Shp2 E76K Research Articles

Targeting M2-Tumor Associated Macrophages By Arginase-1 and PD-L1 in Regulating Juvenile Myelomonocytic Leukemia (JMML) Development and Relapse

Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. Macrophages are largely characterized as M1 or M2 types, and TAMs have been shown to express an M2-like phenotype. TAMs endorse tumor progression and contribute to resistance to chemotherapies. However, it is unclear what the composition of M2 macrophages is in patients with Juvenile myelomonocytic leukemia (JMML) and how do these cells mechanistically contribute to JMML and/or relapse after bone marrow transplantation. To study the role of M2- TAMs in JMML development, we first examined the bulk RNA-sequence data in 90 JMML patients. These data demonstrated a significant increase in the expression of arginase-1 (Arg-1) and programmed cell death-1 (PD-1). Furthermore, single cell RNA-sequencing analysis of monocytes/macrophages from 4 JMML patients revealed higher expression of M2- macrophage markers/genes such as IL-10, CD163, MRC1/CD206, TGF-β1 and IL-1R1 compared to M1 macrophage (CD80, CCR7, IL-6, CXCL10, CXCL11 and TNF) expression. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-macrophages express higher levels of arginase and PD-1, which may contribute to the local suppression of immune responses and damage the bone marrow microenvironment (BME) leading to poor engraftment of normal donor cells, resulting in relapse. To study how alterations in bone marrow (BM) macrophages (M1/M2) contribute to JMML development and relapse, we utilized a mouse model bearing Shp2 E76K mutation (Ptpn11 E76K/+) driven by lysosome-cre (Ptpn11 E76K/+; LysM-Cre+, indicated as Shp2* mice hereafter). This model is frequently used to study JMML as it manifests cardinal features of human JMML. In a competitive transplantation experiment using, Shp2* + Boy/J BM cells (1:1 ratio) transplanted into lethally irradiated Shp2* recipient mice, we show that Shp2* mutant cells out compete WT BoyJ cells and result in rapid growth of CD45.2+ Shp2* mutant mature myeloid cells, hematopoietic stem and progenitors (HSC/Ps) and M2- macrophages (F4/80+/CD206+) in the BM and spleen leading to leukemia relapse. To determine if modulating Arg-1 and PD-1/PD-L1 levels in the background of Shp2* mutant leukemic stem cells in Shp2* recipients would alter the overall engraftment and JMML development and relapse, we again performed a competitive transplantation experiment using, Shp2* + Boy/J (BM cells, 1:1 ratio) into Shp2* and WT recipient mice. After 8 weeks post transplantation, we investigated the role of Arg-1 and PD-L1 in Shp2* recipients using pharmacological inhibitors, CB-1158 (Arg-1 inhibitor; 100 mg/kg, orally) + anti-PD-L1 antibody (10 mg/kg, i.p) for 30 days. The Arg-1 + PD-L1 treatment significantly reduced the number of white blood cells, neutrophils, monocytes and improved RBC and platelet counts. The spleen and liver weights were significantly rescued as well. Interestingly, CD45.1 WT donor cells in the PB, BM, and spleen were significantly increased and a significant reduction of Shp2* mutant CD45.2+ mature myeloid cells in the PB, BM, and spleen was observed. Importantly, the frequency and absolute number of leukemic blasts, LSK (Lin-/Sca1+/c-KIT+) cells, short term hematopoietic cells (ST-HSCs), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP) were significantly reduced. Furthermore, the M2- TAMs were significantly reduced in the BM and spleen of Arg-1 + PD-L1 drug treated group compared to vehicle treated mice. Notably the CD8+ T-cells (IFN-γ+ and TNF-α+) were significantly improved in the drug treated mice. These data suggest that the suppression of arginase-1 allows for the arginine levels to increase, which promotes the proliferation of T-cells. Increasing arginine levels also promotes an anti-tumor immune response resulting in the emergence of CD45.1 WT HSCs as opposed to mutant CD45.2 HSCs, suggesting that Arg-1 + PD-L1 treatment is a novel therapeutic approach to treat patients with JMML and for preventing leukemia relapse after BM transplantation. DisclosuresNo relevant conflicts of interest to declare.

Shp2 activation in bone marrow microenvironment mediates the drug resistance of B-cell acute lymphoblastic leukemia through enhancing the role of VCAM-1/VLA-4

B-cell acute lymphoblastic leukemia (B-ALL) is immune to the chemotherapy-induced apoptosis as a result of the protection of bone marrow mesenchymal stromal cells (BMSCs). However, the precise underlying mechanism of such protection remains unclear so far. In this experiment, protein tyrosine phosphatase 2 (Shp2), which was encoded by the PTPN11 gene, was highly expressed in BMSCs of the newly diagnosed and the recurrent B-ALL patients. The plasmid-induced (including Shp2 E76K) Shp2 activation in BMSCs (Shp2-activated BMSCs) markedly increased the BMSCs-mediated resistance of leukemia cells both in vitro and in vivo. Additionally, studies in vitro suggested that, the expression of vascular cell adhesion molecule 1 (VCAM-1) was markedly up-regulated in Shp2-activated BMSCs, and VCAM-1 expression in BMSCs of B-ALL patients was negatively correlated with Shp2 expression. Down-regulation of VCAM-1 in BMSCs using siRNA reversed the resistance of CCRF-SB cells mediated by the Shp2-activated BMSCs. As for the molecular mechanism, the PI3K/AKT pathway mediated the regulation of VCAM-1 by Shp2. Blocking the very late antigen-4 (VLA-4) by antibodies in CCRF-SB cells dramatically reversed the resistance of CCRF-SB cells mediated by the Shp2-activated BMSCs, and decreased the adhesion effects of both CCRF-SB cells and BMSCs. In conclusion, Shp2 activation in BMSCs up-regulates VCAM-1 expression through increasing the PI3K/AKT phosphorylation level, and targeting the VCAM-1/VLA-4 signaling may serve as a clinically relevant mechanism to overcome the BMSCs-mediated chemoresistance of B-ALL cells.

Shp2 regulates leukemic stem cell frequency in MLL-rearranged acute myeloid leukemia.

Shp2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It contains two SH2 domains, a PTP domain, and a carboxyl-terminal region with tyrosine phosphorylation sites. The Shp2 PTP activity is autoinhibited by its N-SH2 domain. Shp2 is activated by docking to specific tyrosine phosphorylated sites via SH2 domains or by gain-of-function (GOF) mutations that disrupt the autoinhibition. GOF Shp2 mutations are most frequently detected in juvenile myelomonocytic leukemia (JMML), representing 35% of cases. Animal model studies using conditional knockin of GOF Shp2 E76K or Shp2 D61G in the hematopoietic compartments have shown that these Shp2 mutants cause JMML-like myeloproliferative disease (MPD), albeit after ~6 months of latency (1,2).

Critical Role of Stat5 in the Maintenance of Leukemic Stem Cells in Ptpn11-Associated Juvenile Myelomonocytic Leukemia

Effective therapeutic interventions for juvenile myelomonocytic leukemia (JMML), a fatal childhood malignancy, are lacking. Relapse is the most frequent cause of treatment failure, most likely due to the persistence of leukemic stem cells (LSCs), a small population of self-renewing precursor cells that give rise to the bulk of tumor cells. This reservoir of tumor cells are responsible for long-term maintenance of leukemia growth, and are also a major source of drug resistance. Clearly, a novel approach focused on the unique properties of LSCs is needed. However, it remains a critical challenge how such cells may be eradicated. JMML is known to be caused by genetic mutations in cell signaling proteins involved in the Ras pathway, among which the phosphatase Ptpn11 (Shp2) is most frequently mutated. Ptpn11 mutations (heterozygous) cause greatly increased catalytic activity. We have recently created conditional knock-in mice with the Ptpn11E76K mutation, the most common Ptpn11 mutation found in JMML. Induction of the Ptpn11E76K/+ mutation in these mice (Ptpn11E76K/+/Mx1-Cre+) leads to JMML-like myeloid malignancy with full penetrance by aberrant activation of stem cells and myeloid progenitors. In an effort to understand the biological properties of LSCs in JMML, we studied leukemic hematopoietic stem cells (Lin-Sca-1+c-Kit+Flk2-CD150+CD48- cells)(referred to as LSCs since they reproduce the same disease in sub-lethally-irradiated transplants) in this mouse model. We found that Stat5 was hyper-activated in LSCs in Ptpn11E76K/+/Mx1-Cre+ mice in the chronic phase. Stat5 hyperactivation is likely to be mediated through Jak2 kinase as Jak2 is highly activated in Shp2 E76K-expressing cells. More importantly, these LSCs appear to rely on hyperactivation of Stat5 for maintenance and self-renewal because deletion of Stat5ab in Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl double mutant mice resulted in massive cell death in LSCs while neither Stat5ab knockout nor Ptpn11E76K/+ knock-in alone mice had these stem cell phenotypes. Apoptotic cells in the stem cell population were 6.38±1.42, 4.06±0.51, 10.52±6.88, and 24.50±10.27% in Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, Stat5abfl/fl/Mx1-Cre+, and Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice, respectively. Consequently, the stem cell pool in Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl double mutants was drastically decreased. Numbers of stem cells per femur were 3.36±1.51, 0.94±0.63, 1.15±0.39, and 0.17±0.20 (x103) in Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, Stat5abfl/fl/Mx1-Cre+, and Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice, respectively. Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice died of pan cytopenia within 4-8 weeks of Stat5 deletion while none of other groups of mice did. Repopulation capabilities of the double mutant stem cells were essentially lost in sub-lethally-irradiated recipient animals. Further mechanistic investigations revealed that tyrosine phosphorylation levels (indicative of activity) of Stat3, one of the substrates of the Shp2 phosphatase, were decreased by ~7.50 fold in Ptpn11E76K/+ LSCs as compared to Ptpn11+/+ control cells due to the enhanced dephosphorylation by the hyperactive Shp2 E76K mutant. Thus, diminished Stat3 activity sensitized Ptpn11E76K/+ LSCs to Stat5 depletion-induced cell death. Taken together, this study suggests a crucial role of Stat5 in the maintenance of LSCs in Ptpn11-associated JMML. The synthetic lethality induced by loss of Stat5 in Ptpn11-mutated JMML raises the possibility that clinically-used inhibitors of upstream Jak2 kinase may be effective in eradicating LSCs in this particular subtype of JMML. DisclosuresNo relevant conflicts of interest to declare.

Critical Role of the Gab2/PI3K/mTOR Pathway in the Pathogenesis of Ptpn11 (Shp2) Mutation-Induced Myeloproliferative Disease.

Abstract 2856▪▪This icon denotes a clinically relevant abstractGermline and somatic mutations (heterozygous) in Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling processes, have been identified in juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disease (MPD), and pediatric acute leukemias. These mutations cause hyperactivation of Shp2 catalytic activity and enhance the binding of mutant Shp2 to signaling partners. Ptpn11 mutations are sufficient to drive the development of JMML-like MPD and acute leukemias in mice, suggesting that they play a causal role in the pathogenesis of hematological malignancies. However, the mechanisms by which Ptpn11 mutations induce these malignancies are not completely understood and the signaling partners that mediate the pathogenic effects of Ptpn11 mutations have not been explored. We previously generated a line of conditional knock-in mice with Ptpn11E76K mutation, the most common and most active Ptpn11 mutation found in JMML and acute leukemias. Induced knock-in of this mutation in hematopoietic cells resulted in MPD with full penetrance as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors (J. Exp. Med., 2011). Recently, we discovered that the interaction between Shp2 E76K and Gab2, a prominent interacting protein of Shp2 and a scaffolding protein important for cytokine-induced PI3K/Akt/mTOR signaling, was greatly enhanced, and that mTOR was highly activated in Ptpn11E76K/+ MPD cells. To address the role of Gab2 and mTOR in the pathogenesis of Ptpn11E76K/+ mutation-induced MPD, Ptpn11E76K/+/Gab2-/- double mutant mice were generated and their phenotypes were compared with those of Ptpn11E76K/+ single mutant mice. MPD phenotypes were markedly attenuated in Ptpn11E76K/+/Gab2-/- double mutant mice. Overproduction of myeloid cells in the bone marrow was alleviated, and splenomegaly was diminished in the double mutants. Myeloid cell infiltration in the liver also decreased. Cytokine (IL-3 and GM-CSF) sensitivity of myeloid progenitors was significantly decreased in Ptpn11E76K/+/Gab2−/− mice as compared to that in Ptpn11E76K/+ mice. Hyperactivation of HSCs and excessive myeloid differentiation caused by Ptpn11E76K mutation were largely corrected by deletion of Gab2. Furthermore, we treated Ptpn11E76K/+ mice with Rapmycin, a specific and potent mTOR inhibitor, which substantially diminished MPD phenotypes. Collectively, this study reveals the essential role of the Gab2/PI3K/mTOR pathway in mediating the pathogenic effects of Ptpn11E76K/+ mutation and suggests that Gab2 and mTOR are potential therapeutic targets for the treatment of Ptpn11-associated hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

Genetic disruption of the PI3K regulatory subunits, p85 , p55 , and p50 , normalizes mutant PTPN11-induced hypersensitivity to GM-CSF

Juvenile myelomonocytic leukemia is a lethal disease of children characterized by hypersensitivity of hematopoietic progenitors to granulocyte macrophage-colony stimulating factor. Mutations in PTPN11, the gene encoding the protein tyrosine phosphatase Shp2, are common in juvenile myelomonocytic leukemia and induce hyperactivation of the phosphoinositide-3-kinase pathway. We found that genetic disruption of Pik3r1, the gene encoding the Class IA phosphoinositide-3-kinase regulatory subunits p85α, p55α and p50α, significantly reduced hyperproliferation and hyperphosphorylation of Akt in gain-of-function Shp2 E76K-expressing cells. Elevated protein levels of the phosphoinositide-3-kinase catalytic subunit, p110δ, in the Shp2 E76K-expressing Pik3r1-/- cells suggest that p110δ may be a crucial mediator of mutant Shp2-induced phosphoinositide-3-kinase hyperactivation. Consistently, treatment with the p110δ-specific inhibitor, IC87114, or the clinical grade pan-phosphoinositide-3-kinase inhibitor, GDC-0941, reduced granulocyte macrophage-colony stimulating factor hypersensitivity. Treatment with the farnesyltransferase inhibitor, tipifarnib, showed that Shp2 E76K induces hyperactivation of phosphoinositide-3-kinase by both Ras-dependent and Ras-independent mechanisms. Collectively, these findings implicate Class IA phosphoinositide-3-kinase as a relevant molecular target in juvenile myelomonocytic leukemia.

Inhibition of cellular Shp2 activity by a methyl ester analog of SPI-112

The protein tyrosine phosphatase (PTP) Shp2 ( PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2 E76K mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2 E76K-dependent cell survival, which is associated with inhibition of Shp2 E76K PTP activity, Shp2 E76K-induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-γ (IFN-γ)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.

Genetic Disruption of the PI3K Regulatory Subunit, p85α Partially Normalizes Gain-of-Function PTPN11-Induced Hypersensitivity to GM-CSF in Hematopoietic Progenitors.

Abstract 3968Poster Board III-904Juvenile Myelomonocytic Leukemia (JMML) is a lethal myeloproliferative disorder (MPD) of children, characterized by hyperproliferation of myelomonocytic cells and hypersensitivity to Granulocyte-Monocyte Colony-Stimulating Factor (GM-CSF). Most patients show hyperactivation of Ras via one or more mutations, including in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. It has been demonstrated that gain-of-function mutant Shp2 (Shp2 E76K and Shp2 D61Y) causes hyperactivation of the Mitogen-Activated Protein Kinase (MAPK) pathway. Additionally, we have previously shown that macrophage progenitor cells transduced with Shp2 D61Y or Shp2 E76K showed elevated levels of phospho-Akt, both at baseline and following 1 hour of GM-CSF stimulation, indicating a role for the Phospho-Inositol-3-Kinase (PI3K)/Akt pathway in the phenotype of elevated proliferation and survival in mutant Shp2-expressing cells (Yang, et al, 2008). However, it remains to be elucidated how PI3K contributes to the phenotype of increased proliferation and survival in cells bearing gain-of-function mutations in Shp2. Class IA PI3K is a lipid kinase heterodimer consisting of one of three catalytic subunits (p110α, p110β, or p110δ) and one of two regulatory subunits (p85α or p85β). It has been demonstrated that knocking out the main regulatory subunit, p85α, abrogated the hyperproliferative phenotype in mast cell progenitors bearing an oncogenic mutation in Kit in a model of another MPD, Systemic Mastocytosis (Munugalavadla, et al, 2007). In order to examine whether eliminating expression of p85α would cause a similar correction in cells expressing gain-of-function mutant Shp2, we performed timed matings of mice heterozygous for the knock-out of Pik3r1, which encodes the p85α subunit as well as its isoforms, p55α and p50α, since homozygous Pik3r1-/- is lethal in utero. WT and Pik3r1-/- fetal liver cells were isolated from 14.5 day embryos and transduced with either WT Shp2 or mutant Shp2 E76K. Transduced cells were subjected to serum deprivation followed by a 24-hour treatment with increasing doses of GM-CSF, and proliferation was then measured with H3-thymidine incorporation assays. We found that the absence of all the Pik3r1 isoforms resulted in a significant but incomplete correction of GM-CSF hypersensitivity in Shp2 E76K-expressing cells. To further investigate this observation, WT Pik3r1 and Pik3r1-/- macrophage progenitors, transduced with WT Shp2 or mutant Shp2 E76K as described above, were serum- and growth factor-deprived and then stimulated for 1 hour with GM-CSF. Western blot analysis showed that there was an expected increase in phospho-Akt in WT Pik3r1 cells following GM-CSF stimulation and that this increase was larger in Shp2 E76K-expressing cells than in WT Shp2-expressing cells, as previously observed. Upon genetic disruption of Pik3r1, Akt activation was reduced in both WT Shp2- and Shp2 E76K-expressing cells; however, the phospho-Akt in the Shp2 E76K-expressing cells was not reduced to WT levels. The phospho-Akt levels mirrored the proliferation pattern displayed by these cells in the H3-thymidine incorporation assays, where a modest reduction in proliferation in Pik3r1-/-, Shp2 E76K cells corresponded to the modest reduction in phospho-Akt levels in the same cells. Additionally, we found that Pik3r1-/-, Shp2 E76K cells also showed a blunted increase in phospho-Erk levels following GM-CSF stimulation compared to the WT Pik3r1, Shp2 E76K cells, indicating that knocking out Pik3r1 affects the MAPK pathway as well, which likely also contributes to the reduction in proliferation seen in Pik3r1-/-, Shp2 E76K cells following GM-CSF stimulation. Based on these data, we conclude that: (1) gain-of-function Shp2 activity results in dysregulated PI3K signaling, contributing to the leukemic phenotype of increased proliferation and survival; (2) PI3K signaling is reduced but not completely normalized in the absence of the main regulatory subunit, p85α and its isoforms, in gain-of-function mutant Shp2-expressing cells; and (3) there is cross-talk between the PI3K and MAPK pathways in the presence of Shp2 activating mutations, which is revealed by knocking out Pik3r1. Disclosures:No relevant conflicts of interest to declare.

The C-Terminal Zinc Finger of GATA2 Is Necessary for Normalization of Aberrant Myelomonocytic Differentiation Induced by Activating PTPN11 Mutations.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation despite being subjected to conditions that typically support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression revealed that GATA2 expression, needed for mast cell differentiation, is dramatically reduced, while, surprisingly, PU.1 expression is unchanged in cells expressing activating Shp2 mutants. In addition to lineage-specific transcription factors such as PU.1, however, c-jun also promotes monocytic differentiation by functioning as a co-activator of PU.1. Thus, we hypothesized that activating Shp2 mutations (Shp2D61Y or Shp2 E76K) induce increased c-jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression in hematopoietic progenitors. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (GATA2del330–407), which is needed for disruption of the PU.1-c-jun interaction, would normalize aberrant myelomonocytic differentiation induced by activating Shp2 mutants. Consistent with our hypothesis, quantitative RT-PCR studies revealed 5-fold higher c-jun levels in cells expressing Shp2D61Y or Shp2E76K compared to cells expressing WT Shp2. We next utilized retroviral co-transduction of murine bone marrow LDMNCs to generate six groups:1.pMIEG3-Shp2WT plus pCD4 (empty vector);2.pMIEG3-Shp2WT plus pCD4-GATA2;3.pMIEG3-Shp2WT plus pCD4-GATA2del330-407;4.pMIEG3-Shp2E76K plus pCD4;5.pMIEG3-Shp2E76K plus pCD4-GATA2; and6.pMIEG3-Shp-2E76K plus pCD4-GATA2del330-407.Transduced cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2E76K plus pCD4 produced significantly more CFU-M than cells co-transduced with WT Shp2 plus pCD4. Upon co-transduction with GATA2, the number of CFU-M generated from Shp2E76K-expressing cells was significantly reduced and was similar to that observed in cells expressing WT Shp2. In contrast, co-transduction of GATA2del330-407 failed to normalize the number of CFU-M produced by Shp2E76K-expressing cells. Quantitative RT-PCR verified ectopic GATA2 and GATA2del330-407 expression in the co-transduced cells. These findings demonstrate that restoration of GATA2 expression normalizes the propensity toward monocytic differentiation induced by Shp2E76K. The lack of correction conferred by GATA2del330-407 in combination with the observed increased c-jun expression support a model in which GATA2 and c-jun compete for binding to PU.1 to direct cell differentiation decisions in hematopoietic progenitors bearing activating Shp2 mutants. Collectively, these findings imply that normalization of transcription factor expression may provide a novel approach to differentiation-mediated therapy in JMML.

Effects of a Leukemia-associated Gain-of-Function Mutation of SHP-2 Phosphatase on Interleukin-3 Signaling

Mutations in SHP-2 phosphatase that cause hyperactivation of its catalytic activity have been identified in human leukemias, particularly juvenile myelomonocytic leukemia, which is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce hematopoietic malignancies are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in both catalytic-dependent and -independent manners and that overexpression (5-6-fold) of wild type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through accelerated dephosphorylation of STAT5. These results raised the possibility that SHP-2-associated leukemias are not solely attributed to the increased catalytic activity of GOF mutant SHP-2. GOF mutant SHP-2 must have gained additional capacities. To test this possibility, we investigated effects of a GOF mutation of SHP-2 (SHP-2 E76K) on hematopoietic cell function and IL-3 signal transduction by comparing with those of overexpressed WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. The E76K mutation in the N-terminal Src homology 2 domain increased interactions of mutant SHP-2 with Grb2, Gab2, and p85, leading to hyperactivation of IL-3-induced Erk and phosphatidylinositol 3-kinase (PI3K) pathways. In addition, despite the substantial increase in the catalytic activity, dephosphorylation of STAT5 by SHP-2 E76K was dampened. Furthermore, catalytically inactive SHP-2 E76K with an additional C459S mutation retained the capability to increase the interaction with Gab2 and to enhance the activation of the PI3K pathway. Taken together, these studies suggest that in addition to the elevated catalytic activity, fundamental changes in physical and functional interactions between GOF mutant SHP-2 and signaling partners also play an important role in SHP-2-related leukemigenesis.

Increased Proliferation and Altered Growth Factor Dependence of Human Mammary Epithelial Cells Overexpressing the Gab2 Docking Protein

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.

Structural and Signaling Requirements for Transformation Mediated by Shp2 Mutants.

The SH2-containing tyrosine phosphatase Shp2 (PTPN11) is required for normal growth factor and cytokine signaling. Germline Shp2 mutations cause ~50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ~35% of sporadic JMML, and at lower incidence in various chronic and acute leukemias. JMML patients without Shp2 mutations have either activating Ras mutations or homozygotic inactivation of Nf1. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and leukemia mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activating. We previously showed that leukemia-associated mutants E76K or D61Y, but not wild type (WT) Shp2 could transform bone marrow (BM) or fetal liver cells and give rise to cytokine-independent myeloid colony formation. Leukemia-associated mutants yielded much higher number and larger size of colonies than NS-associated mutants (e.g., N308D, N308S, E76D) whereas, mutants associated with both NS and leukemia (T73I, E139D, Q506P) evoked intermediate numbers. Transplantation of E76K− or D61Y− (but not parental virus or WT Shp2) transduced BM into lethally irradiated recipients evoked a fatal JMML-like disorder. To understand the structural requirements of Shp2 for transformation, we made a series of second site mutations in Shp2 E76K and expressed these mutants in murine BM. Mutation of the canonical arginine residue to methionine in the FLVRES motif in either N-SH2 or C-SH2 domain of Shp2 E76K ablated myeloid transforming ability. Mutation of the conserved arginine residue within the Shp2 E76K phosphatase domain signature motif to methionine, which diminishes phosphatase activity, also eliminated myeloid transformation. Tyrosine residues 542 and 580 in the C-terminus of Shp2 are known to undergo phosphorylation in response to many growth factors and required for Grb2 binding. Mutation of tyrosine 542 to phenylalanine markedly reduced transformation, whereas tyrosine 580 mutation alone had little effect. However, mutation of both tyrosines eliminated Shp2 E76K-evoked transformation, suggesting that Grb2-binding to Shp2 could be important for transformation. We observed that Gab2, a major Shp2 SH2 domain binding protein, was absolutely required for transformation by Shp2 E76K but not activating RasV12. Gab3, another member of Gab family protein mainly expressed in hematopoietic cells, was not critical for Shp2 E76K-evoked transformation. We also found that Stat5 was important for Shp2 E76K-mediated transformation. Mast cells derived from E76K− and D61Y− transplanted mice BM exhibited increased proliferation and enhanced activation of Erk, p38MAPK, Akt and Stat5 in response to IL-3. These results provide some mechanistic insights into how Shp2 mutants could transform primary myeloid cells and result in JMML-like MPD.

Effects of a Leukemia-Associated Gain-of-Function Mutation of SHP-2 Phosphatase on IL-3 Signaling.

Mutations in SHP-2 phosphatase that cause its hyper-activation have been identified in human leukemias, in particular, juvenile myelomonocytic leukemia (JMML) that is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce leukemia are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in catalytic-dependent and -independent manners and that overexpression (5-to-6 fold) of wild-type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through dephosphorylation of STAT5. This raised the possibility that SHP-2-associated JMML was not solely attributed to the increased catalytic activities of SHP-2 GOF mutants. The SHP-2 mutants must have gained other additional functions. To test this possibility, we investigated effects of a GOF mutation (E76K, the most frequent SHP-2 mutation seen in JMML) of SHP-2 on IL-3 signal transduction in great detail by comparing signaling activities of SHP-2 E76K to WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. E76K mutation in the N-terminal Src homology 2 domain significantly increased binding of the mutant SHP-2 to Grb2 and Gab2, two adaptor proteins coupling downstream Erk and PI3 kinase pathways to the proximity of the IL-3 receptor. As a result, IL-3-induced Erk and PI3 kinase pathways were highly activated by SHP-2 E76K mutation. In addition, Jak2 kinase activation was also markedly enhanced and due to the E76K mutation the substrate specificity of SHP-2 toward STAT5 was changed. Dephosphorylation of STAT5 by SHP-2 E76K was alleviated. These studies suggest that in addition to elevated catalytic activity, profound changes in protein-protein interaction capacities of GOF mutant SHP-2 play an important role in the pathogenesis of SHP-2-related leukemias.