Human MA9/NRas cells were transduced to allow Tet-inducible shRNA expression marked by dsRed. Knockdown of CDC42 was achieved by 2 distinct shRNA sequences (sh170 and sh347) on treatment with 2 mg/ml doxycycline (2 DOX). Nonspecific shRNA targeting Renilla-luciferase served as the control (shREN). (A) CDC42 knockdown led to decreased CFU-C in methylcellulose and (B) increased apoptosis as measured by AnnexinV⁺ staining, with greater effect on apoptosis seen later at day 7 in culture. (C) CDC42 knockdown inhibited human MA9/NRas cell growth in liquid culture. (D) Wright-Giemsa stain showed typical blast morphology in shRenilla control cultures treated with doxycycline (DOX; left panel), whereas sh170 DOX cultures showed increased differentiation and vacuolization (right panel). Scale bar, 50 μm. (E) Bioluminescent imaging obtained at 4 weeks posttransplant showed decreased in vivo leukemia progression on CDC42 knockdown. (F) Kaplan-Meier analysis showed prolonged survival from knockdown of CDC42 (P < .01). A primary human patient AML sample bearing a t(X;11) translocation encoding the MLL-SEPT6 fusion was transduced with lentiviral vectors expressing CDC42 shRNA (sh471) vs nontargeting control and the Venus fluorescent protein, then injected into NSG mice. (G) FACS analysis at day 3 showed 16.8% and 23.3% Venus+ cells in the nontargeting control (NT) and sh471 transduction cultures, respectively. (H) BM was harvested and analyzed for Venus⁺ cells at week 4 posttransplant to reveal the loss of MLL-SEPT6 AML graft on CDC42 knockdown (P < .002). FSC, forward scatter; N.S., not significant.