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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
    Affiliations
    Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905
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  • Shari L. Sutor
    Affiliations
    Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905
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  • Catherine J. Huntoon
    Affiliations
    Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905
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  • Larry M. Karnitz
    Correspondence
    To whom correspondence should be addressed: Division of Oncology Research, Mayo Clinic, Gonda 19, 200 First St. S.W., Rochester, MN 55905. Tel.: 507-284-3124; Fax: 507-293-0107
    Affiliations
    Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905

    Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905
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  • Author Footnotes
    * This work was supported in part by the Mayo Clinic Ovarian Cancer SPORE Grant P50 CA136393 (through the National Institutes of Health) and Mayo Clinic Pobanz Family Predoctoral Research Fellowship (to P. M. J.).
Open AccessPublished:February 19, 2014DOI:https://doi.org/10.1074/jbc.M114.551143
      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.

      Introduction

      BRCA1 and BRCA2 mutations are found in ∼20% of high grade epithelial ovarian cancers, the histotype that accounts for two-thirds of ovarian cancer deaths (
      • Hennessy B.T.
      • Timms K.M.
      • Carey M.S.
      • Gutin A.
      • Meyer L.A.
      • Flake 2nd, D.D.
      • Abkevich V.
      • Potter J.
      • Pruss D.
      • Glenn P.
      • Li Y.
      • Li J.
      • Gonzalez-Angulo A.M.
      • McCune K.S.
      • Markman M.
      • Broaddus R.R.
      • Lanchbury J.S.
      • Lu K.H.
      • Mills G.B.
      Somatic mutations in BRCA1 and BRCA2 could expand the number of patients that benefit from poly (ADP ribose) polymerase inhibitors in ovarian cancer.
      ,
      • Bast Jr., R.C.
      • Mills G.B.
      Personalizing therapy for ovarian cancer: BRCAness and beyond.
      ,
      Cancer Genome Atlas Research Network
      Integrated genomic analyses of ovarian carcinoma.
      ). These tumor suppressors maintain genomic stability by facilitating homologous recombination (HR),
      The abbreviations used are: HR
      homologous recombination
      PARP
      poly(ADP-ribose) polymerase
      CTD
      C-terminal domain
      DR-GFP
      direct repeats of green fluorescent protein
      SFB
      S-peptide, FLAG, streptavidin-binding peptide.
      a process that repairs double-stranded DNA breaks and restores stalled replication forks (
      • Roy R.
      • Chun J.
      • Powell S.N.
      BRCA1 and BRCA2: different roles in a common pathway of genome protection.
      ). During HR repair, BRCA1 coordinates the early steps of this repair pathway by interacting with proteins that process DNA into single-stranded DNA tracts, which are then coated with RAD51, in a process facilitated by BRCA2 (
      • Holloman W.K.
      Unraveling the mechanism of BRCA2 in homologous recombination.
      ). These RAD51-single-stranded DNA filaments then serve as substrates during the search for homologous sequences to complete the HR repair process. Correspondingly, cells with defects in BRCA1 or BRCA2 exhibit impaired HR repair (
      • Bast Jr., R.C.
      • Mills G.B.
      Personalizing therapy for ovarian cancer: BRCAness and beyond.
      ,
      • Turner N.
      • Tutt A.
      • Ashworth A.
      Hallmarks of ‘BRCAness’ in sporadic cancers.
      ), which causes sensitivity to DNA-damaging agents that inflict lesions repaired by HR. One such lesion is an interstrand DNA cross-link, which is induced by cross-linkers such as cisplatin and other platinum salts that are the backbone of ovarian cancer therapies (
      • Bast Jr., R.C.
      • Mills G.B.
      Personalizing therapy for ovarian cancer: BRCAness and beyond.
      ). Similarly, cells exhibiting HR defects are also selectively sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors (
      • Martin S.A.
      • Lord C.J.
      • Ashworth A.
      DNA repair deficiency as a therapeutic target in cancer.
      ,
      • McCabe N.
      • Turner N.C.
      • Lord C.J.
      • Kluzek K.
      • Bialkowska A.
      • Swift S.
      • Giavara S.
      • O'Connor M.J.
      • Tutt A.N.
      • Zdzienicka M.Z.
      • Smith G.C.
      • Ashworth A.
      Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition.
      ,
      • Ratner E.S.
      • Sartorelli A.C.
      • Lin Z.P.
      Poly (ADP-ribose) polymerase inhibitors: on the horizon of tailored and personalized therapies for epithelial ovarian cancer.
      ). Consistent with these findings, PARP inhibitors have shown impressive activity in BRCA1/2-mutant ovarian cancers (
      • Fong P.C.
      • Boss D.S.
      • Yap T.A.
      • Tutt A.
      • Wu P.
      • Mergui-Roelvink M.
      • Mortimer P.
      • Swaisland H.
      • Lau A.
      • O'Connor M.J.
      • Ashworth A.
      • Carmichael J.
      • Kaye S.B.
      • Schellens J.H.
      • de Bono J.S.
      Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers.
      ,
      • Fong P.C.
      • Yap T.A.
      • Boss D.S.
      • Carden C.P.
      • Mergui-Roelvink M.
      • Gourley C.
      • De Greve J.
      • Lubinski J.
      • Shanley S.
      • Messiou C.
      • A'Hern R.
      • Tutt A.
      • Ashworth A.
      • Stone J.
      • Carmichael J.
      • Schellens J.H.
      • de Bono J.S.
      • Kaye S.B.
      Poly(ADP)-ribose polymerase inhibition: Frequent durable responses in BRCA carrier ovarian cancer correlating with platinum-free interval.
      ,
      • Audeh M.W.
      • Carmichael J.
      • Penson R.T.
      • Friedlander M.
      • Powell B.
      • Bell-McGuinn K.M.
      • Scott C.
      • Weitzel J.N.
      • Oaknin A.
      • Loman N.
      • Lu K.
      • Schmutzler R.K.
      • Matulonis U.
      • Wickens M.
      • Tutt A.
      Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial.
      ,
      • Ledermann J.
      • Harter P.
      • Gourley C.
      • Friedlander M.
      • Vergote I.
      • Rustin G.
      • Scott C.
      • Meier W.
      • Shapira-Frommer R.
      • Safra T.
      • Matei D.
      • Macpherson E.
      • Watkins C.
      • Carmichael J.
      • Matulonis U.
      Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer.
      ).
      Although only 20% of ovarian cancers harbor BRCA1 or BRCA2 mutations, nearly 50% of ovarian cancers show defects in HR repair (
      • Hennessy B.T.
      • Timms K.M.
      • Carey M.S.
      • Gutin A.
      • Meyer L.A.
      • Flake 2nd, D.D.
      • Abkevich V.
      • Potter J.
      • Pruss D.
      • Glenn P.
      • Li Y.
      • Li J.
      • Gonzalez-Angulo A.M.
      • McCune K.S.
      • Markman M.
      • Broaddus R.R.
      • Lanchbury J.S.
      • Lu K.H.
      • Mills G.B.
      Somatic mutations in BRCA1 and BRCA2 could expand the number of patients that benefit from poly (ADP ribose) polymerase inhibitors in ovarian cancer.
      ). This phenomenon has been attributed to loss of BRCA1 expression (due to BRCA1 promoter methylation) and rare mutations in other genes that directly participate in HR (
      Cancer Genome Atlas Research Network
      Integrated genomic analyses of ovarian carcinoma.
      ,
      • Turner N.
      • Tutt A.
      • Ashworth A.
      Hallmarks of ‘BRCAness’ in sporadic cancers.
      ,
      • Banerjee S.
      • Kaye S.B.
      • Ashworth A.
      Making the best of PARP inhibitors in ovarian cancer.
      ). Despite this progress, it remains unclear whether the full spectrum of genetic alterations that cause HR defects has been identified.
      A high throughput exome sequencing effort of 316 high grade serous ovarian cancers by The Cancer Genome Atlas Research Network uncovered a series of genes that are recurrently mutated (
      Cancer Genome Atlas Research Network
      Integrated genomic analyses of ovarian carcinoma.
      ). One of the most frequently altered genes was CDK12, which was mutated in ∼3% of these tumors, a prevalence similar to that observed for somatic mutations in BRCA1 (3.5%) and BRCA2 (3.2%) in this series. Interestingly, although CDK12 is not known to directly participate in HR, a recent study showed that depleting CDK12 reduced levels of BRCA1 and other proteins (e.g. ATR, FANCI, and FANCD2) involved in responses to DNA damage (
      • Blazek D.
      • Kohoutek J.
      • Bartholomeeusen K.
      • Johansen E.
      • Hulinkova P.
      • Luo Z.
      • Cimermancic P.
      • Ule J.
      • Peterlin B.M.
      The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.
      ). In this study, the mechanistic basis for reduced BRCA1 expression was ascribed to the ability of CDK12 to phosphorylate Ser-2 in the heptapeptide repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) located in the C-terminal domain (CTD) of RNA polymerase II, an event that regulates mRNA elongation (
      • Blazek D.
      • Kohoutek J.
      • Bartholomeeusen K.
      • Johansen E.
      • Hulinkova P.
      • Luo Z.
      • Cimermancic P.
      • Ule J.
      • Peterlin B.M.
      The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.
      ,
      • Bartkowiak B.
      • Liu P.
      • Phatnani H.P.
      • Fuda N.J.
      • Cooper J.J.
      • Price D.H.
      • Adelman K.
      • Lis J.T.
      • Greenleaf A.L.
      CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1.
      ). Although Ser-2 CTD phosphorylation is generally attributed to CDK9 (
      • Brès V.
      • Yoh S.M.
      • Jones K.A.
      The multi-tasking P-TEFb complex.
      ), a kinase that is closely related to CDK12, these studies firmly established that 1) CDK12 also phosphorylates this site and 2) CDK12 regulates a small subset of genes, including BRCA1.
      Notably, however, the functional effects of the CDK12 mutations identified in ovarian cancers have not been evaluated. Also, it remains unclear whether CDK12 mutations affect HR in ovarian cancer cells and whether disabling CDK12 sensitizes cells to DNA-damaging agents and PARP inhibitors. Here we show that 1) nearly all 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 not only disrupts HR but also 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.

      DISCUSSION

      A recent reanalysis of the large scale exome sequencing data of ovarian tumors found that CDK12 is frequently biallelically mutated in these tumors (
      • Carter S.L.
      • Cibulskis K.
      • Helman E.
      • McKenna A.
      • Shen H.
      • Zack T.
      • Laird P.W.
      • Onofrio R.C.
      • Winckler W.
      • Weir B.A.
      • Beroukhim R.
      • Pellman D.
      • Levine D.A.
      • Lander E.S.
      • Meyerson M.
      • Getz G.
      Absolute quantification of somatic DNA alterations in human cancer.
      ), raising the possibility that CDK12 is a tumor suppressor. Notably, however, the effects of these mutations on CDK12 kinase activity have not been previously assessed, thus limiting our understanding of how they may impact ovarian cancer biology. Here we show that the vast majority of kinase domain mutations abrogate catalytic activity. This finding, taken in conjunction with the finding that CDK12 mutations occur in both alleles (
      • Carter S.L.
      • Cibulskis K.
      • Helman E.
      • McKenna A.
      • Shen H.
      • Zack T.
      • Laird P.W.
      • Onofrio R.C.
      • Winckler W.
      • Weir B.A.
      • Beroukhim R.
      • Pellman D.
      • Levine D.A.
      • Lander E.S.
      • Meyerson M.
      • Getz G.
      Absolute quantification of somatic DNA alterations in human cancer.
      ), suggests that ovarian tumors harboring inactivating CDK12 mutations have severely impaired CDK12 function.
      Our studies to address the consequences of disabling CDK12 in ovarian cancer cells confirmed that CDK12 depletion diminished BRCA1 levels in ovarian cancer cells. We note, however, that it is unlikely that BRCA1 is the only CDK12 target that contributes to effective HR. For example, depletion of ATR, which is also reduced by CDK12 knockdown (
      • Blazek D.
      • Kohoutek J.
      • Bartholomeeusen K.
      • Johansen E.
      • Hulinkova P.
      • Luo Z.
      • Cimermancic P.
      • Ule J.
      • Peterlin B.M.
      The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.
      ), also disrupts HR (
      • Huntoon C.J.
      • Flatten K.S.
      • Wahner Hendrickson A.E.
      • Huehls A.M.
      • Sutor S.L.
      • Kaufmann S.H.
      • Karnitz L.M.
      ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status.
      ). Similarly, CDK12 depletion also reduced the levels of FANCI and FANCD2 (
      • Blazek D.
      • Kohoutek J.
      • Bartholomeeusen K.
      • Johansen E.
      • Hulinkova P.
      • Luo Z.
      • Cimermancic P.
      • Ule J.
      • Peterlin B.M.
      The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.
      ), two proteins that, when down-regulated, cause sensitivity to PARP inhibitors (
      • McCabe N.
      • Turner N.C.
      • Lord C.J.
      • Kluzek K.
      • Bialkowska A.
      • Swift S.
      • Giavara S.
      • O'Connor M.J.
      • Tutt A.N.
      • Zdzienicka M.Z.
      • Smith G.C.
      • Ashworth A.
      Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition.
      ), a hallmark of defective HR.
      Our further analyses of CDK12 revealed that depleting CDK12 reduced RAD51 foci formation and HR repair, two phenotypes that are associated with HR defects. Additionally, the kinase-dead CDK12 K756R mutant and the tumor-associated CDK12 G909R mutant did not restore impaired RAD51 foci formation, thus demonstrating that CDK12 catalytic activity is required for this function and showing that the catalytically impaired, tumor-associated CDK12 mutants also disrupt HR. Consistent with these findings, we then showed that depleting CDK12 sensitized ovarian cancer cells to the DNA cross-linking agent cisplatin, a representative of a drug class that is the backbone of ovarian cancer treatments. Finally, we showed that CDK12 depletion sensitized multiple ovarian cancer cell lines to PARP inhibition, an exciting therapeutic approach that relies on the synthetic lethality of PARP inhibition in cells with defects in HR repair.
      While this manuscript was under revision, another study found that CDK12 depletion sensitized cells to PARP inhibition in a genome-wide synthetic lethality screen (
      • Bajrami I.
      • Frankum J.R.
      • Konde A.
      • Miller R.E.
      • Rehman F.L.
      • Brough R.
      • Campbell J.
      • Sims D.
      • Rafiq R.
      • Hooper S.
      • Chen L.
      • Kozarewa I.
      • Assiotis I.
      • Fenwick K.
      • Natrajan R.
      • Lord C.J.
      • Ashworth A.
      Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity.
      ). Our results extend these observations by showing that the majority of tumor-associated CDK12 kinase domain mutations actually inactivate the catalytic activity of CDK12 and demonstrating that CDK12 catalytic activity is required for optimal homologous recombination.

      Acknowledgments

      We thank the Mayo Clinic Optical Morphology Laboratory and Advanced Genomics Technology Center.

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