Cyclin-dependent kinase 9 (CDK9) regulates elongation of transcription through phosphorylation of RNA polymerase II (pSer2-RNAPII), and its short-term inhibition downregulates genes with short-lived transcripts and labile proteins. We developed a novel and selective CDK9 inhibitor, AZD4573, with nanomolar potency and physicochemical properties suitable for IV administration and short exposure.
Initial transcriptomic and proteomic analyses were performed on MCF7 breast cancer cells treated with AZD4573 for 4h or 8h to determine which genes were most rapidly modulated by CDK9 inhibition. Of the 1,290 genetically-linked, cancer-associated genes analyzed on these platforms, MCL1 ranked amongst the top as most robustly and sustainably suppressed at both the mRNA and protein level with AZD4573 treatment. MCL1 is an anti-apoptotic member of the BCL2 gene family and is frequently amplified and/or over-expressed in various cancers, conferring a survival advantage to the tumor cell. Mcl1 has also been notoriously difficult to drug with small molecules, although inhibitors are now in early clinical development. Transient inhibition of CDK9 with AZD4573, however, provides a means to indirectly target Mcl1 and induce apoptosis in Mcl1-addicted tumors.
In human cancer cell line panel screens, AZD4573 demonstrated the ability to induce rapid caspase activation (6h) and loss of viability (24h) across a diverse set of hematological cancers (median caspase EC50=30nM, GI50=11nM) but with minimal effect on solid tumors (median EC50 & GI50 >30μM). The activity of AZD4573 in the cell line panels displayed a strong correlation with that of the Mcl1 inhibitor, AZD5991 (r2 = 0.837), suggesting transient CDK9 inhibition induces cell death through an Mcl1-dependent mechanism. Furthermore, knockdown of BAK and/or BAX in sensitive cell lines rescued the cells from AZD4573-mediated caspase induction and cell death, underscoring the mechanistic involvement of the intrinsic apoptosis pathway. In the AML cell line, MV411, AZD4573 led to a rapid dose- and time-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in caspase induction and loss of cell viability. In contrast, protein levels of the other anti-apoptotic proteins, Bcl2 and BclxL, remained unchanged out to 6h post-AZD4573 treatment of MV411 cells.
With intermittent dosing of AZD4573, in vivo efficacy was observed in multiple hematological tumor xenograft models. AZD4573 also exhibited the ability to drive deeper and more durable regressions in combination with multiple targeted agents. Based on our findings, AZD4573 could be an effective treatment option, either as single agent or in combination, for patients with hematological malignancies. Thus, we have initiated a first-time-in-human study with AZD4573 in October 2017 (NCT03263637).
Citation Format: Justin Cidado, Theresa Proia, Scott Boiko, Maryann San Martin, Steven Criscione, Douglas Ferguson, Wenlin Shao, Lisa Drew. AZD4573, a novel CDK9 inhibitor, rapidly induces cell death in hematological tumor models through depletion of Mcl1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 310.