Ptpn11) Phosphatase

EFFECTS OF ACTIVATING MUTATIONS IN SHP-2 (PTPN11) PHOSPHATASE

ON HEMATOPOIETIC CELL DEVELOPMENT

DAN XU

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Adviser: Dr. Cheng-kui Qu

Department of Pathology

CASE WESTERN RESERVE UNIVERSITY

May, 2011 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

______

candidate for the ______degree *.

(signed)______(chair of the committee)

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. Dedication

This thesis is dedicated to my mother, Taiqin Wang.

Contents

List of Tables ...... 3

List of Figures ...... 4

Acknowledgments ...... 6

Abstract ...... 8

Chapter I: Introduction ...... 10

Structure, regulation and functions ...... 11

Shp-2 mutations in human diseases ...... 15

Molecular mechanisms of how Shp-2 gain-of-function (GOF) mutations result in pathogenic effects ...... 17

Hematopoietic stem cell development ...... 21

Methods to determine stem cell activities ...... 22

Chapter II: Shp-2 D61G mutation induces myeloproliferative disease by aberrant activation of hematopoietic stem cells ...... 26

Introduction ...... 26

Results and Discussion ...... 28

Supplementary Information ...... 56

Chapter III: Non-Lineage Restricted Effects of Ptpn11 E76K mutation on Malignant Transformation of JMML ...... 72

Introduction: ...... 72

Results and Discussion ...... 73

Supplementary Information ...... 98

Chapter IV: Future directions ...... 105

Appendix: Experimental Methods ...... 111

Generation of Ptpn11D61G/+ and Ptpn11D61G/+/Gab2-/- mice ...... 111

Generation of Ptpn11E76K knock-in mice...... 111

Flow cytometric analysis and cell sorting ...... 113

Apoptosis and cell-cycle analysis ...... 114

Generation of BM-derived mast cells and macrophages ...... 115

CFU-S and transplantation assays ...... 115

Phosphatase assay...... 116

Colony-forming unit assay...... 117

Statistical analysis ...... 118

Bibliography ...... 119

List of Tables

Table 2-1. Transplantation of Ptpn11D61G/+ whole BM cells or LSK cells…...... 48

Table S2-1. Absolute numbers of hematopoietic cells in WT and Ptpn11D61G/+ mice...... 68

Table S2-2. Peripheral blood parameters in WT and Ptpn11D61G/+ transplants ... 69

Table S2-3. Peripheral blood parameters in the competitive repopulation assay recipients ...... 70

Table S2-4. Peripheral blood parameters in Ptpn11D61G/+/Gab2-/- mice ...... 71

Table S3-1. Peripheral blood hematology of Ptpn11E76K knock-in mice (6-8 weeks following pI-pC treatment) ...... 99

Table S3-2. Absolute numbers of hematopoietic cells in the BM of Ptpn11E76K knock-in mice...... 104

List of Figures

Figure 2-1. Aberrant hematopoietic cell development in Ptpn11D61G/+ mice...... 32

Figure 2-2. Enhanced cycling and decreased apoptosis in Ptpn11D61G/+ LSK cells...... 35

Figure 2-3. Greater short-term and long-term repopulating activities of Ptpn11D61G/+ BM cells...... 40

Figure 2-4. MPD is reproduced in Ptpn11D61G/+ BM cell-reconstituted primary and secondary recipients...... 46

Figure 2-5. MPD phenotypes induced by Ptpn11D61G/+ mutation are substantially attenuated by deletion of Gab2...... 51

Figure 2-6. An important role of Gab2 in mediating the pathogenic effects of Ptpn11D61G/+ mutation in stem cells...... 54

Figure S2-1. MPD phenotypes are reproduced in secondary recipients...... 57

Figure S2-2. Cytokine hypersensitivity of Ptpn11D61G/+ mutant LSK cells...... 59

Figure S2-3. Enhanced GM-CSF-induced Erk activation in Ptpn11D61G/+ ...... 61

Figure S2-4. Absolute numbers of LSK cells are largely restored in the BM of Ptpn11D61G/+/Gab2-/- mice...... 59

Figure S2-5. Gab1 and Gab3 levels are not significantly altered in Gab2-/- or Ptpn11D61G/+/Gab2-/- LSK cells...... 65

Figure S2-6. An important role of Gab2 in mediating the excessive myeloid expansion induced by Ptpn11D61G mutation...... 67

Figure 3-1. Ptpn11E76K/+ mutation initially induces MPD in mice with full penetrance...... 78

Figure 3-2. Ptpn11E76K/+ mutation activates HSCs by enhancing growth factor signaling...... 85

Figure 3-3 Non-lineage specific effects of Ptpn11E76K/+ mutation on malignant transformation of MPD...... 89

Figure 3-4 Leukemias induced by Ptpn11E76K mutation not only derived from HSCs but also from lineage committed progenitors...... 97

Figure S3-1 Generation of conditional Ptpn11E76K knock-in mice ...... 99

Figure S3-2 Characterization of Ptpn11E76K knock-in mice...... 98

Acknowledgments

In the process of finishing the writing of this thesis, I felt time flies, and it became quite clear to me that in the past five years, I could not have completed my Ph.D thesis project without the generous help of my colleagues and friends. I would like to thank those who made this thesis possible.

I owe my deepest gratitude to Dr. Cheng-kui Qu and Wen-Mei Yu. I enjoyed my stay in the Qu Lab and the interactions with the lab members. When

I entered Dr. Qu‟s lab, I was in the beginning stage of understanding science in

Tyrosine Phosphatases and Hematopoiesis. With the unbelievably patience, Dr.

Qu has directed me to make tremendous progress in understanding these fields.

He is always there whenever I need his advices and instructions, no matter how busy his schedule is. I can clearly recall the times when Dr.Qu showed me how to count the CFU assay hand by hand, when he taught me how to design a simple experiment, and when he instructed me how to choose an appropriate research strategy to sharpen my logical thinking. The longer I worked with him, the more I realize what a good PI means for the success of a graduate student!

While Dr. Qu emphasizes more on improving my critical thinking, Wen Mei focuses more on developing my experimental techniques. She guided me from a simple Western Blot, through immunoprecipitation, to a sophisticated multiple color FACS analysis. These learning experiences greatly improved my technical

6 competency. Thus, I would like to express my sincere thanks to Dr.Qu and Wen

Mei for their advice, guidance and help during my Ph.D studies.

I am grateful to my thesis committee members, Drs. David Kaplan, Kevin

Bunting, Zhenghe Wang and Clive Hamlin. I appreciate their critical comments and generous help on my thesis project. In addition, I would like to express my sincere thanks to them for their encouragement, guidance and support during my

Ph.D studies.

I would like to thank all the people in the Division of Hematology/Oncology including members of Qu lab, Gerson Lab, Bunting Lab, Koon Lab and Ma lab. I enjoyed the intellectually stimulating environment in the Division of

Hematology/Oncology, and feel lucky that I had the opportunity to carry out my graduate studies in this wonderful place.

Finally, I would like to thank several of my friends, Yuan Lin, Fangjing

Wang, Wenqian Hu, Ying Wang and Wei Wang, for scientific discussions during my Ph.D studies.

Effects of Activating Mutations in Shp-2 (Ptpn11) Phosphatase on Hematopoietic Cell Development

Abstract

DAN XU

Germline and somatic gain-of-function mutations in the tyrosine phosphatase PTPN11 (Shp-2) are associated with juvenile myelomonocytic leukemia (JMML), a myeloproliferative disease (MPD) of early childhood, which often progresses to blast crisis/overt leukemia. In my study, I have demonstrated that germline mutation Ptpn11D61G in mice aberrantly accelerates hematopoietic stem cell (HSC) cycling, increases stem cell pool, and elevates short-term and long-term repopulating capabilities, leading to the development of MPD. The

MPD is reproduced in primary and secondary recipient mice transplanted with

Ptpn11D61G/+ whole bone marrow cells or purified Lineage- Sca-1+c-Kit+ cells, but not lineage committed progenitors. The pathogenic effects of Ptpn11D61G mutation on HSCs are attributable to enhanced cytokine/growth factor signaling.

The aberrant HSC activities caused by Ptpn11D61G mutation are largely corrected by deletion of Gab2, a Shp-2 interacting protein/target that plays an essential role in cell signaling. More importantly, MPD phenotypes are markedly ameliorated in

Ptpn11D61G/+/Gab2-/- double mutant mice. Together, these studies suggest that

8 oncogenic Ptpn11 induces MPD by aberrant activation of HSCs and identify

Gab2 as an important mediator for the pathogenic effects of Ptpn11 mutations. In my another study, we created a conditional knock-in mouse model with the most common and the most potent PTPN11 activating mutation (E76K) found in JMML.

Unlike Ptpn11D61G mutation, Ptpn11E76K mutation evokes frank T cell lymphoid leukemia/lymphoma (T-ALL), acute myeloid leukemia (AML) and B cell lymphoid leukemia (B-ALL) with full penetrance following a chronic phase of MPD.

Ptpn11E76K mutation aberrantly activates hematopoietic stem cells (HSCs) and lineage progenitors by enhancing growth factor/cytokine signaling. Remarkably,

Ptpn11E76K mutation induces leukemia-initiating/propagating cell development not only in HSCs but also in lineage committed progenitors. These findings reveal non-lineage specific effects of Ptpn11E76K mutation on malignant transformation of JMML and provide the first evidence that an activating mutation in Ptpn11 phosphatase plays a causal role in malignant leukemias. The unique disease evolution pattern of Ptpn11E76K knock-in mice represents the natural course of

JMML. The novel Ptpn11E76K mouse model will be valuable for studying disease progression and preclinical trials aiming at controlling malignant transformation in

JMML.

Chapter I: Introduction

Shp-2 (encoded by PTPN11), a protein tyrosine phosphatase (PTP), is a critical regulator of hematopoiesis. Previous studies have demonstrated that Shp-

2 plays a positive role in hematopoietic cell development and function. Recently, germline or somatic mutations in Shp-2 that cause hyperactivation were identified in the developmental disorder Noonan syndrome and various childhood leukemias (Loh et al., 2004; Tartaglia et al., 2004; Tartaglia et al., 2001; Tartaglia et al., 2003). Fifty percent of the patients with Noonan syndrome, 35% of juvenile myelomonocytic leukemia (JMML) (Loh et al., 2004; Shimada et al., 2004;

Tartaglia et al., 2003), and 6% of B cell acute lymphoblastic leukemia cases carry activating mutations in Shp-2 (Tartaglia et al., 2004). Indeed, genetic animal models have demonstrated that Shp-2 mutations play a causal role in the development of these human diseases. For example, a single Shp-2 D61G mutation is sufficient to induce Noonan syndrome and JMML-like myeloid proliferative diseases (MPD) in mice (Araki et al., 2004). However, the molecular mechanisms by which Shp-2 activating mutations induce these diseases, in particular, the effects of the Shp-2 mutations on hematopoietic stem cells, have not been well understood. Signaling partners that mediate the pathogenic effects of PTPN11 mutations have not been well characterized. Lack of such knowledge set a barrier to design new therapeutics for the Shp-2 related diseases.

In this chapter, I summarized the recent studies on biological functions of

Shp-2, the regulations of Shp-2 activities, the pathogenic mechansims of Shp-2 mutations, hematopoietic cell development and methods to determine stem cell activities.

Structure, regulation and functions

Shp-2, a ubiquitously expressed protein tyrosine phosphatase, plays a critical role in cell signaling and cellular processes. Shp-2 contains two tandem

SH2 domains (N-SH2 and C-SH2), a classic tyrosine phosphatase domain at C- terminus, and a C-terminal tail with two important tyrosine resides (Tyr542 and

Tyr580) and some other functional motifs (Ahmad et al., 1993; Feng et al., 1993;

Freeman et al., 1992; Vogel et al., 1993). The SH2 domain, a 100-amino-acid motif, specifically recognizes phosphorylated tyrosine residues on other molecules and mediates protein-protein interactions. Biochemical and structural studies indicate that, in the basal state, the N-SH2 domain is wedged into the

PTP domain and forms a “backside loop” to prevent substrate accessing. Upon binding to an appropriate phosphotyrosyl (p-Tyr) peptide, N-SH2 alters its conformation and releases the catalytic domain from auto-inhibition (Barford and

Neel, 1998; Hof et al., 1998; Neel et al., 2003). In addition, mutagenesis and protease resistance studies suggest that phosphorylation of Tyr542 and Tyr580 in the C-terminal tail also regulates Shp-2 activity. Phosphorylated Tyr542 and

Tyr580 could be engaged to the N-SH2 and C-SH2 domains, respectively, in an intramolecular manner to stimulate Shp-2 activity (Lu et al., 2001).

Important biological functions of Shp-2 have stemmed from genetic studies. Shp-2 plays an essential role in hematopoiesis and lymphopoiesis.

Homozygous deletion of the N-SH2 domain of Shp-2 (amino acids 46-110) (Shp-

2/) in mice results in embryonic lethality with a defect in mesodermal patterning

(Arrandale et al., 1996; Saxton et al., 1997). To dissect Shp-2 functions in the development of various cell lineages, Shp-2/ ES cell lines was isolated, and the effects of N-SH2 deletion of Shp-2 on cell development were examined using an in vitro ES cell differentiation assay developed by the Keller‟s group (Keller,

1995). ES cells were cultured in the semi-solid methylcellulose medium and grown into cellular spheres termed embryoid bodies (EB). In the EB structure, wild-type (WT) ES cells differentiate into various cell lineages, such as neural, hematopoietic, muscle, and endothelial cells, indicating that this model system is a useful tool to study the early development of embryonic cells. Using this assay, it was shown that loss of Shp-2 function caused suppression of ES cell differentiation to erythroid and myeloid progenitors, suggesting a positive role of

Shp-2 in hematopoietic development (Qu et al., 1997). This conclusion was also sustained by chimeric animal studies. To dissect Shp-2 functions in vivo, chimeric mice were generated by aggregating Shp-2/ ES cells with WT embryos.

Blood cell development in these chimeric animals was determined. No Shp-2 mutant ES cell-derived progenitors for erythroid or myeloid lineages were detected in the fetal liver and bone marrow of chimeric animals by the CFU assay,

12 and primitive hematopoiesis was defective in Shp-2/ yolk sacs (Qu et al., 1998).

In addition, Shp-2 deficiency caused multiple developmental defects in many other tissues, characterized by short hind legs, aberrant limb features, split lumbar vertebrae, abnormal rib patterning, and pathological changes in the lung, the intestine, and the skin (Qu et al., 1998). Using the RAG-2–deficient blastocyst complementation assay, an essential role of Shp-2 in lymphopoiesis has been demonstrated (Qu et al., 2001). Chimeric mice generated by injecting Shp-2/

ES cells into Rag-2–deficient blastocysts had no detectable mature T and B cells, serum immunoglobulin M, or even Thy-1 positive or B220 positive precursor lymphocytes (Qu et al., 2001). Notably, reintroduction of WT Shp-2 into Shp-2/

ES cells restored both primitive and definitive hematopoietic potential (Chan et al.,

2003).

Recently, Feng‟s group has generated a series of tissue-specific Shp-2 knockout mice using Cre-Lox system to determine the biological role of Shp-2 in other organs. The results demonstrated that this enzyme acts predominantly in one pathway versus the others in a cell, depending on the cellular context. To determine the putative functions of Shp-2 in the adult brain, they selectively deleted Shp-2 in postmitotic forebrain neurons by crossing CaMKIIalpha-Cre transgenic mice with a conditional Shp-2 mutant (Shp-2 (flox)) strain. Surprisingly, a prominent phenotype of the mutant (CaMKIIalpha-Cre: Shp-2 (flox/flox) or

CaSKO) mice was the development of early-onset obesity, with increased serum levels of leptin, insulin, glucose, and triglycerides. These results suggest that a primary function of Shp-2 in postmitotic forebrain neurons is to control energy

13 balance and metabolism. Consistent with previous in vitro data, they found that

Shp-2 down-regulates Jak2/Stat3 activation by leptin in the hypothalamus.

However, Jak2/Stat3 down-regulation is offset by a dominant Shp-2 promotion of the leptin-stimulated Erk pathway, leading to induction rather than suppression of leptin resistance upon Shp-2 deletion in the brain. These results demonstrated that this phosphatase is a critical signaling component of leptin receptor ObRb in the hypothalamus (Zhang et al., 2004). Mice with neuronal deletion of Shp-2 developed obesity and diabetes and the associated pathophysiological complications that resemble those encountered in humans, including hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin and leptin resistance, vasculitis, diabetic nephropathy, urinary bladder infections, prostatitis, gastric paresis, and impaired spermatogenesis (Krajewska et al., 2008). In addition, deletion of Shp-2 tyrosine phosphatase in muscle leads to dilated cardiomyopathy, insulin resistance, and premature death (Princen et al., 2009).

Conditional deletion of Shp-2 in neural progenitor cells leads to early postnatal lethality, reduced proliferation of progenitor cells in the ventricular zone, and impaired corticogenesis. In vitro analyses suggest that Shp-2, partly through control of Bmi-1 expression, mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs. Furthermore, through reciprocal regulation of the Erk and Stat3 signaling pathways, Shp-2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, (Ke et al., 2007). Together, these results identify Shp-2 as a critical signaling molecule

14 in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.

Deletion of Shp-2 in the thymus by Lck-Cre results in reduced expansion of CD4 (+) T cells and a significant block in thymocyte differentiation/proliferation at the beta selection step. Furthermore, authors observed decreased TCR signaling in vitro in mature Shp-2-/- T cells. Mechanistically, Shp-2 acts to promote

TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp-2-/- T cells (Nguyen et al., 2006). These results provide physiological evidence that Shp-2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.

Shp-2 mutations in human diseases

Consistent with the notion that Shp-2 phosphatase plays a positive role in hematopoietic cell development, somatic mutations in Shp-2 that cause hyperactivation of its catalytic activity have been identified in juvenile myelomonocytic leukemia (JMML). JMML is a rare, lethal childhood myeloproliferative disorder (MPD) characterized by leukocytosis with prominent monocytosis, macrocytic anemia, hepatosplenomegaly and hypersensitivity of hematopoietic progenitors to granulocyte-macrophage colonystimulating factor

(GM-CSF) and IL-3 (Arico et al., 1997; Emanuel et al., 1996). JMML often transforms to Frank leukemia. Also, germline Shp-2 mutations have been found

15 in the developmental disorder called Noonan Syndrome (NS). Noonan Syndrome

(NS) occurs at an incidence of about 1:1,000 – 1:2,500 live births (Nora et al.,

1974) and is characterized by unusual craniofacial features, congenital heart defects, proportionate short stature, cryptorchidism in males, and a range of secondary manifestations (Allanson, 1987).

Fifty percent of the patients with Noonan syndrome, 35% of JMML (Loh et al., 2004; Shimada et al., 2004; Tartaglia et al., 2003) and 6% of B cell acute lymphoblastic leukemia cases carry mutations in Shp-2 (Tartaglia et al., 2004) .

Essentially all Shp-2 mutations found in NS and/or sporadic leukemia (e.g. D61G,

D61Y, E76D, and E76K) occur in residues located at the N-SH2/PTP domain interface. Such mutations presumably impair the auto-inhibition of enzyme activity, increasing basal catalytic activity while retaining the ability to bind N-SH2 ligands in vitro and cause hyperactivity (Tartaglia et al., 2001).

Neel lab has generated two mouse models (Ptpn11D61G and Ptpn11D61Y).

Homozygous D61G mutation is embryonic lethal. Heterozygous Shp-2 D61G mice (Ptpn11D61G/+) can survive but display short stature, craniofacial abnormalities similar to the hallmarks of Noonan syndrome (Araki et al., 2004).

About 50% of Ptpn11D61G/+ embryos had multiple cardiac defects. Moreover,

Ptpn11D61G/+ mice develop JMML-like myeloid proliferative disease (MPD), including enlarged spleen, increased white blood counts, and higher population of Mac-1/Gr-1(Araki et al., 2004).

Another mouse model expressing leukemia-associated mutant Ptpn11D61Y in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD), featuring leukocytosis, anemia, hepatosplenomegaly, and factor-independent colony formation by bone marrow (BM) and spleen cells (Chan et al., 2009).

Repopulating activity is decreased in the mutant mice, and mice that do engraft with Ptpn11D61Y stem cells fail to develop MPD. Ptpn11D61Y common myeloid

(CMP) and granulocytemonocyte (GMP) progenitors produce cytokine- independent colonies in a cell autonomous manner and demonstrate elevated

Erk and Stat5 activation in response to granulocyte-macrophage colony- stimulating factor (GM-CSF) stimulation (Chan et al., 2009). This study provides a mouse model for Ptpn11-evoked MPD and show that this disease results from cell-autonomous and distinct lineage-specific effects of mutant Ptpn11 on multiple stages of hematopoiesis.

Molecular mechanisms of how Shp-2 gain-of-function (GOF) mutations result in pathogenic effects

Over the past years, significant progresses have been made in understanding the cellular and molecular mechanisms of how Shp-2 mutations cause its pathological effects. Shp-2 is involved in multiple cell signaling processes, such as the RAS-MAP kinase, JAK/STAT, PI3K/AKT, NF-ĸB, and

NFAT pathways (Neel et al., 2003; Tonks, 2006; Xu and Qu, 2008). Shp-2 generally plays a positive role in transducing signals initiated from receptor and

17 cytosolic kinases, particularly RAS Pathway. GOF mutations of Shp-2 usually exert pathogenic effects through activating Erk and Akt activities. Consistently,

JMML patients without Shp-2 mutations usually have either homozygous deficiency of NF1 (a RASGAP) or activating NRAS or KRAS mutations, leading to the hyperactivation of the Erk pathway (Bollag et al., 1996; Largaespada et al.,

1996; Le et al., 2004; Zhang et al., 1998). The remaining 15–20% of JMML patients with unknown lesions also likely has mutations in other components of the RAS/ERK pathway, such as SOS mutations. SOS mutations have also been identified in Noonan syndrome (Roberts et al., 2007; Tartaglia et al., 2007).

These data imply a general involvement of the RAS/ERK pathway is leukemogenesis.

Several studies have clearly demonstrated that Erk and Akt are the key components mediating pathogenic effects of Shp-2 mutations. In BM macrophages isolated from Ptpn11D61G mice, an enhanced GM-CSF-mediated

Erk and Akt phosphorylation was observed (Chan et al., 2005). An enhanced IL-3 mediated Akt and Erk activation has been shown in BM-derived mast cells and pro-B cell line Ba/F3 expressing Shp-2 D61Yor Shp-2 E76K (Mohi et al., 2005; Yu et al., 2006). Moreover, administrating inhibitors of the downstream effecters of Erk or Akt pathways such as Mek and Tor significantly reduced factor-independent transformation by Shp-2 mutation (Mohi et al., 2005; Yu et al., 2006).

Tremendous efforts have been made to identify the immediate downstream binding partners of Shp-2. One key targeting molecule appears to be the scaffolding adaptor Gab2. Using immunoaffinity purification followed by

Mass Spectrum, Gu et al cloned Gab2 as a direct binding partner of Shp-2 (Gu et al., 1998). Upon cytokine or growth factor stimulation, Gab2 is recruited to Shp-2 through the SH2 domain, and is phosphorylated at its Tyrosine sites. Expression of Gab2 mutants which block its binding activity with Shp-2 resulted in reduced activity of cytokine-induced c-fos promoter and diminished transactivation

-/- mediated by Elk1 and STAT5 (Gu et al., 1998). Indeed, the Gab2 mice is refractory to myeloid transformation by leukemogenic Shp-2 mutant such as

E76K. E76K mutation could transform BM from wild type mice, resulting in cytokine-independent myeloid colonies, but E76K was unable to transform

Gab2−/− BM cells (Mohi et al., 2005).

In addition, Gab2 is required for Bcr-Abl oncogenic tyrosine kinase induced transformation (Sattler et al., 2002). Y177 is an auto-phosphorylation site in Bcl-Abl. Its mutation prevented the induction of a myeloproliferative disorder in a murine CML transduction-transplantation model (He et al., 2002; Million and

Van Etten, 2000; Zhang et al., 2001a). Phosphorylated Y177 recruits Grb2, and subsequently Shp-2 and Gab2 (Sattler and Griffin, 2001). Gab2 deficiency causes a resistance to the BCR/ABL-induced transformation in bone marrow myeloid progenitors and a diminished transformation in lymphoid due to an increased apoptosis (Sattler et al., 2002). Consistently, Gab2 deficiency impairs

PI3K/Akt and Ras/Erk activation induced by BCR/ABL in primary myeloid and lymphoid cells, indicating Gab2 is a critical mediator of these two pathways

(Sattler et al., 2002).

Intensive studies in Shp-2 field focus on modeling the pathogenic effects of Shp-2 mutations and elucidate the detailed mechanisms of how Shp-2 mutations exert its pathogenic effects. Although the effects of Shp-2 mutations on several types of hematopoietic cells have been well documented, how it affects

HSC functions and what are the underlying mechanisms are not known. In the

Chapter II, I examined the effects of Shp-2 D61G mutation on HSCs. I found that germline mutation Ptpn11D61G in mice aberrantly accelerates hematopoietic stem cell (HSC) cycling, increases the stem cell pool, and elevates short-term and long-term repopulating capabilities, leading to the development of MPD. MPD is reproduced in primary and secondary recipient mice transplanted with

Ptpn11D61G/+ whole bone marrow cells or purified Lineage-Sca-1+c-Kit+ cells, but not lineage committed progenitors, indicating the pathogenic effects of

Ptpn11D61G mutation initiate at stem cell level. The deleterious effects of

Ptpn11D61G mutation on HSCs are attributable to enhancing cytokine/growth factor signaling. The aberrant HSC activities caused by Ptpn11D61G mutation are largely corrected by deletion of Gab2, a prominent interacting protein and target of Shp-2 in cell signaling. As a result, MPD phenotypes are markedly ameliorated in Ptpn11D61G/+/Gab2-/- double mutant mice. Our data suggest that oncogenic

Ptpn11 induces MPD by aberrant activation of HSCs. This study also identifies

Gab2 as an important mediator for the pathogenic effects of Ptpn11 mutations.

Another outstanding question is whether Shp-2 mutation is sufficient to lead to leukemias. Previous studies have demonstrated that single Shp-2 GOF mutations can induce cytokine hypersensitivity, JMML-like myeloproliferative

20 disease and Noonan syndrome. However, no animal model of Shp-2 GOF mutations could develop leukemias, a malignant progression of JMML, so whether Shp-2 GOF mutations are sufficient to cause malignant leukemias and cancers were under debate. In Chapter III of this thesis, I present a novel mouse model generated by us with the most common and most potent Shp-2 mutation

(E76K) in JMML. Unlike other mouse models published before, Ptpn11E76K conditional knock-in mice developed T cell acute lymphoid leukemia/lymphoma

(T-ALL), acute myeloid leukemia (AML) and B cell acute lymphoid leukemia (B-

ALL) following chronic MPD. My studies in chapter III provide the first evidence that an activating mutation in Ptpn11 phosphatase plays a causal role in acute leukemias and novel mouse model mimicking the natural course of JMML.

Hematopoietic stem cell development

Hematopoietic stem cells (HSCs) are characterized by their ability to self-renew and differentiate into all the mature blood cells including myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK- cells). HSC can be divided into long-term-HSC (LT-HSC), capable of self-renewal throughout the life of an animal, and short-term-HSC (ST-HSC) that can self-renew and give rise to multiple blood lineages only for a limited interval (typically less than 12 weeks in mouse). HSCs, triggered by extracellular clues at different locations, can constitute the entire hematopoietic system. First, some HSCs stop their self-renew cycle

21 and are committed to common lymphoid progenitors (CLP) and common myeloid progenitors (CMP). CLPs evolve into the lymphoid system, including T cells and B cells.

Transplantation with CLPs produces only lymphoid fates and not any myeloerythroid fate in vivo (Karsunky et al., 2008; Kondo et al., 1997). CMPs develop into the myeloerythroid hierarchy; transplantation of CMPs reconstitute all myeloerythroid but not any lymphoid progeny (except for rare B cells). CMPs produce both granulocyte- macrophage progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP)

(Akashi et al., 2000). GMPs give rise to granulocytes, monocytes, and cells of the macrophage lineage, as well as all types of dendritic cells, while MEPs give only red cells and platelets, and GMP gave rise only to granulocytes, monocytes, and cells of the macrophage lineage, as well as all types of dendritic cells (Akashi et al., 2000; Forsberg et al., 2006; Karsunky et al., 2008; Nakorn et al., 2003; Traver et al., 2000).

Methods to determine stem cell activities

Many methods have been developed to isolate stem cell population. Weisman lab has earlier defined the methods for purification of stem cells by fluorescence- activated cell sorting (FACS) based on the expression of stem cell antigen-1 (Sca-1), c- kit, and lack of expression of lineage markers (Spangrude et al., 1988). More recently, the same lab proposed the SLAM antigens (CD150+ CD244- CD48- cells) as useful markers to further purify HSC and the purified cell population has been demonstrated to have the capacity of reconstituting lethally irradiated animals (Kiel et al., 2005).

Various assays have been developed to analyze stem cell activities both in vitro and in vivo. Colony-forming cell (CFC) assay is a widely used in vitro assay to detect functional potential of HSCs and progenitor cells in a given population. Cells will be grown on semisolid agar- or, more commonly, well-defined methylcellulose-based culture media, cultured for 7 days and observed under microscope. Different cell types form lineage-restricted colonies with distinct appearances: erythroidrestricted burst- forming units-erythroid (BFU-E), which are more immature than the colony-forming unitserythroid (CFU-E); megakaryocyte-restricted CFU-Mk; colony-forming units- granulocytes (CFU-G), colony-forming units-monocytes/macrophages (CFU-M); and colonyforming units-granulocytes/macrophages (CFU-GM). The most immature

(multipotent) CFC measurable contains granulocytes, erythrocytes, macrophages, and often megakaryocytes (CFU-GEMM) (Purton and Scadden, 2007). B and T lymphocyte in vitro CFC potential are more difficult to assess, usually requiring specialized coculture systems, but now there are commercially available methylcellulose-based colony assays to measure pre-B cells.

In vivo, the spleen colony-forming unit (CFU-S) assay has been widely used to assess the short-term repopulating ability of HSCs in a relative short period (about 2 weeks). Once cells are injected into an irradiated recipient, they will home to spleen, proliferate and form a macroscopic colonies containing l05-l07 cells mainly consisting of erythrocytes, granulocytes, or megakaryocytes (Becker et al., 1963; Till and Mc, 1961;

Wu et al., 1967). This assay has been widely used to evaluate not only short-term repopulating activities of HSC but also some precursor cells.

There are various types of long-term repopulating assays, and the most common assay is the competitive repopulation assay (Harrison, 1980). This assay measures the functional potential of the unknown source of HSCs against a set known number of

HSCs (usually whole bone marrow cells from congenic wild-type mice). While providing information about the repopulating capacity of HSC in a given population compared to the competing bone marrow, this study it cannot tell whether the different repopulating capacity is due to difference in the number of HSCs or their quality (progeny produced per HSC).

The hematopoietic stem cell limiting dilution assay is used to measure frequency of HSCs. This assay is essentially a variation of the competitive reconstitution assay. A series of dilutions of “test” cells are transplanted into lethally irradiated recipients together with a set number of competing bone marrow cells. In each cell dose, a contribution of higher than 5% from test cells in the recipient animals is considered as long term engraftment of hematopoietic stem cells and a contribution of lower than 5% from test cells is considered as negative reconstitution. The frequency of negative reconstitution in each dose of competing cells is subject to Poisson statistical analysis

(L-Cal) to calculate the frequency of HSC (Purton and Scadden, 2007). The best source of donor test cells is whole bone marrow cell pupulation. This avoids potential problems associated with discrepancy between HSC phenotype and function. David Scadden‟s

3 6 lab reported that each recipient mouse received cell doses ranging from 8 x10 to 2 x10

5 whole bone marrow cells together with 2 x10 competing congenic bone marrow cells and such a cell population range is sufficient to detect both reductions (Purton et al.,

2006) and increases (Janzen et al., 2006) in HSC numbers in a “test” cell population.

Alternatively, LSK+ cells which are enriched for hematopoietic stem cells are also used as the source of test cells. Using LSK cells may cause low rate of long-term reconstituted mice, so very large numbers of recipient mice are required for accurate

5 results. Ema et al used LSK+ CD34-cells together with 2x10 competitor bone marrow cells; 48 of 135 transplanted mice showed multilineage reconstitution at 12 or 16 weeks posttransplant, respectively (Ema et al., 2006; Ema et al., 2005). However, the limiting dilution assay using LSK+CD34- cells as test cells still have some problems, such as low number and purity of sorted LSK+CD34- cells. Regardless what cell types are tested, reconstitution of test cells in the recipient mice are usually determined at around 16 weeks following the transplantation.

Chapter II: Shp-2 D61G mutation induces

myeloproliferative disease by aberrant activation of

hematopoietic stem cells

Introduction

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disease

(MPD) of young children. It is characterized by cytokine hypersensitivity of myeloid progenitors and is often associated with mutations in the RAS pathway

(Emanuel, 2008; Lauchle et al., 2006). 35% patients with JMML have activating mutations in the tyrosine phosphatase Ptpn11 (Shp-2), a known positive regulator of the Ras pathway; 20% patients have activating mutations in RAS

(N-RAS or K-RAS); 15% patients have mutations inactivating NF1, a GTPase activating protein that negatively regulates RAS pathway (Emanuel, 2008;

Lauchle et al., 2006); 10-15% of patients bear a mutation in E3 ubiquitin ligase c-

CBL (Loh et al., 2009). Ptpn11, RAS, NF1, and c-CBL mutations are usually mutually exclusive in patients, and often play a causal role in the pathogenesis of

JMML. Single disease mutations, such as Ptpn11D61G/Y, K-RasG12D, Nf1 deficiency, and Cbl deficiency are sufficient to induce cytokine hypersensitivity in myeloid progenitors and JMML-like MPD in animal models (Araki et al., 2004;

Braun et al., 2004; Chan et al., 2009; Chan et al., 2004; Chan et al., 2005; Le et al., 2004; Rathinam et al., 2008; Zhang et al., 2001b).

Ptpn11 (Shp-2) is a ubiquitously expressed protein tyrosine phosphatase and is involved in multiple cell signaling processes, such as the RAS-MAP kinase,

JAK/STAT, PI3K/AKT, NF-B, and NFAT pathways (Neel et al., 2003; Tonks,

2006; Xu and Qu, 2008). In spite of Shp-2 activity in protein dephosphorylation,

Shp-2 often positively regulates cell signaling transduction pathway initiated from receptor and cytosolic kinases, and is essential for cellular development process.

Particularly, it is the case for RAS pathway, cytokine (IL-3) signaling pathway and hematopoietic cell development (Qu et al., 2001; Qu et al., 1997; Qu et al., 1998).

The underlying mechanism is being intensively investigated. Several proteins have been identified as Shp-2 interacting partners, some of which are direct substrates of Shp-2 enzymatic activity. The scaffolding proteins Gab1 and Gab2 are prominent targets of Shp-2 (Gu and Neel, 2003; Nishida and Hirano, 2003), and they form stable complexes with Shp-2 to play critical roles in growth factor/cytokine signal transduction, especially in RAS and PI3K/AKT pathways

(Gu and Neel, 2003; Nishida and Hirano, 2003). However, none of the putative substrates identified to date can fully account for the overall positive signaling effects of Shp-2 on the many biological processes.

The most common target of genetic mutations in JMML is Shp-2 (Loh et al., 2004; Tartaglia et al., 2003). These mutations, such as congenital mutation

D61G and somatic mutation E76K, often disrupt the inhibitory intramolecular interactions between the N-terminal SH2 (N-SH2) and catalytic domains, resulting in hyperactivation of Shp-2 (Keilhack et al., 2005; Tartaglia et al., 2003) and enhanced interactions with Shp-2 partners, such as Gab1 and Gab2

(Kontaridis et al., 2006; Yu et al., 2006).. However, the biochemical, molecular and cellular mechanisms by which gain-of-function (GOF) mutations in Ptpn11 induce JMML are poorly defined. Particularly, it is not clear how germline and somatic mutations in Ptpn11 impact hematopoietic stem cells (HSC) function.

In this study, we used the Ptpn11D61G knock-in mouse model (Araki et al.,

2004) to analyze the effects of germline Ptpn11 GOF mutations on HSC functions. This mutation leads to development of MPD, which might result from aberrantly enhanced HSC activity. The MPD could be reproduced after primary and secondary transplantation with Ptpn11D61G/+ whole bone marrow (BM) cells or purified Lineage- Sca-1+c-Kit+ (LSK) cells, but not lineage committed progenitors. The deleterious effects of Ptpn11D61G mutation on HSCs were attributable to enhancing cytokine/growth factor signaling. The phenotypes in

Ptpn11D61G mice were markedly ameliorated in Ptpn11D61G/+/Gab2-/- double mutant mice, suggesting Gab2 is an important mediator for the pathogenic effects of Ptpn11D61G mutation.

Results and Discussion

1) Germline mutation Ptpn11D61G increases HSC and lineage progenitor

populations

Ptpn11D61G/D61G mice die at embryonic day 13.5-15.5 due to heart developmental defects, and Ptpn11D61G/+ mice develop JMML-like MPD characterized by excessive myeloid expansion in the BM and spleen (Araki et al.,

2004). To investigate the mechanism by which GOF mutations in Ptpn11 induce

MPD, we determined effects of Ptpn11 mutation on HSCs. Flow cytometry analysis showed that the frequency (Figure 2-1A, B) and the absolute number

(Table S2-1) of Lineage negative Sca-1 and c-Kit double positive (LSK) cells were doubled in the bone marrow (BM) of Ptpn11D61G/+ mice. The difference in the percentages (Figure 2-1C, D) of LSK cells between mutant and WT mice was even more dramatic in the spleen. Furthermore, we performed multiparameter

FACS analyses to quantify the population of phenotypic long-term HSCs (LT-

HSC), short-term HSCs (ST-HSC), and later stage progenitor populations, including common myeloid progenitors (CMPs), common lymphoid progenitors

(CLPs), granulocyte-macrophage progenitors (GMPs), and megakaryocyte- erythroid progenitors (MEPs) (Fleming et al., 2008; Tothova et al., 2007) (Figure

2-1E, G). The percentage of LT-HSCs, ST-HSCs, GMPs and MEPs were significantly increased in Ptpn11D61G/+ mice (Figure 2- 1F, H, Table S2-1), while the percentage of CLPs was relatively decreased (Figure 2- 1H, Table S2-1). The increase in primitive hematopoietic cell population and later stage myeloid progenitors suggests that Ptpn11D61G/+ mutation promotes expansion of both

HSCs and the myeloid lineage.

Figure 2-1 A-F

Figure 2-1 G & H

Figure 2-1. Aberrant hematopoietic cell development in Ptpn11D61G/+ mice.

(A and B) BM cells freshly harvested from femurs and tibias (both hind limbs) from 5-7 month old Ptpn11D61G/+ and WT littermates (n=10 per group) were assayed by multiparameter flow cytometry analysis to determine frequencies of hematopoietic cell populations of various stages and lineages. The cells were stained with antibodies against lineage markers, c-Kit, Sca-1, CD34, Flk2,

CD16/32, and IL-7R (CD127). The proportion of BM cells corresponding to the

HSC-containing LSK (Lineage-Sca-1+c-Kit+) cell population was quantified. (C and D) Frequencies of LSK cells in spleens of Ptpn11D61G/+ (n=5) and WT (n=4) littermates were determined as above. (E) BM LSK cells were sub-fractioned according to CD34 and Flk2 expression to yield phenotypic assessments of LT-

HSC (LSK, Flk2-CD34-) and ST-HSC (LSK, Flk2-CD34+) fractions. (G) More differentiated BM LK cell (Lineage-c-Kit+Sca-1- ) population was sub-fractioned based on CD16/32 and CD34 expression to identify CMP, GMP, and MEP progenitors (left panel). CLP progenitors were identified by gating L-KlowSlow

(Lineage-c-Kit low Sca-1low) cells followed by assessing CD127 expression (right panel). (F and H) Frequencies of LT-HSC, ST-HSC, CMP, GMP, MEP, and CLP populations in BM cells were quantified (n=10 per group).

2) Ptpn11D61G mutation promotes cell cycling and decreases apoptosis in

HSCs

To determine the mechanism by which Ptpn11D61G/+ mutation causes HSC expansion, we analyzed cell cycle status of LSK cells in Ptpn11D61G/+ mice and in

WT littermates. This analysis revealed an approximately 2-to-3-fold reduction in the percentage of LSK cells at G0 phase and an increase in the percentages of

D61G/+ cells at the G1 and S/G2/M phase in Ptpn11 mice (Figure 2-2A, B), suggesting an enhanced entry from quiescence (G0 phase) into the cell cycle in the mutant mice. Cell survival of HSC was also examined, and apoptotic LSK cells in Ptpn11D61G/+ mice were markedly decreased (Figure 2-2C, D). Thus, the expansion of the stem cell population in mutant mice appears to be attributed to both the enhanced cell proliferation and decreased apoptosis.

3) Ptpn11D61G mutation enhances short-term and long-term repopulating

activities of BM cells

To examine the effects of Ptpn11D61G/+ mutation on HSC functions, both short- term and long-term in vivo repopulating activities of BM hematopoietic cells were determined. To determine short-term repopulating activities, we performed

Spleen colony-forming unit (CFU-S) assays, a well established approach to evaluate short-term repopulating activity of hematopoietic cells (Morrison and

Weissman, 1994). In Ptpn11D61G/+ cell transplanted mice, number of CFU-S was increased by 3-fold in day 12 (Figure 2-3A, B), and the size of CFU-S colonies

Figure 2-2

Figure 2-2. Enhanced cycling and decreased apoptosis in Ptpn11D61G/+ LSK cells.

(A) BM cells freshly harvested from 5-7 month old Ptpn11D61G/+ (n=6) and WT

(n=5) littermates were assayed by multiparameter flow cytometry analysis to determine cell cycle status of HSC-enriched LSK cells as described in Methods.

(B) Percentages of LSK cells in G0, G1, and S/G2/M phases identified based on

Pyronin Y and Hoechst staining profiles were determined. (C and D) BM cells from Ptpn11D61G/+ and WT littermates (n=5 per group) were analyzed by multiparameter flow cytometry analysis to determine apoptosis (Annexin V positive cells) in the LSK population as described in Methods.

35 was also markedly increased. These data clearly indicate that Ptpn11D61G/+ HSCs have an increased short-term repopulating activity.

To assess long-term repopulating activity of Ptpn11D61G/+ mutant BM cells, competitive repopulating assays were then performed. BM cells (test cells) from

Ptpn11D61G/+ or WT littermates (CD45.2+) were mixed with competitor BM cells from congenic BoyJ mice (CD45.1+) at the ratio of 1:1. Mixed cells were transplanted into lethally irradiated BoyJ recipients. After 4, 8, 12, and 16 weeks,

FACS analyses were performed to determine the relative contributions from test cells and competitor cells to the hematopoiesis reconstitution in recipients mice.

WT test cells contribute to about 60% of the reconstitution, which is little bit higher than expected (50%). This might be due to the slight difference in the genetic backgrounds of test and competitor cells (F4 WT and Ptpn11D61G/+ mice from the backcrosses to the C57BL6 background were used in this study since

F5 Ptpn11D61G/+ mice were completely lethal at the embryonic stage) (Araki et al.,

2009). In the recipients of Ptpn11D61G/+ test cells, percentages of test cell-derived nucleated cells (CD45.2+) in the peripheral blood were greatly increased at all time points (Figure 2-3C). Lineage analyses showed that the increased contribution from Ptpn11D61G/+ test cells was primarily attributed to excess expansion of myeloid cells (Mac/Gr-1 double positive cells) (data not shown). 16 weeks post transplantation, recipients were sacrificed, and BM cells (Figure 2-3D) and spleen cells (data not shown) were analyzed, similar increases in the percentages of CD45.2+ cells and enhanced myelopoiesis in the CD45.2+ population (Figure 2-3E) were observed in Ptpn11D61G/+:BoyJ transplants.

Conversely, the contribution of Ptpn11D61G/+ cells to lymphoid lineages was decreased in the BM (Figure 2-3E) and spleen (data not shown), implicating an impact of Ptpn11D61G/+ mutation on lineage determination. To further evaluate effects of Ptpn11D61G/+ mutation on long-term stem cell repopulating activity, secondary transplantation was performed using BM cells harvested from primary recipients. Sixteen weeks after secondary transplantation, Ptpn11D61G/+ recipients manifested a substantial increase in test cell reconstitution in the BM (Figure 2-

3F) and spleen (data not shown). Increased test cell-derived myelopoiesis was also observed in the BM (Figure 2-3G) and spleen (data not shown). These data clearly indicate that Ptpn11D61G/+ mutation confers a significant competitive advantage and a greater long-term repopulating ability to BM stem cells.

To further examine the effects of D61G mutation on LSK cells population, we analyzed the percentage of LSK cells (CD45.2+) derived from test cells in both primary and secondary transplantation. As shown in Figure 2-3H, the percentage of Ptpn11D61G/+ cell-derived LSK cells (CD45.2+) in the BM and spleen (Figure 2- 3I) of primary recipients was significantly higher than that of the

LSK cells derived from WT test cells. Similar observations were made during secondary transplantation (Figure 2-3J, K). These data suggest the increased repopulation activity and the enhanced myelopoiesis of Ptpn11D61G/+ mutant cells occur largely at the HSC level.

Figure 2-3 A-E

Figure 2-3 F-K

Figure 2-3. Greater short-term and long-term repopulating activities of

Ptpn11D61G/+ BM cells.

(A and B) BM cells (1x105) freshly harvested from 3 month old Ptpn11D61G/+ and

WT littermates were injected into sub-lethally irradiated C57BL/6 mice (n=9 per group). Twelve days following the transplantation, colonies on the spleen (CFU-S) were counted. (C) BM cells (test cells) (1x106) harvested from WT or

Ptpn11D61G/+ littermates (CD45.2+) were transplanted with the same number of

BoyJ (CD45.1+) BM cells (competitor cells) into lethally irradiated BoyJ (CD45.1+) mice (n=5 per group). Test cell reconstitution (CD45.2+) was determined at 4, 8,

12, and 16 weeks by FACS analyses of peripheral blood cells of the recipient mice. (D) Sixteen weeks following transplantation, percentages of test cell- derived CD45.2+ cells in the whole BM population were determined (n=5 per group). (E) Contributions of test cells (CD45.2+) to each lineage (Mac-1, Gr-1,

B220, Ter119, and CD4 positive cells) in the BM were also quantified (n=5 per group). (F) BM cells (2x106) harvested from the primary recipients were transplanted into lethally irradiated BoyJ (CD45.1+) mice. Sixteen weeks after the transplantation, BM cells of the secondary recipients were analyzed for the contribution of test cells in the BM (n=5 per group). (G) Reconstitution from test cells in each lineage of the BM was determined as described above (n=5 per group). (H and I) BM cells harvested from WT or Ptpn11D61G/+ littermates were transplanted with the same number of BoyJ BM cells into lethally irradiated BoyJ mice (n=5 per group) as above. Sixteen weeks following the transplantation, recipient mice were sacrificed and frequencies of LSK cells renewed from test

40 cells (CD45.2+) in the BM (H) and spleen (I) were quantified by multiparameter

FACS analyses as described in Figure 2-1. BM cells (2x106) harvested from primary recipient mice were transplanted into lethally irradiated BoyJ mice.

Sixteen weeks following transplantation, frequencies of LSK cells renewed from test cells (CD45.2+) were quantified in the BM (J) and spleen (K) of the secondary recipient mice.

4) MPD phenotypes are reproduced in primary and secondary recipient

mice transplanted with Ptpn11D61G cells

To determine whether the pathogenic effects of Ptpn11 GOF mutations are transplantable, we transplanted Ptpn11D61G/+ or WT BM cells (CD45.2+) into

BoyJ recipients (CD45.1+) that were lethally irradiated. By 16 weeks after transplantation, nearly 100% peripheral blood cells from recipient mice are

CD45.2+, indicating that the entire hematopoietic system in recipient mice was reconstituted by donor cells. Recipient mice receiving Ptpn11D61G/+ cells developed MPD features, including increased white blood cell (WBC) counts which were mainly due to excess expansion of neutrophils (Figure 2-4A, B, Table

S2-2), enlarged spleens (Figure 2-4C), and elevated percentages of Mac-1/Gr-1 double positive myeloid cells in the BM (Figure 2-4D) and spleen (Figure 2- 4E).

BM cells harvested from primary recipients were further used for secondary transplantation and similar results were obtained (Figure S2-1A-D).

We then monitor the MPD development in recipient mice transplanted with

BM cells mixed from Ptpn11D61G/+ mice (or Wt mice) and BoyJ mice at 1:1 ratio.

MPD phenotypes were reproduced in recipient mice transplanted with

Ptpn11D61G/+ cells (Figure 2-4F-H). These recipient mice displayed elevated WBC

(mainly neutrophils) counts (Figure 2-4F, G, Table S2-3) and splenomegaly

(Figure 2-4H). Further, BM cells harvested from primary recipients were transplanted into secondary recipient mice, and secondary recipient mice of

Ptpn11D61G/+:BoyJ cells also developed MPD (Figure S2-1E, F). Collectively, these data demonstrate the deleterious effects of Ptpn11D61G/+ mutation is

42 transplantable and cell autonomous. Furthermore, the long-term transplantability evidenced by both primary and secondary recipients suggest that the pathogenic effects of Ptpn11D61G/+ mutation initiate at stem cell level.

To further evaluate the pathogenic capabilities of Ptpn11D61G/+ mutant

HSCs, we performed limiting dilution using BM cells from Ptpn11D61G/+ which was diluted by BM cells from BoyJ mice (Table 2-1). 16-to-24 weeks after transplantation, Ptpn11D61G/+ mutant BM cells mixed with BoyJ cells at 1:5 and

1:25 ratio could still result in MPD in 2/5 and 1/5 recipient mice respectively

(Table 2-1). A lower ratio between Ptpn11D61G cells and BoyJ cells is insufficient to cause MPD phenotypes in recipient mice, which is likely because too few mutant long-term repopulating stem cells were actually transplanted into the recipients. We next determined whether the MPD phenotypes were transplanted via HSCs or lineage progenitors. Purified LSK cells, CMPs, and GMPs were transplanted into recipient mice with or without competitor cells of the same cell types. The results clearly showed that Ptpn11D61G/+ mutant LSK cells outcompeted normal LSK cells even at the ratio of 1:2.5 (Table 2-1). Only LSK cells, not CMP or GMP progenitors, could confer the disease phenotypes in recipient mice. These data together indicate that the pathogenic effects of

Ptpn11D61G/+ mutation start at stem cells.

Figure 2-4 A-E

Figure 2-4 F, G & H

Figure 2-4. MPD is reproduced in Ptpn11D61G/+ BM cell-reconstituted primary and secondary recipients.

BM cells (1x106) freshly harvested from 3 month old Ptpn11D61G/+ and WT littermates (CD45.2+) were directly transplanted into lethally irradiated BoyJ

(CD45.1+) mice (non-competitive repopulation assay) (n=3 and 7 for WT and

Ptpn11D61G/+ groups, respectively). White blood cell (WBC) counts in peripheral blood (A), percentages of neutrophils and lymphocytes (B), spleen weights (C), and percentages of donor cell-derived myeloid (Mac-1+/Gr-1+) cells in BM (D) and spleen (E) of the recipients were determined at 16 weeks or the indicated time points following the transplantation. BM cells (1x106) harvested from 3 month old WT or Ptpn11D61G/+ littermates were transplanted with the same number of BoyJ BM cells into lethally irradiated BoyJ mice (n=5 per group).

Sixteen weeks following transplantation, WBC counts (F), percentages of neutrophils and lymphocytes (G), and spleen weights (H) of the recipient mice were determined.

5) Increased response of Ptpn11D61G stem cells/multipotent progenitors to

IL-3

Cytokine hypersensitivity of myeloid progenitors is a hallmark of JMML

(Emanuel, 2008; Lauchle et al., 2006). To determine whether Ptpn11D61G/+ LSK cells have increased cytokine sensitivity, we cultured LSK cells from WT and

Ptpn11D61G mice (5x103/well) in the presence of IL-3. At day 7, number of

Ptpn11D61G/+ mutant cells increases by 300-to-400-fold while Wt cell number only increases by 40-fold (Figure S2-2A). Accompanying this increased cell number of

Ptpn11D61G/+ cells is a higher percentage of Mac-1 single positive and Mac-1/Gr-1 double positive myeloid cells in culture, indicating an enhanced myeloid differentiation induced by IL-3 (Figure S2-2B, C). Thus, Ptpn11D61G/ mutation results in IL-3 hypersensitivity in HSCs in a cell autonomous manner.

Ptpn11-associated MPD appears to be attributable to aberrantly enhanced sensitivity of cell proliferation and myeloid differentiation to cytokine/growth factor, particularly IL-3, but the underlying molecular mechanisms are poorly understood.

To perform large scale biochemical analysis, we generated BM-derived mast cells from 12 week old WT and Ptpn11D61G/+ mice. Upon IL-3 treatment, IL-3- induced tyrosine phosphorylation of cellular proteins was increased in

Ptpn11D61G/+ cells (Published in Xu et al., 2010); Activation of Erk and Akt kinases was substantially elevated in Ptpn11D61G/+ cells (Published in Xu et al.,

2010). Moreover, Ptpn11D61G mutation increased the Erk activity upon GM-CSF stimulation in macrophages (Figure S2-3).

Table 2-1. Transplantation of Ptpn11D61G/+ whole BM cells or LSK cells.

6) Important role for Gab2 in mediating the pathogenic effects of

Ptpn11D61G mutation

We found that Ptpn11D61G/+ mutation dramatically enhances the interaction between Shp-2 and Gab2, a prominent interacting protein of Shp-2 and an important scaffolding protein for cytokine/growth factor signaling(Gu et al., 2003;

Nishida and Hirano, 2003) (Published in Xu et al., 2010). These observations raise a possibility that Gab2 may mediate the pathogenic effects of Ptpn11D61G/+ mutation. To test this possibility, we next generated Ptpn11D61G/+/Gab2-/- double mutant mice from Ptpn11D61G/+ (Araki et al., 2004) and Gab2+/- (Nishida et al.,

2002) mice. 5-7 months after birth, MPD was fully developed in Ptpn11D61G/+ mice

(Araki et al., 2004). Although Gab2 deficiency did not rescue the embryonic lethality induced by homozygous Ptpn11D61G/D61G mutation (Araki et al., 2004) or the developmental defects (Noonan syndrome) induced by Ptpn11D61G/+ mutation

(Araki et al., 2004) (data not shown), it significantly alleviated the MPD phenotypes of Ptpn11D61G/+/Gab2-/- double mutants. Compared to

Ptpn11D61G/+mice, double mutants have a lower WBC counts (Figure 2-5A, Table

S2-4) and lower percentage of myeloid cells in the BM (Published in Xu et al.,

2010), and a diminished splenomegaly (Figure 2-5B). Furthermore, myeloid progenitors from Ptpn11D61G/+/Gab2-/- mice, compared to that of Ptpn11D61G/+ cells, showed a significantly attenuated basal and stimulated colony formation by

IL-3 or GM-CSF (Published in Xu et al., 2010, Figure 2-5C).

Figure 2-5

Figure 2-5. MPD phenotypes induced by Ptpn11D61G/+ mutation are substantially attenuated by deletion of Gab2.

Ptpn11D61G/+ mice were used to cross Gab2+/- mice to generate

Ptpn11D61G/+/Gab2+/- mice. The double heterozygous mice were then intercrossed to produce WT, Gab2-/-, Ptpn11D61G/+, and Ptpn11D61G/+/Gab2-/- mice. These mice were analyzed at 5-7 months after birth. WBC counts (A, n=10-13), spleen weights (B, n=5-8), (C) BM cells (2×104 cells/ml) freshly harvested from 4 genotypes of mice were assayed for colony forming units (CFUs) in 0.9% methylcellulose IMDM medium containing 30% FBS, glutamine (10-4 M), β- mercaptoethanol (3.3×10-5 M), GM-CSF at the indicated concentrations. After 7 days of culture at 37 °C in a humidified 5% CO2 incubator, hematopoietic cell colonies (primarily CFU-GM) were counted under an inverted microscope.

Experiments described in C were performed three times and similar results were obtained in each. Results shown are the mean ± SEM of triplicates from one experiment. P values for comparisons between Ptpn11D61G/+/Gab2-/- and

Ptpn11D61G/+ mice were determined by Student‟s t tests. Statistical significance among four groups in all panels was verified by two-way ANOVA followed by

Bonferroni or one-way ANOVA followed by Tukey‟s posttest.

To determine effects of Gab2 deletion at the stem cell level, we analyzed

HSC homeostasis in the double mutants and in Ptpn11D61G/+ single mutants. In the double mutants, the frequency (Figure 2-6A) and absolute number (Figure

S2-4) of LSK cells were significantly decreased in the BM and spleen (data not shown). Cell cycle analyses showed that Gab2 deletion can largely rescue the aberrant LSK cell cycle distribution, especially cell quiescence (Figure 2-6B) and programmed cell death in LSK cells (Figure 2-6C). To assess the effect of Gab2 deletion on cytokine response of LSK cells, LSK cells were sorted and cultured in

IL-3.Compared to those of Ptpn11D61G/+ cells, cell expansion (Figure 2-6D) and myeloid differentiation (Figure 2-6E) of Ptpn11D61G/+/Gab2-/- cells in response to

IL-3 were markedly attenuated. These data, together with the ameliorated phenotypes in Ptpn11D61G/+/Gab2-/- mice (Figure 2-6A-C) support the notion that

Gab2 is an important mediator for the pathogenic effects of Ptpn11D61G mutation.

To further verify the role of Gab2, we knocked down Gab2 from

Ptpn11D61G/+ BM cells and evaluated the effect of acute knockdown of Gab2 on attenuating myeloid over-production. As seen in Figure S2- 6B, both cytokine- independent and cytokine (IL-3 and GM-CSF)-stimulated colony formations were significantly decreased in Gab2 knockdown cells. In addition, the size of the colonies derived from Gab2 knockdown cells was much smaller (Figure S2-6C).

These results reaffirm the important role of Gab2 in mediating Ptpn11D61G mutation-induced pathogenesis, implying a potential therapeutic benefit of targeting Gab2 or Gab2-dependent pathways.

Figure 2-6

Figure 2-6. An important role of Gab2 in mediating the pathogenic effects of

Ptpn11D61G/+ mutation in stem cells.

WT, Gab2-/-, Ptpn11D61G/+, and Ptpn11D61G/+/Gab2-/- mice were generated as described in Figure 2-6. The mice were analyzed for the percentage of LSK cells in the BM (A, n=6 per group), cell cycle status (B, n=5-8), and apoptosis (C, n=5 per group) as described in Figure 2-1. (D) LSK cells were sorted from these mice and cultured in IL-3 (0 or 2 ng/ml) and 10% FBS-containing IMDM media (5x103 cells/well). After 7 days of in vitro culture, total cell numbers were determined

(n=3 per group). (E) Percentages of myeloid (Mac-1+/Gr-1+) cells differentiated from the sorted LSK cells in the presence of IL-3 (2 ng/ml) were assessed by

FACS analysis (n=6 per group). P values for comparisons between

Ptpn11D61G/+/Gab2-/- and Ptpn11D61G/+ mice were determined by Student‟s t tests.

Statistical significance among four groups in all panels was verified by two-way

ANOVA followed by Bonferroni or one-way ANOVA followed by Tukey‟s post-test.

My studies in this chapter demonstrated that Shp-2 D61G mutation resulted in increased HSC population and enhanced HSC repopulating ability, which leads to the development of MPD in animal model. Through both primary and secondary transplantation, the pathogenic effects of D61G are transplantable by whole BM cells, LSK cells, but not myeloid progenitors, establishing Ptpn11D61G-induced MDP as a stem cell disease. For therapeutic purpose, I examined the effects of deleting Gab2 in vitro and in vivo, which is an immediate downstream scaffolding protein for Shp-2. Knockdown of Gab2 reduced the colony formation ability of Ptpn11D61G/+ BM cells. Consistently,

Ptpn11D61G/+/Gab2-/- double mutant mice displayed remarkable ameliorated MPD phenotypes. These findings indicate that Gab2 is essential in the pathogenesis of

Ptpn11 GOF mutation-induced MPD, suggesting that targeting Gab2 could be a potential therapeutic strategy for the JMML caused by GOF mutations in Shp-2.

Supplementary Information

Figure S2-1

Figure S2-1. MPD phenotypes are reproduced in secondary recipients.

BM cells harvested from Ptpn11D61G/+ and WT littermates were directly transplanted into lethally irradiated BoyJ mice as described in Figure 4. Sixteen weeks following the transplantation, BM cells were harvested and pooled from the primary recipients and transplanted into lethally irradiated BoyJ mice.

Peripheral WBC (A and B), spleen weight (C), and percentages of donor-derived myeloid (Mac-1+Gr-1+) cells (D) in the BM of the secondary recipient mice were analyzed 18 weeks post secondary transplantation (n=5). (E) BM cells harvested from WT or Ptpn11D61G/+ littermates were transplanted with the same number of

BoyJ BM cells into lethally irradiated BoyJ mice. Sixteen weeks following the transplantation, BM cells were harvested and pooled from the primary recipients and transplanted into lethally irradiated BoyJ mice. WBC (E) and spleen (F) of the secondary recipient mice were examined 16 weeks post the secondary transplantation (n=5 per group).

Figure S2-2

Figure S2-2. Cytokine hypersensitivity of Ptpn11D61G/+ mutant LSK cells.

(A) LSK cells (5x103) sorted from 4-6 month old WT and Ptpn11D61G/+ mice were cultured in IL-3 (0 or 2 ng/ml) and 10% FBS-containing IMDM media for 7 days.

Total cell numbers were determined (n=3 per group). (B and C) Percentages of myeloid (Mac-1+/Gr-1+) cells differentiated from the sorted LSK cells in the presence of IL-3 (2 ng/ml) were assessed by flow cytometry (n=8 per group). P values for comparisons between Ptpn11D61G/+ and control cells were determined by Student‟s t tests.

Figure S2-3

Figure S2-3. Enhanced GM-CSF-induced Erk activation in Ptpn11D61G/+

BM-derived macrophages were generated as described in Methods. The macrophages were starved in serum and cytokine-free medium for 5 hours and then stimulated with GM-CSF (10 ng/ml) for the indicated periods of time. Whole cell lysates were prepared and examined for Erk activation by immunoblotting with anti-phospho-Erk Ab. Blots were stripped and reprobed with anti-Erk to check protein loading.

Figure S2-4

Figure S2-4. Absolute numbers of LSK cells are largely restored in the BM of

Ptpn11D61G/+/Gab2-/- mice.

WT, Gab2-/-, Ptpn11D61G/+, and Ptpn11D61G/+/Gab2-/- mice were generated as described in Figure2- 6. The mice (n=6 per group) were analyzed for absolute numbers of LSK cells in BM as described in Figure2- 1. P values for comparisons between Ptpn11D61G/+/Gab2-/- and Ptpn11D61G/+ mice were determined by

Student‟s t tests.

Figure S2-5

Figure S2-5. Gab1 and Gab3 levels are not significantly altered in Gab2-/- or

Ptpn11D61G/+/Gab2-/- LSK cells.

LSK cells were sorted from WT, Gab2-/-, Ptpn11D61G/+, and Ptpn11D61G/+/Gab2-/- mice. RNA was extracted and real-time PCR was performed to determine mRNA levels of Gab1, Gab2, and Gab3.

Figure S2-6

Figure S2-6. An important role of Gab2 in mediating the excessive myeloid

D61G expansion induced by Ptpn11 mutation.

(A) Mouse Gab2 shRNA lentiviral plasmids were purchased from Open

Biosystems (Huntsville, AL). Lentiviral vectors expressing Gab2 shRNAs were generated and used to infect mouse embryonic fibroblasts. Forty-eight hours after infection, RNA was extracted and real-time PCR was performed to determine Gab2 mRNA levels in the infected cells. (B) BM cells harvested from

4-6 months old Ptpn11D61G/+ mice were transduced with lentivirus expressing

Gab2 shRNA-1 or control lentivirus. Transduced cells (GFP positive) were sorted by flow cytometry and assayed for CFU-GM (2x104/ml) in the absence or presence of GM-CSF (0.1 ng/ml) or IL-3 (1 ng/ml) as described in Figure 2-6D, E.

Table S2-1. Absolute numbers of hematopoietic cells in WT and Ptpn11D61G/+ mice.

Table S2-2. Peripheral blood parameters in WT and Ptpn11D61G/+ transplants

Table S2-3. Peripheral blood parameters in the competitive repopulation assay recipients

Table S2-4. Peripheral blood parameters in Ptpn11D61G/+/Gab2-/- mice

Chapter III: Non-Lineage Restricted Effects of Ptpn11

E76K mutation on Malignant Transformation of JMML

Introduction:

Juvenile myelomonocytic leukemia (JMML) is a rare, lethal myeloproliferative disorder (MPD) of early childhood which often transforms to frank leukemia. Recent studies indicate that approximately 75-85% of JMML cases result from gain-of-function mutations in Ras pathway such as NRAS,

KRAS, or PTPN11 or homozygous loss-of-function mutations in NF1 (Mohi and

Neel, 2007; Schubbert et al., 2007). Somatic mutations in Ptpn11 are the most frequent (~35%) cause of JMML. Ptpn11 mutations are also found in 10% of myeloid dysplastic syndrome, 6% of B-ALL, 2% of AML (Loh et al., 2004;

Tartaglia et al., 2004), and solid tumors (Keilhack et al., 2005). Although previous studies have demonstrated that Ptpn11D61G/Y mutations induce MPD in mice

(Araki et al., 2004; Chan et al., 2009), whether Ptpn11 GOF mutations play a causal role in leukemias and cancers, and the molecular and cellular mechanisms by which Ptpn11 gain-of-function (GOF) mutations induce JMML are still unknown.

To address these questions, we generated a conditional knock-in mice with the most common and most potent Ptpn11 activating mutation (E76K) found in JMML. In this chapter, my results demonstrate that the Ptpn11E76K mutation evokes T cell acute lymphoid leukemia/lymphoma (T-ALL), acute myeloid leukemia (AML) and B cell acute lymphoid leukemia (B-ALL) following chronic

MPD. Results in this chapter also demonstrate that Ptpn11E76K mutation induces leukemic stem cell development not only in HSCs but also in lineage committed progenitors.

Results and Discussion

1) Generation of inducible knock-in mice expressing the Ptpn11 E76K mutation.

E76K mutation is the most common and potent Ptpn11 activating mutation in human leukemia. To better model human disorders caused by this mutation, we introduced the E76K mutation and a selective marker “neo cassette” into

Exon 3 and intron 2, respectively. The chimeric mice generated from heterozygous knock-in (Ptpn11E76K neo/+) ES cells were first crossed with HPRT-

Cre transgenic mice to remove the neo cassette to produce Ptpn11E76K/+ mice.

Genotyping results of early stage embryos indicated that Ptpn11E76K/+ mice died at around embryonic day 11.5 (E.11.5) (Figure S3-2A, B), which may explain why no germline Ptpn11E76K mutation has been identified in the patients.

Development of Ptpn11E76K/+ embryos was retarded (Figure S3-2B); E11.5

73 embryos showed enlarged heart tubes and craniofacial structures. These features were consistent with the phenotypes in Noonan syndrome cause by homozygous Ptpn11D61G mutation (Figure S3-2B).

Surprisingly, when we crossed the chimeric mice with regular C57BL6 mice to produce Ptpn11E76K heterozygous mice with the neo cassette

(Ptpn11E76K neo/+), we found alive and healthy Ptpn11E76K neo/+ pups (Figure S3-

2C), which raised a question whether inserted neo cassette with a stop codon prevents expression of Ptpn11 E76K allele. To test this possibility, we generated wild-type (WT) and Ptpn11E76K neo/+ mouse embryonic fibroblasts (MEF) from

E.14.5-E.16.5 embryos and infected the cells with recombinant adenovirus expressing Cre recombinase. Before adeno-cre infection, expression of Shp-2 in

Ptpn11E76K neo/+ MEFs was approximately 50% of that in WT cells; 48 hrs after infection, the expression of Shp-2 is similar in Ptpn11E76K neo/+ and WT MEFs

(Figure S3-2D). These results indicated that the targeted allele did not express

Shp-2 E76K due to the insertion with neo-STOP cassette, and removal of the neo cassette by Adeno-Cre restored Shp-2 expression in the targeted allele.

Although the mechanism for the inactivation of the targeted allele by neo-STOP cassette is still unclear, it provides us an opportunity to generate an inducible mouse model and study the pathogenesis of Ptpn11E76K induced diseases in adult animals.

Figure 3-1 A-D

Figure 3-1 E & F

For panel E, histology slides were prepared by Dan Xu and pictures were taken by Howard J. Meyerson

Figure 3-1 G&H

Figure3-1. Ptpn11E76K/+ mutation initially induces MPD in mice with full penetrance.

(A) Schematic diagram of the Ptpn11E76K neo conditional allele created. The neo cassette with a stop codon in intron 2 disrupts the targeted allele. This allele is reactivated and expresses Shp2 E76K upon deletion of the neo cassette by Cre

DNA recombinase. (B) The neo cassette was nearly completely deleted in the

BM cells (upper panel) and splenocytes (lower panel) in Ptpn11E76K conditional knock-in mice. Ptpn11E76K neo/+/Mx1-Cre+ and Ptpn11E76K neo/+/Mx1-Cre- mice were generated. Four-week-old mice were treated by intraperitoneal injection of a total of 5 doses of polyinosine-polycyticyclic acid (pI-pC) (350 ug/mouse) administered every other day over 10 days. Four weeks after pI-pC treatment, genomic DNA was extracted from the BM and spleen. PCR detection of the neo segment was performed using the primers shown in the diagram. (C) Shp-2 catalytic activity was substantially increased in Ptpn11E76K knock-in mice. Spleen lysates prepared from Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, and

Ptpn11E76K neo/+/Mx1-Cre- mice 6 weeks post pI-pC treatment were assayed for

Shp-2 catalytic activity by immunocomplex phosphatase assays as described in

Methods. Shp-2 levels in the tissue lysates were examined by anti-Shp2 immunoblotting. Blots were stripped and re-probed with β-actin antibody to check protein loading. (D) Ptpn11E76K knock-in mice showed splenomegaly. Spleen weights of Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, and Ptpn11E76K neo/+/Mx1-

Cre- mice were quantified 6-8 weeks after pI-pC treatment (n=8 per group). (E)

Myeloid hyper-proliferation in the spleen and BM of Ptpn11E76K knock-in mice.

Spleens and femurs isolated from Ptpn11E76K/+/Mx1-Cre+ and Ptpn11+/+/Mx1-

Cre+ mice 8 weeks post pI-pC injection were processed for histopathological examination (Hematoxylin and eosin staining). (F) Percentage of Mac-1+/Gr-1+ myeloid cells was significantly increased in Ptpn11E76K knock-in mice. BM cells and splenocytes isolated from Ptpn11E76K/+/Mx1-Cre+ and Ptpn11+/+/Mx1-Cre+ littermates (n=5 per group) were assayed for Mac-1+/Gr-1+ cells by FACS analyses 8 weeks after pI-pC injection. (G, H) Cytokine-independent myeloid colony formation and greatly increased responses of myeloid and lymphoid progenitors to cytokines in Ptpn11E76K knock-in mice. BM cells (2 x104/ml) harvested from Ptpn11E76K/+/Mx1-Cre+ and Ptpn11+/+/Mx1-Cre+ littermates (n=3 per group) were assessed by myeloid (G) and Pre-B (H) colony assays 6 weeks after pI-pC injection as described in Methods. Representative CFU-Pre-B and

CFU-GM colonies growing in IL-7-containing medium are shown (H).

To determine the pathogenic effects of somatic Ptpn11E76K mutation on hematopoietic cell development, we generated inducible knock-in (Ptpn11E76K neo/+/Mx1-Cre+) mice by crossing Ptpn11E76K neo/+ mice with Mx1-Cre transgenic mice (Kuhn et al., 1995). Four-weeks-old Ptpn11E76K neo/+/Mx1-Cre+ mice were treated with polyinosinic-polycytidylic acid (pI-pC) to induce Cre expression to delete the neo cassette and induce the expression of Shp-2 E76K in hematopoietic cells (Figure 3-1A). Neo deletion efficiency, assessed by PCR, was nearly 100% in bone marrow (BM) cells and splenocytes (Figure 3-1B); the expression of Shp-2, assessed by Western Blotting, was almost restored in spleen lysates (Figure 3-1C); Shp-2 phosphatase activity, assessed by

Immunocomplex phosphatase assays, was substantially increased in spleen lysates of pl-pC-treated Ptpn11E76K/+/Mx1-Cre+ mice compared to that of control mice (Figure 3-1C), which was consistent with previous observations that E76K mutation hyper-activates Shp-2 (Bentires-Alj et al., 2004; Tartaglia et al., 2003).

2) Ptpn11E76K mutation causes JMML-like MPD in mice

Ptpn11E76K/+/Mx1-Cre+ mice quickly developed MPD within 4 weeks post pI-pC injection. The mice showed high white blood cell (both neutrophils and lymphocytes) counts (Table S3-1). Splenomegaly (Figure 3-1D), hepatomegaly and enlarged thyme. Histopathological examination revealed hyper proliferation of myeloid cells in both spleen and BM (Figure 3-1E). Mac-1+/Gr-1+ double positive myeloid cells were markedly increased in Ptpn11E76K/+/Mx1-Cre+ mice in

BM and spleen (Figure 3-1F). Colony assays indicated that mutant myeloid

(Figure 3-1G) and lymphoid progenitors (Figure 3-1H) were greatly increased in responses to GM-CSF/IL-3 and IL-7, respectively. Furthermore, myeloid progenitors from Ptpn11E76K knock-in mice yielded growth factor-independent colony formation (Figure 3-1G, H).

To further understand the mechanism by which Ptpn11E76K mutation induces MPD, we assessed the pathological effects of Ptpn11E76K mutation on

HSC function. We found that Lineage-Scal-1+c-Kit+ (LSK) cells that are enriched for HSCs were decreased by 3-fold in the BM of Ptpn11E76K/+/Mx1-Cre+ mice

(Figure 3-2A and Table S3-2). LSK cells in the spleen, however, were greatly increased (Figure 3-2B and Table S3-2). Additional multi-parameter FACS analyses (Fleming et al., 2004; Tothova et al., 2007; Xu et al.) showed that highly pure HSCs (Lin-Scal-1+c-Kit+CD150+CD41-CD48-) (Figure 3-2C and Table S3- 2), phenotypic long-term HSCs (LT-HSCs), and short-term HSCs (ST-HSCs) (Figure

3-2D and Table S3-2) were all significantly decreased in the BM in Ptpn11E76K knock-in mice. Later stage progenitor populations, such as common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte- macrophage progenitors (GMPs), and megakaryocyte erythroid progenitors

(MEPs), were also markedly decreased in mutant mice (Figure 3-2E and Table

S3-2). Cell cycle analyses demonstrated a 2-fold decrease in the percentage of quiescent HSCs at the G0 phase, and a 2-fold increase in cells at the S and G2/M phases (Figure 3-2F ), indicating an excess activation of HSC, which may exhaust the population of HSC.

Figure 3-2 A-E

Figure 3-2 F&G

Figure 3-2 H&I

Figure 3-2. Ptpn11E76K/+ mutation activates HSCs by enhancing growth factor signaling.

BM cells freshly harvested from Ptpn11E76K/+/Mx1-Cre+ and Ptpn11+/+/Mx1-Cre+ littermates 8 weeks following pI-pC treatment were assayed by multiparameter

FACS analyses to determine frequencies of hematopoietic cell populations of various stages and lineages as described in Methods. (A, B) Frequency of HSC- enriched LSK cells in the BM was decreased in Ptpn11E76K/+/Mx1-Cre+ mice compared to that in Ptpn11+/+/Mx1-Cre+ mice (n=8 per group) whereas frequency of LSK cells in the spleen was greatly increased in Ptpn11E76K/+/Mx1-Cre+ mice

(n=6 per group). (C, D) Frequencies of pure HSCs, LT-HSCs, and ST-HSCs (n=4 per group) in the BM of Ptpn11E76K/+/Mx1-Cre+ mice were decreased compared to those in Ptpn11+/+/Mx1-Cre+ mice. (E) Frequencies of CMP, GMP, and MEP populations in the BM were decreased in Ptpn11E76K/+/Mx1-Cre+ mice compared to those in Ptpn11+/+/Mx1-Cre+ mice (n=7 per group). (F) Decreased HSC quiescence and increased HSC cycling in Ptpn11E76K knock-in mice. BM cells were assayed by multiparameter FACS analyses to determine cell cycle status of

HSC-enriched LSK cells as described in Methods. Percentages of LSK cells at

G0, G1, and S/G2/M phases identified based on Pyronin Y and Hoechst staining profiles were determined (n=5 per group). (G) Apoptosis in LSK cells was decreased in Ptpn11E76K knock-in mice. BM cells from Ptpn11E76K/+/Mx1-Cre+ and Ptpn11+/+/Mx1-Cre+ mice (n=4 per group) were analyzed by multiparameter

FACS analyses to determine apoptotic cells (Annexin V positive cells) in the LSK population as described in Methods. (H, I) Growth factor signaling in HSC-

85 enriched LSK cells was enhanced by Ptpn11E76K/+ mutation. Lineage- BM cells from Ptpn11E76K/+/Mx1-Cre+ and Ptpn11+/+/Mx1-Cre+ mice (n=3 per group) were purified and starved for 1 hour in IMDM medium. Cells were then stimulated with

SCF (50 ng/ml) for 5 min, fixed, permeabilized, and stained with antibodies against Sca-1, c-Kit, and phospho-Erk or phospho-Akt. Percentages of the cells stained positive for phospho-Erk or phospho-Akt in the gated LSK population were determined by multiparameter FACS analyses as described in Methods.

Analyses of cell survival showed that the percentage of apoptotic cells in

Ptpn11E76K/+ mutant LSK cells was greatly decreased (Figure 3-2G), which is consistent with elevated activities of Erk (Figure 3-2H) and Akt (Figure 3-2I) kinases in the mutant LSK cells in response to stem cell factor (SCF) stimulatioin.

3) Ptpn11E76K knock-in mice developed various types of leukemias after

MPD stage.

12-50 weeks after pI-pC treatment, most Ptpn11E76K knock-in mice rapidly became moribund and progressed to frank leukemia including T-ALL, B-ALL and

AML after a similar chronic MPD stage (Figure 3-3A, B). About 33% mutant mice developed T-ALL including thymic lymphoma which are characterize by 50%~70%

CD4+/CD8+ and CD44+ cells in BM and extremely high CD4+/CD8+ and c-

Kit+/CD44+ cell populations in the Lymphoid blasts (CD45high/SSClow), indicating T cell differentiation is blocked at around the Pro-T stage (Figure 3-3C). Terminal deoxynucleotidyl transferase (TdT) staining showed high positive lymphoid blasts throughout the BM and other organs, confirming the T-ALL found in Ptpn11E76K knock-in mice (Figure 3-3G). 22% of mice developed AML, which was characterized by over 15/20% of myeloid blasts and greater than 15 /20% of

Mac-1+/c-Kit+ cells in peripheral blood/BM (Figure 3-3D). Mac-1+/Gr-1low or Mac-

1+/Gr-1- poorly differentiated cells were also substantially increased (Figure 3-3D).

Low frequency of B-ALL was found in Ptpn11E76K knock-in mice. FACS analysis revealed that a large B lymphoid blasts (CD45lowSSClow) in PB, BM and spleen

87 and 60~70% B220+ in BM containing 70-80% Scal-1+ cells but negative for the mature B lineage marker IgM (Figure 3-3E). Pathological examination confirmed leukemic cell invasion into hematopoietic and non-hematopoietic organs (Figure

3-3F).

I performed transplantation experiments to test whether leukemia found in

Ptpn11E76K knock-in mice is transplantable, a key feature of leukemias. Different numbers of BM cells from T-ALL mutant mice were transplanted into sub-lethally- irradiated NOD-SCID mice. Recipients rapidly reproduced T-ALL within 3-4 weeks post transplantation. 200 donor BM cells still can reproduce, T-ALL suggesting a very high frequency of leukemic stem cells in T-ALL caused by

E76K mutation (Figure 3-3H).

4) Leukemias induced by Ptpn11E76K mutation not only derived from

HSCs but also from lineage committed progenitors.

To determine at what stage of hematopoiesis, leukemias were derived in

Ptpn11E76K knock-in mice from MPD. I purified LSK cells, CMPs, and CLPs from the MPD stage Ptpn11E76K/+/Mx1-Cre+ mice 8-10 weeks post pI-pC treatment.

These purified cells were mixed with 1x105 BM cells from congenic BoyJ mice

(carrier cells) and then transplanted into BoyJ mice. 6 months post transplantation, 30% of Ptpn11E76K mutant LSK cell transplants developed MPD followed by malignant transformation to leukemias (Figure 3-4A). However, none of the CMP and CLP transplants developed any diseases because of the failure

Figure 3-3 A&B

For Panel B, mouse dissection and organ isolation were performed by Dan Xu, and pictures were taken by Wen-Mei Yu

Figure 3-3 C, D&E

Figure 3-3 F

These Histology Slides were prepared by Dan Xu and the pictures were taken by Howard J. Meyerson. 91

Figure 3-3 G&H

Figure 3-3 Non-lineage specific effects of Ptpn11E76K/+ mutation on malignant transformation of MPD.

(A) Ptpn11E76K mutant mice progressed to frank leukemias from chronic MPD.

Kaplan– Meier survival curve in a cohort of Ptpn11E76K/+/Mx1-Cre+ mice (n=31) and a cohort of Ptpn11+/+/Mx1-Cre+ (n=72) mice following pI-pC treatment.

Disease distribution in the 27 moribund/dead Ptpn11E76K/+/Mx1-Cre+ mice is shown on the right panel. (B) Ptpn11E76K knock-in mice that progressed to frank leukemias showed increased blasts in peripheral blood, hepatosplenomegaly, thymic tumor, enlarged lymph nodes, and lung and liver infiltration. (C) T-ALL developed in Ptpn11E76K mice was demonstrated by flow cytometric profiling of

BM cells. BM cells collected from moribund Ptpn11E76K/+/Mx1-Cre+ mice and

Ptpn11+/+/Mx1-Cre+ control mice were immunostained with antibodies against

CD45, CD4, CD8, CD44, and c-Kit. The side scatter (SSC)lowCD45high/med blast population was sub-fractioned according to CD44 and c-Kit or CD4 and

CD8 expressions to determine CD4+/CD8+ cells and CD44+/c-Kit+ blasts. (D)

Flow cytometric profiling of BM and peripheral blood cells in AML developed in

Ptpn11E76K mice. BM and peripheral blood cells harvested from terminally ill

Ptpn11E76K/+/Mx1-Cre+ mice and Ptpn11+/+/Mx1-Cre+ control mice were immunostained with antibodies against CD45, Mac-1, Gr-1, and c-Kit. The

SSCmedCD45high population was sub-fractioned according to c-Kit and Mac-1 expressions to determine Mac-1+/c-Kit+ blasts. (E) B-ALL in Ptpn11E76K mice was shown by flow cytometric profiling of BM cells. BM cells collected from moribund

Ptpn11E76K/+/Mx1-Cre+ mice and Ptpn11+/+/Mx1-Cre+ control mice were

93 immunostained with antibodies against CD45, B220, IgM, and Sca-1. B220+/Sca-

1+ cells, B220+/IgM+ cells, and the blast (SSClowCD45low) population were shown.

(F) Leukemia cell invasion into hematopoietic and none-hematopoietic tissues in

Ptpn11E76K knock-in mice following progression to overt leukemias. Tissues harvested from terminally diseased Ptpn11E76K/+/Mx1-Cre+ mice were processed for histopathological examination (Hematoxylin and eosin staining). (G) Terminal deoxynucleotidyl transferase (TdT)- positive lymphoid blasts in the BM, thymus, and liver of Ptpn11E76K/+/Mx1-Cre+ mice in which MPD had evolved into T-ALL.

(H) Transplantability of the leukemias developed in Ptpn11E76K mice. BM cells collected from Ptpn11E76K/+/Mx1-Cre+ mice with T-ALL or AML were transplanted into sub-lethally (200 rads) irradiated NOD-SCID mice at the indicated cell doses.

Recipient mice were monitored on a daily basis for approximately 100 days.

Moribund mice were sacrificed. Diagnosis for each mouse was made by multiparameter FACS analyses, blood smear, and tissue histopathological examinations as described above.

94 of reconstitution. These data indicated that the leukemia found in Ptpn11E76K mutant mice are derived from HSCs (Figure 3-4A).

To further determine whether Ptpn11E76K mutation in lineage committed progenitors can also induce leukemias, we generated pan hematopoietic cell and lineage-specific Ptpn11E76K knock-in mice including Ptpn11E76K/+/Vav-Cre+ (pan hematopoietic cells), Ptpn11E76K/+/LCK-Cre+ (CD4-/CD8- stage T cells),

Ptpn11E76K/+/CD19-Cre+ (pro/pre-B stage B cells) and Ptpn11E76K/+/LySM-Cre+

(granulocyte macrophage progenitors). Notably, 20% of Ptpn11E76K/+/Vav-Cre+ mice developed T-ALL after a chronic MPD stage. 46% of Ptpn11E76K/+/LCK-Cre+ mice, 22% of Ptpn11E76K/+/CD19-Cre+ mice and 40% of Ptpn11E76K/+/LysM-Cre+ mice still developed T-ALL, B-ALL and AML (Figure 3-4B). Collectively, these results suggested that Ptpn11E76K mutation induced various leukemias not only from HSCs but also from lineage progenitors.

In this chapter, I showed that Ptpn11E76K mutant mice rapidly developed

MPD followed by malignant transformation into T cell acute lymphoid leukemia/lymphoma (T-ALL), acute myeloid leukemia (AML), and B cell ALL (B-

ALL), provided the first evidence that an activating mutation in Ptpn11 phosphatase plays a causal role in acute leukemias and established a novel mouse model mimicking the natural course of JMML. This mutation first promotes growth factor/cytokine signaling, thereby enhancing cell survival and proliferation in HSCs and lineage progenitors, resulting in MPD. HSCs or certain lineage progenitors are later transformed to leukemic stem cells (LSCs), triggering the onset of a variety of leukemias.

Figure 3-4 A&B

Figure 3-4 Leukemias induced by Ptpn11E76K mutation not only derived from

HSCs but also from lineage committed progenitors.

(A) HSC-enriched LSK cells but not lineage committed progenitors (CMPs and

CLPs) isolated from Ptpn11E76K knock-in mice at the MPD stage reproduced

MPD followed by evolution into frank leukemias in recipient mice. LSK cells,

CMPs, and CLPs were purified from Ptpn11E76K/+/Mx1-Cre+ mice 8 weeks following pI-pC treatment. These cells (2.5x103 cells, 5x103, and 1.6x104/per mouse for LSK cells, CMPs, and CLPs, respectively) were mixed with 1x105 BM cells from BoyJ mice and then transplanted into lethally irradiated (1100 rads)

BoyJ mice. Recipient mice were monitored on a daily basis for 8 months.

Moribund mice were sacrificed and diagnosed as described above. (B) Non- lineage specific effects of Ptpn11E76K/+ mutation on LSC development. Ptpn11E76K neo/+ mice were used to cross Vav-Cre, LCK-Cre, CD19-Cre, and LysM-Cre transgenic mice to generate pan hematopoietic cell, T lymphoid-specific, B lymphoid-specific, and granulocyte-macrophage progenitor-specific Ptpn11E76K knock-in mice, respectively. The mice were monitored for 12 months for leukemia development as described above.

Supplementary Information

Figure S3-1

Figure S3-1 Generation of conditional Ptpn11E76K knock-in mice.

E76K mutation (GAA to AAA) in Ptpn11E76K neo/+ mice was confirmed. Genomic

DNA extracted from tail snips from Ptpn11E76K neo/+ mice was sequenced.

Figure S3-2 A&B

For Panel B, genotyping was performed to identify the wt and mutant embryos by Dan Xu. Pictures were taken by Wen-Mei Yu.

100

Figure S3-2 C&D

101

Figure S3-2. Characterization of Ptpn11E76K knock-in mice.

(A) Global Ptpn11E76K/+ mutation induces embryonic lethality. Chimeric mice generated from Ptpn11E76K neo/+ ES cells were crossed with HPRT-Cre transgenic mice. Resulting embryos at various stages were identified by PCR genotyping. (B)

Developmental defects of Ptpn11E76K/+ embryos. Representative E.9.5 and E.11.5 embryos of Ptpn11+/+/HPRT-Cre+ and Ptpn11E76K/+/HPRT-Cre+ genotypes produced from intercrosses between Ptpn11E76K neo/+ and HPRT-Cre+ mice. (C)

Ptpn11E76K neo/+ mice are viable and futile. Chimeric mice generated from

Ptpn11E76K neo/+ ES cells were crossed with C57BL6/J mice. Resulting embryos at various stages were identified by PCR genotyping. (D) Insertion of the neo cassette in intron 2 inactivates the targeted Ptpn11 allele. Ptpn11E76K neo/+ and

WT mouse embryonic fibroblasts (MEFs) were generated from E.14.5 - E.16.5 embryos produced from crossing of Ptpn11E76K neo/+ with C57BL6 mice. These cells were infected with recombinant adenovirus expressing Cre recombinase.

Cellular Shp-2 levels were examined prior to and 48 hours post Cre infection by

Western blotting.

102

Table S3-1. Peripheral blood hematology of Ptpn11E76K knock-in mice (6-8 weeks following pI-pC treatment)

103

Table S3-2. Absolute numbers of hematopoietic cells in the BM of Ptpn11E76K knock-in mice.

104

Chapter IV: Future directions

My results have demonstrated that Shp-2 mutations have pathogenic effects on the onset of MDP and leukemia. An outstanding question is whether the pathogenic effects of Ptpn11 mutations depend on their enhanced Shp-2 catalytic activities resided in the PTP domain? Previous studies have clearly demonstrated that the catalytic activities of different Shp-2 mutants correlate well with their pathogenic results. While 60% of Ptpn11D61G heterozygous mice are viable (these mice subsequently develop MPD),

Ptpn11D61G/D61G homozygous mice are lethal at the embryonic stage. E76K mutation results in a higher catalytic activity of Shp-2 than D61G mutation, which correlates with a more malignant phenotype in E76K knock-in mice and in patients. Ptpn11E76K mutation causes embryonic lethality with full penetrance.

Seventy percent of Ptpn11E76K conditional knock-in heterozygous mice progress to T-ALL, B-ALL and AML following a stage of chronic MPD (3-8 months).

However, an equally important but less well explored alternative explanation is that these mutations, compared to the wild-type enzyme, might gain novel functions, such as binding to new protein targets or triggering different signaling pathways, which might be independent on the catalytic domain and its phosphatase activity.

To determine whether the pathogenic effects of Shp-2 mutant rely on their higher catalytic activities, an ongoing project in our lab is to generate the

105 transgenic mice with a pathogenic E76K mutation in N-terminal domain and a

C459S mutation in the C-terminal PTP domain which will abolish the catalytic activities of Shp-2. To generate the Shp-2-E76K-C459S knock-in plasmid, site mutagenesis will be made in the construct previously used to make E76K knock- in mice. The availability of E76K knock-in construct will facilitate this process.

After micro-injection and germline transmission, the E76K-C459S mice will be monitored to examine their hematopoietic effects and compared to E76K knock- in mice which service as a control.

First, if E76K-C459S mice don‟t develop MPD/leukemia, it means that pathogenic effects of E76K mutation depend on its enhanced phosphatase activities. Second, observing similar phenotypes to E76K knock-in mice indicates that the gain-of-function of E76K mutation reside in N-terminal domain other than

PTP domain. Although either of these two results will be conclusive, the third possible result is more complex. If the E76K mutation causes pathogenic effects through both higher catalytic activities and triggering novel signaling pathways, we might see a slightly milder phenotype. Thus, unless the E76K-C459S mice can totally rescue the phenotypes of E76K knock-in mice (first outcome), we need to investigate what new substrates Shp-2 E76K recognize. A complimentary method is discussed below.

Our results indicate that Gab2 plays a key role in mediating the effects of

Ptpn11D61G/+ mutation on cell signaling. Gab2 is the most prominent substrate of

Shp-2 in cytokine signaling. It forms a stable complex with Shp-2 in response to cytokines. Previous studies have shown that this scaffolding protein plays an

106 essential role in coupling downstream pathways to the proximity of cytokine receptors. Indeed, we and others observed increased phosphorylation of Gab2 in both D61G and E76K mutants compared with wt, but a more quantitative assay need to be developed to determine the extent of phosphorylation in Gab2 by

Shp-2 E76K (and Shp-2 D61G). However, it is possible that the E76K mutation might trigger new signaling pathway by recognizing new substrates wild type

Shp-2 can‟t. To directly examine what substrate Shp-2 E76K and wild type Shp-2 recognize, two experiments will be performed, 1) microarray using RNAs extract from HSCs in Wt, Ptpn11E76K/+mice (MPD stage) and 2) substrate trapping.

HSCs (LSK cells) will be isolated using FACS, RNAs will be extracted with

Qiagen Kit according the manufacturer instructions, standard Affix array platform will be used, and array will be normalized using the mean value in each array.

Compared to wild type array, genes with expression change >2 fold will be considered as up-regulation and <0.5 fold will be considered as down-regulation.

Both up-regulated subset and down-regulated genes set will be analyzed using

Biobase for the enrichment of Gene Ontology. These predicted changes in pathways will be confirmed by experiments. RT-PCR will be used to confirm the changes in gene expression, and biochemical methods will also be designed according to the predicted changes in Gene Ontology. The advantage of such a global analysis is that all genes will be examined and a great amount of information will be obtained in one experiment. But a limitation of high-through analysis is that it is difficult to distinguish “causes” and “effects”. For example, when leukemia samples and wild type samples are compared, the dominantly

107 enriched Gene Ontology will be Cell cycle, Cell dividing, Mitosis, Apoptosis, et al.

In other words, the “causes” of the leukemogenesis are overwhelmed by the secondary effects. To provide more direct clues for how Shp-2 E76K mutation causes leukemia, Substrate-trapping will be used as a complimentary method discussed below.

During the early course of phosphatase studies, diverse protein-tyrosine phosphatase (PTP) mutants have been designed to find the physiological substrates of tyrosine phosphatases. These mutants do not possess any catalytic activities but appear to bind to their tyrosine phosphorylated substrates tightly, and are hence named as PTP “substrate-trapping” mutants and can be used as tools to pull down their respective substrates from heterogeneous extracts.

These substrate-trapping mutants are epitomized by C/S mutants in whom the semi-conservative active cysteine residue of the signature PTP motif is mutated to a serine residue and D/A mutants in which the aspartate 452 is replaced by alanine to block the catalytic process. Generally, D/A mutants provide a tighter binding affinity to substrates than C/S mutants, and have been most widely used as substrate-trapping tools.

To perform substrate-trapping experiments, I will stably overexpress Shp-

2-E76K-D/A mutant and SHP-2-D/A mutant in Ba/F3 cell line, an interleukin-3 (IL-

3)-dependent murine hematopoietic cell line using the protocol generated in our lab. The expression level of Shp-2-E76K-D/A will be confirmed by Western-blot.

Shp-2-D/A will be used as control. Ba/F3 cells will be stimulated with IL-3 before immunoprecipitation with anti-Shp-2 antibody. The co-precipitated substrates will

108 be separated a 10% SDS-PAGE gel. Because Shp-2 is a phosphatase and the substrate-tapping mutants are catalytically-inactive, the co-precipitated substrates, if trapped by the Shp-2 D/A mutant, likely contain tyrosine phosphorylated proteins. Thus, a monoclonal anti-phosphotyrosine antibody will be used to perform Western-blotting to detect the putative Shp-2 substrates.

Binds detected only in Shp-2-E76K-D/A or Shp-2-D/A are particularly interesting, because they might contain substrates that are differentially recognized by Shp-2

E76K or wild type SHP-2. The identity of these substrates will be determined using mass spectrometry. The identified substrates will be further confirmed by biochemical methods.

All the experiments described above, if performed successfully, should provide insights into the mechanisms underlying Shp-2 mutations‟ pathogenic effects. It also provides a platform to investigate a more detailed question: why

Ptpn11D61G/+ mice develop MDP while Ptpn11E76K/+ mice develop a variety of leukemia after a chronic MPD phase. To determine whether the more malignant effects of Shp-2 E76K mutation depends on its higher catalytic activities compared to Ptpn11D61G/+, Shp-2-D61G- C459S mice can be generated to compare with Shp-2-E76K- C459S mice. If Shp-2-D61G- C459S and Shp-2-

E76K-C459S mice develop much similar phenotypes, it suggests that the more malignant effects of E76K mutation are due to its higher PTP catalytic activity. If

SHP-2-E76K-C459S mice still display a more severe phenotype, it means that

E76K and D61G mutation may have different downstream substrates. To identify the different pathways Shp-2 D61G and Shp-2 E76K may trigger and different

109 substrates that they may recognize, as describe before, microarray and substrate trapping can be performed.

Taken together, these proposed experiments will provide unprecedented detailed mechanisms underlying the pathogenic effects of various Shp-2 mutants.

It may reveal new drug targets for Shp-2 mutation-related diseases.

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Appendix: Experimental Methods

Generation of Ptpn11D61G/+ and Ptpn11D61G/+/Gab2-/- mice

Ptpn11D61G /+ mice (Araki et al., 2004) were originally imported from Beth

Israel Deaconess Medical Center. Gab2+/- mice (Nishida et al., 2002) were obtained from Dr. Toshio Hirano of Osaka University, Japan. Gab2+/- mice had been backcrossed with C57BL/6 mice at least 8 generations(Zhang et al., 2007).

Ptpn11D61G/+ mice were backcrossed with C57BL/6 mice for 3 generations and then used to cross Gab2+/- mice to generate Ptpn11D61G/+/Gab2+/- mice.

Ptpn11D61G/+/Gab2+/- mice were intercrossed to produce various types of mice (4th generation backcross to the C57BL6 background) for this study. Backcrossing of

Ptpn11D61G/+ or Ptpn11D61G/+/Gab2+/- mice to C57BL/6 mice could not be continued because of the complete penetrance of embryonic lethality in F5

Ptpn11D61G/+ mice (Araki et al., 2009). C57BL/6 (CD45.2+) mice and congenic strain B6.SJL-PtprcaPep3b/BoyJ (BoyJ, CD45.1+) mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME) and subsequently bred in house.

Generation of Ptpn11E76K knock-in mice.

Ptpn11 allele was targeted by homologous recombination. The targeting vector was constructed using the „recombineering‟ technique as previously described. In brief, a mini targeting vector with a loxP flanked neo cassette and

111 the mutation GAA (E) to AAA (K) at the amino acid 76 encoding position in exon

3 of Ptpn11 was first generated. This mini vector was then used to construct the targeting vector. The targeting vector was linearized and electroporated into D1 mouse embryonic stem (ES) cells derived from F1 hybrid blastocysts of 129S6 x

C57BL/6J. G418 resistant ES clones were isolated and screened for homologous recombination by nested PCR using primers outside the construct paired with primers inside the neo cassette. Positive clones were further confirmed by PCR genotyping (data not shown) and sequencing for the mutation (GAA to AAA) in genomic DNA (data not shown). Two individual ES cell clones, containing a correctly targeted Ptpn11 allele, were used to generate chimeric mice. Germline transmitted chimeric mice were obtained and used to cross C57BL6/J to generate heterozygous mice with the neo cassette (Ptpn11E76K neo/+). E76K mutation in F1 Ptpn11E76K neo/+ mice was verified by sequencing the targeted site of genomic DNA (Figure S3-1). These mice were backcrossed with

C57BL6/J mice for 3-6 generations for experiments. Mice used for transplantation analyses were 8th to 10th generation backcross to C57BL6/J background. Ptpn11E76K neo/+ mice were used to cross HPRT-Cre transgenic mice

(Jackson laboratory, Bar Harbor, ME) to remove the floxed neo cassette to generate Ptpn11E76K/+ mice. Ptpn11E76K neo/+ mice were also used to cross Mx1-

Cre transgenic mice (Jackson laboratory) to produce Ptpn11E76K neo/+/Mx1-Cre+ animals. No differences in the two lines of mutant mice derived from the original two ES cell clones were observed. All mice were kept under specific pathogen- free conditions in the Animal Resources Center at Case Western Reserve

112

University. All animal procedures complied with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal

Care and Use Committee.

Flow cytometric analysis and cell sorting

Multiparameter fluorescent activated cell sorting (FACS) analysis was performed to determine populations of hematopoietic stem cell (HSC)-enriched

Lineage-Sca-1+c-Kit+ (LSK) cells, pure HSCs (Lineage-Sca-1+c-Kit+CD41-CD48-

CD150+), long-term HSCs (LT-HSCs, Lineage-Sca-1+c-Kit+Flk2-CD34-), short- term HSCs (ST-HSCs, Lineage-Sca-1+c-Kit+Flk2-CD34+), and lineage progenitors, such as common myeloid progenitors (CMPs, Lineage-c-Kit+Sca-1-

CD16/32medCD34+), common lymphoid progenitors (CLPs, Lineage-c-Kit low Sca-

1lowCD127+), granulocyte-macrophage progenitors (GMPs, Lineage-c-Kit+Sca-1-

CD16/32highCD34+), and megakaryocyte-erythroid progenitors (MEPs, Lineage-c-

Kit+Sca-1-CD16/32med/low CD34-). Bone marrow (BM) cells freshly harvested from femurs and tibias were first stained with anti–Flk2-biotin and subsequently stained with antibodies labeled with various fluorochromes: streptavidin-APC-Cy7, c-Kit11APC, Sca-1-PE, CD34-Pacific Blue, CD16/32-PE-Cy7, CD127 (IL-7R)-

PE-Cy5 (eBioscience, San Diego, CA), and FITC-labeled antibodies for lineage markers Mac-1, Gr-1, Ter119, CD4, CD8a, CD3e, and B220 (BD Biosciences,

San Jose, CA). Specific cell populations were gated based on immunophenotypes for quantification or cell sorting as previously reported

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(Fleming et al., 2008; Kiel et al., 2005; Schindler et al., 2009; Tothova et al.,

2007). Fluorescence minus one (FMO) was used for setting the gating on control samples. For intracellular signaling analysis, Lineage− cells were purified from

BM and starved for 1 hour in IMDM medium. Cells were then stimulated with

SCF (50 ng/ml) for 5 min, fixed, permeabilized, and stained with antibodies against Sca-1, c-Kit and phospho-Erk or phospho-Akt (Cell Signaling Technology,

Inc., Beverly, MA) as previously reported (Chan et al., 2009; Kalaitzidis and Neel,

2008). Percentages of the cells stained positive for phospho-Erk or phospho-Akt in the gated LSK population were determined by multiparameter FACS analyses.

Apoptosis and cell-cycle analysis

Fresh BM cells were stained with biotin-labeled antibodies to lineage markers (Gr-1, Mac-1, B220, Ter119, CD4, CD8, and CD3), followed by staining with streptavidin-conjugated APC-Cy7, anti-c-Kit-APC, and anti-Sca-1-FITC. The cells were subsequently stained with anti-Annexin V-PE and 7-amino- actinomycin D (7-AAD) using the BD Annexin V-PE Apoptosis Detection Kit I (BD

Biosciences, San Jose, CA). Apoptosis was analyzed by quantification of the

Annexin V/7-AAD positive cell population by FACS. For HSC cell cycle analysis,

Pyronin Y and Hoechst 33342 staining and population gating were performed as previously reported (Cheng et al., 2000; Shen et al., 2008).

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Generation of BM-derived mast cells and macrophages

BM cells were cultured in RPMI1640 medium supplemented with 10%

FBS and mouse recombinant IL-3 (10 ng/ml) for 3-4 weeks. Mast cell phenotype was confirmed by FACS analysis with antibodies specific for c-Kit and FcRII/RIII.

At the time of use, greater than 98% of the cultured cells were mast cells. To generate BM-derived macrophages, BM cells were cultured in Dulbecco modified

Eagle medium (DMEM) supplemented with 10% FBS and 20% L-cell conditioned medium (as a source of mouse colony-stimulating factor 1). After 24 and 48 hours, non-adherent cells were collected and seeded into new tissue culture plates. Following 5 to 7 days of culture, cells were confirmed as macrophages as more than 90% of semi-adherent cells were positive for Mac-1 and F4/80.

CFU-S and transplantation assays

The colony-forming unit-spleen (CFU-S) assay was carried out as described (Morrison and Weissman, 1994). In brief, BM cells (1x105) were injected through the lateral tail vein into sub-lethally irradiated (9.5 Gy) C57BL/6 mice. Twelve days following the transplantation, recipients were sacrificed and spleens were dissected and fixed with Telleyesniczky‟s solution. Colonies on the spleen were counted under a dissecting microscope. For non-competitive repopulation assays, 1x106 BM cells from 12 week old wildtype (WT) or

Ptpn11D61G/+ littermates (CD45.2+) were directly injected through the lateral tail

115 vein into lethally irradiated (11.0 Gy) BoyJ (CD45.1+) recipients. Donor cell reconstitution was monitored by FACS analyses of peripheral blood cells at 4, 8, and 16 weeks post transplantation. Sixteen weeks post transplantation recipients were sacrificed and used as donors for the subsequent transplantation cycle. For competitive repopulation assays, 1x106 BM cells (test cells) from 12 week old WT or Ptpn11D61G/+ littermates (CD45.2+) were transplanted with the same number of

BoyJ (CD45.1+) BM cells (competitor cells) into lethally irradiated BoyJ (CD45.1+) recipients. Test cell reconstitution was determined at 4, 8, 12, and 16 weeks by

FACS analyses of peripheral blood. For secondary transplantations, BM cells

(2x106) harvested from 3-5 primary recipient mice were pooled and transplanted into lethally irradiated secondary recipients. Contributions of test cells to the hematopoietic system were determined at 18 to 24 weeks following the secondary transplantation.

Phosphatase assay.

Spleen tissues were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1%

Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotin, and 1 mM phenylmethylsulfonylfluoride). The lysates (500 μg) were

Immunoprecipitated with anti-Shp-2 antibody (Santa Cruz Biotechnology, Inc.,

Santa Cruz, CA). Immune complexes were washed three times in RIPA buffer and once in a buffer containing 20 mM HEPES (pH 7.4) and 150 mM NaCl. The immunocomplexes were then incubated at 30°C for 1 hour in a phosphatase

116 assay buffer containing 25 mM HEPES (pH7.4), 5 mM dithiothreithol, 50 mM

NaCl, 2.5 mM EDTA, and the substrate 25 mM 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma-Aldrich, St. Louis, MO). Hydrolysis of the substrate was measured by absorbance at 405 nm.

Colony-forming unit assay.

For the myeloid progenitor assay, BM cells (2104 cells/ml) freshly harvested from mice were assayed for colony forming units (CFUs) in 0.9% methylcellulose IMDM medium containing 30% fetal bovine serum (FBS), glutamine (10-4 M), -mercaptoethanol (3.310-5 M), and IL-3 or GM-CSF at various concentrations. After 7 days of culture at 37 C in a humidified 5% CO2 incubator, hematopoietic cell colonies (primarily CFU-GM) were counted under an inverted microscope. For Pre-B colony assays, BM cells (with 5x104/ml) were plated in methylcellulose-based medium containing 10 ng/ml recombinant human

IL-7 (Stem Cell Technologies, Vancouver, BC, Canada). After 7 days of culture at

37 C in a humidified 5% CO2 incubator, CFU-Pre-B colonies were counted under an inverted microscope.

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Statistical analysis

Data are presented as mean ± SEM. Statistical significance was assessed using an unpaired, two-tailed Student‟s t test. P values of <0.05 were considered to be significant. Statistical significance among four groups was determined by one-way ANOVA followed by Tukey's post-test or or two-way ANOVA followed by

Bonferroni post-test.

118

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