CDK9 inhibition enhances the suppressive effects of cytarabine in vitro and in vivo. (A) U937 cells were plated in methylcellulose in the presence of control (DMSO), atuveciclib (Atuv; 0.5 µM), or cytarabine (Ara-C; 5 ng/mL), or a combination of atuveciclib and cytarabine for 7 days. CFU-L was assessed in clonogenic assays in methylcellulose. Data are expressed as a percentage of DMSO control-treated cells. Shown are the means + SE of 4 independent experiments. **P < .01 using a 1-way ANOVA with Tukey’s multiple comparisons test. (B) MV4-11 cells were seeded in methylcellulose in the presence of control (DMSO), atuveciclib (0.3 µM), or cytarabine (10 ng/mL) or a combination of atuveciclib and cytarabine, for 7 days. CFU-L was assessed in clonogenic assays in methylcellulose. Data are expressed as a percentage of DMSO control-treated cells. Shown are the means + SE of 4 independent experiments. *P < .5, ***P < .001 using a 1-way ANOVA with Tukey’s multiple comparisons test. (C) Tumor volumes from an AML xenograft model are shown. MV4-11 cells were subcutaneously injected into the left flank of athymic nude mice. Once mice had measurable tumors, mice were randomized into 5 animals per group treated with control, cytarabine, atuveciclib, and the combination of atuveciclib and cytarabine. Mice were treated for a total of 12 days (days 21-33 postinjection). Start and end of treatment are indicated by arrows on the graph. (D) Survival analysis of mice that are described in panel C. Survival was determined as time to euthanasia. (E) For analysis of CDK9 target gene expression after treatment, a separate cohort of mice was treated as described in panel C for 3 days. Tumors were harvested and frozen, and then RNA was extracted. Gene expression for MYC, and PIM1 was measured by qRT-PCR, using GAPDH for normalization. Data are expressed as fold increase over the average of the expression levels in tumors from control mice, and shown are means + SE of 4 separate mice per group. (F) Immunoblotting analyses for p-rpS6 detection were conducted on frozen tumor lysates from a separate cohort of mice treated as per panel C for 3 days. Cell lysates from tumors for the indicated treatment conditions were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The immunoblots with antibodies against the phosphorylated forms of rpS6 or against total rpS6 were analyzed in parallel by SDS-PAGE. Densitometry was performed and calculated using the total rpS6 for each respective mouse for normalization. Each lane represents an individual tumor.