Oncogenic Nras Increases Hematopoietic Stem Cell Proliferation and Self-Renewal Through a Bimodal Effect

Abstract 119 Accumulating evidence suggests that most leukemias are initiated by rare leukemic stem cells (LSC) that are transformed from the normal hematopoietic stem cells and progenitors (HSC/P) by genetic lesions that lead to activation of oncogenes and inactivation of tumor suppressor genes. However, the signaling mechanisms by which these genes transform HSC/P into LSC are poorly understood. Activating mutations of NRAS and KRAS are highly prevalent in acute myeloid leukemia (AML), some myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). In addition other leukemia associated genetic lesions, such as the BCR-ABL fusion, PTPN11 mutations, FLT3 internal tandem duplications, and NF1 inactivation all deregulate Ras signaling. We previously developed a mouse strain that conditionally expresses an oncogenic NrasG12D allele from the endogenous locus. This consistently resulted in an indolent MPD with delayed onset and prolonged survival in Mx1-cre, NrasG12D/+ mice (referred to as NrasG12D). Oncogenic NrasG12D, however, cooperated with the MOL4070LTR retrovirus to induce AMLs that share molecular and morphologic features with human M4/M5 AML. Here we report that NrasG12D directly affects HSC/P functions. While normal HSCs must remain quiescent to maintain the long term self-renewal capacity and mutations that drive HSC into cycle often lead to HSC depletion, NrasG12D increased HSC proliferation but at the same time increased the self-renewal and competitiveness of HSCs. Serial transplantations revealed that NrasG12D HSCs were able to give higher level of reconstitution than wild-type (WT) HSCs and gave rise to long term multi-lineage reconstitution in lethally irradiated mice after up to four rounds of transplantation while WT HSCs failed to reconstitute beyond two rounds. These effects were not associated with the development of leukemia suggesting oncogenic Nras dys-regulates HSC at a pre-leukemic stage and therefore plays an important role in leukemia initiation. Using histone-2B-GFP (H2B-GFP) label-retaining assays, we further detected a “bimodal” effect of NrasG12D on HSCs: NrasG12D induced a subpopulation of rapid “cycling” HSCs that lost GFP labeling and reconstitution activity faster than WT HSC but another HSC subpopulation that remained more “quiescent” than WT HSCs and retained higher reconstitution when transplanted to irradiated mice. The canonical Ras effector, ERK, was not activated in NrasG12D HSC/Ps and inhibition of ERK with a MEK inhibitor, PD325901, did not have any effect on the Nras induced increase of HSC proliferation. Stat5, on the other hand, was significantly activated in NrasG12D HSC/Ps and heterozygous knockout of Stat5ab abolished the increased proliferation in NrasG12D HSCs, suggesting that Stat5 signaling mediates at least part of the Nras induced increase in HSC proliferation. Nras is thus the first signaling pathway that simultaneously increases HSC proliferation, self-renewal and competitiveness without inducing frank leukemogenesis. This is likely through a “bimodal” effect of Nras signaling on HSC cell cycle regulation. Our studies also identified Stat5 as a novel therapeutic target to inhibit early events in Ras mediated leukemic transformation. Disclosures: No relevant conflicts of interest to declare.