Journal: eLife

Article Title: Repression of PRMT activities sensitize human homologous recombination-proficient ovarian and breast cancer cells to PARP inhibitor treatment

doi: 10.7554/eLife.99225

Figure Lengend Snippet: ( A ) Illustration of the “induced PARPi sensitivity by epigenetic modulation” strategy for treatment of cancer patients with HR-proficient tumors. Due to frequent defection of one or more DDR pathways, increased replication stress, and higher endogenous DNA damage levels, cancer cells highly rely on the DDR pathways for survival compared with normal cells. During tumorigenesis, to solve these crises, certain DDR-related genes are pathologically upregulated in cancer cells. Epigenetic mechanisms represent a major strategy used by cancer cells to maintain or restore their abnormal need for DDR gene expression, resulting in a targetable vulnerability. This ‘Achilles’ heel’ can be targeted by epigenetic modulators. ( B ) Examples of the clinical trials based on the ‘induced PARPi sensitivity by epigenetic modulation’ strategy. The combinations of PARPis with the epi-drugs or small molecules that indirectly modulate DDR gene transcription were evaluated in the clinic. ( C ) Illustration of the drug combination screen approach used in this study. ( D ) The 74 epigenetic modifying enzyme inhibitors in the panel that was used for the drug combination screen were classified based on their mechanisms of action and target developmental levels. ( E ) The combination index (CI) values were generated from a drug combination screen between olaparib and each epigenetic inhibitor in OVCAR8 and MDA-MB-231 cell lines. The CI quantitatively depicts synergism (CI <0.83), additive effect (CI = 0.83–1.2), and antagonism (CI >1.2). ( F ) Five weighted features were collected and used to estimate the priority scores. ( G ) The epigenetic inhibitors were ranked based on their priority scores, and the top prioritized inhibitors are highlighted. Color indicates mechanism of action. ( H ) The heatmap shows average CI values between PARPis (olaparib or rucaparib) and PRMTis (GSK3368715, GSK3235025, or GSK3203591) in OVCAR8, OVCAR3, MDA-MB-231, and MDA-MB-468 cell lines. Color (red, synergism; blue, antagonism) intensity (light to dark) indicates increasing average CI values for each combination. ( I ) An example of the sensitivity of cancer cells to PARPi alone, PRMTi alone, and a PARPi and PRMTi combination. Only results from OVCAR8 cells are shown here. The results from other cell lines are provided in . For each treatment, the upper panel shows crystal violet staining of a colony formation assay; the lower left panel shows a quantified survival fraction; and the lower right panel shows the CI values. Fa, fraction affected.

Article Snippet: Cell line ( Homo sapiens ) , MDA-MB-468 , ATCC , Cat: #HTB-132; RRID: CVCL_0419 , .

Techniques: Gene Expression, Clinical Proteomics, Generated, Staining, Colony Assay

Journal: eLife

Article Title: Repression of PRMT activities sensitize human homologous recombination-proficient ovarian and breast cancer cells to PARP inhibitor treatment

doi: 10.7554/eLife.99225

Figure Lengend Snippet: ( A ) A comet assay was used to measure DNA damage in cancer cells treated with DMSO, olaparib (5 μM, 96 hr), GSK3368715 (5 μM, 96 hr), GSK3235025 (5 μM, 96 hr), or combinations (representative images from OVCAR8). Scale bars, 10 μm. ( B ) The extent of DNA damage was quantified using the tail moment in comet assays of OVCAR8, OVCAR3, MDA-MB-231, and MDA-MB-468 cell lines treated with DMSO, olaparib, GSK3368715, GSK3235025, or combinations. Data are presented as means ± SDs, *p<0.05 determined by two-tailed Student’s t tests. ( C ) Western blot analyses of γH2AX in cancer cells treated with DMSO, olaparib, GSK3368715, GSK3235025, or combinations. ( D ) Caspase-3/7 activity was measured using a caspase-Glo 3/7 assay in OVCAR8 and MDA-MB-231 cells treated with DMSO, olaparib, GSK3368715, GSK3235025, or combinations. Data are presented as means ± SDs, n=3 biological replicates, *p<0.05 determined by two-tailed Student’s t tests. ( E ) Western blot analyses of γH2AX in OVCAR8 and MDA-MB-231 cells in which PRMT1 and PRMT5 were independently knocked out using lentiviral CRISPR/Cas9. ( F ) A comet assay was used to measure olaparib treatment-induced DNA damage in cells in which PRMT1 and PRMT5 were independently knocked out using lentiviral CRISPR/Cas9 (representative images from OVCAR8). Scale bars, 10 μm. ( G ) The extent of olaparib treatment-induced DNA damage was quantified using the tail moment in comet assays of OVCAR8, MDA-MB-231, and MDA-MB-468 cell lines in which PRMT1 and PRMT5 were independently knocked out using lentiviral CRISPR/Cas9. Data are presented as means ± SDs, *p<0.05 determined by two-tailed Student’s t tests. ( H ) and ( I ) Correlations between the expression levels of PRMT1/PRMT5 and ‘50 hallmark’ molecular signatures in the cancer cell lines from the DepMap ( H ) and primary tumor specimens from the TCGA (ovarian cancer was represented as an example, I ) cohort. ( J ) Correlations between the expression levels of PRMT1/PRMT5 and ‘50 hallmark’ molecular signatures across primary tumor specimens from the TCGA cohort. For each cancer type, the correlations between PRMT1/5 expression and ‘50 hallmark’ molecular signatures were ranked 0–1 based on the p-values, with 1 indicating the most significant signature. Red and blue circles indicate DNA repair and Myc signatures, respectively. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original file for western blots displayed in .

Article Snippet: Cell line ( Homo sapiens ) , MDA-MB-468 , ATCC , Cat: #HTB-132; RRID: CVCL_0419 , .

Techniques: Single Cell Gel Electrophoresis, Two Tailed Test, Western Blot, Activity Assay, Caspase-Glo Assay, CRISPR, Expressing

Journal: eLife

Article Title: Repression of PRMT activities sensitize human homologous recombination-proficient ovarian and breast cancer cells to PARP inhibitor treatment

doi: 10.7554/eLife.99225

Figure Lengend Snippet: ( A ) Left panels: A comet assay was used to measure DNA damage in OVCAR8, OVCAR3, MDA-MB-231, and MDA-MB-468 cell lines treated with DMSO, olaparib, GSK3368715, GSK3235025, or combinations. Scale bars, 10 μm. Right panels: The extent of DNA damage was quantified using the tail moment in comet assays of OVCAR8, OVCAR3, MDA-MB-231, and MDA-MB-468 cell lines treated with DMSO, olaparib, GSK3368715, GSK3235025, or combinations. Data are presented as means ± SDs, *p<0.05 determined by two-tailed Student’s t tests. ( B ) Left panels: A comet assay was used to measure olaparib treatment-induced DNA damage in OVCAR8, MDA-MB-231, and MDA-MB-468 cell lines in which PRMT1 and PRMT5 were independently knocked out using lentiviral CRISPR/Cas9. Scale bars, 10 μm. Right panels: The extent of olaparib treatment-induced DNA damage was quantified using the tail moment in comet assays of OVCAR8, MDA-MB-231, and MDA-MB-468 cell lines in which PRMT1 and PRMT5 were independently knocked out using lentiviral CRISPR/Cas9. Data are presented as means ± SDs, *p<0.05 determined by two-tailed Student’s t tests.

Article Snippet: Cell line ( Homo sapiens ) , MDA-MB-468 , ATCC , Cat: #HTB-132; RRID: CVCL_0419 , .

Techniques: Single Cell Gel Electrophoresis, Two Tailed Test, CRISPR

Journal: eLife

Article Title: Repression of PRMT activities sensitize human homologous recombination-proficient ovarian and breast cancer cells to PARP inhibitor treatment

doi: 10.7554/eLife.99225

Figure Lengend Snippet: ( A ) The expression levels of aDMA and sDMA in cancer cell lines in which PRMTs were inhibited by chemical compounds. The expression levels of aDMA and sDMA in cancer in OVCAR8, OVCAR3, MDA-MB-231, and MDA-MB-468 cell lines treated with GSK3368715 or GSK3235025. To ensure identical experimental conditions, the protein samples analyzed in these western blots were identical to those used in ; accordingly, the same loading controls are presented in both figures. ( B ) The expression levels of aDMA and sDMA in cancer cell lines in which PRMTs were inhibited by genetic approach. The expression levels of aDMA and sDMA in cancer in OVCAR8 and MDA-MB-231 cell lines in which PRMT1 or PRMT5 was knocked out by CRISPR/Cas9. To ensure identical experimental conditions, the protein samples analyzed in these western blots were identical to those used in ; accordingly, the same loading controls are presented in both figures. ( C ) Dot blots of CPD levels in MDA-MB-231 cells in which ERCC1 was knocked out using lentiviral CRISPR/Cas9. Methylene blue staining was used as the loading control. Figure 5—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 5—figure supplement 1—source data 2. Original file for western blots displayed in .

Article Snippet: Cell line ( Homo sapiens ) , MDA-MB-468 , ATCC , Cat: #HTB-132; RRID: CVCL_0419 , .

Techniques: Expressing, Western Blot, CRISPR, Staining, Control