Cyclin-dependent kinases 12 and 13 (CDK12 and 13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and 13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and 13 covalent inhibitor, THZ531. Co-crystallization with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super–enhancer–associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and 13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

Summary Although poly (ADP-ribose) polymerase (PARP) inhibitors are active in homologous recombination (HR)-deficient cancers, their utility is limited by acquired resistance following restoration of HR. Here, we report that dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs) 1, 2, 5 and 9, additionally has potent activity against CDK12, a transcriptional regulator of HR. In BRCA-mutated triple-negative breast cancer (TNBC) cells and patient-derived xenografts (PDXs), dinaciclib ablates restored HR and reverses PARP inhibitor resistance. Additionally, we show that de novo resistance to PARP inhibition in BRCA1-mutated cell lines and a PDX derived from a PARP inhibitor-naïve BRCA1 carrier is mediated by residual HR and is reversed by CDK12 inhibition. Finally, dinaciclib augments the degree of response in a PARP inhibitor-sensitive model, converting tumor growth inhibition to durable regression. These results highlight the significance of HR disruption as a therapeutic strategy and support the broad use of combined CDK12 and PARP inhibition in TNBC.

The bromodomain protein Brd4 regulates the transcription of signal-inducible genes. This is achieved by recruiting the positive transcription elongation factor P-TEFb to promoters by its P-TEFb interaction domain (PID). Here we show that Brd4 stimulates the kinase activity of P-TEFb for phosphorylation of the C-terminal domain (CTD) of RNA polymerase II over basal levels. The CTD phosphorylation saturation levels, the preferences for pre-phosphorylated substrates, and the phosphorylation specificity for Ser5 of the CTD however remain unchanged. Inhibition of P-TEFb by Hexim1 is relieved by Brd4, although no mutual displacement with the Cyclin T-binding domain of Hexim1 was observed. Brd4 PID shows a surprising sequence motif similarity to the trans-activating Tat protein from HIV-1, which includes a core RxL motif, a polybasic cluster known as arginine-rich motif, and a C-terminal leucine motif. Mutation of these motifs to alanine significantly diminished the stimulatory effect of Brd4 and fully abrogated its activation potential in presence of Hexim1. Yet the protein was not found to bind Cyclin T1 as Tat, but only P-TEFb with a dissociation constant of 0.5 μM. Our data suggest a model where Brd4 acts on the kinase subunit of P-TEFb to relieve inhibition and stimulate substrate recognition.