Abstract 5978: Cyclin dependent kinase 9 inhibitor AZD4573 induces cell death through DNA damage accumulation in breast cancer cells in vitro

Background: Cyclin dependent kinase 9 (CDK9) regulates progression of RNA polymerase II through phosphorylation. Inhibition of CDK9 induces transcription-replication conflicts (T-R conflicts), leading to replication stress. AZD4573, a highly selective CDK9 inhibitor, showed anti-tumor effects in acute myeloid leukemia in vitro and in vivo. Recently it is being evaluated in phase 2 clinical trial (NCT04630756). However, the anti-tumor effect of AZD4573 is unknown in breast cancer. Thus, we investigated the anti-tumor effect of AZD4573 in breast cancer cells and explored the underlying mechanism. Methods: To evaluate the antitumor effect in vitro, MTT assays were preformed using increasing concentration of AZD4573 (doses range : 0-100 nM). The IC50 values were calculated using Sigma Plot software. Cell cycle analysis was performed using flow cytometry analysis. The expression of signal transduction molecules was determined using quantitative PCR, western blotting, and immunofluorescence. Apoptotic cell death was verified by annexin-V assay. DNA strand breaks and repair efficacy were evaluated through alkaline comet assay. siRNA knock-down system was used to confirm the action mechanism. Results: SK-BR-3, HCC70, HCC1937 and ZR-75-1 were sensitive to AZD4573 with IC50 ranging from 9.16-25.81 nM, whereas T47D and HCC1428 were less-sensitive (IC50 >100 nM), as assessed by MTT assay. After treatment of AZD4573, sensitive and less-sensitive cells showed increased T-R conflicts in 10 minutes. T-R conflicts were maintained in SK-BR-3, HCC70, HCC1937 and ZR-75-1 for 3 hours, but T-R conflicts disappeared immediately in T47D and HCC1428 cells. Moreover, expression of γ-H2AX, cleaved PARP and cleaved caspase-3 was increased, indicating the induction of apoptosis in sensitive cells. Contrary to these results, γ-H2AX induction and apoptosis were not observed in T47D and HCC1428 cells. siRNA knock-down of CDK9 in HCC1937 resulted in an increase of T-R conflicts, γ-H2AX, with induction of PARP cleavage and caspase-3 cleavage. Conclusion: AZD4573 induced T-R conflicts and subsequent DNA damage. Accumulation of DNA damage caused caspase-3-dependent apoptotic cell death in sensitive breast cancer cells. However, less-sensitive breast cancer cells resolved T-R conflicts immediately and maintained genomic stability. These data suggest that AZD4573 induced significant anti-tumor effects by apoptotic cell death through accumulation of DNA damage, following T-R conflicts. Citation