FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2556688586> ?p ?o ?g. }

• W2556688586 endingPage "2381" @default.
• W2556688586 startingPage "2367" @default.
• W2556688586 abstract "•Dinaciclib is a potent inhibitor of CDK12 and disrupts homologous recombination•Residual HR is a cause of de novo PARP inhibitor resistance in BRCA-mutated cancer•Dinaciclib sensitizes resistant BRCA-mutated breast cancer models to PARP inhibition•In HR-deficient cancer, dinaciclib augments the response afforded by PARP inhibition Although poly(ADP-ribose) polymerase (PARP) inhibitors are active in homologous recombination (HR)-deficient cancers, their utility is limited by acquired resistance after 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-naive 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. Although poly(ADP-ribose) polymerase (PARP) inhibitors are active in homologous recombination (HR)-deficient cancers, their utility is limited by acquired resistance after 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-naive 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. Poly(ADP-ribose) polymerase (PARP) inhibition has emerged as a compelling strategy for BRCA-deficient or otherwise homologous recombination (HR)-repair-deficient cancers (Scott et al., 2015Scott C.L. Swisher E.M. Kaufmann S.H. Poly (ADP-ribose) polymerase inhibitors: recent advances and future development.J. Clin. Oncol. 2015; 33: 1397-1406Crossref PubMed Scopus (264) Google Scholar). However, the broad utility of these drugs has been limited by their lack of activity in HR-proficient cancers, as well as acquired resistance of initially responding tumors, often mediated by restoration of HR (Bouwman and Jonkers, 2014Bouwman P. Jonkers J. Molecular pathways: how can BRCA-mutated tumors become resistant to PARP inhibitors?.Clin. Cancer Res. 2014; 20: 540-547Crossref PubMed Scopus (113) Google Scholar). Additionally, a proportion of BRCA-mutated cancers display de novo (primary) resistance, potentially mediated by hypomorphic isoforms of BRCA1 (Hill et al., 2014Hill S.J. Clark A.P. Silver D.P. Livingston D.M. BRCA1 pathway function in basal-like breast cancer cells.Mol. Cell. Biol. 2014; 34: 3828-3842Crossref PubMed Scopus (39) Google Scholar), tumor heterozygosity (King et al., 2007King T.A. Li W. Brogi E. Yee C.J. Gemignani M.L. Olvera N. Levine D.A. Norton L. Robson M.E. Offit K. et al.Heterogenic loss of the wild-type BRCA allele in human breast tumorigenesis.Ann. Surg. Oncol. 2007; 14: 2510-2518Crossref PubMed Scopus (75) Google Scholar), or preexisting alterations in the DNA damage response that may confer residual HR activity (Bouwman et al., 2010Bouwman P. Aly A. Escandell J.M. Pieterse M. Bartkova J. van der Gulden H. Hiddingh S. Thanasoula M. Kulkarni A. Yang Q. et al.53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers.Nat. Struct. Mol. Biol. 2010; 17: 688-695Crossref PubMed Scopus (720) Google Scholar). These challenges have prompted interest in combining PARP inhibitors with agents capable of disrupting HR in cancer cells as an approach to sensitize BRCA wild-type cancers to PARP inhibition, and also to overcome de novo and acquired resistance in BRCA-mutated cancers. Because complex mechanisms of HR restoration confer resistance to PARP inhibitors in BRCA-mutated cells, simultaneous suppression of multiple HR genes together with PARP inhibition may be a preferred strategy for resensitizing resistant cells to these agents. In this regard, cyclin-dependent kinase (CDK) 12, an RNA polymerase II C-terminal domain (CTD) kinase, has recently been identified as an essential regulator for the transcription of various DNA damage response (DDR) and DNA repair genes, particularly those involved in the HR and Fanconi anemia (FA) pathways (Bartkowiak et al., 2010Bartkowiak 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.Genes Dev. 2010; 24: 2303-2316Crossref PubMed Scopus (275) Google Scholar, Blazek et al., 2011Blazek 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.Genes Dev. 2011; 25: 2158-2172Crossref PubMed Scopus (305) Google Scholar, Liang et al., 2015Liang K. Gao X. Gilmore J.M. Florens L. Washburn M.P. Smith E. Shilatifard A. Characterization of human cyclin-dependent kinase 12 (CDK12) and CDK13 complexes in C-terminal domain phosphorylation, gene transcription, and RNA processing.Mol. Cell. Biol. 2015; 35: 928-938Crossref PubMed Scopus (122) Google Scholar). Somatic inactivating mutations in CDK12 have been observed in a subset of epithelial ovarian carcinomas, resulting in compromised HR (Joshi et al., 2014Joshi P.M. Sutor S.L. Huntoon C.J. Karnitz L.M. 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.J. Biol. Chem. 2014; 289: 9247-9253Crossref PubMed Scopus (141) Google Scholar). Furthermore, short hairpin RNA (shRNA)-mediated depletion of CDK12, or its cyclin K binding partner, from BRCA and CDK12 wild-type ovarian cancer or other transformed cell lines has been shown to suppress HR gene expression and sensitize cells to cisplatin-induced interstrand crosslinks and PARP inhibition (Bajrami et al., 2014Bajrami I. Frankum J.R. Konde A. Miller R.E. Rehman F.L. Brough R. Campbell J. Sims D. Rafiq R. Hooper S. et al.Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity.Cancer Res. 2014; 74: 287-297Crossref PubMed Scopus (242) Google Scholar, Blazek et al., 2011Blazek 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.Genes Dev. 2011; 25: 2158-2172Crossref PubMed Scopus (305) Google Scholar, Joshi et al., 2014Joshi P.M. Sutor S.L. Huntoon C.J. Karnitz L.M. 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.J. Biol. Chem. 2014; 289: 9247-9253Crossref PubMed Scopus (141) Google Scholar). These observations have led to interest in the development of pharmacological inhibitors of CDK12 to act as sensitizers to PARP inhibitors, as well as to standard DNA-damaging agents. Here, we show that dinaciclib, a known inhibitor of CDKs 1, 2, 5, and 9 (Parry et al., 2010Parry D. Guzi T. Shanahan F. Davis N. Prabhavalkar D. Wiswell D. Seghezzi W. Paruch K. Dwyer M.P. Doll R. et al.Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor.Mol. Cancer Ther. 2010; 9: 2344-2353Crossref PubMed Scopus (372) Google Scholar) that has produced documented responses in breast cancer (Mita et al., 2014Mita M.M. Joy A.A. Mita A. Sankhala K. Jou Y.M. Zhang D. Statkevich P. Zhu Y. Yao S.L. Small K. et al.Randomized phase II trial of the cyclin-dependent kinase inhibitor dinaciclib (MK-7965) versus capecitabine in patients with advanced breast cancer.Clin. Breast Cancer. 2014; 14: 169-176Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), has previously unreported potent activity against CDK12. We studied dinaciclib as a CDK12 inhibitor in models of triple-negative breast cancer (TNBC), an aggressive breast cancer subset associated with poor outcome and absence of defined molecular targets. Dinaciclib reduces HR gene expression in BRCA wild-type TNBC cells and sensitizes these cells to PARP inhibition. We have further investigated the activity of dinaciclib in concert with PARP inhibition in BRCA-mutated TNBC cell lines and patient-derived xenograft (PDX) models, and demonstrate reversal of de novo and acquired PARP inhibitor resistance. Finally, in a BRCA-mutated model in which long-term tumor growth control is achieved by PARP inhibitor monotherapy, the addition of dinaciclib converts the outcome to deep and prolonged tumor regression. Collectively, these data support the combination of dinaciclib with PARP inhibition in both BRCA wild-type and mutant TNBCs. To identify potential inhibitors of CDK12, we made use of its recently elucidated crystal structure. Although the kinase domain of CDK12 shares significant primary sequence homology with CDK9, a panel of small-molecule CDK9 inhibitors was previously shown to have substantially reduced potency against CDK12 in in vitro biochemical assays (Bösken et al., 2014Bösken C.A. Farnung L. Hintermair C. Merzel Schachter M. Vogel-Bachmayr K. Blazek D. Anand K. Fisher R.P. Eick D. Geyer M. The structure and substrate specificity of human Cdk12/Cyclin K.Nat. Commun. 2014; 5: 3505Crossref PubMed Scopus (119) Google Scholar). To further interrogate this result, we aligned the CDK12 crystal structure 4NST with the CDK9 crystal structure 3BLQ (Baumli et al., 2008Baumli S. Lolli G. Lowe E.D. Troiani S. Rusconi L. Bullock A.N. Debreczeni J.E. Knapp S. Johnson L.N. The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation.EMBO J. 2008; 27: 1907-1918Crossref PubMed Scopus (259) Google Scholar). Although the two kinases share extensive tertiary structural homology (root-mean-square deviation [RMSD] = 0.83 Å; Figure 1A), inspection of secondary structure elements demonstrated a variance in the C-terminal portion of each kinase domain (Figures 1B and S1A). CDKs that regulate transcriptional elongation have a unique extension helix that lies C-terminal to the canonical CDK kinase domain. In CDK12, this extension helix interacts with the ATP binding site and is initiated by a DCHEL motif beginning at amino acid 1038. The interaction of the C-terminal extension helix with the nucleotide binding site of CDK12 is mediated by the H1040 and E1041 residues, and loss of the helix severely disrupts activity of the kinase (Bösken et al., 2014Bösken C.A. Farnung L. Hintermair C. Merzel Schachter M. Vogel-Bachmayr K. Blazek D. Anand K. Fisher R.P. Eick D. Geyer M. The structure and substrate specificity of human Cdk12/Cyclin K.Nat. Commun. 2014; 5: 3505Crossref PubMed Scopus (119) Google Scholar). CDK9 shares a similar C-terminal extension helix, but does not share the initiating 1038DCHEL motif (Figure 1B). Because this structural variation occurs in close proximity to the site of binding for small-molecule inhibitors of CDK9, we hypothesized that it may be responsible for the lack of shared specificity with CDK12. In silico modeling of flavopiridol, a well-described potent CDK9 inhibitor, into the ATP binding site of CDK12 revealed a significant steric clash between the benzene ring of bound flavopiridol and the H1040 residue of the DCHEL motif of CDK12. To determine whether this occlusion was a shared feature of other compounds that tightly bind CDK9, we modeled dinaciclib, a CDK9 inhibitor that had not been tested against CDK12, into the CDK12 ATP binding site. In contrast with flavopiridol, there does not appear to be steric hindrance between the CDK12 H1040 aromatic ring and the pyridine-N-oxide ring of dinaciclib (Figure 1B). We predicted that this favorable interaction would afford potent CDK12 inhibitory activity to dinaciclib. The addition of 10× or 1,000× concentration of dinaciclib to 0.2 μM cyclin K-CDK12 or cyclin T-CDK9 holoenzyme complexes reduced CDK12 activity by approximately 20-fold and CDK9 activity by 12- to 25-fold (Figure 1C). Compared with previously reported results of similar assays using other CDK9 inhibitors (Bösken et al., 2014Bösken C.A. Farnung L. Hintermair C. Merzel Schachter M. Vogel-Bachmayr K. Blazek D. Anand K. Fisher R.P. Eick D. Geyer M. The structure and substrate specificity of human Cdk12/Cyclin K.Nat. Commun. 2014; 5: 3505Crossref PubMed Scopus (119) Google Scholar), dinaciclib demonstrates strong inhibition of CDK12 kinase activity. Concentration series were then performed to determine half-maximal inhibitory concentration (IC50) values against CDK12 and other CDK family members (Figures 1D and S1B). Whereas flavopiridol had only modest activity against CDK12 with potency compared with CDK9 reduced by more than 10-fold (Bösken et al., 2014Bösken C.A. Farnung L. Hintermair C. Merzel Schachter M. Vogel-Bachmayr K. Blazek D. Anand K. Fisher R.P. Eick D. Geyer M. The structure and substrate specificity of human Cdk12/Cyclin K.Nat. Commun. 2014; 5: 3505Crossref PubMed Scopus (119) Google Scholar), dinaciclib demonstrated robust inhibitory activity against both kinases, with IC50 in the 40–60 nM range, making it the most potent known inhibitor of CDK12. Furthermore, mutation of the H1040 site to glycine, or mutation of either the DCHEL motif or the adjacent polybasic region to alanine conferred sensitivity of CDK12 to flavopiridol, consistent with the predictions of structural modeling. In contrast, these three CDK12 mutations had no effect on the IC50 of dinaciclib (Figure 1D). We next characterized the transcriptional effects of dinaciclib treatment on TNBC cells. Eukaryotic gene transcription is regulated by a coordinated sequence of phosphorylation events along the CTD of RNA polymerase II. CDK9 is recruited to the 5′ ends of gene bodies, where it primarily phosphorylates CTD-Ser5, releasing the assembled transcription complex from promoter-proximal pausing and initiating transcription (Eick and Geyer, 2013Eick D. Geyer M. The RNA polymerase II carboxy-terminal domain (CTD) code.Chem. Rev. 2013; 113: 8456-8490Crossref PubMed Scopus (282) Google Scholar, Ghamari et al., 2013Ghamari A. van de Corput M.P. Thongjuea S. van Cappellen W.A. van Ijcken W. van Haren J. Soler E. Eick D. Lenhard B. Grosveld F.G. In vivo live imaging of RNA polymerase II transcription factories in primary cells.Genes Dev. 2013; 27: 767-777Crossref PubMed Scopus (102) Google Scholar). CDK12 is predominantly associated with the 3′ ends of genes, where it has been shown to coordinate transcript elongation and processing largely by phosphorylation of CTD-Ser2 (Bartkowiak et al., 2010Bartkowiak 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.Genes Dev. 2010; 24: 2303-2316Crossref PubMed Scopus (275) Google Scholar, Blazek et al., 2011Blazek 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.Genes Dev. 2011; 25: 2158-2172Crossref PubMed Scopus (305) Google Scholar, Eick and Geyer, 2013Eick D. Geyer M. The RNA polymerase II carboxy-terminal domain (CTD) code.Chem. Rev. 2013; 113: 8456-8490Crossref PubMed Scopus (282) Google Scholar). Treatment of MDA-MB-231 cells with low nanomolar concentrations of dinaciclib for 6 hr resulted in concentration-dependent reduction in phospho-CTD levels, with greater effects on Ser2 compared with Ser5 phosphorylation (Figure 2A). Whereas CDK9-mediated phosphorylation of RNA polymerase II occurs globally across transcripts (Garriga and Graña, 2004Garriga J. Graña X. Cellular control of gene expression by T-type cyclin/CDK9 complexes.Gene. 2004; 337: 15-23Crossref PubMed Scopus (142) Google Scholar), CDK12 predominantly associates with the 3′ ends of genes involved with DNA damage and repair (Blazek et al., 2011Blazek 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.Genes Dev. 2011; 25: 2158-2172Crossref PubMed Scopus (305) Google Scholar). Gene expression analysis of RNA collected from MDA-MB-231 cells after 12 hr of dinaciclib exposure showed a significant reduction in expression of only a limited number of genes, in contrast with the global transcriptional repression that has been reported with potent CDK9 inhibitors (Lam et al., 2001Lam L.T. Pickeral O.K. Peng A.C. Rosenwald A. Hurt E.M. Giltnane J.M. Averett L.M. Zhao H. Davis R.E. Sathyamoorthy M. et al.Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.Genome Biol. 2001; 2Crossref Google Scholar) (Figure 2B). Pathway analysis revealed that the differentially expressed genes were significantly enriched for those involved in HR repair and DNA damage-sensing (Figures 2C and S2A), with representation from multiple genes previously reported to be repressed by disruption of CDK12 activity (Blazek et al., 2011Blazek 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.Genes Dev. 2011; 25: 2158-2172Crossref PubMed Scopus (305) Google Scholar) (Figure 2D). We confirmed these results via qPCR using primers for BRCA1 and RAD51 (Figure 2E). Consequently, the expression of multiple proteins involved in HR was decreased in dinaciclib-treated cells, demonstrated in both concentration- and time-dependent experiments, with substantial reduction of these proteins by 24 hr (Figures 2F and 2G). Importantly, the transcriptional effects of dinaciclib could not be attributed to a block in cell cycle progression, because we observed only minimal cell cycle perturbations in asynchronous or hydroxyurea-synchronized cells (Figures 2H and S2B). Taken together, these data suggest that dinaciclib acts primarily as a transcriptional CDK inhibitor in TNBC cells, and that the transcriptional consequences of dinaciclib exposure are predominantly associated with its inhibition of CDK12. We reasoned that the transcriptional effects of dinaciclib that we observed would severely impair HR, as reported in multiple myeloma cells (Alagpulinsa et al., 2016Alagpulinsa D.A. Ayyadevara S. Yaccoby S. Shmookler Reis R.J. A cyclin-dependent kinase inhibitor, dinaciclib, impairs homologous recombination and sensitizes multiple myeloma cells to PARP inhibition.Mol. Cancer Ther. 2016; 15: 241-250Crossref PubMed Scopus (45) Google Scholar). To test this prediction, we assessed functional metrics of HR in BRCA wild-type TNBC cells. Irradiated MDA-MB-231 cells pretreated with dinaciclib showed significant concentration-dependent reduction in the recruitment of BRCA1 and RAD51 to sites of double-strand DNA breaks (Figures 3A and 3B ). To directly measure HR, we utilized U2OS cells with stable integration of the DR-GFP reporter. Transfection of the I-SceI restriction enzyme resulted in 13.3% and 3.6% GFP-positive cells following vehicle or dinaciclib treatment, respectively (Figures 3C and S3A). The profound disruption of HR suggested that dinaciclib could sensitize HR-proficient cells to PARP inhibition. We found that dinaciclib treatment sensitized a panel of BRCA wild-type TNBC cell lines to the PARP inhibitor veliparib (Figures 3D and S3B). In the presence of dinaciclib, the IC50 to veliparib was reduced between 2.5- and 12.5-fold (Table S1). To provide further evidence that the effects of dinaciclib are mediated by CDK12 inhibition, we utilized CRISPR/Cas9-mediated knockout of CDK12 in MDA-MB-468 and BT549 cells (Figure S4A). Knockout of CDK12 caused reduced expression of BRCA1, BRCA2, and RAD51, which compromised RAD51 focus formation after γ-irradiation, and resulted in substantial sensitization to veliparib. Importantly, treatment with dinaciclib did not further sensitize CDK12-depleted cells to veliparib. CDK9 knockout did not reduce HR gene expression (Figure S4A). Consistent with previously published results, CDK9 knockout over several days was lethal to TNBC cells (Wang et al., 2015Wang Y. Zhang T. Kwiatkowski N. Abraham B.J. Lee T.I. Xie S. Yuzugullu H. Von T. Li H. Lin Z. et al.CDK7-dependent transcriptional addiction in triple-negative breast cancer.Cell. 2015; 163: 174-186Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). We therefore used low concentrations of flavopiridol (Figure S4B), which reduced phosphorylation of Ser5, but not Ser2, of the CTD, and we observed no impact on HR gene expression or RAD51 focus formation after DNA damage. In contrast with dinaciclib or CDK12 knockout, flavopiridol did not sensitize TNBC cells to veliparib- or olaparib-mediated PARP inhibition. Many mechanisms of acquired PARP inhibitor resistance have shared a common feature in that they restore RAD51 loading and rescue HR repair. We hypothesized that the multifocal disruption of HR resulting from CDK12 inhibition could potentially resensitize BRCA-mutated cells that have developed resistance to PARP inhibition. We made use of a previously generated PARP-inhibitor-resistant clone of the BRCA1-mutated MDA-MB-436 cell line, in which heterozygous mutation of the TP53BP1 gene and stabilization of a hypomorphic BRCT-domain-mutated BRCA1 protein results in rescue of DNA end resection, RAD51 loading, and HR (Johnson et al., 2013Johnson N. Johnson S.F. Yao W. Li Y.C. Choi Y.E. Bernhardy A.J. Wang Y. Capelletti M. Sarosiek K.A. Moreau L.A. et al.Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance.Proc. Natl. Acad. Sci. USA. 2013; 110: 17041-17046Crossref PubMed Scopus (182) Google Scholar). Dinaciclib treatment substantially reduced protein levels of both RAD51 and the hypomorphic BRCA1 mutant protein, as well as formation of RAD51 foci following irradiation, and resensitized the resistant cells to PARP inhibition (Figure 3E; Table S1). To test the ability of dinaciclib to reverse acquired PARP inhibitor resistance in vivo, we generated a PDX model derived from a TNBC patient carrying a germline S1970∗ BRCA2 mutation. This heavily pretreated patient achieved stable disease for approximately 10 months on combined cisplatin and olaparib followed by olaparib alone (Balmaña et al., 2014Balmaña J. Tung N.M. Isakoff S.J. Graña B. Ryan P.D. Saura C. Lowe E.S. Frewer P. Winer E. Baselga J. Garber J.E. Phase I trial of olaparib in combination with cisplatin for the treatment of patients with advanced breast, ovarian and other solid tumors.Ann. Oncol. 2014; 25: 1656-1663Crossref PubMed Scopus (133) Google Scholar), before disease progression. After brief intervening chemotherapy, a biopsy was performed when new hepatic metastases developed, which was used for establishment of the PDX 12-58 model (Figure 3F) (Tao et al., 2014Tao J.J. Castel P. Radosevic-Robin N. Elkabets M. Auricchio N. Aceto N. Weitsman G. Barber P. Vojnovic B. Ellis H. et al.Antagonism of EGFR and HER3 enhances the response to inhibitors of the PI3K-Akt pathway in triple-negative breast cancer.Sci. Signal. 2014; 7: ra29Crossref PubMed Scopus (105) Google Scholar). Although targeted sequencing did not demonstrate evidence of a BRCA2 reversion mutation (Figure S5A), the model was refractory to cisplatin as well as veliparib (Figures 3G and 3H), requiring animal euthanasia at approximately 40 days for progressive tumor growth, suggesting alternative mechanisms governing resistance. However, the combination of dinaciclib and veliparib resulted in tumor growth inhibition lasting at least 60 days (Figure 3H). End-of-experiment histology revealed no abnormalities in lung, liver, gastrointestinal (GI) tract, and bone marrow of combination-treated mice, with similar appearance of organs harvested from vehicle-treated mice and only modest staining for gamma-H2AX (γ-H2AX) in marrow (Figure S5B). To further study the selectivity of combination treatment for transformed cells, we exposed human mammary epithelial cells (HMECs) to olaparib in the absence or presence of dinaciclib (Figure S5C). In contrast with transformed cells, dinaciclib improved the viability of HMECs treated with olaparib; this protective effect was likely due to the much greater degree of G2 arrest observed, which should preclude PARP-inhibitor-mediated cytotoxicity that typically occurs in S phase. In addition to acquired PARP inhibitor resistance, there is a high rate of de novo resistance to PARP inhibition in BRCA-mutated tumors. To address the utility of CDK12 inhibition in this setting, we first identified and characterized BRCA-mutated cell lines with primary PARP inhibitor resistance. Relative PARP inhibitor sensitivity was determined for a panel of BRCA1-mutated TNBC cell lines using hormone-receptor-positive and non-transformed breast cell lines as a reference standard for insensitivity to PARP inhibition. Whereas MDA-MB-436 and HCC1395 displayed exquisite sensitivity to PARP inhibition, SUM149 and HCC1937 were relatively insensitive to PARP inhibitor treatment, either with veliparib or with olaparib (Figures 4A and S6A). To determine whether the variability in sensitivity to PARP inhibition was due to differences in susceptibility to apoptosis, we performed mitochondrial BH3 profiling on the BRCA-mutated cell lines. No significant differences were observed (Figure S7), suggesting that resistance to PARP inhibition in SUM149PT and HCC1937 was not due to an anti-apoptotic phenotype. In addition to PARP inhibition, HR-deficient tumors are sensitive to the accumulation of DNA interstrand crosslinks (ICLs). To our surprise, all four BRCA1-mutated cell lines displayed marked sensitivity to cisplatin, regardless of PARP inhibitor sensitivity (Figure 4A). Furthermore, we observed that treatment with another DNA crosslinking agent, mitomycin C, resulted in accumulation of chromosomal aberrations in both PARP-inhibitor-resistant and -sensitive BRCA1-mutated lines, whereas veliparib produced chromosomal aberrations in only MDA-MB-436 and HCC1395 cells (Figure 4B). While sensitivity to PARP inhibition is associated with defects in HR, the repair of ICLs requires the activity of multiple DNA repair pathways, including nucleotide excision repair (NER) and the Fanconi anemia (FA) pathway, in addition to HR. BRCA1 function is essential for both HR and the FA pathway, and BRCA1-deficient tumors have been observed to also carry NER defects. We hypothesized that the BRCA1-mutated cell lines SUM149PT and HCC1937 may have selectively retained functional HR while maintaining a defect in NER, as described for a subset of BRCA1-mutated ovarian carcinomas (Ceccaldi et al., 2015Ceccaldi R. O’Connor K.W. Mouw K.W. Li A.Y. Matulonis U.A. D’Andrea A.D. Konstantinopoulos P.A. A unique subset of epithelial ovarian cancers with platinum sensitivity and PARP inhibitor resistance.Cancer Res. 2015; 75: 628-634Crossref PubMed Scopus (84) Google Scholar), or the FA pathway, as in Brca1−/− 53BP1−/− mouse embryonic fibroblasts (MEFs) that display resistance to PARP inhibition, but sensitivity to crosslinking agents (Bunting et al., 2012Bunting S.F. Callén E. Kozak M.L. Kim J.M. Wong N. López-Contreras A.J. Ludwig T. Baer R. Faryabi R.B. Malhowski A. et al.BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair.Mol. Cell. 2012; 46: 125-135Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). We first assessed NER proficiency in SUM149 and HCC1937, and failed to detect an NER defect in either cell line (Figure S6B). We next determined HR and FA pathway proficiency by monitoring the recruitment of repair factors immediately downstream of BRCA1 in both pathways. Recent work has demonstrated that, in addition to its role in RAD51 loading following end resection in HR, BRCA1 is required for the removal of stalled replication machinery and subsequent recruitment of the FA complex to the site of crosslinks (Schlacher et al., 2012Schlacher K. Wu H. Jasin M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.Cancer Cell. 2012; 22: 106-116Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). We therefore measured RAD51 and FANCD2 foci formation as surrogate markers for repair activity downstream of the role of BRCA1 in the HR and FA pathways, respectively (Figure 4D). Following PARP inhibitor treatment, a significant increase in RAD51 foci was observed in SUM149PT and HCC1937, suggesting the presence of functional HR. Strikingly, none of the BRCA1-mutated lines displayed recr" @default.
• W2556688586 created "2016-11-30" @default.
• W2556688586 creator A5000605077 @default.
• W2556688586 creator A5002819777 @default.
• W2556688586 creator A5004300577 @default.
• W2556688586 creator A5011404891 @default.
• W2556688586 creator A5013805263 @default.
• W2556688586 creator A5015821699 @default.
• W2556688586 creator A5019284234 @default.
• W2556688586 creator A5022460700 @default.
• W2556688586 creator A5028674372 @default.
• W2556688586 creator A5029447788 @default.
• W2556688586 creator A5032396015 @default.
• W2556688586 creator A5044648110 @default.
• W2556688586 creator A5045419282 @default.
• W2556688586 creator A5048072263 @default.
• W2556688586 creator A5048688857 @default.
• W2556688586 creator A5053524802 @default.
• W2556688586 creator A5058975989 @default.
• W2556688586 creator A5063661228 @default.
• W2556688586 creator A5068603027 @default.
• W2556688586 creator A5069532291 @default.
• W2556688586 creator A5069889485 @default.
• W2556688586 creator A5073423716 @default.
• W2556688586 creator A5082410534 @default.
• W2556688586 creator A5083605021 @default.
• W2556688586 creator A5087165116 @default.
• W2556688586 creator A5087308492 @default.
• W2556688586 creator A5088192892 @default.
• W2556688586 creator A5088828593 @default.
• W2556688586 date "2016-11-01" @default.
• W2556688586 modified "2023-10-10" @default.
• W2556688586 title "CDK12 Inhibition Reverses De Novo and Acquired PARP Inhibitor Resistance in BRCA Wild-Type and Mutated Models of Triple-Negative Breast Cancer" @default.
• W2556688586 cites W1611726375 @default.
• W2556688586 cites W1986471561 @default.
• W2556688586 cites W1992661182 @default.
• W2556688586 cites W1994898053 @default.
• W2556688586 cites W2006305979 @default.
• W2556688586 cites W2014599726 @default.
• W2556688586 cites W2031819190 @default.
• W2556688586 cites W2042134072 @default.
• W2556688586 cites W2044202291 @default.
• W2556688586 cites W2045153367 @default.
• W2556688586 cites W2047119008 @default.
• W2556688586 cites W2073569331 @default.
• W2556688586 cites W2074045994 @default.
• W2556688586 cites W2074304165 @default.
• W2556688586 cites W2077037832 @default.
• W2556688586 cites W2096380982 @default.
• W2556688586 cites W2101519297 @default.
• W2556688586 cites W2102967423 @default.
• W2556688586 cites W2103608656 @default.
• W2556688586 cites W2114860131 @default.
• W2556688586 cites W2117800899 @default.
• W2556688586 cites W2120806256 @default.
• W2556688586 cites W2123814549 @default.
• W2556688586 cites W2128327044 @default.
• W2556688586 cites W2131511000 @default.
• W2556688586 cites W2131670056 @default.
• W2556688586 cites W2134134362 @default.
• W2556688586 cites W2140642045 @default.
• W2556688586 cites W2149765709 @default.
• W2556688586 cites W2171705583 @default.
• W2556688586 cites W2213491014 @default.
• W2556688586 doi "https://doi.org/10.1016/j.celrep.2016.10.077" @default.
• W2556688586 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5176643" @default.
• W2556688586 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27880910" @default.
• W2556688586 hasPublicationYear "2016" @default.
• W2556688586 type Work @default.
• W2556688586 sameAs 2556688586 @default.
• W2556688586 citedByCount "196" @default.
• W2556688586 countsByYear W25566885862017 @default.
• W2556688586 countsByYear W25566885862018 @default.
• W2556688586 countsByYear W25566885862019 @default.
• W2556688586 countsByYear W25566885862020 @default.
• W2556688586 countsByYear W25566885862021 @default.
• W2556688586 countsByYear W25566885862022 @default.
• W2556688586 countsByYear W25566885862023 @default.
• W2556688586 crossrefType "journal-article" @default.
• W2556688586 hasAuthorship W2556688586A5000605077 @default.
• W2556688586 hasAuthorship W2556688586A5002819777 @default.
• W2556688586 hasAuthorship W2556688586A5004300577 @default.
• W2556688586 hasAuthorship W2556688586A5011404891 @default.
• W2556688586 hasAuthorship W2556688586A5013805263 @default.
• W2556688586 hasAuthorship W2556688586A5015821699 @default.
• W2556688586 hasAuthorship W2556688586A5019284234 @default.
• W2556688586 hasAuthorship W2556688586A5022460700 @default.
• W2556688586 hasAuthorship W2556688586A5028674372 @default.
• W2556688586 hasAuthorship W2556688586A5029447788 @default.
• W2556688586 hasAuthorship W2556688586A5032396015 @default.
• W2556688586 hasAuthorship W2556688586A5044648110 @default.
• W2556688586 hasAuthorship W2556688586A5045419282 @default.
• W2556688586 hasAuthorship W2556688586A5048072263 @default.
• W2556688586 hasAuthorship W2556688586A5048688857 @default.
• W2556688586 hasAuthorship W2556688586A5053524802 @default.
• W2556688586 hasAuthorship W2556688586A5058975989 @default.
• W2556688586 hasAuthorship W2556688586A5063661228 @default.
• W2556688586 hasAuthorship W2556688586A5068603027 @default.

• next