Fig 2: BRD4 transcriptionally regulates mismatch repair genes (A) UCSC Genome Browser was used to show BRD4 ChIP sequencing signal profiles in the MLH1 gene locus. Primers for ChIP-qPCR validation were indicated. (B) ChIP-qPCR of BRD4 in SKOV3 treated with vehicle or 1 µM AZD5135 for 48 hours. Data is shown as mean±SEM from each of three independent replicates. Two-tailed t-tests: *, p<0.05; **, p<0.01; ***, p<0.001. (C) ChIP sequencing showed BRD4 binding at the MLH1, MSH2, MSH6, and PMS2 loci in MM1.S cells treated with DMSO or the indicated dose of JQ1 in GSE44931 data set. (D) ChIP sequencing showed BRD4 binding at the MLH1, MSH2, MSH6, and PMS2 loci in SUM149 and JQ1-resistant (JQ1R) SUM149 cells treated with DMSO or 10 µM JQ1 in GSE131102 data set. BRD4, bromodomain containing 4; ChIP, chromatin immunoprecipitation; MLH1, mutL homologue 1; MSH2, mutS homologue 2; MSH6, mutS homologue 6; PMS2, PMS1 homolog 2, mismatch repair system component; qPCR, quantitative PCR; DMSO, Dimethyl sulfoxide.

Fig 3: BRD4 and mismatch repair proteins expression are correlated across cancer lineages (A–B) mRNA expression (A) and protein levels (B) data in TCGA pan-cancer were obtained and the correlations of mRNA expression between BRD4 with MLH1, MSH2, MSH6, and PMS2 (A), and the correlations of protein levels of BRD4 with MSH2 and MSH6 were performed by Pearson’s correlation analyses (B). Color indicates coefficients calculated by Pearson’s correlation test (*, p<0.05; **, p<0.01; ***, p<0.001). (C) Proteomics data in CPTAC data was obtained and Pearson’s correlation analyses of BRD4 with MLH1, MSH2, MSH6, and PMS2 was performed. Color indicates coefficients calculated by Pearson’s correlation test (*, p<0.05; **, p<0.01; ***, p<0.001). (D) Representative IHC images (200×) of BRD4, MLH1, MSH2, MSH6, and PMS2 (left) and correlation between BRD4 IHC score and the sum of MLH1, MSH2, MSH6, and PMS2 IHC scores (right) in high-grade serous ovarian cancer tissues. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; BRD4, bromodomain containing 4; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; CPTAC, Clinical Proteomic Tumor Analysis Consortium; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; IHC, immunohistochemistry; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; MLH1, mutL homologue 1; mRNA, messenger RNA; MSH2, mutS homologue 2; MSH6, mutS homologue 6; OV, ovarian cancer; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PMS2, PMS1 homolog 2, mismatch repair system component; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; RPPA, reverse phase protein arrays; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma.

Fig 4: Graphical abstract. pMMR tumors with adequate mismatch repair proteins (MLH1, PMS2, MSH2, MSH6, etc.) are resistant to ICBs. BRD4 inhibition transcriptionally decreases the expression of MMR genes in cancer cells, thus promotes cancers acquiring a mutational profile that could sensitize patients to ICBs. MMR, mismatch repair; pMMR, proficient MMR; dMMR, deficient MMR; BRD4, bromodomain containing 4; ICB, immune checkpoint blockade.

Fig 5: JMJD6 enhances HOTAIR expression to maintain the stemness of LCSCs and promotes their radioresistance. A Quantitative analysis of JMJD6 and BRD4 expression in liver cancer and adjacent normal tissues measured by immunohistochemistry. *p < 0.05 compared with adjacent normal tissues. B The expression of JMJD6, BRD4 and ERK2 (MAPK1) in Hep3B and Huh7 CSCs with JMJD6 silencing or BRD4 silencing detected by Western blot, relative to GAPDH. *p < 0.05 compared with shCtl-treated cells. C The stemness of LCSCs with JMJD6 silencing or BRD4 silencing detected by microsphere formation assay. *p < 0.05 compared with shCtl-treated cells. D After exposure to X-ray, the cell colony formation in the presence of shJMJD6 or shBRD4, *p < 0.05 compared with shCtl-treated cells. E The enrichment of JMJD6 and BRD4 in the promoter region of HOTAIR after JMJD6 silencing, as examined by ChIP assay, relative to Input. The relative enrichment on the Y-axis represents JMJD6 or BRD4/IgG. *p < 0.05 compared with shCtl-treated cells. F Detection of expression of JMJD6 and LSD1 in cells following silencing of JMJD6, *p < 0.05 compared with shCtl-treated cells. G The expression of ERK2 (MAPK1) and JMJD6 after JMJD6 depletion or in combination with HOTAIR overexpression, as examined by Western blot and RT-qPCR. *p < 0.05 compared with shCtl + oe-NC-treated cells. #p < 0.05 compared with shJMJD6 + oe-NC-treated cells. H The microsphere formation assay was adopted to measure LCSC stemness after JMJD6 depletion or in combination with HOTAIR overexpression. *p < 0.05 compared with shCtl + oe-NC-treated cells. #p < 0.05 compared with shJMJD6 + oe-NC-treated cells. I Cell colony formation after JMJD6 depletion or in combination with HOTAIR overexpression following 6 Gy X-ray treatment examined by clonogenic assay. *p < 0.05 compared with shCtl + oe-NC-treated cells. #p < 0.05 compared with shJMJD6 + oe-NC-treated cells. J, RIP assay validated the interaction of JMJD6/BRD4 complex with LSD1 (relative enrichment in Y-axis represents IP-LSD1/IgG, * p < 0.05 compared with IgG group. Measurement data were expressed as mean ± standard deviation. Results between two groups were compared by unpaired t test. The data comparison between multiple groups was performed by one-way ANOVA with Tukey’s post-hoc test. Cellular experiments were repeated three times