Ovarian Cancer-associated Mutations Disable Catalytic Activity of CDK12, a Kinase That Promotes Homologous Recombination Repair and Resistance to Cisplatin and Poly(ADP-ribose) Polymerase Inhibitors

Poorval M Joshi,Shari L Sutor,Catherine J Huntoon,Larry M Karnitz

doi:10.1074/jbc.m114.551143

Poorval M Joshi, Shari L Sutor + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m114.551143

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Abstract

Mutations in the tumor suppressors BRCA1 and BRCA2, which encode proteins that are key participants in homologous recombination (HR) repair, occur in ∼20% of high grade serous ovarian cancers. Although only 20% of these tumors have mutations in BRCA1 and BRCA2, nearly 50% of these tumors have defects in HR. Notably, however, the underlying genetic defects that give rise to HR defects in the absence of BRCA1 and BRCA2 mutations have not been fully elucidated. Here we show that the recurrent somatic CDK12 mutations identified in ovarian cancers impair the catalytic activity of this kinase, which is involved in the transcription of a subset of genes, including BRCA1 and other DNA repair genes. Furthermore, we show that disabling CDK12 function in ovarian cancer cells reduces BRCA1 levels, disrupts HR repair, and sensitizes these cells to the cross-linking agents melphalan and cisplatin and to the poly(ADP-ribose) polymerase (PARP) inhibitor veliparib (ABT-888). Taken together, these findings suggest that many CDK12 mutations are an unrecognized cause of HR defects in ovarian cancers.

Highlights

We show that disabling CDK12 function in ovarian cancer cells reduces BRCA1 levels, disrupts homologous recombination (HR) repair, and sensitizes these cells to the cross-linking agents melphalan and cisplatin and to the poly(ADP-ribose) polymerase (PARP) inhibitor veliparib (ABT-888)
(K975E) severely disrupted kinase activity (Fig. 1, B and C). These results show that many, but not all, CDK12 kinase domain mutations found in ovarian cancers inactivate its catalytic activity
To determine whether CDK12 depletion affected HR, we examined the assembly of RAD51 foci at sites of double-strand breaks induced by ionizing radiation and the ability of the cells to carry out HR repair of a genomically integrated DR-GFP substrate, which is composed of tandem inactive green fluorescent protein (GFP) fragments that flank an I-SceI restriction enzyme site [32]

Summary

Background

We show that disabling CDK12 function in ovarian cancer cells reduces BRCA1 levels, disrupts HR repair, and sensitizes these cells to the cross-linking agents melphalan and cisplatin and to the poly(ADP-ribose) polymerase (PARP) inhibitor veliparib (ABT-888) Taken together, these findings suggest that many CDK12 mutations are an unrecognized cause of HR defects in ovarian cancers. We show that 1) most ovarian tumor-associated CDK12 kinase domain mutations severely impede catalytic activity, 2) cells expressing catalytically inactive CDK12 have defects in HR, and 3) disabling CDK12 in ovarian cancer cells disrupts HR and robustly sensitizes cells to DNA cross-linking agents and PARP inhibitors Taken together, these observations suggest that ovarian cancer-associated CDK12 mutations cause HR defects, a finding that may help identify tumors that will respond to specific therapeutic interventions, including PARP inhibitors

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION