Biochemical Dissection of SHP-2 and C-CBL in Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare childhood myeloproliferative neoplasm/myelodysplastic syndrome associated with mutations in the Ras/mitogen activated protein kinase (MAPK) signaling pathway. PTPN11 is the most commonly mutated gene in JMML, accounting for 35% of cases, followed by gain-of-function mutations in KRAS and NRAS and loss-of-function mutations in NF1. The protein tyrosine phosphatase (PTP) SHP-2 encoded by the gene PTPN11 is known to play a critical role in hematopoiesis and development, and studies in Drosophila and C. elegans have identified the SHP-2 homologs corkscrew and ptp-2 as crucial mediators of Ras and growth factor receptor signaling. SHP-2 is an SH2-domain containing non-receptor PTP whose activity is basally auto-inhibited by the occlusion of its catalytic cleft by its N-SH2 domain. The most frequently identified PTPN11 mutation results in an E76K substitution that is predicted to interfere with the N-SH2/PTP domain interface, thereby disrupting the auto-inhibited conformation. SHP-2 is unusual for a PTP in that it positively regulates growth factor signaling, and the mutations identified in JMML are gain-of-function mutations resulting in a basal increase in catalytic activity. A hallmark feature of JMML is hypersensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF) in an in vitro colony-forming assay (CFU-GM). It has been demonstrated that the catalytic activity of SHP-2 is required for its transforming ability, as substitution of critical catalytic residues abolishes E76K-mediated CFU-GM hypersensitivity. Utilizing PTP substrate-trapping technology, we have used tandem affinity purification and mass spectrometry to identify several putative substrates of SHP-2 E76K, including the protein parafibromin. Examination of changes in global phosphotyrosine mediated by SHP-2 E76K revealed a role for Src family kinases (SFK) in SHP-2 signaling downstream of the GM-CSF receptor, although CFU-GM transformation assays using hematopoietic progenitors from SFK-null murine models suggested that this relationship was not essential for leukemogenesis. Instead, high overexpression of SHP-2 in the human TF-1 leukemia cell line demonstrated a potential role for SHP-2 in promoting megakaryocytic differentiation via inhibition of the SFK Lyn. Lastly, the identification of mutations in C-CBL in a number of hematologic malignancies led us to discover and characterize the role of similar mutations in JMML