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. 2020 Feb 10;37(2):157-167.e6.
doi: 10.1016/j.ccell.2019.12.015. Epub 2020 Jan 30.

EZH2 Inhibition Sensitizes CARM1-High, Homologous Recombination Proficient Ovarian Cancers to PARP Inhibition

Sergey Karakashev  1 , Takeshi Fukumoto  1 , Bo Zhao  1 , Jianhuang Lin  1 , Shuai Wu  1 , Nail Fatkhutdinov  1 , Pyoung-Hwa Park  1 , Galina Semenova  1 , Stephanie Jean  2 , Mark G Cadungog  2 , Mark E Borowsky  2 , Andrew V Kossenkov  1 , Qin Liu  3 , Rugang Zhang  4
Affiliations

EZH2 Inhibition Sensitizes CARM1-High, Homologous Recombination Proficient Ovarian Cancers to PARP Inhibition

V体育ios版 - Sergey Karakashev et al. Cancer Cell. .

Abstract

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts. VSports手机版.

Keywords: BAF155; CARM1; EZH2 inhibitors; MAD2L2 (REV7); PARP inhibitors; SWI/SNF; epithelial ovarian cancer; homologous recombination (HR); non-homologous end-joining (NHEJ); polycomb repressive complex 2 (PRC2); shieldin V体育安卓版. .

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Conflict of interest statement (V体育ios版)

Declaration of Interests S. K. and R. Z. are co-inventors on a patent application covering the use of EZH2 inhibitors in CARM1-expressing cancers VSports最新版本. All other authors declare no competing interests.

Figures

Figure 1:
Figure 1:. CARM1-dependent sensitization of PARP inhibitor by EZH2 inhibitor.
A-B, Synergy analysis for PARP inhibitor Olaparib and EZH2 inhibitor GSK126 in (A) CARM1-high A1847 parental and (B) CARM1 knockout A1847 cells. Cells were treated with the indicated concentration of Olaparib and GSK126 for 72 hrs. The combination index (CI) value was determined as detailed in the materials and methods. CI value indicate: <1 synergism, =1 additive effect, >1 antagonism. C-D, Olaparib dose-response curves of (C) parental A1847 and (D) CARM1 knockout A1847 treated with the indicated concentration of EZH2 inhibitors GSK126 or Tazemetostat or vehicle DMSO control based on colony formation assays. E-F, The indicated cells were treated with 0.4 μM Olaparib, 2.5 μM GSK126 or a combination for 72 hrs. The apoptotic cells were (E) quantified by Annexin V staining or (F) examined for expression of cleaved PARP p85 or cleaved caspase 3 by immunoblot. Data represent mean ± SEM. n = 3 independent experiments. p values were calculated using a two-tailed t-test. See also Figure S1.
Figure 2:
Figure 2:. CARM1-dependent upregulation of MAD2L2 by EZH2 inhibitor
A, EZH2 and H3K27me3 ChIP-seq and RNA-seq tracks of the MAD2L2 gene locus in parental and CARM1 knockout A1847 cells. B-C, Expression of MAD2L2 (B) mRNA and (C) protein in CARM1-high parental A1847 cells with or without CARM1 knockout and treated with or without GSK126. D, Expression of MAD2L2 in CARM1-low OVCAR3 cells with or without ectopic CARM1 expression treated with or without GSK126 determined by qRT-PCR. E-G, Parental and CARM1 knockout A1847 cells treated with vehicle control or 10 μM GSK126 were subjected to ChIP analysis for the MAD2L2 promoter using antibodies against (E) EZH2, (F) H3K27me3, (G) BAF155 or an isotype matched IgG control. H, Negative correlation between CARM1 and MAD2L2 expression in HGSOC specimens based on a laser-capture and microdissected dataset (Mok et al., 2009). I-L, A1847 cells were infected with a lentivirus encoding shRNA targeting the 3’ untranslated region (UTR) of the BAF155 gene together with a retrovirus encoding a wild-type BAF155 or a BAF155 R1064K mutant. Drug-selected cells were (I) examined for expression of the indicated proteins by immunoblot or subjected to ChIP analysis for the MAD2L2 promoter using antibodies against (J) EZH2, (K) H3K27me3, (L) BAF155 or an isotype matched IgG control. Data represent mean ± SEM. n = 3 independent experiments unless otherwise stated. p values were calculated using a two-tailed t-test. See also Figures S2.
Figure 3:
Figure 3:. EZH2 inhibitor selectively increases NHEJ activity in CARM1-high cells.
A, Schematics of the dual NHEJ and HR reporter assay (Arnoult et al., 2017). B-C, NHEJ activity in (B) CARM1-high A1847 cells with or without CARM1 knockout or (C) CARM1-low OVCAR3 cells with or without ectopic CARM1 expression and treated with or without 10 μM GSK126 for 72 hr as determined by the percentage of eGFP cells in the dual NHEJ and HR reporter assay. D-E, A1847 cells were infected with a lentivirus encoding shRNA targeting the 3’ untranslated region (UTR) of the BAF155 gene together with a retrovirus encoding a wild-type BAF155 or a BAF155 R1064K mutant. Drug-selected cells were examined for (D) NHEJ and (E) HR activity in the dual NHEJ and HR reporter assay as determined by percentages of eGFP or mCherry positive cells. F, EZH2 inhibitor GSK126 increases DNA end protection as determined by DNA combing assay. Schematics of the assay is shown in the top and representative fibers from each of the indicated conditions are shown in the inserted images. The CIdU/IdU ratio was quantified as a surrogate for DNA end protection (n = 100 cells). Scale bar = 5 μm. Data represent mean ± SEM. n = 3 independent experiments unless otherwise stated. p values were calculated using a two-tailed t-test. See also Figure S3.
Figure 4:
Figure 4:. Sensitization to PARP inhibitor by EZH2 inhibitor is NHEJ and MAD2L2 dependent.
A-C, CARM1-high A1847 cells expressing shKU70 or shControl were (A) validated for KU70 knockdown by immunoblot, (B) examined for Olaparib dose-response curves with or without 2.5 μM GSK126, or (C) quantified for apoptosis by Annexin V staining in cells treated with vehicle control, 0.4 μM Olaparib, 2.5 μM GSK126 or a combination for 72 hr. Red dash line indicates 50% of growth inhibition. D-H, A1847 cells expressing the indicated shMAD2L2s or shControl and treated with or without GSK126 were (D) validated for MAD2L2 knockdown by immunoblot, examined for (E) NHEJ or (F) HR activities, (G) examined for Olaparib dose-response curves with or without 2.5 μM GSK126, or (H) quantified for apoptosis by Annexin V staining in cells treated with vehicle control, 0.4 μM Olaparib, 2.5 μM GSK126 or a combination for 72 hr. Data represent mean ± SEM. n = 3 independent experiments unless otherwise stated. p values were calculated using a two-tailed t-test.
Figure 5:
Figure 5:. Sensitization to PARP inhibitor by EZH2 inhibitor correlates with an increase in chromosomal abnormalities and mitotic catastrophe.
A-B, Parental control and CARM1 knockout A1847 cells were treated with vehicle control, 0.4 μM Olaparib, 2.5 μM GSK126 or a combination for 72 hr. The treated cells were (A) subjected to metaphase chromosome spread, and (B) chromosomal abnormalities were quantified for the indicated groups (n =20 metaphase spreads). Arrows point to examples of chromosomes with missing arms or fusion chromosomes. Scale bar = 10 μm. C-D, (C) Parental control and (D) CARM1 knockout A1847 cells treated with vehicle control, 0.4 μM Olaparib, 2.5 μM GSK126 or a combination for 72 hr were subjected to time-lapse video microscopic analysis for mitosis. Cell nuclei were visualized by staining for DNA using siR-DNA. Scale bar = 10 μm. Time is expressed as minutes: seconds. Data represent mean ± SEM. p values were calculated using a two-tailed t-test. See also Movies S1–8.
Figure 6:
Figure 6:. EZH2 inhibitor sensitizes CARM1-high tumors to PARP inhibitor in vivo.
A-B, CARM1-high A1847 cells were unilaterally injected into the ovarian bursa sac of immunocompromised mice (n = 5 mice per group). Randomized mice were treated with vehicle control, Olaparib (50 mg per kg daily by i.p.), GSK126 (25 mg per kg daily by i.p.), or a combination for an additional 3 weeks. At the end of treatment, the mice were euthanized. (A) Reproductive tracts with tumors from the indicated treatment groups were dissected and (B) tumor volumes were measured as a surrogate for tumor burden. ANOVA with post-hoc multiple comparisons statistical analysis revealed that the combination is synergistic (p = 0.028). C, Kaplan–Meier survival curves for the indicated groups. p value was calculated by log-rank test. D, Expression of CARM1 and a loading control β-actin in the indicated CARM1-high and CARM1-low HGSOC PDXs was determined by immunoblot. A1847 and OVCAR3 were used as CARM1-high and CARM1-low controls, respectively. E-H, Mice with the indicated orthotopic (E-F) CARM1-high and (G-H) CARM-low PDXs were randomized into four different treatment groups and treated with vehicle control, Olaparib (50 mg per kg daily by i.p.), GSK126 (25 mg per kg daily by i.p.), or a combination. Tumor weight was measured as a surrogate for tumor burden at the end of the treatment. ANOVA with post-hoc multiple comparisons statistical analysis revealed that the combination is synergistic in CARM1-high (p = 0.006), but not CARM1-low (p = 0.394), PDXs. I-K, Tumors dissected from CARM1-high PDX #1 with the indicated treatments were (I) examined for MAD2L2 mRNA expression by qRT-PCR, or (J) subjected to immunological staining for apoptosis marker cleaved caspase 3, cell proliferation marker Ki67 or H3K27Me3 on serial sections and (K) the histological score (H-score) of the indicated markers was quantified from three separate fields from five tumors from five individual mice in each of the indicated treatment groups. Scale bar = 50 μm. L-M, Cells isolated from fresh CARM1-high or low PDXs post the indicated treatments were (L) subjected to metaphase chromosome spread, and (M) chromosomal abnormalities were quantified for the indicated groups (n>20 metaphase spreads). Arrows point to examples of chromosomes with missing arms or fusion chromosomes. Scale bar = 10 μm. Data represent mean ± SEM. p values were calculated using a two-tailed t-test unless otherwise stated. See also Figure S4.
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