Abstract 4176: Activated NRAS mediates self-renewal capacity in AML

NRAS mutations are found in 11-14% of AML (Schubbert et al. Nat Rev Cancer 2007, Bowen et al. Blood 2006), implicating NRAS signaling pathways as potential targets for clinical intervention. However, the specific effects of targeting these pathways on AML physiology are currently unclear. We employ a manipulatable murine model of AML to elucidate these phenomena for therapeutic applications. This model harbors a tetracycline repressible, activated NRAS (NRASG12V) transgene along with an Mll/AF9 transgene. Primary leukemia cells are then transplanted into SCID mice and, upon development of clinically evident leukemia, NRASG12V transgene expression is repressed with doxycycline. NRASG12V repression leads to apoptosis and disease remission (Kim et al. Blood 2009). To dissect the critical signaling networks directing remission, immunophenotypic markers, along with the phosphorylation status of critical signaling intermediates, were analyzed by flow cytometry after NRASG12V repression. This analysis revealed that NRAS withdrawal led to an enrichment of less differentiated Mac1/Gr1 negative cells indicating that, in addition to well-described effects on proliferation, NRAS affects leukemia differentiation status. Furthermore, we only found modifications in known NRAS signaling effectors in the less differentiated, Mac1 negative leukemia cells. Therefore, in addition to affecting the differentiation status of the leukemia, oncogene withdrawal leads to cell-type specific signaling alterations. We hypothesize that these signaling alterations mediate remission-induction by altering the self-renewal capacity of the leukemia at the gene expression level. Parallel RNA sequencing and gene expression microarrays were performed to address this hypothesis and revealed that the gene expression pattern of NRASG12V-expressing cells is enriched with hematopoietic self-renewal genes (Krivtsov et al. Nature 2006, Ivanova et al. Science 2002, Gene Set Enrichment Analysis per Subramanian et al. PNAS 2005). Withdrawal of the oncogene led to loss of this self-renewal gene expression program and an enrichment of genes expressed in terminally differentiated, mature cells. Furthermore, the NRASG12V-dependent gene expression program mimics the Mll/AF9-determined, Myb-mediated self-renewal program seen in a similar AML model (Zuber et al. Genes & Dev 2011). These data suggest that the Mll/AF9-mediated self-renewal program requires the continued expression of NRASG12Vand support a novel r