Fig 2: Effect of P4 on protein abundance of CASP3 (A), Bax (B), CCND2 (C), CYP11A1 (D), CYP19A1 (E), and StAR (F) in pTr cells after 48 h treatment. The results represent the average ± SEM of three independent experiments; * indicates values significantly different from that of the control group (p < 0.05); ** indicates values significantly different from that of the control group (p < 0.01). Original Western blot bands and intensity ratio are shown in Figure S1 and Table S1.

Fig 3: RUNX1/ETO Regulates CCND2 Expression by Promoting AP-1 Activity(A) Log fold change of three JUN shRNA construct levels in in vivo screens in Kasumi-1 and SKNO-1 cells. ***p < 0.001; **p < 0.01 compared with no dox controls.(B) UCSC screenshot showing JUND and RUNX1/ETO binding to the CCND2 locus with and without RUNX1/ETO knockdown in Kasumi-1 cells.(C–E) (C) Manual ChIP validation of JUND binding at the CCND2 promoter with and without RUNX1/ETO knockdown in Kasumi-1 cells. shRE, RUNX1/ETO shRNA; shNTC, non-targeting control shRNA. n = 3; mean ± SD; *p < 0.05 compared with shNTC. (D) Changes in transcript levels of JUN and FOS members upon RUNX1/ETO knockdown in Kasumi-1 cells as assessed by RNA-seq. Mean ± SD; n = 3. ***p < 0.001; *p < 0.05 compared with shNTC. (E) Expression of dnFOS transcript in Kasumi-1 cells lentivirally transduced with dnFOS or control (Ctrl) vector. Cells were incubated for 5 days with and without dox. n = 3; mean ± SD; ***p < 0.001.(F) Impact of dnFOS induction by doxycycline in Kasumi-1 cells on relative CCND2 transcript levels measured by qPCR normalized to GAPDH. n = 3; mean ± SD; *p < 0.05.(G) Immunoblot showing CCND2 protein levels in Kasumi-1 cells upon dnFOS induction for 5 days. dnFOS#1 and dnFOS#2, FOS overexpressing clones 1 and 2, respectively; Ctrl, normal Kasumi-1 cells.(H) Scheme depicting a model for the regulation of CCND2 by RUNX1/ETO. Depletion of RUNX1/ETO enhances interaction between -30 kb and TSS, increases H3K9 acetylation and occupation of the -30 kb element by RUNX1 and RNA Pol II, impairs AP1 binding at the promoter, and stalls RNA Pol II at the TSS, leading to reduced CCND2 transcription.See also Figure S3.

Fig 4: CDK4/6 Inhibition Impairs Proliferation of Primary AML Cells and Increases Median Survival In Vivo(A) Impact of palbociclib on proliferation of primary AML blasts. Blasts were co-cultured on MSC feeder layers with and without 300 nM palbociclib for 72 hr. Mean ± SD; n = 3 for both t(8;21) and non-t(8;21) AML patient samples. *p < 0.05 compared with no palbociclib.(B) Phase contrast photographs showing primary t(8;21) AML blasts from patient sample LK111 in co-culture with MSCs with and without (CV) palbociclib. Standard bar, 200 µm.(C) Proliferation of a t(8;21) AML sample from a relapsed patient on MS-5 feeders upon palbociclib treatment for 72 hr. Mean ± SD; n = 3 technical replicates.(D and E) Cell cycle distribution (D) and changes in apoptotic subG1 cell fractions (E) of primary AML blasts obtained from two t(8;21) patients (patient samples LK19 and LK111) on MSC after 96 hr incubation with and without (CV) palbociclib.(F) Clonogenic growth of three different t(8;21) patient samples after MSC co-culture for 96 hr. Indicated palbociclib concentrations were added either only to co-culture medium (Palbociclib in co-culture) or to both co-culture and semisolid medium (Palbociclib in co-culture & CFA). Colony numbers are relative to no palbociclib. Mean ± SD; n = 3. ***p < 0.001; *p < 0.05 compared with no palbociclib.(G) Bioluminescent images of RG mice transplanted with luciferase-expressing (luc+) Kasumi-1 cells after 21 days of treatment with control vehicle (CV) or palbociclib.(H) Luminal flux of bioluminescence for CV (n = 8) or palbociclib-treated animals (n = 9). Treatment blocks are indicated at the bottom of the graph. **p < 0.01; *p < 0.05 compared with no palbociclib by one-way ANOVA using a D'Agostino and Pearson test; mean ± SEM.(I) Survival curve for RG mice transplanted with luc+ Kasumi-1 cells. Significance was tested by log rank test.(J) Percentage of Kit+ RUNX1/ETO9a (AE9a) GFP+ cells in mice after CV or palbociclib treatment as determined by fluorescence-activated cell sorting (FACS).(K) Survival curve for Bl6 mice transplanted with KIT+ AE9a GFP+ cells. Significance was tested by log rank test. n = 5 for both groups with one censored animal in the palbociclib group due to death unrelated to palbociclib treatment.(L) Model depicting the RUNX1/ETO-promoted G1 cell cycle progression and leukemic propagation by direct transcriptional activation of CCND2 and CDK6, which can be blocked by either CCND2 knockdown or by pharmacologic inhibition of CDK4/6-CCND complexes.See also Figure S7.

Fig 5: The CDK4/6 Inhibitor Palbociclib Inhibits Growth of RUNX1/ETO-Expressing Leukemic Cells(A) Dose-response curve for proliferation of SKNO-1 and Kasumi-1 cells treated for 72 hr with the indicated palbociclib concentrations. Mean ± SD; n = 6.(B) Dose-response curve for inhibition of colony formation by palbociclib. Colonies formed in presence of palbociclib were counted 14 days after seeding. Mean ± SD; n = 3.(C) Growth curves of t(8;21) cell lines during long-term treatment with palbociclib. Mean ± SD; n = 3.(D) Dose-response curve for proliferation of CD34+ cord blood cells expressing truncated RUNX1/ETO9a treated with palbociclib for 72 hr. Mean ± SD; n = 3.(E) Cell cycle distribution of Kasumi-1 and SKNO-1 cells after 72 hr treatment with the indicated palbociclib doses. Mean ± SD; n = 3. ***p < 0.001; *p < 0.05 compared with no palbociclib (CV).(F) Cell cycle distribution as indicated by Pyronin Y and Hoechst33342 staining of Kasumi-1 and SKNO-1 cells with and without 50 nM palbociclib for 24 hr. ***p < 0.001; **p < 0.01 compared with no palbociclib (CV).(G) Impact of CDK4/6 inhibition on senescence in Kasumi-1 and SKNO-1 cells as indicated by staining for SA-ßGAL. Top panels, stained cells after 7 days with 50 nM palbociclib; bottom panel, quantitation of SA-ßGAL-positive cell numbers. Mean ± SD; n = 5 technical replicates. Scale bar, 50 µm.(H) Immunoblots showing dose-dependent impact of 72 hr palbociclib treatment on indicated protein levels in Kasumi-1 and SKNO-1 cells.(I) GSEA for correlation between palbociclib, RUNX1/ETO, and CCND2 knockdown signatures derived from RNA-seq. NES, normalized enrichment score.(J) Hallmarks of cancer pathways shared between palbociclib treatment, CCND2, and RUNX1/ETO knockdown. Enriched pathways were identified by GSEA.See also Figure S6.