Oncogenic Nras Grants Apoptosis Resistance to Hematopoietic Stem Cells

Abstract

Genetic lesions hijack the regulatory programs of hematopoietic stem cells and progenitors (HSC/P) to transform them into leukemic stem cells (LSC). These initiating mutations confer a competitive advantage to promote clonal expansion of pre-leukemic stem cells (pre-LSC). Pre-LSCs are believed to serve as a reservoir for leukemia relapses and a cure for leukemia likely depends on the eliminating of pre-LSCs. Better understanding of how mutations dysregulate HSCs to transform them into pre-LSCs will identify new therapeutic targets to eliminate pre-LSCs.Activating RAS mutations are highly prevalent in hematopoietic malignancies. We have previously shown that a single allele of oncogenic NrasG12D promotes clonal expansion in hematopoietic stem cells (HSCs) through increased proliferation and self-renewal, but the role apoptosis plays in the clonal dominance of NrasG12D HSCs remains unclear. Here we report that oncogenic NRasG12D protects HSCs from cellular stress. Upon cytokine starvation and γ-irradiation, HSCs fromMx1-cre; LSL-NrasG12D/+ mice display reduced apoptosis as measured by Annexin V staining and Caspase 3/7 activation. This NRasG12D-mediated HSC survival is not dependent on autophagy since Bafilomycin A, an inhibitor previously shown to inhibit autophagy in HSCs, did not rescue the phenotype. Moreover, NrasG12D HSCs exhibits decreased levels of cellular reactive oxygen species (ROS), and restoration of ROS levels with buthionine sulfoximine significantly blocked the survival of NrasG12D HSCs.We next sought to identify the signaling activated by NRasG12D to promote HSC survival. Although our previous studies show that STAT5 is required for NRasG12D-mediated HSC proliferation, STAT5 is dispensable for NRasG12D-mediated HSC survival. We then determined whether inhibition of the canonical Ras effector pathways blocks NRasG12D-mediated HSC survival. To our surprise, inhibition of PI3K/AKT, MEK/ERK, or mTOR signaling did not abrogate the pro-survival effect of NRasG12D in HSCs. However, inhibition of protein kinase C (PKC) with two structurally independent inhibitors rescued the pro-survival phenotype of NrasG12D/+ HSCs. PKC inhibition also led to increased ROS levels in NrasG12D mutant HSCs, suggesting that NRasG12D reduces ROS and protects HSCs from stress through activation of PKC signaling. Taken together, we discover that in addition to increasing HSC proliferation, oncogenic NRasG12D promotes HSC survival under stress conditions. Furthermore, we reveal a pathway NRasG12D relies on to evade apoptosis and manage reactive oxygen species in HSCs independent of the STAT5 signaling that governs cell proliferation. Targeting PKC signaling, alone or in combination with STAT5 signaling, may have therapeutic benefit in eliminating pre-LSCs. DisclosuresNo relevant conflicts of interest to declare.