Fig. 1: Treatment with CDK9 inhibitors dose-dependently induced apoptosis, decreased chromatin accessibility and depleted oncogene expressions in sAML cells.

A HEL92.1.7 (HEL) and SET-2 cells were treated with the indicated concentrations of BAY-1143572 (BAY) or NVP2 for 48 h. At the end of treatment, cells were washed with 1× PBS and stained with annexin V and To-PRO-3 iodide. The % of annexin V-positive, apoptotic cells were determined by flow cytometry. Curves represent the mean of three experiments ± S.E.M. B SET-2 and ruxolitinib-persister/resistant SET-2-RuxP cells were treated with the indicated concentrations of BAY-1143572 or NVP2 for 48 h. Following this, the % of annexin V-positive, apoptotic cells were determined by flow cytometry. Curves represent the mean of three experiments ± S.E.M. C PD, CD34+ sAML cells were treated with the indicated concentrations of BAY-1143572 or NVP2 for 48 h. Following this, cells were washed with 1× PBS and stained with propidium iodide (PI). The % of PI-positive, non-viable cells were determined by flow cytometry. Horizontal black lines represent the mean loss of viability in the PD sAML samples. D SET-2 cells were treated with 5 µM of BAY-1143572 or 250 nM of NVP for 16 h. Total nuclei were isolated and ATAC-Seq analysis was performed utilizing Tn5 transposase and next generation sequencing. The total number of gained and lost peaks in the treated versus untreated cells was determined utilizing diffReps. E IGV plot of ATAC-Seq peak densities in SET-2 cells treated with BAY-1143572 or NVP2 for 16 h. Significantly altered down peaks (≥1.25-fold down relative to untreated and p-value <0.05) are noted by blue boxes. The red bar indicates the position of the MYC super enhancer locus (Enhancer regions 1–5). The approximate locations of the individual enhancer peaks are E1 = 130,548 kb; E2 = 130, 559 kb; E3 = 130, 595 kb; E4 = 130, 604 kb and E5 = 130, 679 kb. F SET-2 cells were treated with 250 nM of NVP2 for 8 h. Total RNA was harvested and utilized for reverse transcription. The resulting cDNA was utilized for quantitative PCR with TaqMan probes as indicated. The relative expression of each mRNA was normalized to the expression of GAPDH and compared to the untreated control cells. G Immunoblot analysis of SET-2 and SET-2-RuxP cells following 18 h of treatment with NVP2. The expression levels of GAPDH in the cell lysates served as the loading control.