Endogenous Oncogenic Nras Signaling Promotes Proliferative Exhaustion of Hematopoietic Stem Cells Through a Dose-Dependent Hyperactivation of ERK,

Abstract 3997

Both monoallelic and biallelic oncogenic NRAS mutations are identified in human leukemias, suggesting a dose-dependent role of oncogenic NRAS in leukemogenesis. We recently characterized conditional mouse models that express one or two copies of constitutively active Nras G12D in the hematopoietic compartment (Nras G12D/+ and Nras G12D/G12D). Nras G12D/G12D results in stronger downstream signaling than Nras G12D/+ and consequently distinct hematopoietic phenotypes. In particular, we found that somatic expression of Nras G12D/G12D but not Nras G12D/+ leads to an acute myeloproliferative neoplasm (MPN). The development of acute MPN is associated with cytokine-evoked hyperactivation of ERK but not Stat5 and Akt in hematopoietic stem/progenitor cells (HSPCs). Interestingly, genetically altered HSCs appear to be required for initiation and maintenance of chronic and acute MPD phenotypes mediated by Nras G12D/+ and Nras G12D/G12D, respectively. Furthermore, Nras G12D/G12D-mediated signaling promotes excessive HSC proliferation and leading to HSC exhaustion. Exhaustion of HSCs is tightly associated with diminished MPN phenotypes in non-engineered recipient mice into which Nras G12D/G12D HSCs are transferred by bone marrow transplantation. To investigate the molecular mechanisms underlying the exhaustion of Nras G12D/G12D HSCs, we performed a microarray analysis using highly purified control, Nras G12D/+, and Nras G12D/G12D HSCs. To our surprise, neither p16INK4a and p15INK4b, critical cell senescence genes, or other genes or pathways known to involve in HSC self-renewal were differentially expressed in Nras G12D/G12D HSCs. In contrast, gene ontology analysis revealed significant differential expression of genes in the MEK/ERK pathway in Nras G12D/G12D HSCs. This result is consistent with our phospho-flow study showing that Nras G12D/G12D selectively hyperactivates ERK in HSPCs. It has been documented that prolonged ERK signaling is a potent inducer of differentiation in cultured PC12 cells and inhibition of normal ERK signaling by a chemical inhibitor increases self-renewal in mouse embryonic stem cells. Thus, we are currently testing whether hyperactivation of ERK promotes depletion of Nras G12D/G12D HSCs through promoting HSC differentiation and whether blocking hyperactivation of ERK restores the normal HSC function and prevents MPN development.