Fig 2: CDK12 kinase activity is not required for HQ461-induced CCNK degradation.

Fig 3: HQ461-mediated CCNK degradation causes cell death and impairs CDK12 function.(A) Depletion of CCNK by CRISPR reduces colony formation in parental A549 cells but not in A549 cells expressing an sgRNA-resistant CCNK cDNA. (B) Western blotting to examine levels of POLII CTD Serine 2 phosphorylation and total POLII CTD in parental A549 cells or A549 CDK12 G731E or G731R knock-in cells following HQ461 treatment. (C) RT-qPCR to examine levels of BRCA1, BRCA2, ATR, and ERCC4 mRNAs in parental A549 cells or A549 CDK12 G731E or G731R knock-in cells following 10 µM HQ461 treatment for 16 hr. Error bars represent standard deviations (SD) from three technical replicates. * represents p<0.05 (T-test, two tail, paired). (D) A model of HQ461’s mechanism of action.

Fig 4: HQ461 promotes CCNK polyubiquitination and degradation through DDB1-CUL4-RBX1.(A) Western blotting of CDK12 and CCNK from A549 cells treated with HQ461. Dilutions of untreated sample (0 hr) were included to allow quantitative assessment of the reduction of protein levels. (B) Effect of CDK12 G731E and G731R mutations on CCNK and CDK12 protein levels in A549 cells treated with HQ461. (C) Effect of bortezomib and MLN4924 on CCNK and CDK12 protein levels in A549 cells treated with HQ461. (D) HQ461 triggers in vivo polyubiquitination of CCNK in complex with wild-type CDK12 but not CDK12 G731E or G731R. The asterisk indicates a non-specific band. (E) Wild-type CDK12 kinase domain but not G731E or G731R mutant is sufficient for mediating HQ461-dependent destabilization of CCNK.

Fig 5: Structure-activity relationship of HQ461 analogs.Half maximal CCNK-degradation concentrations are reported for each analog. CCNK degradation was measured using a CCNK-luc reporter.