Instead, CARM1 knockout altered cell cycle distribution characterized by an increase in G2 phase (Figure S3G), which is known to favor HR-mediated DSB repair (Rothkamm et al., 2003). imaging of CARM1 knockout A1847 cells treated with 0.4 M Olaparib. NIHMS1569396-supplement-6.avi (4.8M) GUID:?E72D9C47-90A1-4321-A98A-646BBD625563 7: Supplemental Movie 7, related to Figure 4Live-cell time-lapse microscopy imaging of CARM1 knockout A1847 cells treated with 2.5 M GSK126. NIHMS1569396-supplement-7.avi (5.6M) GUID:?425AD6F7-C774-46AE-A630-6D618A28B1E2 8: Supplemental Movie 8, related to Figure 4Live-cell time-lapse microscopy imaging of CARM1 knockout A1847 cells treated with a combination of 0.4 M Olaparib and 2.5 M GSK126. NIHMS1569396-supplement-8.avi (7.5M) GUID:?1DCCBFB1-D537-49B5-9F21-AD59CFD9BAD4 9. NIHMS1569396-supplement-9.pdf (3.8M) GUID:?8B6FD1C6-815B-43DE-947E-C5F285EC6B15 Summary 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 inhibitor (PARPi) in a CARM1-dependent manner. CARM1 promotes silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex around the promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARPi treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARPi in both orthotopic and patient-derived xenografts. Graphical Abstract In Brief Karakashev et al. show that CARM1 promotes EZH2-mediated epigenetic silencing of the shieldin complex protein MAD2L2. Inhibition of EZH2 induces MAD2L2 expression and non-homologous end joining in CARM1-high, homologous recombination proficient ovarian carcinoma cells, sensitizing them to PARP inhibitors. Introduction High-grade serous ovarian cancer (HGSOC) is the most common and fatal subtype of epithelial ovarian cancer (EOC). By inhibiting single-strand DNA break repair, PARP inhibitors (PARPi) are synthetically lethal in homologous recombination (HR)-deficient malignancy cells (Lord and Ashworth, 2017). Indeed, PARPi such as Olaparib have been approved for treatment and maintenance in HGSOC with HR deficiency such as those caused by mutations with substantial clinical benefits (Konstantinopoulos et al., 2015; Moore et al., 2018). However, there is a major unmet clinical need to expand PARPi power into HR-proficient HGSOCs that account for Promazine hydrochloride ~50% of HGSOCs (Konstantinopoulos et al., 2015). CARM1 (also known as PRMT4) is an arginine methyltransferase that asymmetrically dimethylates arginine residues on protein substrates implicated in a number of pathways, including epigenetic regulation of gene transcription (Wang et al., 2014; Wu and Xu, 2012). amplification/overexpression occurs in ~20% of HGSOCs, and CARM1-high HGSOCs are typically HR-proficient and mutually unique with mutations (Karakashev et al., 2018). EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which silences its target genes by generating a lysine 27 trimethylation epigenetic mark on histone H3 (H3K27me3) (Cao and Zhang, 2004). CARM1 functions as an oncogene in breast malignancy by methylating the BAF155 subunit of the SWI/SNF complex (Wang et al., 2014). In addition, inhibition of EZH2 activity is usually a therapeutic vulnerability in cells with functional deficiency in the SWI/SNF complex (Hohmann and Vakoc, 2014). However, despite the mutual exclusivity between amplification/overexpression and mutations in HGSOCs, whether EZH2 inhibition Promazine hydrochloride sensitizes CARM1-high HGSOCs to PARPi has not been explored. DNA double strand break (DSB) is usually repaired by either error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ) pathways (Ceccaldi et al., 2016). The choice between these two DSB repair pathways is regulated by a number of factors such as cell cycle and DSB end structure (Ceccaldi et al., 2016). For example, HR requires end resection to generate a 3 overhang, while NHEJ can join unresected LAMB2 antibody ends. MAD2L2 (also known as REV7) is usually a subunit of the shieldin complex that plays a critical Promazine hydrochloride role in the choice between HR and NHEJ DSB repair (Boersma et al., 2015; Ghezraoui et al., Promazine hydrochloride 2018; Gupta et al., 2018; Noordermeer et al., 2018; Tomida et al., 2018; Xu et al., 2015). The MAD2L2-made up of shieldin promotes NHEJ by protecting DNA ends from resecting. In BRCA-deficient cells, loss of shieldin complex impairs NHEJ and drives PARP Promazine hydrochloride inhibitor resistance. Despite the role of shieldin in promoting NHEJ and its loss in mediating PARP inhibitor resistance in BRCA-deficient cells (Boersma et al., 2015; Dev et al., 2018; Ghezraoui et al., 2018; Gupta et al., 2018; Noordermeer et al., 2018; Xu et al., 2015), whether MAD2L2-made up of shieldin complex can be explored for sensitizing PARP inhibitor in HR-proficient cells has not been investigated. Results EZH2 inhibitor sensitizes CARM1-high cells to a PARP inhibitor. amplification/overexpression is typically mutually unique with genetic alterations that cause HR defects such as mutations in HGSOCs (Physique S1ACB) and expression of positively correlates with copy number gain or amplification in the TCGA HGSOC dataset.