Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors.

CDK9, is a serine/threonine kinase from the CDK family, of which multiple members are involved in the regulation of either cell-cycle progression or gene transcription. CDK9 extends the AR half-life and activity through its N-terminal phosphorylation (Ser81). Unphosphorylated AR is exported from the nucleus, where it is ubiquitinated and degraded by the proteasome. Inhibition of CDK9 therefore may offer an indirect route for AR modulation in CRPC. Additionally, CDK9 and cyclin T1 form the positive transcription elongation factor b (P-TEFb), which facilitates phosphorylation of Ser2 of the C-terminal domain of RNA polymerase II (RNA Pol II), ultimately initiating transcriptional elongation. P-TEFb inhibition enables selective downregulation of oncogenes by decreasing RNA Pol II Ser2 phosphorylation, resulting in universal RNA Pol II stalling that disproportionately downregulates sites of heavy transcriptional recruitment and transcriptional cooperativity at superenhancers within transcriptional condensates or insulated environments. Consequently, transcriptional CDK inhibitors, including those that preferentially inhibit CDK9, have shown strong potential as therapeutic agents owing to their ability to selectively downregulate oncogenic transcription programs and target tumors addicted to transcription factors such as AR or MYC.

Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.