NRAS/Nras expression is required for maintenance of AML harboring oncogenic mutations. (A) Human AML cell lines with mutations in NRAS (OCI-AML3, HL60, THP-1), KRAS (NB4), or FLT3 (MV4-11 and MOLM-14) were infected with control shRenilla targeting Renilla luciferase and 2 independent NRAS shRNAs shown in panel B. Note depletion of NRAS-mutant AML cells expressing either NRAS hairpin. Max, maximum. (B) Western blot demonstrating N-Ras protein knockdown in each of the human AML cell lines using shNRAS.30, 34, and shRenilla control. (C) Western blot demonstrating N-Ras protein knockdown in primary murine Nras^G12D AML #6695 expressing shNras.26, shNras.30, or the control shRenilla. We infected AML #6695 with shNras.26, shNras.30, or shRenilla at equivalent multiplicities of infection (input mCherry-positive cells 17% to 29% of total AML), bulk transplanted into recipient mice (n = 4 mice per shRNA), and measured the percentage of mCherry-positive blasts (D) in BM and spleen (E) 3 weeks later. Note the selective dropout of shNras-expressing cells with 2 independent shRNAs (P < .0001). (F) Survival of mice transplanted with Nras^G12D AML #6695 (red lines, n = 10) or with a control AML generated in a WT mouse (black lines, n = 6). AMLs were infected with shNras.30 (solid lines) or control shRenilla (broken lines) and sorted to near purity before transplant. Nras knockdown prolongs survival in Nras-mutant AML (P = .0052). (G) Nras message knockdown in primary mouse AML cells before transplant (shNras.30) assessed by quantitative polymerase chain reaction. Early relapse (day 62) shNras.30 mCherry-positive leukemia (relapse) partially restored Nras expression relative to Renilla controls.