CDK9 Inhibition Sensitizes TP53 Mutated AML to Standard Chemotherapy

Background: The prognosis of AML patients remains poor with cure rates (5-year survivals) continuing to be 35% to 40% for patients younger than 60 years of age and 5% to 15% for patients older than 60. Prognosis is especially poor for those leukemias characterized by mutations or deletions of the TP53 gene since the activity of most chemotherapy agents relies on the p53 protein to trigger apoptosis. Inhibiting CDK9 is a strategy to trigger apoptosis downstream of p53, making mutated p53 less relevant. Dinaciclib has been previously evaluated in small human clinical trials and next-generation CDK9 inhibitors are currently under investigation in patients with hematologic malignancies. Here, we combine dinaciclib, a potent CDK9 inhibitor, with standard azacitidine and venetoclax to demonstrate inhibition of CDK9 is an effective approach to inducing apoptosis in TP53 mutated AML.

Methods: To determine the effect of inhibiting CDK9 on apoptosis of p53 mutated AML, we treated both p53 mutated AML cell lines, THP1, NOMO1, and U937, and wild type (WT) p53 cell lines, MV4-11 and HL-60, with azacitidine, venetoclax, and dinaciclib, as single-agents and in combination. We performed viability assays by CellTiter-Glo (CTG) to examine cell death and determine the IC50 and optimal exposure time of these drugs. After determining the IC50 values, we examined cell viability at various exposure times between 4 and 24 hours. AML cell lines and peripheral blood mononuclear cells (PBMCs) were treated under these conditions as single, dual, and triple-drug combinations and apoptosis was measured through flow cytometry with Annexin-V and 7-AAD staining and through the quantification of apoptotic proteins (c-PARP, caspase-3, MCL-1, and survivin) with immunoblotting.

Results: Treatment of THP1 cells for 24 hours with azacitidine, venetoclax, and dinaciclib as monotherapy demonstrated a dose-dependent decrease in cell viability with IC50 values of 237 nM, 7158 nM, and 6.3 nM respectively. Given these results, we selected concentration ranges of 20 nM-3000 nM azacitidine, 1500 nM-20000 nM venetoclax, and 5-12 nM dinaciclib for our cell viability and synergy experiments. We tested these concentrations at various time points and determined an exposure time of 8-hours provided optimal cell death in the triple-drug combination. When used in the p53 mutant cell line, THP1, and p53 WT cell line, MV4-11, the standard chemotherapy combination of azacitidine and venetoclax was found to have an additive effect (synergy scores of -0.98 and 1.4, respectively using the HSA reference model). Synergy was increased in these cell lines when dinaciclib was added to azacitidine and venetoclax (synergy scores of 29.6 and 12.9, respectively with 12 nM dinaciclib using the HSA reference model). When this drug combination at an exposure of 8-hours was tested in PBMCs of healthy donors, they were notably less sensitive and had lower synergy scores (1.2 with 12 nM dinaciclib using the HSA reference model) than the AML cell lines, suggesting normal cells are less sensitive to CDK9 inhibition. Based on our synergy results, we selected concentrations of 200 nM azacitidine, 6000 nM venetoclax, and 10 nM dinaciclib and treated the p53 mutant cell line THP1 at these concentrations with an exposure time of 8-hours to measure apoptosis. The addition of dinaciclib resulted in increased apoptosis (14.8%) when compared to the two-drug combination of azacitidine and venetoclax (2.0%). When used as a triple-drug combination, these agents readily killed both p53 mutated and WT p53 AML cell lines, with less cytotoxicity in normal PBMCs of healthy donors.

Conclusions: We have shown that the addition of the CDK9 inhibitor dinaciclib to azacitidine and venetoclax individually or in combination results in enhanced cytotoxicity of p53 mutant and p53 WT AML cell lines, even at low concentrations. These results suggest that the inhibition of CDK9 sensitizes AML cells independent of TP53. This action was less pronounced in normal PBMCs. We have ongoing experiments testing this drug combination on normal human CD34+ cells to better determine a therapeutic window to maximize apoptosis in leukemia cells while causing the least cytotoxicity in normal human cells, which should lead to validation studies in vivo.

Sykes:Clear Creek Bio: Consultancy, Current equity holder in private company.