Ras-Pathway Inhibition With Targeted Therapies Abrogates Self-Renewal In Acute Myelogenous Leukemia

Hyperactivated Ras-pathways serve as oncogenic drivers in multiple human tumors including acute myelogenous leukemia (AML) (Ahearn et al. Nat Rev Mol Cell Biol 2011). The specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Recently, we have shown that NRAS^G12V–activated signaling pathways are critical to leukemia stem cell maintenance (Sachs et al. submitted). To elucidate which Ras-activated signaling molecules mediate self-renewal in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRAS^G12V) and develops AML (Kim et al. Blood 2009). Primary leukemia cells were treated with therapeutic agents targeting Ras-activated signaling pathways. We used PD325901 to inhibit the Mek-Erk pathway, GDC0941 to inhibit the Pi3k pathway, and RAD001 to inhibit the mTor pathway. Using MTS assays, we identified the IC₅₀ dose for each of these agents. Inhibitor-treated leukemia cells were submitted for RNA sequencing in order to investigate the effects of these agents on leukemia gene expression. Previously, we identified a list of NRAS^G12V responsive genes in our model. In these studies, we identified that PD325901-treatment most closely recapitulates the effect of NRAS^G12V inhibition on this comprehensive list of RAS-responsive genes. However, when we study the effects of these inhibitors on the subset of RAS-responsive genes that mediate leukemia self-renewal, we find that both PD325901 and RAD001 independently recapitulate the effects of NRAS^G12V withdrawal on this subset of genes implicating the Mek and mTor pathways in leukemia self renewal. Next, we treated primary leukemia cells with the IC₅₀ dose of each drug and plated them in colony forming assays. We found that Mek or mTor inhibition, but not Pi3k inhibition, abrogated secondary colony formation corroborating our gene expression analyses and showing that, at doses that have equivalent effects on cell growth, only the Mek and mTor pathways are important for leukemia cell stem cell maintenance. These studies provide potential targets for leukemia stem cell-specific therapies.