Activated NRAS Mediates Self-Renewal Capacity in AML by Facilitating the Mll/AF9-Specified Gene Expression Signature

Abstract 5116 RAS is a known oncogene in AML (Schubbert et al. Nat Rev Cancer 2007, Bowen et al. Blood 2006), however the specific effects of targeting RAS-activated pathways on AML physiology are unclear. NRAS G12V transgene repression in an NRAS G12V / Mll/AF9 transgenic murine AML model leads to apoptosis and disease remission (Kim et al. Blood 2009). To better understand possibilities for and implications of therapeutic targeting of RAS-activated pathways, we inactivate NRAS G12V in this mouse model and characterize the subsequent signaling and transcriptional response. We profiled signaling intermediates and markers of apoptosis and cell cycle using mass cytometry, a next-generation flow cytometry technology, which simultaneously measured the levels of 32 antibody-labeled proteins in single cells. These analyses revealed specific signaling changes that varied with the surface immunophenotype of the AML cells and highlighted relevant RAS-directed signaling pathways. Parallel RNA sequencing and gene expression microarrays revealed that NRAS G12V -expressing cells express hematopoietic self-renewal genes and repression of NRAS G12V leads to loss of this program. Importantly, the NRAS G12V -dependent gene expression program mimics the Mll/AF9 -determined, Myb-mediated self-renewal program reported by Zuber et al. (Zuber et al. Genes Dev 2011). These data suggest a novel role for RAS in AML self-renewal capacity by maintaining the Mll/AF9 -mediated self-renewal program. Furthermore, inhibition of the PI3K-mTOR pathway decreases viability, in vitro colony formation, and recapitulates the effects of NRAS G12V inactivation on self renewal-associated genes, implicating this pathway as the mediator of RAS-directed leukemia self renewal capacity. These data provide rationale for therapeutic targeting of leukemia stem cells via PI3K-mTOR pathway inhibition and for using this mouse model as a tool to test such therapeutic approaches. Since a variety of AML-specific genetic mutations lead to perturbations in RAS signaling, these results may be generalizable to AML with a broader range of mutations. Disclosures: Largaespada: Discovery Genomics, Inc. : Consultancy, Equity Ownership; NeoClone Biotechnology, Inc. : Consultancy, Equity Ownership.