The long-term goal of Project 4 is to validate the biological significance of discovered mutations so thatspecific molecular targets can be used to guide the treatment of patients with acute myeloid leukemia (AMI).Mutations in genes involved in cytokine signaling (e.g. PL73, KRAS, NRAS, KIT) can be identified in nearly50% of AML cases. We hypothesized that every AML tumor harbors at least one mutation in a cytokinesignaling gene, and that these mutations cooperate to cause disease progression. Focused high-throughputexonic re-sequencing of expression-prioritized receptor tyrosine kinase (RTK), cytoplasmic tyrosine kinase(CTK), and Ras-MAPK pathway genes has been performed on 94 selected primary de novo AML patientsamples. Sequence data has been analyzed and novel, non-synonymous mutations have been identified inmultiple genes in the Janus kinase (JAK) family, including JAK1, JAK3, and TYK2. We propose the followingSpecific Aims:
Specific Aim 1 : We will validate the biological significance of somatic Janus kinase(JAK) family mutations by assessing the growth and differentiation of primary hematopoietic cellsexpressing mutant JAK1 and TYK2, and we will characterize the mechanisms by which thesesomatic mutations contribute to leukemic transformation. Our laboratory has validated the functionalsignificance of several myeloid leukemia-associated oncogenes using biochemical, cell culture, and mousemodel assays. We will express JAK1, TYK2, and JAK3 mutations in cell lines and primary murine bonemarrow assays to determine the effect of these mutations on growth and survival of hematopoietic cells. Theeffects of these mutations on the subcellular localization of the mutant proteins themselves, and associatedproteins will be examined using confocal microscopy, biochemical analysis, and localization-tagged mutants.
Specific Aim 2 : We will validate the biological significance of high-priority germline single nucleotidepolymorphisms (SNPs) in JAK family genes TYK2 and JAK3 by characterizing their functions usingcell culture and animal model systems. We have found known and previously unidentified nonsynonymousSNPs in our AML discovery set. To prioritize these SNPs for further study, we will collaboratewith Project 5 to determine the frequency of these SNPs in control populations. We will express high-prioritynon-synonymous SNPs in cell lines and in murine bone marrow transduction-transplantation assays tocharacterize their role in transformation. We will also perform biological validation experiments withmutations and SNPs in cooperation as they are found in our patients to rigorously assess the contribution ofdiscovered mutations to disease pathogenesis.
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