-CSF stimulation.

3.4.3 Increased expression of LYN kinase in CBL mutant cells.

Although JAK2 is the primary kinase activated downstream of GM-CSF receptor, members of the SRC family kinase are also stimulated in response to GM-CSF (Corey et al., 1993; Perugini et al., 2010; Wei et al., 1996). Specifically, LYN has been shown to directly associate with GM-CSFR βc (Li et al., 1995), and to play an important role in mediating the anti-apoptotic effects of GM-CSF in polymorphonuclear leukocytes (Wei et al., 1996; Yousefi et al., 1996). LYN also associates with GM-CSFRα and is involved in the survival signal required for factor-independent growth of cells expressing the FIΔ

FIGURE 3.3. Expression of CBL-Y371H and CBL-C384R mutants results in elevated levels of JAK2 kinase. A, Lysates collected from TF-1 cells expressing WT and CBL mutants were probed for phosphoY1007/1008 JAK2, and reprobed for total JAK2 and tubulin. B, Blots were quantified using ImageJ software. Values presented are ratios of total JAK2 to tubulin, normalized to CBL-WT at time zero. C, TF-1 cells were treated with proteasomal and lysosomal inhibitors, MG-132 and chloroquine (CQ), prior to stimulation. (n=4)

GM-CSFR βc mutant (Perugini et al., 2010). Considering that TF-1 cells expressing CBL-Y371H and CBL-C384R show enhanced survival, while exhibiting lower expression of GM-CSFR βc, we were interested to determine whether activation or expression of LYN was altered in CBL mutant expressing cells. Lysates collected from GM-CSF stimulated cells were probed with an antibody specific for the activatory phosphotyrosine residue of LYN, pY396-LYN. Increased LYN phosphorylation was observed in cells expressing CBL-Y371H and CBL-C384R mutants (Figure 3.4A – top panel – lanes 5-12), compared to those expressing wild type CBL (lanes 1-4). Reprobing for total LYN revealed that CBL mutant cells have increased levels of LYN (p56) (Figure 3.4A – middle panel – lanes 5-12, Figure 3.4B). This observed increase in LYN expression leads to elevated phospho-LYN signal, which may contribute to the enhanced survival of CBL mutant TF-1 cells.

3.4.4 Enhanced phosphorylation of SHP2 in CBL mutant cells

The protein tyrosine phosphatase, SHP2 plays an essential role in activation of RAS pathway downstream of the GM-CSF receptor (Welham et al., 1994). Upon GM-CSF stimulation SHP2 is recruited to the receptor, where it becomes phosphorylated and activated. The phosphatase activity of SHP2 is required for its positive regulation of Ras signalling. About 35% of JMML patients harbor a mutation in SHP2 (Chan et al., 2008; Schubbert et al., 2007) leading to deregulation of the RAS pathway downstream of GM-CSF receptor. In order to determine the effects of CBL mutations on GM-CSF signalling, SHP2 was immunoprecipitated from lysates of GM-CSF stimulated TF-1 cells expressing WT-CBL, and Y371H or C384R CBL mutants. Phosphotyrosine immunoblotting of SHP2 immunoprecipitates revealed that SHP2 phosphorylation was increased in cells expressing the CBL mutants (Figure 3.5 – lanes 6-9, 10-12). Treatment of the cells with proteasomal (MG-132) and lysosomal (chloroquine) inhibitors prior to stimulation resulted in a more pronounced increase in SHP2 phosphorylation in CBL-Y371H and CBL-C384R mutants relative to wild type CBL controls (Figure 3.5).

FIGURE 3.4. Increased expression of LYN kinase in CBL-Y371H and CBL-C384R mutant TF-1 cells. A, Whole cell lysates collected from TF-1 cells expressing CBL-WT, and CBL mutants, were immunoblotted with antibodies specific for pTyr396-LYN. Blots were reprobed for total LYN (p56) and tubulin. B, Blots were quantified using ImageJ software. Values presented are ratios of total LYN to tubulin, normalized to CBL-WT at time zero. (n=6)

FIGURE 3.5. Mutant forms of CBL elevate GM-CSF induced phosphorylation of SHP2. TF-1 cells were either incubated with MG-132 and chloroquine for 2 hours prior to stimulation (+MG-132/CQ), or were left untreated (-MG-132/CQ). Lysates collected from TF-1 cells expressing HA tagged CBL-WT, CBL-Y371H, and CBL-C384R were utilized in SHP2 immunoprecipitations. Immunoblots were probed with pTyr antibody, 4G10, and re-probed for SHP2.

3.4.5 Expression of CBL mutants result in elevated S6 phosphorylation and enhanced factor-free survival.

Investigation of signalling effectors further downstream of the GM-CSF receptor, revealed an enhanced and constitutive phosphorylation of S6 in TF-1 cells expressing CBL-Y371H and CBL-C384R (Figure 3.6A, lanes 5-12), compared to wild type CBL expressing controls (lanes 1-4). No significant differences were observed in the phosphorylation of STAT5 and ERK 1/2 between wild type CBL and mutant CBL samples (Figure 3.6B). The observed pattern of elevated signalling in Cbl mutant expressing cells suggests that CBL-Y371H and CBL-C384R mutants lead to an enhancement of the PI-3K signalling downstream of the GM-CSF receptor. In order to determine whether the expression of the CBL linker and RING finger mutants result in enhanced survival and growth, XTT assays were performed. TF-1 cells expressing CBL-Y371H and CBL-C384R mutants displayed enhanced survival in the absence of GM-CSF relative to cells expressing WT-CBL (Figure 3.7). The increase in the survival of the mutant expressing cells decreases with increasing concentration of GM-CSF. Cell counting assays were also performed to confirm these results (data not shown). These results indicate that JMML associated CBL mutations result in enhanced survival of TF-1 cells at low and limiting doses of GM-CSF.

3.4.6 Constitutive and enhanced phosphorylation of CBL mutants is dependent on a SRC family kinase.

CBL has been shown to become phosphorylated downstream of numerous oncogenic protein tyrosine kinases (BCR/ABL and vSrc), receptor tyrosine kinases (PDGF-R, FLT- 3 and c-KIT), and cytokine receptors (TPO, EPO) (reviewed in (Tsygankov et al., 2001)). For example CBL is tyrosine phosphorylated downstream of the βc chain in response to IL-3 (Anderson et al., 1997; Barber et al., 1997) and GM-CSF (Naccache et al., 1997; Odai et al., 1995).

FIGURE 3.6. Expression of CBL mutants results in constitutive phosphorylation of S6. TF-1 cells expressing CBL-WT, CBL-Y371H and CBL-C384R were depleted of cytokines overnight and stimulated with GM-CSF. Lysates collected were probed with A, phospho- and total S6 or B, phospho- and total STAT5 and ERK 1/2 antibodies.

FIGURE 3.7. Expression of JMML associated CBL mutants results in enhanced survival of TF-1 cells in the abscence of GM-CSF. TF-1 cells expressing WT-Cbl, or Cbl mutants Y371H or C384R were cytokine starved overnight. Starved cells were plated in 96-well plates and incubated for 48 hours, prior to the addition of XTT reagent. Reduction of XTT reagent was measured using a spectrometer to indirectly determine the survival of the TF-1 cells. (n=4) ** p≤0.01, *** p≤0.001

The carboxy terminal region of CBL contains three major tyrosine phosphorylation sites; Tyr700, Tyr731, and Tyr774 (Feshchenko et al., 1998). The phosphorylation of these tyrosines leads to the recruitment of SH2 domain containing effectors. Phosphorylated Tyr700 of CBL binds the guanine nucleotide exchange factor, VAV (Marengere et al., 1997). Phosphorylated Tyr731 acts as the binding site for the p85 regulatory sub-unit of PI-3Kinase (Fukazawa et al., 1995; Liu et al., 1997a), and members of the CRK adaptor protein family have been shown to bind both phosphorylated Tyr700 and Tyr774 (Andoniou et al., 1996; Reedquist et al., 1996; Sawasdikosol et al., 1996). These CBL tyrosine phosphorylation sites play an important role in CBL mediated signalling. Linker region mutations of CBL have been shown to lead to enhanced tyrosine phosphorylation of CBL (Fernandes et al., 2010; Rathinam et al., 2010; Thien et al., 2005a). We were interested to determine whether expression of the linker region Y371H, and the RING finger C384R mutations would result in altered CBL tyrosine phosphorylation. CBL was immunoprecipitated from wild type and mutant CBL TF-1 lysates and phosphorylation was assessed by pTyr immunoblotting (Figure 3.8). CBL- Y371H (lanes 5-6) and CBL-C384R (lanes 8-9) were highly phosphorylated compared to CBL wild type controls (lanes 1-3). In the absence of GM-CSF stimulation both CBL mutants showed constitutive phosphorylation (lanes 4 & 7). Although numerous members of the SRC family kinases, including LYN (Grishin et al., 2000; Meng et al., 1998; Tezuka et al., 1996), are capable of phosphorylating CBL, there is evidence that Janus Kinase family members may also be responsible for tyrosine phosphorylation of CBL (Uddin et al., 1996). To determine whether inhibition of JAK2 or SRC family kinases could abolish the enhanced and constitutive phosphorylation of CBL, TF-1 cells expressing wild type and mutant CBL were treated with JAK2 specific inhibitor, TG101348, and the SRC family kinase inhibitor, Dasatinib, prior to stimulation. The constitutive and enhanced phosphorylation of CBL-Y371H and CBL-C384R mutants (lanes 22-27) persisted in TG101348 treated samples (Figure 3.9), whereas pre-treatment with Dasatinib abolished the elevated phosphorylation of CBL mutants (lanes13-18) to levels comparable to wild type CBL controls (lanes 10-12).

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FIGURE 3.8. CBL-Y371H and CBL-C384R mutants show enhanced phosphorylation in response to GM-CSF stimulation. TF-1 cells expressing HA tagged WT-CBL, CBL-Y371H, and CBL-C384R were cytokine depleted overnight. The cells were then stimulated with GM-CSF for the indicated times. CBL was immunoprecipitated using the 12AC5 anti-HA antibody. Precipitated complexes were resolved on an SDS-PAGE gel, and immunoblotting with pTyr antibody was performed. Blots were reprobed with HA specific antibodies.

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FIGURE 3.9. Dasatinib treatment abolishes the enhanced phosphorylation of CBL-Y371H and CBL-C384R. TF-1 cells expressing WT and mutant CBL were incubated with dasatinib and TG101348 for two and four hours, respectively, prior to GM-CSF stimulation. Cells were lysed and HA immunoprecipitations were performed. Blots were probed with pTyr antibodies, and reprobed with HA specific antibodies.

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These results indicate that downstream of the GM-CSF receptor, CBL is phosphorylated by a SRC family kinase, potentially LYN, and mutations in the linker region or the RING finger domain of CBL lead to not only enhanced but also constitutive phosphorylation of CBL.

3.4.7 TG101348 mediated inhibition of enhanced phosphorylation of GM-CSFR βc in CBL mutant expressing cells

We were interested to determine how the inhibition of JAK2, or SRC family kinases downstream of GM-CSFR would modulate the elevated phosphorylation of GM-CSFR βc observed in CBL mutant expressing cells (Figure 3.1B, Figure 3.10 – lanes 1-9). TF- 1 cells were treated with JAK2 inhibitor, TG101348, and/or SRC family kinase inhibitor, dasatinib, prior to stimulation, and GM-CSFR βc immunoprecipitation. TG101348 treatment resulted in a significant inhibition of GM-CSF induced phosphorylation of GM- CSFR βc in both wild type CBL and CBL mutant expressing cells, resulting in an equally down-modulated level of the phosphorylated receptor (Figure 3.10 – lanes 10-18). This was expected, as JAK2 is known to be the kinase responsible for tyrosine phosphorylation of the GM-CSFR βc in response to stimulation (Muto et al., 1995; Sakamaki et al., 1992; Watanabe et al., 1996). Interestingly, inhibition of Src family kinases did not result in the inhibition of GM- CSFR βc phosphorylation, however, it did result in equivalent level of GM-CSFR βc phosphorylation in wild type CBL and CBL mutant samples (Figure 3.10 – lanes 19-27). Treatment with both TG101348 and Dasatinib inhibited GM-CSFR βc phosphorylation (Figure 3.10 – lanes 28-36) to similar levels observed in samples treated with only TG101348.

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FIGURE 3.10. Inhibition of JAK2 activity via treatment with TG101348 abolishes the enhanced phosphorylation of GM-CSFR β in CBL mutant expressing cells. TF-1 cells expressing WT-CBL, CBL-Y371H and CBL-C384R were cytokine depleted and treated with 2 mM TG101348 for 4 hours or 0.1 mM Dasatinib for 2 hours prior to GM-CSF stimulation. GM-CSFRb specific immunoprecipitations were performed on collected lysates. Immunoblotting was preformed using pTyr and GM-CSFR βc specific antibodies.

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3.5 DISCUSSION

Hypersensitivity to GM-CSF is one of the defining characteristics of juvenile myelomonocytic leukemia. Until recently, genes which were identified to be mutated in JMML patients (NF1, SHP2, NRAS, KRAS) were directly involved in the RAS signalling pathway. The Loh and Maciejewski groups found that 10-15% of JMML patients have mutations in CBL and that these mutations are associated with acquired uniparental disomy (Loh et al., 2009; Makishima et al., 2009; Muramatsu et al., 2010). The majority of CBL mutations identified clustered in the linker region and RING finger of CBL. Both regions play an important role in the E3 ligase activity of CBL. The identification of these JMML associated CBL mutations raised a number of questions about the role of CBL downstream of GM-CSF receptor, whether CBL is involved in RAS signalling downstream of GM-CSFR and how mutations in CBL can lead to hypersensitivity to GM- CSF. In order to address these questions we utilized the TF-1 erythroleukemia cell line, which is GM-CSF responsive (Kitamura et al., 1989) and has a low endogenous expression of CBL. To examine the functional consequences of CBL linker region mutations, we transformed cells with CBL-Y371H, which is the most common CBL mutation identified in patients (Loh et al., 2009). We also employed the CBL-C384R RING finger mutant, since this residue is also frequently mutated in JMML patients (Loh et al., 2009; Makishima et al., 2009). We found that expression of these CBL mutants in TF-1 cells leads to enhanced and prolonged phosphorylation of GM-CSFR βc in response to stimulation. Expression of both major kinases downstream of GM-CSFR, JAK2 and LYN, were elevated in CBL mutant cells. Assessment of downstream signalling revealed enhanced phosphorylation of SHP2 and S6 in CBL-Y371H and CBL-C384R expressing cells relative to WT-CBL controls. TF-1 cells expressing CBL mutations showed enhanced survival in the absence of GM-CSF. We also observed elevated and constitutive phosphorylation of CBL-Y371H and CBL-C384R mutants, which was inhibited upon treatment with the SRC family kinase inhibitor, Dasatinib. Interestingly treatment with Dasatinib also led to equalization of GM-CSFR βc phosphorylation between wild type CBL and CBL mutant expressing cells. Inhibition of JAK2 activity via TG101348

105 treatment, however, resulted in complete inhibition of GM-CSFR βc phosphorylation in CBL mutant and WT-CBL control cells. Expression of CBL-Y371H and CBL-C384R mutants result in prolonged and elevated receptor tyrosine phosphorylation, which supports the observed enhancement in survival (Itoh et al., 1998). Surprisingly, we also found that in CBL mutant cells there is an overall lower expression of the GM-CSFR βc. Collectively these data indicated that there is likely enhanced tyrosine kinase activity in CBL-Y371H and CBL-C384R mutant cells. Consistent with this hypothesis, we found that in CBL mutant cells there was elevated expression of JAK2 kinase, leading to increased levels of phosphorylated and activated JAK2. CBL linker region and RING finger play an important role in the E3 ligase activity of CBL; and Y371H and C384R mutations disrupt the E3 ligase activity of CBL (Thien et al., 2001). Phosphorylation of CBL-Y371 has been shown to be required for the optimal activation of E3 ligase activity (Dou et al., 2012; Kassenbrock et al., 2004). C384 is a conserved cysteine residue involved in the coordinating one of the zinc ions in the RING finger (Borden et al., 1996). The ubiquitination of target proteins by CBL targets them for proteasomal degradation; therefore, disruption of E3 ligase activity of CBL-Y371H and CBL-C384R mutants will result in a decrease in the ubiquitination of CBL targets downstream of the GM-CSF receptor. There are at least two possible mechanisms that account for loss of CBL E3 ligase activity affecting GM- CSFR and Jak2. Martinez-Moczygemba and Huston, using the IL-5 receptor as a model for βc subunit sharing cytokine receptors (IL-3/IL-5/GM-CSF), have shown that after stimulation, the βc sub-unit becomes ubiquitinated. This ubiquitination event leads to the proteasomal degradation of the cytoplasmic tail of the βc subunit, which attenuates downstream signalling (Martinez-Moczygemba et al., 2001; Martinez- Moczygemba et al., 2007). They also found that the ubiquitination of the βc subunit may be mediated by CBL, as the E3 ligase was co-immunoprecipitated with the βc subunit. The loss of E3 ligase activity of CBL-Y371H and CBL-C384R mutants, may prevent GM-CSFR βc ubiquitination and proteasomal degradation of the cytoplasmic region of the receptor, and culminate in the observed enhancement of GM-CSFR βc phosphorylation and elevated levels of JAK2. However, this is unlikely, as we have

106 observed total levels of GM-CSFR βc to be lower in CBL mutant cells. Alternatively, JAK2 may be regulated via CBL mediated ubiquitination (Nagao et al., 2011; Sanada et al., 2009). Therefore, the expression of CBL mutants Y371H and C384R may result in decreased ubiquitination of JAK2, culminating in elevated levels of JAK2 as well as enhanced JAK2 mediated phosphorylation of GM-CSFR βc. Similar to JAK2, various members of the SRC family kinases (Andoniou et al., 2000), including LYN have been shown to be ubiquitinated (Bhattacharyya, 2001) by CBL in a number of studies (Kaabeche et al., 2004; Kyo et al., 2003). The enhanced expression of LYN in TF-1 cells expressing CBL-Y371H and CBL-C384R indicate that CBL downstream of GM-CSFR mediates LYN ubiquitination and degradation. LYN mediates the tyrosine phosphorylation of CBL downstream of β1 integrin (Meng et al., 1998), granulocyte-colony stimulating factor (G-CSF) (Grishin et al., 2000; Wang et al., 2002), and the B-cell antigen receptor (Nishizumi et al., 1998; Tezuka et al., 1996). We have observed enhanced tyrosine phosphorylation of CBL-Y371H and CBL-C384R mutants, which is abolished upon treatment with SRC family kinase inhibitor, Dasatinib. This indicates that LYN is likely the kinase responsible for tyrosine phosphorylation of CBL downstream of the GM-CSFR and the enhanced phosphorylation of CBL mutants may be a functional consequence of the elevated levels of LYN in CBL mutant expressing cells. Furthermore Futami et al. has shown that the SHP2 dephosphorylates LYN-Tyr507 downstream of G-CSFR (Futami et al., 2011), which is required for the optimal activation of LYN, therefore, enhanced phosphorylation of SHP2 in CBL mutant expressing cells may contribute to the elevated level of LYN phosphorylation downstream of GM-CSFR. Phosphorylation of Tyr-731 of CBL leads to the activation of PI-3K pathway via recruitment of the p85 regulatory subunit of PI-3K (Feshchenko et al., 1998; Liu et al., 1997a). The Corey group has shown that LYN couples to the PI-3K pathway in a CBL dependent manner whereby activated LYN binds and phosphorylates CBL allowing for the recruitment of p85 to the complex (Dombrosky-Ferlan et al., 1997; Grishin et al., 2000). LYN has also been shown to activate the PI-3K pathway downstream of GM-CSFR α (Perugini et al., 2010) activating cell survival. We have shown that mutation of CBL resulting in a loss of E3 ligase activity leads to aberrant

107 regulation of LYN downstream of GM-CSFR, which ultimately leads to constitutive and enhanced phosphorylation of S6, suggesting elevated PI-3K pathway activity. Interestingly basal activation of S6 has been observed in mononuclear cells isolated from JMML patients (Kotecha et al., 2008). Furthermore, in the absence of LYN the anti- apoptotic effects of GM-CSF are abolished (Wei et al., 1996; Yousefi et al., 1996). At low concentrations of GM-CSF, the signalling pathways activated downstream of the receptor lead to cell survival only, whereas stimulation with higher doses results in cell proliferation and cell survival (Guthridge et al., 2006). We have shown that expression of CBL mutants results in enhanced survival, especially at low doses, or in complete absence of GM-CSF, potentially indicating that CBL linker and RING finger mutants lower the threshold concentration of GM-CSF, required to induce cell survival. Together, these results suggest that in CBL-Y371H and CBL-C384R expressing cells, the modulation of the PI3-K pathway due to the increase in LYN levels may be contributing to the observed enhancement of survival in the absence of GM-CSF (Figure 3.11). CBL phosphorylation on Tyr700 recruits Crk family of adaptor proteins (Andoniou et al., 1996; Reedquist et al., 1996; Sawasdikosol et al., 1996). Crk family proteins can engage guanine nucleotide exchange factors SOS and C3G, leading to activation of the RAS signalling pathway (Matsuda et al., 1994). Furthermore, CBL has been shown to interact with Grb2 (Fukazawa et al., 1995; Panchamoorthy et al., 1996) downstream of GM-CSFR (Odai et al., 1995), potentially forming a Cbl/Grb2/Crk/Shc complex (Smit et al., 1996), which can lead to modulation of RAS pathway signalling. Based on this mechanism, the enhanced phosphorylation of Tyr700 of CBL-Y371H and CBL-C384R mutants can potentially lead to elevated RAS pathway activity. We have also observed an increase in SHP2 phosphorylation in CBL-Y371H and CBL-C384R expressing cells, potentially due to the increase in JAK2 levels. SHP2 phosphorylation leads to the activation of the phosphatase, which is required for initiation of Ras signalling (Chan et al., 2008; Schubbert et al., 2007). Therefore, CBL linker and RING finger mutations can potentially lead to enhancement of the Ras signalling cascade via multiple pathways (Figure 3.11). However, we fail to see increased phosphorylation of Erk 1/2 in CBL

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FIGURE 3.11. CBL linker and RING finger mutants lead to elevated GM-CSF signalling and enhanced survival. Top panel represents normal GM-CSF signalling. Loss of E3 ligase activity of CBL- Y371H and CBL-C384R mutants (lower panel) leads to increased levels of JAK2 and LYN kinases downstream of GM-CSFR βc, and enhanced phosphorylation of GM-CSFR βc, CBL, SHP2 and S6. The dashed arrows represent potential mechanisms by which CBL mutants may modulate signalling downstream of GM-CSF signalling, leading to enhanced PI-3K signalling and elevated survival. Gray/faded arrows represent lost E3 ligase activity of the CBL mutants. Purple pentagon with T represents increase in total protein. Das – dasatinib, TG – TG101348.

109 mutant expressing cells. We hypothesize that this may be due to the low dose of GM- CSF used to stimulate the TF-1 cells. It is possible that at these lower concentrations, GM-CSF activation of Ras signalling pathway could lead to cross talk to the PI-3K pathway (Rodriguez-Viciana et al., 1994), leading to cell survival. The mechanism by which these mutations lead to elevated GM-CSF signalling and enhanced survival depends on the role of CBL as both an E3 ligase as well as an adaptor protein. CBL-Y371H and CBL-C384R mutants compromise the E3 ligase activity of CBL. We have shown that these loss-of-function mutations lead to increased levels of JAK2 and LYN kinases downstream of the GM-CSFR, potentially due to loss of ubiquitination of the kinases, or the inhibition of GM-CSFR βc ubiquitination and cytoplasmic domain degradation. These events contribute to the elevated levels of GM- CSF signalling observed in CBL mutant expressing TF-1 cells. Sanada et al. has shown that Cbl-/- LSK cells show a mild cytokine hypersensitivity, but transformation of Cbl-/- LSK cells with Cbl linker region mutants significantly enhances the cytokine hypersensitivity, indicating a gain-of-function of mutants which can not be ascribe to a simple loss of CBL E3 ligase activity (Sanada et al., 2009). Inhibition of Cbl-b function by mutant Cbl has been proposed as a possible mechanism. However, it is possible that functionality of CBL as an adaptor protein may be contributing to the gain-of- function of the mutants. Our group, as well as Fernandes et al., have shown that expression of CBL linker and RING finger mutants result in enhanced tyrosine phosphorylation of CBL (Fernandes et al., 2010). This increase in CBL phosphorylation culminates in enhanced PI-3K and Ras pathway activation. We found that treatment with Src family kinase inhibitor Dasatinib results in loss of CBL phosphorylation. Interestingly, Dasatinib treatment equalized GM-CSFR βc phosphorylation in wild type and CBL mutant expressing TF-1 cells. Therefore, Dasatinib treatment may be a way to decouple the gain-of-function capacity of CBL mutants from the loss of E3 ligase activity. Considering that treatment with JAK2 inhibitor, TG101348, results in complete inhibition of GM-CSFR βc phosphorylation, Dasatinib may provide a treatment option for the enhanced signalling driven by CBL gain-of-function mutations.

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Prior studies have shown that expression of CBL linker region and RING finger mutants inhibit stimulation-induced ubiquitination of the EGF-R (Levkowitz et al., 1999; Sanada et al., 2009), FLT3, c-KIT, and JAK2 (downstream of EPO-R) (Sanada et al., 2009). Niemeyer et al. has shown that expression of CBL linker region mutants in Ba/F3-EPO-R cells results in an enhanced phosphorylation of ERK1/2, AKT and S6 (Niemeyer et al., 2010). In Ba/F3 cells expressing the FLT3 receptor tyrosine kinase, expression of CBL-Y371 mutants leads to elevated FLT3 phosphorylation, as well as AKT and STAT5 (Fernandes et al., 2010). While these studies are useful to probe how CBL mutations perturb FLT3 signaling in AML, no studies have examined the role of CBL JMML mutations in GM-CSF dependent signaling pathways to date. Our analysis reveals that expression of CBL-Y371H or CBL-C384R in TF-1 cells generates GM-CSF hypersensitivity that affects proximal steps in receptor activation including GM-CSFR bc, JAK2 and LYN coupling to PI 3 kinase activation. Unlike the Ba/F3 results, we failed to observe effects on ERK1/2 or STAT5 activation. Whether this is a function of the unique receptor systems or distinct cell lines remains to be determined. In conclusion, these studies have confirmed that CBL plays a negative regulatory role downstream of GM-CSFR. We have shown that the enhancement of GM-CSF signalling observed upon loss of CBL E3 ligase activity is due to increased stability of JAK2 and LYN. These results provide in vitro support for further investigation into the applicability of JAK2 and Src family kinase inhibitors for use in treatment of myeloid malignancies associated with CBL linker region and RING finger mutations.

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4 DISCUSSION AND FUTURE DIRECTIONS

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The SH2B1 Adaptor Protein Negatively Regulates EPO Signalling

Summary of Results

In order to identify SH2 domain containing effectors that bind to phospho-Tyr343 of the EPO-R, we employed a COLT screen. We identified the SH2B1 adaptor protein as a novel EPO-R interactor. We confirmed the association of SH2B1 with EPO-R via co- immunoprecipitation experiments utilizing several hematopoietic cell lines as well as primary murine splenic erythroblasts. Interestingly, we found that SH2B1 preferentially binds to the EPO-R instead of JAK2. SH2B1 was demonstrated to bind to EPO-R pTyr343 and pTyr401 in an SH2-dependent manner. Furthermore, we found that SH2B1 was also capable of binding to the inactive unphosphorylated receptor. The membrane proximal region of the EPO-R and the amino terminus of SH2B1 mediate this constitutive interaction. SH2B1 becomes serine/threonine phosphorylated in response to EPO in a dose and time dependent manner. In order to determine the signalling consequences of the SH2B1/EPO-R interaction, we employed siRNA to knock down of endogenous SH2B1 in Ba/F3-EPO-R cells. Depletion of SH2B1 results in elevation of EPO induced phosphorylation of JAK2, EPO-R and ERK 1/2, indicating that SH2B1 is a negative regulator of EPO signalling.

Outstanding Questions and Future Directions

Preferential binding of SH2B1 to EPO-R

SH2B1 was initially identified as a Jak2 binding partner, making it an attractive target on our COLT screen. SH2B1 binds to Jak2 downstream of the growth hormone and leptin receptors, but fails to directly associate with the receptor itself. Therefore, we were surprised to find that in context of EPO signalling, SH2B1 preferentially binds to the EPO-R independently of Jak2. The SH2 domain of SH2B1 binds to Jak2 pTyr813, which is in an YXXL motif (Kurzer et al., 2004), as are EPOR pTyr343 and pTyr401. Determining the binding affinity of the SH2 domain of SH2B1 to Jak2 pTyr813 versus

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EPO-R pTyr343 and pTyr401 will provide insight into the preferential binding of SH2B1 to EPO-R. As previously discussed, SH2B1 is also capable of binding to inactive Jak2. Amino acids 269 – 555 of SH2B1 mediate the interaction with inactive Jak2 (Rui et al., 2000a). In contrast, we have identified SH2B1 amino acids 1 – 266 to be the minimal region required for constitutive association with the EPO-R. Comparison of the binding affinity of these regions of SH2B1 for inactive Jak2 or inactive EPO-R may provide further evidence to explain why SH2B1 favours binding to EPO-R rather than Jak2. The region of SH2B1 required for constitutive association with EPOR contains the SH2B1 dimerization domain, whether this could allow for recruitment of a larger proportion of SH2B1 to the EPO-R in comparison to Jak2, requires further investigation. Functional consequences of the interaction of SH2B1 with inactive and activated EPO-R, via differing mechanisms, could be similar to those described with respect to Jak2 (Li et al., 2007b). Association of SH2B1 with inactive EPO-R can lead to elevated concentration of the adaptor protein in the vicinity of the receptor. Upon EPO stimulation, this would allow for a rapid and robust binding of the SH2B1 via its SH2 domain to the phosphorylated receptor.

Functional consequences SH2B1 phosphorylation in response to EPO stimulation

As discussed earlier, various agonists can lead to differential tyrosine or serine/threonine phosphorylation of SH2B1. Our dephosphorylation experiments using CIAP suggest that SH2B1 is serine/threonine phosphorylated in response to EPO. Utilizing the serine/threonine specific phosphatase, PP2A, will confirm EPO mediated Ser/Thr phosphorylation of SH2B1. PKC and ERK1/2 are both activated by EPO and have been shown to Ser/Thr phosphorylate SH2B1 (Lanning et al., 2011; Rui et al., 1999c). Pre-stimulation treatment with PKC inhibitors, bisindolymaleimide I or LY333531, or MEK inhibitors, U0126 or PD98059, would determine which kinase is responsible for the EPO mediated phosphorylation of SH2B1. Serine phosphorylation of SH2B1 regulates shuttling of the adaptor protein between the nucleus and the cytoplasm. In order to determine whether EPO stimulation results in modulation of the cellular localization of SH2B1, immunofluorescence (IF) studies can be utilized.

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Employing the SH2B1-S154,157,161,165A mutant (Maures et al., 2011) in the IF studies would clarify the mechanistic role of EPO induced SH2B1 serine phosphorylation. Inhibition of SH2B1 nucleocytoplasmic shuttling prevents the transcription of genes required for neural differentiation (Maures et al., 2009). This calls into question the functional consequences of EPO mediated serine phosphorylation of SH2B1. To address this, we can again utilize the SH2B1-S154,157,161,165A mutant, which is unresponsive to stimulation-induced nuclear localization, and determine whether loss of critical SH2B1 serine residues has an effect on EPO induced gene expression.

Investigating the mechanism of negative regulation of EPO signalling by SH2B1

We have shown that the depletion of SH2B1 results in enhanced EPO stimulated phosphorylation of EPO-R, Jak2 and Erk1/2. However, further investigations are required to determine the mechanism by which SH2B1 negatively regulates EPO signalling. One potential mechanism is the SH2-dependent recruitment of SH2B1 to the EPO-R competing with the binding of Ship1 and Shp2 to pTyr401. Both Ship1 and Shp2 contribute to recruitment of Grb2-Sos to the receptor leading to the activation of the MAPK pathway. SH2B1 could also be recruiting phosphatases and other negative regulatory effectors to the EPO-R and into close proximity to JAK2, leading to inhibition of signalling. Flag- or His- tagged SH2B1 expressed in Ba/F3-EPO-R cells can be utilized in affinity chromatography/mass spectrometry studies, to identify potential SH2B1 binding partners.

Erythroid phenotype of SH2B1 deficient mice

We have observed that SH2B1 binds to the EPO-R in primary splenic erythoblasts, calling into question the physiological role of SH2B1 in erythroid homeostasis and development. Erythroid phenotyping of SH2B1-/- mice would include determining steady state erythroid parameters such as hematocrit levels, mean corpuscular volume, hemoglobin levels and red blood cell counts. Furthermore, the developmental capacity of SH2B1-/- erythroid progenitors can be evaluated by BFU-E and CFU-E colony assays.

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Erythroblast differentiation can be assessed via Ter119/CD71 flow cytometric analysis. Deficiencies in erythropoiesis are not always apparent in resting mice. Our group has demonstrated that induction of erythroid stress (via treatment with 5-FU or phenylhydrazine) is frequently required to dissect an underlying subtle erythroid phenotype (Halupa et al., 2005a). Repeated attempts to obtain SH2B1 deficient mice from Dr. Morris White were unsuccessful. Erythropoiesis involves a coordinated balance of proliferative and differentiation signals. ERK 1/2 have been shown to be potent mediators of EPO stimulated mitogenic signals (Haq et al., 2002), and knock down of SH2B1 leads to enhanced Erk 1/2 activation. This provides the basis for the hypothesis that SH2B1 may be contributing a switch from proliferative capacity to differentiation and maturation of erythroid progenitors.

Investigating functional redundancies of SH2-B family members

The SH2B family member Lnk (SH2B3) has also been shown to be a negative regulator of EPO signalling via modulating JAK2 activity (Tong et al., 2005). Studies utilizing Lnk- /- mice have shown that in the absence of Lnk, JAK2-V617F mediated myeloproliferative disease is enhanced. This calls into question whether loss of SH2B1 would also result in a more aggressive JAK2-V617F mediated disease. SH2B1 knock down and over- expression studies using Ba/F3-EPO-R-JAK2-V617F cells would provide insight into the potential role of SH2B1 in mediating oncogenic JAK2-V617F signalling. In order to test the potential in vivo consequences of SH2B1 on JAK2-V617F mediated myeloproliferative disease, bone marrow transplants using donor SH2B1-/- BM cells transduced with JAK2-V617F could be utilized. SH2B1 has been shown to regulate actin based cell motility (Diakonova et al., 2007), which may be a confounding factor in homing of SH2B1-/- BM cells in BMT assays. To overcome this potential limitation, SH2B1-/- mice can be crossed with the inducible JAK2-V617F knock-in mice (Akada et al., 2010; Tiedt et al., 2008) as an alternative to BMT assays. Due to its role in GH and leptin signalling, it was assumed that SH2B1 would also act as a positive regulator of EPO signalling (Tong et al., 2005). However, our study

116 has shown that like its other family member, SH2B1 is also a negative regulator of EPO signalling. Furthermore, the ability of SH2B family members to heterodimerize, makes it critical to examine the role of each adaptor protein in the context of other family members. For example, SH2B2β, an isoform of SH2B2 lacking the SH2 domain, has been shown to bind to SH2B1 and abolish SH2B1 enhancement of GH induced JAK2 activity (Li et al., 2007a). Dimerization of SH2B1 with either family member may provide mechanisms by which SH2B1 negatively regulates EPO signalling. Previously, our group has shown that CBL is a negative regulator of erythropoiesis (Richmond et al., 2008). Dimerization of SH2B1 with SH2B2 can potentially mediate the recruitment of CBL to the EPO-R (Wakioka et al., 1999b), providing another potential mechanism by which SH2B1 may be modulating EPO signalling. Alternatively, heterodimerization of SH2B1 with SH2B3 could increase the concentration of SH2B3 at the receptor leading to a more potent inhibition of JAK2 by SH2B3. Since all three members of the SH2B family have been shown to negatively regulate EPO signalling, functional redundancy among the family members should be examined. SH2B1-/- SH2B2-/- double knock out mice have been established, however their erythropoietic compartment has not been studied. SH2B1 and SH2B3 are the family members most predominantly expressed in the hematopoietic compartment, therefore, SH2B1-/- SH2B3-/- double knock out mice may present with the most prominent erythroid phenotype (Li et al., 2006). Ultimately analysis of a triple knock out of all SH2B family members would provide the most conclusive answer to the functional role of SH2B family of adaptor proteins in erythropoiesis. It is also important to examine whether loss of multiple SH2B family members could lead to elevated myeloproliferative disease mediated by TEL-JAK2 or JAK2-V617F. SH2B3-/- mice spontaneously develop a CML-like MPD at 12 – 18 months of age (Bersenev et al., 2010). It would be of interest to determine whether concurrent loss of SH2B1 could result in earlier development of this CML-like disease.

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CBL linker region and RING finger mutations lead to enhanced GM-CSF signalling via elevated levels of JAK2 and LYN.

Summary of Results

In order to elucidate the potential role of CBL linker region and RING finger mutations in development of GM-CSF hypersensitivity, we expressed two of the most common JMML associated CBL mutations in the TF-1 erythroleukemia cell line. We found that expression of CBL-Y371H and CBL-C384R mutants resulted in elevated and prolonged phosphorylation of GM-CSFR βc. Interestingly, we found that the expression of GM- CSFR βc was decreased in CBL mutant cells. Investigations into the mechanism of increased βc phosphorylation in CBL mutant cells revealed enhanced expression of JAK2 and LYN kinases. Further studies into the dysregulation of GM-CSF signalling by CBL-Y371H and CBL-C384R mutants showed an increase in phosphorylation of SHP2. We observed constitutive activation of S6 in CBL mutant cells, indicating enhanced activation of the PI-3K signalling pathway. Since activation of PI-3K downstream of GM-CSFR leads to cell survival, it was not surprising to find that expression of CBL-Y371H and CBL-C384R resulted in elevated survival in the absence of GM-CSF. We have also demonstrated that CLB-Y371H and CBL-C384R mutants are constitutively and highly phosphorylated. We also confirmed that a member of the SRC family of kinases, potentially LYN, is responsible for phosphorylating CBL. Treatment with the SRC family kinase inhibitor, Dasatinib, equalized GM-CSFR βc phosphorylation in wild type CBL and CBL mutant expressing cells. These studies establish the role of CBL downstream of the GM-CSF receptor, and add insight into the contribution of CBL linker and RING finger mutants to the development of GM-CSF hypersensitivity.

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Outstanding questions and Future directions

Decreased expression of GM-CSFR βc in CBL mutant expressing cells

Our studies have indicated that the expression of CBL linker and RING finger mutations result in elevated phosphorylation of GM-CSFR βc in response to GM-CSF stimulation. We were surprised to find that total levels of GM-CSFR βc were lower in CBL mutant cells, as our initial hypothesis was that due to the loss of E3 ligase activity, GM-CSFR βc levels would be elevated. The enhancement of GM-CSFR βc phosphorylation despite decreased expression can potentially lead to elevated signalling and response to very low doses of GM-CSF. Furthermore the prolonged phosphorylation of the receptor may contribute to the observed increase in cell survival in the absence of GM- CSF. Stimulation induced ubiquitination of the IL-3/IL-5/GM-CSFR βc cytoplasmic domain plays an important role in attenuation of downstream signalling (Martinez- Moczygemba et al., 2001; Martinez-Moczygemba et al., 2007). Since we have observed enhanced and prolonged phosphorylation of the GM-CSFR, it would be of interest to determine whether the expression of CBL mutants abrogates GM-CSFR βc ubiquitination. GM-CSF binding to the receptor also leads to internalization of the receptor and ultimately delivery to the lysosome for degradation (Martinez-Moczygemba et al., 2007; Nicola et al., 1988). Expression of Cbl linker and RING finger mutants abrogate EGFR internalization (Thien et al., 2001). Similarly T-cells deficient in Cbl and Cbl-b show impaired TCR internalization and trafficking to the lysosome (Naramura et al., 2002). This raises the question of whether expression of CBL-Y371H and CBL- C384R results in impairment of GM-CSFR βc endocytosis. In our studies, we observe the down regulation of GM-CSFR βc following stimulation in CBL mutant cells. However, in order to evaluate whether the kinetics of this process varies between wild type CBL and CBL mutant cells, cell-surface expression of GM-CSFR βc at various time points post stimulation can be determined via flow cytometic analysis. GM-CSFR βc immunofluorescence could also be utilized, which would not only determine whether βc internalization is compromised in CBL mutant cells, but also whether the receptor is properly trafficked to the lysosome for degradation. Alternatively I25 labeled GM-CSF

119 can be utilized to perform ligand internalization assays (Gross et al., 2006; Thien et al., 2001). Use of I25 labeled GM-CSF will allow us to compare the availability of functional cell surface GM-CSFR in CBL mutant expressing cells to that in wild type controls (Hilton et al., 1995). Furthermore, this approach can be utilized to determine whether the rate of internalization of ligand bound GM-CSFR complexes is affected by the expression of CBL mutants. Proper trafficking of GM-CSFR βc to the lysosome in CBL mutant cells is of special interest, since Cbl mediated ubiquitination of EGFR has been shown to be critical for proper endosomal sorting to the lysosome (Longva et al., 2002). Studies utilizing IL-5 receptor have shown that IL-5Rα is down regulated after stimulation; however, whether GM-CSFRα behaves in a similar manner has not been investigated. GM-CSFRα specific immunofluorescence and flow cytometeric studies in cells expressing wild type and mutant CBL would provide a clearer picture on the modulation of GM-CSFRα post-stimulation, and whether CBL mutants play a regulatory role. Alternatively, cell surface biontinylation and streptavidin precipitation, followed by GM-CSFR α and βc immunoblotting at various time points post GM-CSF stimulation would determine the surface expression and internalization of each component of the GM-CSFR complex. Ubiquitination and internalization of βc in response to IL-5 stimulation has been shown to be dependent on JAK2 activity, with over-expression of JAK2 leading to enhanced internalization and degradation of βc (Lei et al., 2011; Martinez-Moczygemba et al., 2007). Therefore, elevated levels of JAK2 in CBL mutant expressing TF-1 cells may be a potential mechanism leading to the observed decrease in GM-CSFR βc levels. Enhanced phosphorylation of GM-CSFR βc in CBL mutant cells may lead to recruitment of other E3 ligases leading to enhanced degradation and internalization. As CBL may not be the only E3 ligase responsible for the ubiquitination of GM-CSFR βc, efforts should be dedicated to determine βc targeting E3 ligases. The decrease in GM-CSFR βc in CBL mutant cells may also be due to changes in expression of GM-CSFR βc transcription factors. Rathinam et al. has shown that in CblC379A/- HSCs there is a decrease in the expression of Myb transcription factor (Rathinam et al., 2010). Interestingly, the region proximal to the GM-CSFR βc promoter

120 contains a putative Myb binding site (van Dijk et al., 1998). It is possible that expression of CBL-Y371H and CBL-C384R mutations may also result in decreased MYB expression, leading to decreased GM-CSFR βc levels. GM-CSFR βc transcription is mediated by the Ets family transcription factors (van Dijk et al., 1998). Further investigation into whether the Ets family of transcriptional factors is differentially expressed in CBL mutant cells may provide insight into the decreased expression of GM-CSFR βc.

Confirming elevated levels of JAK2 and LYN due to decrease in ubiquitination

Our studies suggest that the elevation of signalling observed in CBL-Y371H and CBL- C384R mutant expressing cells is due to an enhanced expression of the major tyrosine kinases downstream of the GM-CSFR, JAK2 and LYN. Multiple studies have shown both JAK2, and LYN to be ubiquitination targets of CBL (Kyo et al., 2003; Nagao et al., 2011). Although we have shown that treatment with proteasomal and lysosomal inhibitors equalizes the expression of JAK2 in wild-type and CBL mutant cells, assessment of JAK2 and LYN ubiquitination would provide a more refined mechanism to explain the elevated levels of the kinases in CBL mutant cells.

Effect of CBL mutants on survival and apoptosis

Although we have shown that expression of CBL linker and RING finger mutants result in elevated survival in the absence of GM-CSF, we were restricted in our assessment of cell survival, as we found TF-1 cells are not obligately factor-dependent. Confirmatory experiments could be performed utilizing the factor-dependent Ba/F3 cell line. Since Ba/F3 cells are IL-3 responsive and therefore endogenously express IL-3/IL-5/GM- CSFR βc, only exogenous expression of GM-CSFRα would be required. CBL mutations in JMML patients have been shown to be associated with UPD, indicating homozygosity of CBL mutations is required for oncogenesis. This is inline with in vitro and mouse model findings that expression of wild-type Cbl abrogates the effects of CBL linker and RING finger mutants (Rathinam et al., 2010; Sanada et al., 2009). Ba/F3 cells have been shown to express Cbl endogenously, therefore, shRNA mediated stable knock

121 down of Cbl in Ba/F3-GM-CSFR cells would provide the optimal in vitro model for cell survival studies. Ba/F3-GM-CSFR-CblKD cells can be utilized to determine whether expression of Cbl linker and RING finger mutants would inhibit apoptosis induced by factor withdrawal. Although endogenous expression of CBL was quite low in TF-1 cells, it may have contributed to the signalling differences between wild type and CBL mutants that we observed. However, Ba/F3-GM-CSFR-CblKD cells may provide a better system to assess the effects of Cbl mutations on GM-CSF signalling, particularly activation of PI-3K pathway, assessed via phospho-Akt and phospho-S6 levels.

Consequences of enhanced phosphorylation of CBL mutants on downstream signalling

It has been suggested that Cbl RING finger mutations result in a gain-of-function, which ultimately leads to enhance activation of the PI-3 kinase pathway (discussed in more detail below). Rathinem et al. have demonstrated Cbl-C379A (human CBL-C381A) becomes highly phosphorylated, particularly on Tyr737 (human Tyr731) in response to Flt3 ligand stimulation. Abolishing the p85-binding site by mutating Tyr737 to a phenylalanine, inhibits the Cbl-C379A induced enhancement of PI-3K activation (Rathinam et al., 2010). We also observe enhanced phosphorylation of CBL mutants in our study. Determining which CBL tyrosine residues in CBL-Y371H and CBL-C384R are hyperphosphorylated in response to GM-CSF could provide interesting insight into dysregulation of signalling by the mutants. This is of particular importance as phosphorylation of the other two major CBL phosphorylation sites (Tyr700 and Tyr774) leads to activation of the RAS signalling pathway, which is most commonly affected in JMML cases.

Animal models of CBL mutant induced myeloproliferative disease

The studies presented in this thesis provide an insight into the potential mechanism by which JMML associated CBL mutations result in GM-CSF hypersensitivity. However, whether these mutations would result in recapitulation of disease in an animal model is an outstanding question. In vivo modeling of CBL-C384R would likely result in a MPD with a long latency, similar to that observed in the CblC379A/- mice (Rathinam et al.,

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2010). The CblC379A/- mice provide a good in vivo model for CBL-C384R, as the two conserved cystines (mouse Cbl-C379, C381, and human CBL-C381, C384) coordinate the same zinc ion in the Cbl RING finger. Although no Cbl linker region mutant knock-in mice have been established, it is tempting to extrapolate that the in vivo modeling of the CBL-Y371H mutation would also resemble CblC379A/- transgenic mice. However, in JMML patients, the CBL-Y371H mutations occur at a much higher frequency than CBL- C384R, which raises the question whether CBL-Y371H mediated myeloproliferative disease would vary from that observed in CblC379A/- mice.

Investigating whether CBL mutantsʼ gain-of-function or potential capacity as a CBL-B dominant negative leads to disease development

Currently the prevailing question in the field is whether CBL linker region and RING finger mutations primarily act as dominant negative inhibitors of CBL-B or whether their associated phenotype is due to a gain-of-function. In order to establish the role of CBL mutants as dominant negative inhibitors of CBL-B function, a comparison of the various Cbl knockout and knock-in animal models is required. Complete loss of Cbl expression in germ line knockouts has been shown to result in an increased HSC pool, as well as hypersensitivity and enhanced repopulation capacity (Rathinam et al., 2008). However Cbl-/- mice do not develop any type of myeloid malignancy. CblC379A/- RING finger mutant mice on the other hand, develop a MPD like disease, which progresses to a fatal leukemia. The latency of disease development in these mice is rather long with a median survival of 47.5 weeks (Rathinam et al., 2010). Comparatively, Cbl/Cbl-b double knockout mice show the most aggressive phenotype. They develop a fatal MPD and have a median survival of only 67 days (Naramura et al., 2010). Naramura et al. hypothesized that the absence of MPD development in Cbl-/- mice, as well as the long latency of the CblC379A/- mice in comparison to the aggressive MPD developed by Cbl/Cbl-b double knockouts, may be due to the compensatory role played by Cbl-b (Naramura et al., 2011a). This indicates that Cbl linker and RING finger mutants may be behaving as dominant negative

123 inhibitors of Cbl-b. However, establishing a mechanism for how Cbl mutants inhibit Cbl- b function requires further investigations. Sanada et al. have shown that expression of Cbl linker region mutations, which result in a loss of E3 ligase activity, display a more profound phenotype when expressed in Cbl-/- cells than in Cbl expressing cells. Furthermore, as discussed above CblC379/A mice develop a MPD, where as Cbl-/- mice do not. This evidence demonstrates that Cbl mutations abrogating E3 ligase functionality do not equate to complete loss of Cbl, and in fact, represent a potential gain-of-function. This resonates with the fact that in patients, mutations leading to complete loss of CBL have not yet been identified. Investigations into the potential mechanism of Cbl mutant gain-of-function, has shown that expression of RING finger mutants results in elevated phosphorylation of Cbl, particularly on Tyr737 (human CBL-Y731), leading to elevated activation of the PI-3 Kinase pathway (Niemeyer et al., 2010; Rathinam et al., 2010; Thien et al., 2005a). The results of our research are congruent with these studies. We have shown that Cbl mutants are highly phosphorylated and result in elevated PI-3K pathway activation, thereby providing evidence for the gain-of-function properties of CBL linker and RING finger mutations. As with our study, those of Sanada et al. and Rathinam et al. were performed in cell lines and mice with wild type expression of Cbl-b. Establishing a CblC379A/-/Cbl-b-/- transgenic mouse line would provide substantial insight into the mechanistic functionality of Cbl RING finger mutations. A more aggressive MPD in CblC379A/-/Cbl-b-/- mice, relative to that observed in Cbl/Cbl-b double knockouts, would prove that Cbl RING finger mutants have gain-of-function properties. Ultimately, the phenotypes observed upon expression of CBL mutants is likely due to a combination of inhibiting CBL-B compensation, and enhancing activation of the PI-3K pathway, which is mediated by elevated CBL phosphorylation (Rathinam et al., 2010). Since enhanced phosphorylation of CBL is associated with the gain-of-function properties of CBL mutations and potentially disease development, then inhibition of CBL phosphorylation may provide an attractive therapeutic option. We have demonstrated that although inhibition of JAK2 kinase abolishes the enhanced phosphorylation of GM-

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CSFR βc in CBL mutant cells, it has no effect on the constitutive hyperphosphorylation of CBL. This calls into question the clinical applicability of JAK2 inhibitors for MPD and MDS/MPD cases that harbor CBL linker and RING finger mutations. However, we have shown that treatment with Dasatinib, not only inhibits the constitutive hyperphosphorylation of CBL mutants, but also results in equalization of GM-CSFR βc phosphorylation between wild type and CBL mutant expressing cells. The myeloid leukemic cell line, GDM-1 is homozygous for the CBL-R420Q RING finger mutation. The Maciejewski group has also shown that Dasatinib inhibits growth of GDM-1 cells while having no effect on cell lines lacking homozygous CBL mutations. Furthermore, they have demonstrated that treatment with dasatinib results in a much more significant inhibition of CFU-GM colony formation of BM cells from AML patients with CBL-R420Q mutations, than those with wild type CBL. Our findings along with that of the Maciejewski group suggest that inhibition of SRC family kinases via Dasatinib treatment may in fact be an effective treatment option in cases where CBL linker and RING finger mutations have been identified. In order to test the potential of Dasatinib in treatment of CBL mutant mediated disease, CblC379A/- mice should be treated with Dasatinib to determine whether disease development can be inhibited. In summation, our studies also provide preliminary evidence regarding the feasibility of Dasatinib treatment of JMML in patients suffering from mutations in the CBL E3 ubiquitin ligase.

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5 CONCLUDING REMARKS

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Cytokines regulate an array of biological functions from directing the development and maturation of hematopoietic lineages to mediating immune responses. Understanding the signalling pathways activated by cytokines and how they are negatively regulated is of great scientific and clinical importance. In the second chapter of this thesis, we investigated how the adaptor protein SH2B1 can interact with and negatively regulate EPO mediated signalling. In the next chapter we demonstrated that disruption of negative regulation of GM-CSF signalling via E3 ligase inactivating mutations in CBL results in aberrant signalling leading to oncogenic hypersensitivity to GM-CSF. The SH2B family provides an exemplary demonstration of how adaptor proteins can modulate different signalling pathways in varying ways. SH2B1 has been found to positively regulate insulin, leptin, GH, PDGF and NGF signalling, however, it behaves as a negative regulator downstream of the EPO-R. The ability of SH2B1 to regulate the signalling mediated by so many stimulants is dependent on its capacity to mediate protein-protein interactions. Similarly, the full oncogenic potential of CBL mutations may not be completely dependent on the loss of E3 ligase activity and enhanced by the gain- of-function of the adaptor protein properties of CBL mutants. Therefore, the inhibitors of protein-protein interactions may hold promise as therapeutics. The use of EPO or erythropoiesis-stimulating agents (ESA) to treat cancer patients suffering from chemotherapy-induced anemia has been under scrutiny, as several trials have shown an association with decreased survival. One can imagine that inhibition of the SH2 dependent recruitment of SH2B1 to the EPO-R, via administration of inhibitor of protein-protein interactions can lead to enhanced EPO signalling and therefore erythropoiesis. This can potentially provide an alternative to ESA for treatment of anemia in oncology patients. In a similar fashion, an inhibitor abrogating the recruitment of p85 to phosp-Tyr731 of CBL linker or RING finger mutants, may abolish the enhanced activation of the PI-3K pathway, providing a potential therapeutic for treatment of myeloproliferative diseases caused by CBL mutations. The studies presented in this thesis not only highlight the importance of investigating negative regulation of cytokine signalling, but also the significance of adaptor protein functionality in modulating cytokine signalling.

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