mTOR and MEK inhibition recapitulate the effects of NRAS^G12V withdrawal on leukemia self-renewal capacity. (A) Primary leukemia cells were treated with RAS-pathway inhibitors and plated in CFAs. Colonies were scored as the number of colonies that developed per 10 000 cells plated. The percentage of colonies that developed from drug-treated cells, relative to vehicle-treated cells from the same experiment, is shown. Each bar represents the average value of 4 to 13 experiments (doxycycline treatment was performed 13 times, RAD001 and PD325901 treatments were performed 4 times, and GDC0941 treatment was performed 8 times; vehicle treatment was performed at each experiment). Error bars represent the standard error of the mean. (B) RNA was extracted from primary leukemia cells after 24-hour in vitro treatment with RAS-pathway inhibitors and submitted for RNA sequencing in triplicate. Doses used were PD325901 20 μM, RAD001 26 μM, and GDC0941 20 μM. Normalized transcript expression levels were used for GSEA, comparing our data set with leukemia-specific self-renewal gene lists, and showed a loss of the MLL-AF9–directed signature in MEK and mTOR-inhibited cells. (C) Schematic figure indicating the NRAS^G12V-driven pathways that mediate leukemic self-renewal based on our findings.