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Biochemical and Functional Analysis of Ras Pathway Mutations in Myeloid Leukemia and Developmental Disorders

  • Author(s): Schubbert, Suzanne
  • Advisor(s): Shannon, Kevin M
  • et al.
Abstract

Noonan syndrome is a common dominant disorder characterized by short stature, facial dysmorphism, cardiac defects, and a predisposition to juvenile myelomonocytic leukemia (JMML). Germline PTPN11 mutations cause ~50% of Noonan syndrome. PTPN11 encodes SHP-2, a protein tyrosine phosphatase that relays signals from activated receptor complexes to Ras and other effectors. Studies of patient samples and in mouse models have demonstrated that hyperactive Ras signaling plays a central role in JMML. Based on the association of JMML with Noonan syndrome and the known role of SHP-2 in Ras signaling, our laboratory and others screened the PTPN11 gene in JMML, and discovered somatic mutations in ~ 35% of cases.

I performed functional studies of mutant SHP-2 proteins associated with JMML and Noonan syndrome. I used primary murine hematopoietic cells to investigate the effects of mutant SHP-2 on proliferation, survival, and differentiation. The most common leukemia-associated amino acid substitution (E76K) induced a hypersensitive pattern of myeloid progenitor colony growth in response to granulocyte-macrophage colony-stimulating factor and interleukin 3 that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells, perturbed erythroid growth, and impaired normal differentiation. In addition, leukemia-associated SHP-2 mutations conferred stronger phenotypes in primary hematopoietic progenitors than a germline mutation found in individuals with Noonan syndrome. Although PTPN11 mutations account for 50% of Noonan syndrome, the genetic lesions in the remaining individuals were unknown. Our collaborators discovered de novo germline KRAS mutations that introduce V14I, T58I, or D153V amino acid substitutions in individuals with Noonan syndrome and P34R and F156L alterations in individuals with cardio-facio-cutaneous syndrome, which has overlapping phenotypic features with Noonan syndrome. I performed biochemical and functional analysis of these novel syndrome-associated K-Ras proteins. Mutant K-Ras proteins demonstrate a range of gain-of-function effects in different cell types, and biochemical analysis supports the idea that the intrinsic Ras guanosine nucleotide triphosphatase (GTPase) activity, the responsiveness of these proteins to GTPase activating proteins, and guanine nucleotide exchange all regulate developmental programs in vivo.

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