Fig 2: BRD2 validates as ARID1A mutant specific lethal hit. a The functional phenotypes of non-overlapping lentiviral shBRD2 vectors (#2 and #5) in the OCCC cell line panel were measured in a long-term colony formation assay. Cells expressing a mixture of nonfunctional scrambled hairpins (SCR) were used as control. The cells were fixed, stained and photographed after 10–12 days. In the cell lines labeled with an asterisk (*) BRD2 was identified as lethal hit. b The indicated ARID1A wildtype and mutant OCCC cell lines were plated in 6-well plates (10,000 cells per well) and exposed to increasing JQ1 (0, 125, 250, and 500 nM) concentrations in triplicate. The cells were fixed, stained with crystal violet and photographed after 10 days. c Colony formation assays (CFA) from b were quantified by crystal violet dye extraction. Shown is relative reduction in crystal violet staining compared to untreated control. Colony formation assays were performed in triplicate. Error bars denote SD. The dotted line denotes the cut-off used (below 0.5 at 250 nM JQ1 concentration) to qualify cell lines as “JQ1 sensitive”. A Fisher Exact test with the abovementioned cutoff at 250 nM JQ1 gave the statistic value of 0.09. P-values (only shown for the “JQ1 sensitive” lines) were calculated with multiple t-tests, asterisk denote the number of digits after the decimal

Fig 3: Direct binding of BRD2 in ARID1B locus. a The ARID1A wildtype line OVCA429 and the ARID1A mutant line HAC2 were cultured for 24 h in 500 nM JQ1 or DMSO vehicle control and subjected to chromatin immunoprecipitation with BRD2 and control antibodies. ChIP sequences were generated by Illumina Hiseq 2000 genome analyzer and aligned to the Human Reference Genome (assembly hg38) and visualized in IGV. Displayed are IGV snapshots of the Transcriptional Start Sites (TSS) of the indicated SWI/SNF members ARID1A, ARID1B, SMARCE1 and SMARCC2. b, c Displayed are the average profiles of ChIP-seq signal for the indicated cell lines in absence and presence of JQ1 at transcriptional start site regions (+/−3 kb) (n = 66035). Shading indicates standard error of average read count profiles. d, e qRT-PCR amplification was performed with primers located in the ARID1B gene. Shown is the percentage of the BRD2 (d) and BRD4 (e) and control IgG chromatin immunoprecipitations over chromatin input qRT-PCR amplification. Error bars denote SD over n = 4 (d) and n = 3 (e) experiments. P-values were calculated with multiple t-tests, asterisk denote the number of digits after the decimal. Primer location is visualized in red in the gene map displayed underneath the graph

Fig 4: BRD4 depletion inhibits DUX4 expression. a–c Expression of BET genes (a), BET proteins (b), and DUX4 and DUX4 targets (c) after BRD2, BRD3, or BRD4 siRNA knockdown in 54-2 FSHD1 myoblasts. Error bars indicate the standard deviation from the mean of three biological replicates. GAPDH serves as a loading control. p values were calculated using a one-way analysis of variance with Dunnett’s post test. *p < 0.05

Fig 5: Compound screen and CRISPR-based persister screen identified BRD2 as a vulnerability of DTEPs(A) Schematic representation of DTP induction and generation of DTEPs in PC9-SS cells.(B) Schematic of small-molecule screen on parental, senescent, DTP, and DTEP cells.(C) Top hits were selected based on the therapeutic window of senescent (Senes)/DTEP vs. parental. The x axis represents log fold change of the AUC score (Senes/DTEP vs. parental). The y axis represents the AUC score of Senes/DTEP cells.(D) CellTiter blue quantification of relevant viability of parental cells or DTEPs (osimertinib and gefitinib induced) treated with BET inhibitors.(E) Schematic of CRISPR-based kinome-wide genetic screen on DTEPs (n = 3 for each arm).(F) Top hits were selected based on the fold depletion of sgRNAs DTEP vs. parental. Genes with 4 sgRNA dropped out were identified as hits.(G) Western blot of BRD2 and β-actin in PC9-SS-iCas9 wild-type (WT) and BRD2KO clones.(H) Relative fold change of persisters number based on cell counting obtained from WT and BRD2KO clones after 14 days of osimertinib exposure.(I) IncuCyte-based proliferation of DTEPs of WT and BRD2KO clones.Error bars in (D), (H), and (I) represent mean ± SD, n = 3 independent experiments. Statistical significance was calculated by 2-tailed Student’s t test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).