Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells

Abstract

PTPN11 encodes the protein tyrosine phosphatase SHP-2, which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS), a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS.We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential, perturbed erythroid growth, and impaired normal differentiation in liquid cultures. In addition, leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments, which supports a primary role of hyperactive Ras in the pathogenesis of JMML. IntroductionThe PTPN11 gene encodes SHP-2, a nonreceptor tyrosine phosphatase (PTPase) that relays signals from activated growth factor receptors to p21 ras (Ras), Src family kinases, and other signaling molecules (for reviews, see Barford and Neel 1 and Neel et al 2 ). SHP-2 contains 2 Src homology 2 (SH2) domains and a catalytic PTPase domain. The SHP-2 crystal structure predicts that binding of the N-SH2 domain to phosphotyrosyl peptides results in a conformational shift that relieves inhibition of the PTPase and activates SHP-2 function. 3 Missense mutations in PTPN11 underlie about 50% of cases of Noonan syndrome (NS), a developmental disorder characterized by cardiac defects, facial dysmorphism, and skeletal malformations. 4 Most of the PTPN11 mutations found in NS introduce amino acid substitutions within the N-SH2 and PTPase domains. 4,5 Molecular modeling and biochemical data infer that exon 3 mutations dominantly activate SHP-2 phosphatase activity by altering critical N-SH2 amino acids that lie on the interface with the PTPase domain. 4,5 Infants with NS show a spectrum of hematologic abnormalities that includes isolated monocytosis as well as myeloid disorders with features of chronic myelomonocytic leukemia that may remit spontaneously. [6][7][8] Patients with NS are also predisposed to juvenile myelomonocytic leukemia (JMML), an aggressive myeloproliferative disorder (MPD) characterized by leukocytosis, tissue infiltration, and hypersensitivity to granulocyte-macrophage colonystimulating factor (GM-CSF). 9,10 Studies of JMML specimens and experiments in mutant strains of mice strongly implicate aberrant Ras signaling in response to GM-CSF and othe...

Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse

“…SHP-2 is required for normal development of both myeloid and lymphoid lineage cells (32,33), and gain-of-function mutations of SHP-2 are associated with childhood leukemias (13,33,34). We found in this work that bone marrow cells from cagA Hs mice exhibit hypersensitivity to IL-3 and GM-CSF, the hallmark of SHP-2 activation (31,35). The finding provides evidence for the hyperactivation of SHP-2 in cagA Hs mice and suggests the role of CagAderegulated SHP-2 in leukemogenesis.…”

Developmental SHP2 dysfunction underlies cardiac hypertrophy in Noonan syndrome with multiple lentigines

“…Both NSML and NS mutations facilitate the open conformation and lead to enhanced interaction of SHP2 with binding partners, including cell membrane receptors and scaffolding adapters (20). However, whereas NS mutations are gain of function (GOF) and potentiate SHP2 phosphatase activity (21)(22)(23)(24)(25)(26), all NSML mutations identified so far affect conserved residues important for PTP catalysis and are loss of function (LOF) for the phosphatase activity (20,(27)(28)(29)(30).…”

Childhood Myelodysplastic Syndrome: Focus on the Approach to Diagnosis and Treatment of Juvenile Myelomonocytic Leukemia

“…Studies of the E76K protein reveal that it displays the highest phosphatase activity, induces profound hypersensitivity to GM-CSF in fetal liver cells, and transforms transduced BaF3 cell lines to cytokine independence. 14,17,18 The D61G mutation is the only one reported to occur in both Noonan syndrome and de novo JMML. Transgenic mouse models with this lesion develop a mild MPN with a latency of 5 months, in contrast to the D61Y, which is a lesion only occurring in JMML in which transplanted mice develop a fatal, JMML-like aggressive disease by 6 to 7 months.…”