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. 2016 Nov 22;17(9):2367-2381.
doi: 10.1016/j.celrep.2016.10.077.

CDK12 Inhibition Reverses De Novo and Acquired PARP Inhibitor Resistance in BRCA Wild-Type and Mutated Models of Triple-Negative Breast Cancer

Shawn F Johnson  1 , Cristina Cruz  2 , Ann Katrin Greifenberg  3 , Sofia Dust  3 , Daniel G Stover  4 , David Chi  1 , Benjamin Primack  5 , Shiliang Cao  1 , Andrea J Bernhardy  6 , Rhiannon Coulson  7 , Jean-Bernard Lazaro  8 , Bose Kochupurakkal  8 , Heather Sun  9 , Christine Unitt  9 , Lisa A Moreau  5 , Kristopher A Sarosiek  1 , Maurizio Scaltriti  10 , Dejan Juric  11 , José Baselga  10 , Andrea L Richardson  9 , Scott J Rodig  9 , Alan D D'Andrea  5 , Judith Balmaña  12 , Neil Johnson  6 , Matthias Geyer  3 , Violeta Serra  13 , Elgene Lim  14 , Geoffrey I Shapiro  15
Affiliations

CDK12 Inhibition Reverses De Novo and Acquired PARP Inhibitor Resistance in BRCA Wild-Type and Mutated Models of Triple-Negative Breast Cancer

Shawn F Johnson et al. Cell Rep. .

Abstract

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.

Keywords: BRCA-associated breast cancer; CDK inhibitor; CDK12; PARP inhibitor; dinaciclib; homologous recombination repair; triple-negative breast cancer.

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Figures

Figure 1
Figure 1. Dinaciclib is a potent inhibitor of CDK12 in addition to CDK9
(A) Tertiary structural alignment of CDK9 and CDK12. (B) Sequence corresponding to the variance in the C-terminal extension helix of the kinase domains of CDK12 and CDK9 (see also Figure S1), as well as structural modeling of the orientations of flavopiridol and dinaciclib in relation to H1040 and E1041 of the CDK12 ATP binding site. The benzene ring of flavopiridol shows a steric clash with H1040 of Cdk12, whereas the pyridine-N-oxide ring of dinaciclib overlaps the aromatic H1040 side chain, resulting in a possible stacking of the aromatic ring systems that stabilizes the interaction and contributes to binding specificity. (C) In vitro kinase assays using pS7-CTD[3] as substrate and 0.2 μM cyclin T-CDK9 and cyclin K-CDK12 holoenzyme complexes alone or with and 10x or 1000x dinaciclib. (D) Concentration series of dinaciclib and flavopiridol for cyclin T1-CDK9 and cyclin K-CDK12 at 0.2 μM kinase concentration. The IC50 values against Cdk9 and CDK12 are comparable for dinaciclib but disparate for flavopiridol. Introduction of the indicated mutations sensitizes CDK12 to flavopiridol. All data are reported as the mean ± SD from three independent experiments.
Figure 2
Figure 2. Dinaciclib is a transcriptional CDK inhibitor that reduces expression of genes in DNA damage response and DNA repair pathways
(A) MDA-MB-231 cells were treated with the indicated concentrations of dinaciclib for 6 hrs, demonstrating reduced phosphorylation at the Ser2 and Ser5 sites of the CTD of RNA pol II. (B) Cells were collected before and after treatment with 10 nM dinaciclib for 12 hrs and changes in transcription were measured using the Affymetrix HG-U133A2 arrays. Analyses were performed in triplicate. 21% of genes were significantly downregulated in dinaciclib-treated vs. untreated samples (P < 0.05). (C) Genes statistically significantly downregulated in response to dinaciclib were analyzed by Ingenuity Pathway Analysis (IPA) software, demonstrating downregulation of DNA damage response and DNA repair pathways. (D) Expression of genes in the “Role of BRCA1 in DNA Damage Response” pathway. (E) Downregulation of expression of BRCA1 and RAD51 mRNAs in cells treated with the indicated concentrations of dinaciclib was confirmed utilizing RT-PCR. (F) Concentration-dependent reduction in expression of BRCA1, BRCA2, RAD51 and FANCD2 in cells treated with dinaciclib for 24 hrs. (G) Time-dependent reduction in expression of BRCA1 and RAD51 in response to dinaciclib. (H) Cell cycle patterns following dinaciclib exposure demonstrate the absence of G1 arrest in MDA-MB-231 cells (see also Figure S2).
Figure 3
Figure 3. Disruption of HR by dinaciclib and sensitization to PARP inhibition of BRCA wild type and BRCA-mutated cells with acquired PARP inhibitor resistance
(A) Cells were treated with vehicle or 10 nM dinaciclib for 18 hrs prior to treatment with 10 Gy γ-irradiation (IR). Six hrs post-IR, cells were analyzed for BRCA1, RAD51 and γ-H2AX focus formation. (B) Quantification of cells with > 5 foci in irradiated cells pretreated with vehicle or dinaciclib at the indicated concentrations. (C) USOS DR-GFP cells were transfected I-SceI in the presence of vehicle or 15 nM dinaciclib. The percentage of GFP-positive cells is significantly reduced (P < 0.0001) in the presence of dinaciclib, consistent with direct inhibition of HR repair (See also Figure S3). (D) BRCA-proficient TNBC cell lines were treated with veliparib in the absence and presence of dinaciclib, demonstrating reduced IC50 values in the presence of dinaciclib. (E) (Left) An MDA-MB-436 PARP inhibitor-resistant derivative (MDA-MB-436-RR2) (Johnson et al., 2013) was treated with veliparib in the absence or presence of dinaciclib, demonstrating reduced IC50 in the presence of dinaciclib. (Middle) Reduced expression of the mutant BRCA1 protein and RAD51 in response to dinaciclib. (Right) MDA-MB-436-RR2 cells were treated with vehicle or dinaciclib at the indicated concentration for 18 hours, subjected to 10 Gy IR and assessed for RAD51 focus formation 6 hours later. (P < 0.0001) (F) Treatment history of the BRCA2 carrier; PDX 12-58 was procured after progression on cisplatin and olaparib (see also Figure S4A) . SD, stable disease. (G) (Left) Mice bearing 12–58 xenografts were treated with vehicle (n = 3) or cisplatin (n = 3) on days 1 and 22 (arrows), with tumor volume measured over 36 days. (Right) Mice were treated with vehicle (n = 4), veliparib (n = 8), dinaciclib (n = 8) or the combination (n = 8). Combination treatment produced significant tumor growth inhibition at day 42 compared to vehicle (P < 0.0001) or monotherapies (P < 0.0001 for both veliparib and dinaciclib). * P < 0.01, ** P < 0.001, ***P < 0.0001 for experimental value vs. control.
Figure 4
Figure 4. Characterization of BRCA1-mutated TNBC cell lines
(A) Panel of indicated cell lines was treated with the indicated concentrations of veliparib or cisplatin and viability assessed after 7 days of treatment. (B) Cells were treated with vehicle, veliparib or mitomycin C and metaphase spreads were prepared; radials quantified in vehicle and drug-treated cells. (C) Cells were treated with vehicle, veliparib or cisplatin and analyzed by immunofluorescence for RAD51 and FANCD2 foci. Graphs show quantification of cells with > 5 foci in vehicle and drug-treated cells.
Figure 5
Figure 5. BRCA1-mutated SUM149PT and HCC1937 cells are sensitized to PARP inhibition by siRNA- or dinaciclib-mediated depletion of the BRCA1-PALB2-BRCA2 axis and RAD51
(A) SUM149PT cells were transfected with the indicated siRNAs targeting BRCA1, BRCA2 or PALB2, followed by veliparib treatment at the indicated concentrations. Colony formation was assessed over a 14-day period. (B) Similar experiments were performed with HCC1937 cells using siRNAs targeting BRCA2 and PALB2. (C) Cells were treated with vehicle (0 nM) or the indicated concentrations of dinaciclib for 24 hrs and nuclear lysates subjected to Western blotting with the indicated antibodies. (D) Cells were pretreated with vehicle or 10 nM dinaciclib for 18 hours followed by 10 Gy IR. RAD51 and γH2AX focus formation was assessed by immunofluorescence 6 hrs after IR. (E) Quantification of RAD51 focus formation 6 hrs after IR in cells pretreated with vehicle (0 nM) or the indicated concentrations of dinaciclib (* P < 0.0001 for dinaciclib vs. vehicle). (F) Cells were treated with the indicated concentrations of veliparib in the absence or presence of dinaciclib, demonstrating reduced IC50 values in the presence of dinaciclib.
Figure 6
Figure 6. Generation and treatment of the PDX127 model from a 185delAG BRCA1 carrier
(A) Treatment history of the BRCA1 carrier; the model was procured prior to exposure to cisplatin/olaparib or olaparib monotherapy. PR, partial response; PD, progressive disease. (B) Mice bearing xenografts were treated with vehicle (n = 4) or cisplatin (n = 6) on the days 1 and 34 (arrows) demonstrating tumor regression in response to platinum-based treatment. (C) Mice bearing xenografts were treated with vehicle (n = 8), olaparib (n = 7), dinaciclib (n = 3) or the combination (n = 7). Combination treatment produced significant tumor growth inhibition compared to vehicle or monotherapies. At day 35, P = 0.018 (*) for combination vs. dinaciclib and P < 0.0001 (**) for combination vs. olaparib. (D) Mice bearing xenografts were treated with vehicle, olaparib, dinaciclib or the combination (n = 6/group). (Left) After 15 days, mice were sacrificed and tumors subjected to immunofluroescence for RAD51 and γ-H2AX foci. (Right) Quantification of cells with > 5 γ-H2AX foci, as well as γ-H2AX-positive cells with > 5 RAD51 foci. For γ-H2AX foci, P values for control vs. dinaciclib, olaparib or the combination are 0.103, 0.013 (*) and 0.005, respectively. P values for dinaciclib or olaparib vs. the combination are < 0.0001 and 0.0076 (**), respectively. For RAD51 quantification in γ-H2AX-positive cells, P values for control vs. dinaciclib, olaparib or the combination are 0.69, 0.04 and 0.74 (NS), respectively. P values for dinaciclib or olaparib vs. the combination are 0.158 and 0.0035 (**), respectively. (E) Tumor RNA from mice in D treated with vehicle or dinaciclib (n = 3/group) was subjected to RT-PCR for BRCA1 and RAD51. P values for vehicle vs. dinaciclib are 0.000068 (**) in both cases. (F) Tumor lysates from mice in D treated with vehicle or dinaciclib were subjected to Western blotting with the indicated antibodies.
Figure 7
Figure 7. Treatment of the 11–26 PDX model harboring somatic BRCA1 R1443* mutation
(A) Mice bearing xenografts were treated with vehicle or veliparib (n = 5/group). (Left) After 15 days, mice were sacrificed and tumors subjected to immunofluorescence for RAD51 and γ-H2AX foci. (Right) Quantification of γ-H2AX-positive cells with > 5 RAD51 foci. P, non-significant. (B) Mice bearing xenografts were treated with vehicle or dinaciclib for 2 doses over 5 days (n = 3/group), after which mice were sacrificed and tumors stained for RAD51. P = 0.059. Bar, 100 μm. (C) Mice bearing xenografts were treated with vehicle (n = 3), dinaciclib (n = 7), veliparib (n = 4) or the combination (n = 10), demonstrating long-term growth control with veliparib and sustained tumor regressions with combination treatment. After 2 months of treatment (day 61), P < 0.001 (*) for combination treatment vs. either monotherapy. (D) Waterfall plot demonstrating % change in tumor volume at the time of sacrifice for individual mice in the four treatment groups. (E) Representative end-of-experiment histology (H & E) and γ-H2AX staining of tumors isolated from mice in the 4 treatment groups. Bar, 100 μm. (F) Quantification of % nuclei staining positively for γ-H2AX at end-of-experiment (* P < 0.05 for combination vs. control treatment).
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References

    1. Alagpulinsa DA, Ayyadevara S, Yaccoby S, Schmookler Reis RJ. A cyclin-dependent kinase inhibitor, dinaciclib, impairs homologous recombination and sensitizes multiple myeloma cells to PARP inhibition. Mol Cancer Ther. 2016;15:241–250. - PMC - PubMed
    1. Bajrami I, Frankum JR, Konde A, Miller RE, Rehman FL, 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;74:287–297. - PMC - PubMed
    1. Balmaña J, Tung NM, Isakoff SJ, Grana B, Ryan PD, Saura C, Lowe ES, Frewer P, Winer E, Baselga J, et al. Phase I trial of olaparib in combination with cisplatin for the treatment of patients with advanced breast, ovarian and other solid tumors. Annals Oncol. 2014;25:1656–1663. - PubMed
    1. Bartkowiak B, Liu P, Phatnani HP, Fuda NJ, Cooper JJ, Price DH, Adelman K, Lis JT, Greenleaf AL. CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev. 2010;24:2303–2316. - PMC - PubMed
    1. Baumli S, Lolli G, Lowe ED, Troiani S, Rusconi L, Bullock AN, Debreczeni JE, Knapp S, Johnson LN. The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation. EMBO J. 2008;27:1907–1918. - PMC - PubMed

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