Ras Effector Pathways Differentially Support Proliferation and Survival of Acute Myeloid Leukemia

Abstract

Mutations that lead to activation of Ras oncogenes and their effector pathways are common in acute myeloid leukemia (AML) making Ras signaling an attractive therapeutic target. To date, efforts to therapeutically target Ras directly have proven difficult and clinical outcomes have been disappointing. The rational development of strategies for targeting Ras effector pathways in AML have been hindered by a limited understanding of the how specific Ras pathways maintain AML proliferation and survival. To determine the roles of specific Ras effector pathways in AML we disrupted signaling through canonical Ras effector pathways (RAF-MEK-ERK, PI3K-AKT-mTOR, and RAL) in murine and human AML cells. Consistent with previously published data (Kim et al. Blood. 2009;113:1086), we found that suppression of NRAS(V12) expression in a tetracycline-repressible NRAS(V12) and Mll-AF9-driven murine AML model (tNM AML) led to AML remission in leukemic mice in vivo and almost completely suppressed leukemic-colony formation in vitro (20-fold reduction, p <0.01). In the tNM model suppression of the NRAS oncogene led to apoptosis of AML cells, but had little effect on cell cycle progression. Surprisingly, pharmacologic inhibition of the major oncogenic Ras effector pathways, RAF-MEK-ERK and PI3K-AKT-mTOR, alone or in combination suppressed AML cell growth by inducing G0/G1 cell cycle arrest in both tNM AML cells and human AML cell lines, but did not promote apoptosis in either. Conversely, pharmacologic inhibition of RALB signaling in both mouse and human AML cells induced apoptosis, but had little effect on cell cycle progression. Notably, these effects were seen in both NRAS mutant (tNM and THP1) and NRAS wild-type (MOLM13) AML cells. In preclinical studies, the RALB inhibitor Dinaciclib and the dual PI3K/mTOR inhibitor BEZ235 synergized to suppress AML growth in vitro, and had a potent anti-leukemic activity against tNM AML in vivo. We are currently evaluating the effects of a dominant negative mutant of RALB, RALB(N28), on AML growth and survival, and investigating whether RALB signaling supports AML survival through TBK1, NF-kB, and BCL2 family members as has been shown for KRAS-driven epithelial cancers (Barbie et al. Nature. 2009;462:108). By elucidating the distinct roles for RAS effector pathways in maintaining AML proliferation and survival, we have uncovered a critical role for RALB signaling in AML survival. This work suggests that combination therapy targeting both Ras effectors that promote proliferation (PI3K/mTOR) and survival (RALB) may provide a rational treatment approach for AML. DisclosuresLargaespada:NeoClone Biotechnology: Consultancy, Equity Ownership; Discovery Genomics, Inc.: Consultancy, Equity Ownership.