Fig 2: Variant distribution of ARID1A, EPHA2, PIK3CA and LAMA1 genes in 795 CCA patients.Lollipop plots showing the distribution of ARID1A, EPHA2, PIK3CA and LAMA1 genes in CCA patients. The most frequent variant alterations are annotated as on top of the plots.

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: JQ1 specifically inhibits in vivo growth of ARID1A mutant OCCC xenografts and PDX models. a, b ARID1A wildtype ES2 cells (5 × 106 in PBS) were subcutaneously injected in the flank of 8–10-weeks-old NSG mice. For the ARID1A mutant SMOV2 xenograft experiments, successfully established SMOV2 engraftments (see methods) were dissected, and tumor pieces were subcutaneously propagated in the flank of new mice. When tumors reached ~200 mm3, mice were randomized into vehicle (DMSO) control or treatment (JQ1) groups (n = 6 mice/group). ES2 xenografts reached the 1500 mm3 endpoint after 14 days of JQ1 administration, SMOV2 xenografts were treated with JQ1 for 21 days. c, d Displayed are the tumorweights of the indicated excised tumors after vehicle or JQ1 treatments. e, f ARID1A wildtype (e) and mutant (f) PDX F3 tumors were established (see methods) and tumor pieces were subcutaneously implanted in the flank of NSG mice. When tumors demonstrated sustained growth, mice were randomized into vehicle (DMSO) control or treatment (JQ1) groups. Treatment with JQ1 was continued for 21 days. For all in vivo experiments, JQ1 (50 mg/kg) or DMSO vehicle were administered intraperitoneally daily and tumor growth was quantified by caliper measurements. Tumor growth was determined as tumor volume treatment day/ tumor volume start treatment. Statistical significance for tumor growth was determined using two-way ANOVA with Bonferoni post-hoc test correction. Error bars denote standard error of mean

Fig 5: BET inhibition reduces ARID1B levels. a OVCA429 ARID1A knockout cell lines were generated with a dual vector doxycycline inducible CRISPR/Cas9 vector system. Wildtype cells, one polyclonal knockout line (ARID1A kopc) and two monoclonal knockout lines (ARID1A ko#12 and #37) were exposed to increasing amounts of JQ1 as indicated. After 48 h drug exposure ARID1B RNA analysis was performed. mRNA levels were normalized to expression of GAPDH. Error bars denote SD. P-values were calculated with multiple t-tests, asterisk denote the number of digits after the decimal. b The OVCA429 cells described in a were subjected to Western blot analysis for the indicated proteins. ACTIN served as a loading control. c Western blot analysis of ARID1B and BRD2 in OVCA429 cells stably infected with lentiviral shRNA vectors targeting BRD2. ACTIN served as a loading control. d Doxycycline inducible lentiviral ARID1B shRNA vectors #3 and #5 were introduced in two ARID1A wildtype and two ARID1A mutant OCCC lines. After stable selection cells were plated in a long-term proliferation assay in the presence of doxycycline. Cells expressing a mixture of nonfunctional scrambled hairpins (SCR) were used as control. The cells were fixed, stained and photographed after 10–14 days. e ARID1B mRNA expression analysis by qRT-PCR in ES2, OVCA429, SMOV2 and HAC2 cells stably expressing the two doxycycline-inducible shARID1B vectors. mRNA levels were normalized to expression of GAPDH. Error bars denote SD. P-values were calculated with multiple t-tests, asterisk denote the number of digits after the decimal