Gordon Chan

The tyrosine phosphatase Shp2 (PTPN11) in cancer

Diverse cellular processes are regulated by tyrosyl phosphorylation, which is controlled by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). De-regulated tyrosyl phosphorylation, evoked by gain-of-function mutations and/or over-expression of PTKs, contributes to the pathogenesis of many cancers and other human diseases. PTPs, because they oppose the action of PTKs, had been considered to be prime suspects for potential tumor suppressor genes. Surprisingly, few, if any, tumor suppressor PTPs have been identified. However, the Src homology-2 domain-containing phosphatase Shp2 (encoded by PTPN11) is a bona fide proto-oncogene. Germline mutations in PTPN11 cause Noonan and LEOPARD syndromes, whereas somatic PTPN11 mutations occur in several types of hematologic malignancies, most notably juvenile myelomonocytic leukemia and, more rarely, in solid tumors. Shp2 also is an essential component in several other oncogene signaling pathways. Elucidation of the events underlying Shp2-evoked transformation may provide new insights into oncogenic mechanisms and novel targets for anti-cancer therapy.

Noonan syndrome cardiac defects are caused by PTPN11 acting in endocardium to enhance endocardial-mesenchymal transformation

Noonan syndrome (NS), the most common single-gene cause of congenital heart disease, is an autosomal dominant disorder that also features proportionate short stature, facial abnormalities, and an increased risk of myeloproliferative disease. Germline-activating mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, cause about half of NS cases; other causative alleles include KRAS, SOS1, and RAF1 mutants. We showed previously that knock-in mice bearing the NS mutant Ptpn11D61G on a mixed 129S4/SvJae X C57BL6/J background exhibit all major NS features, including a variety of cardiac defects, with variable penetrance. However, the cellular and molecular mechanisms underlying NS cardiac defects and whether genetic background and/or the specific NS mutation contribute to the NS phenotype remained unclear. Here, using an inducible knock-in approach, we show that all cardiac defects in NS result from mutant Shp2 expression in the endocardium, not in the myocardium or neural crest. Furthermore, the penetrance of NS defects is affected by genetic background and the specific Ptpn11 allele. Finally, ex vivo assays and pharmacological approaches show that NS mutants cause cardiac valve defects by increasing Erk MAPK activation, probably downstream of ErbB family receptor tyrosine kinases, extending the interval during which cardiac endocardial cells undergo endocardial-mesenchymal transformation. Our data provide a mechanistic underpinning for the cardiac defects in this disorder.

Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells

Src homology 2 domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is a member of the nonreceptor protein-tyrosine phosphatase family, and functions in cell survival, proliferation, migration, and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs, indicating that Ras is functionally downstream of Shp2 in these cells. Thus, Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo.

A germline gain-of-function mutation in Ptpn11 (Shp-2) phosphatase induces myeloproliferative disease by aberrant activation of hematopoietic stem cells

Germline and somatic gain-of-function mutations in tyrosine phosphatase PTPN11 (SHP-2) are associated with juvenile myelomonocytic leukemia (JMML), a myeloproliferative disease (MPD) of early childhood. The mechanism by which PTPN11 mutations induce this disease is not fully understood. Signaling partners that mediate the pathogenic effects of PTPN11 mutations have not been explored. Here we report that germ line mutation Ptpn11(D61G) 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 Ptpn11(D61G/+) whole bone marrow cells or purified Lineage(-)Sca-1(+)c-Kit(+) cells, but not lineage committed progenitors. The deleterious effects of Ptpn11(D61G) mutation on HSCs are attributable to enhancing cytokine/growth factor signaling. The aberrant HSC activities caused by Ptpn11(D61G) 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 Ptpn11(D61G/+)/Gab2(-/-) double mutant mice. Collectively, 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.

SH2 Domain-Containing Protein-Tyrosine Phosphatases

Publisher Summary This chapter deals with the SH2 domain containing protein-tyrosine phosphatases. It begins by presenting a background of history and nomenclature of this phosphatase group. Following this, it discusses its structure, expression, and regulation. Shps have two SH2 domains at their N-termini (N-SH2 and C-SH2): a classical protein-tyrosine phosphatase (PTP) catalytic domain and a C-terminal tail (“C-tail”). Shp2 and its orthologs are expressed ubiquitously, although at variable levels in different tissues. The SH2 domains of Shps target them to phosphotyrosyl-containing (pTyr) proteins. Multiple proteins are known to bind the SH2 domains of mammalian Shp1 and Shp2. Most fall into three distinct categories: receptors (RTKs, cytokine receptors, or integrin cytoplasmic domains), scaffolding adaptors and FRS proteins, and so-called “immune inhibitory receptors” (commonly termed “inhibitory receptors”). Furthermore, the study discusses the biological functions of SH2 domain-containing protein-tyrosine phosphatases, describing the phenotypes of Shp-deficient organisms. Under this, it considers the examples of the motheaten phenotype, invertebrate models of Shp2 deficiency, and functions of vertebrate Shp2. Following this, it states that the most exciting recent discoveries about Shps have focused on their involvement in several different human diseases. Most of this work has centered on the role of Shp2 mutations in developmental disorders and neoplasia. Many of the pathways in which Shps participate, the biological consequences of loss-of-function, and, in the case of Shp2, gain of function mutations in Shps, are well known. Shp structures have been solved to atomic resolution, and the basics of regulation of Shp activity are well understood. However, the question of specificity of Shp target remains and calls for further research.

Erk1 and Erk2 are required for maintenance of hematopoietic stem cells and adult hematopoiesis

Extracellular signal-regulated kinase 1 (Erk1) and Erk2 play crucial roles in cell survival, proliferation, cell adhesion, migration, and differentiation in many tissues. Here, we report that the absence of Erk1 and Erk2 in murine hematopoietic cells leads to bone marrow aplasia, leukopenia, anemia, and early lethality. Mice doubly-deficient in Erk1 and Erk2 show rapid attrition of hematopoietic stem cells and immature progenitors in a cell-autonomous manner. Reconstitution studies show that Erk1 and Erk2 play redundant and kinase-dependent functions in hematopoietic progenitor cells. Moreover, in cells transformed by the oncogenic KRas(G12D) allele, the presence of either Erk1 or Erk2 with intact kinase activity is sufficient to promote cytokine-independent proliferation.

PI3K p110δ uniquely promotes gain-of-function Shp2-induced GM-CSF hypersensitivity in a model of JMML

Although hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110δ, but not PI3K p110α, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage-colony-stimulating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p110δ-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p110δ-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p110δ could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p110δ.

SHP2 is required for BCR-ABL1-induced hematologic neoplasia

BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph+) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation models for CML and BCR-ABL1+ B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia. Ptpn11 deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1+ B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, are essential for BCR-ABL1+, but not WT, pre-B-cell proliferation. The mitogen-activated protein kinase kinase (MEK) / extracellular signal-regulated kinase (ERK) pathway is regulated by SHP2 in WT and BCR-ABL1+ pre-B cells, but is only required for the proliferation of BCR-ABL1+ cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1+ pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1+ and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, represses MXD3/4 to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells.

Cancer: Bad neighbours cause bad blood

Expression of a blood-cancer-associated genetic mutation in the non-blood cells of the bone marrow is sufficient to cause blood cancer in mice. This finding could point to new approaches to treating an often-fatal disease. See Letter p.304 Hereditary mutations in the tyrosine phosphatase SHP2 (encoded by PTPN11), part of the Ras signalling pathway, have been linked to a syndrome leading to an increased risk of developing leukaemia. Previous studies in mouse models have shown that the function of haematopoietic stem cells carrying these mutations is defective, which suggests a cell-autonomous effect. Cheng-Kui Qu and colleagues find that the mutations also affect cells in the bone marrow environment, blocking their normal control on haematopoietic stem cells and thereby promoting the development of leukaemia. Administration of CCL3 receptor antagonists effectively reversed oncogenesis driven by the Ptpn11-mutated bone marrow microenvironment.

Abstract 4296A: Myeloproliferative disease induced by leukemogenic Ptpn11 (Shp-2) phosphatase arises from hematopoietic stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Germline and somatic gain-of-function (GOF) mutations in the tyrosine phosphatase PTPN11 (SHP-2) are associated with juvenile myelomonocytic leukemia (JMML), a clonal myeloproliferative disease (MPD) of early childhood. However, the mechanism by which PTPN11 mutations induce this disease is not fully understood. Here we report that the germline mutation Ptpn11D61G increases mouse hematopoietic stem cell (HSC) activity, as evidenced by accelerated cycling, increased stem cell pool, enhanced self-renewal, and elevated short-term and long-term repopulating capabilities. More importantly, the MPD is reproduced in primary and secondary recipient mice transplanted with Ptpn11D61G mutant marrow cells or sorted Lineage- Sca-1+c-Kit+ cells, indicative of the long-term stem cell origin of the disease. The leukemogenic effect of Ptpn11D61G mutation appears to be attributed to enhancing cytokine signaling. Furthermore, HSC homeostasis disrupted by Ptpn11D61G mutation is partially restored by deletion of Gab2, an important interacting protein/target of SHP-2. MPD phenotypes are markedly ameliorated in Ptpn11D61G/+/Gab2−/− double mutant mice. Together, these studies suggest that SHP-2 associated JMML arises from HSCs and that targeting SHP-2/Gab2 mediated signaling in stem cells may be useful for the treatment of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4296A.