Fig 1: RUNX1/ETO-Expressing AML Cells Are Addicted to CCND2(A) Scheme of the competitive co-culture and transplantation approaches. t(8;21) cells were lentivirally transduced with either a vector linking RPF657 to a non-targeting control shRNA (shNTC) or dTomato to an shRNA targeting either RUNX1/ETO (shRE) or CCND2 (shCCND2-1, −3). Control and knockdown cells were mixed 50:50 followed by co-culture (Kasumi-1 and SKNO-1) or intrahepatic transplantation into newborn RG mice (Kasumi-1). shRE, RUNX1/ETO shRNA; shCCND2, CCND2 shRNA; shNTC, non-targeting control shRNA.(B) Graph showing percentage of shRE-, shCCND2-1- or shCCND2-3 expressing Kasumi-1 and SKNO-1 cells compared with snNTC expressing cells during LTC.(C) Percentage of Kasumi-1 cells with indicated shRNA in transplanted RG mice. BL, starting pool prior to transplantation; RG, cells harvested from transplanted RG mice humanely killed at clinical endpoints. Mean ± SD, n = 5.(D) Proliferation curves for Kasumi-1 and SKNO-1 cells electroporated sequentially every two days with the indicated siRNAs. Mock; non-siRNA electroporated cells; siCCND2, CCND2 siRNA; siMM, mismatch control siRNA. Kasumi-1, n = 3, mean ± SD; SKNO-1, n = 1.(E) Colony formation of Kasumi-1 cells transduced with the indicated siRNA constructs at 12 days post plating. CFU, colony-forming unit. Mean ± SD; Kasumi-1, n = 3; ∗∗p < 0.01; ∗p < 0.05 compared with Mock.(F) Cell cycle distribution of Kasumi-1 and SKNO-1 cells with and without CCND2 knockdown. Mean ± SD; n = 3. Counts at 12 days post plating. Mean ± SD; Kasumi-1, n = 3; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05 compared with siMM.(G) Senescence in Kasumi-1 and SKNO-1 cells as indicated by staining for senescence-associated β-galactosidase (SA-βGAL) after two sequential electroporations with the indicated siRNAs. Top panels, stained cells; bottom panel, quantitation of SA-βGAL+ cell numbers. n = 2 technical replicates; mean ± range. Standard bar, 50 μm.(H) Immunoblots showing the effect of RUNX1/ETO and CCND2 knockdown in Kasumi-1 and SKNO-1 cells on phosphorylation of RB1. Numbers indicate fold changes.(I) Cell cycle distribution of Kasumi-1 cells after 5 days with and without dnFOS induction by doxycycline. Ctrl, empty vector control; dnFOS, dnFOS vector-containing cells. Mean ± SD; n = 3. ∗∗∗p < 0.001 compared with no dox.(J) Impact of dnFOS inductions on cell doubling times (tD). Mean ± SD; n = 3. ∗∗∗p < 0.001 compared with no dox.(K) Impact of dnFOS induction on clonogenicity of Kasumi-1 cells. Colonies were counted 12 days post plating relative to no dox. Mean ± SD; n = 3. ∗p < 0.05 compared with no dox.See also Figure S4.