Fig 2: Therapeutic PIK3CA inhibition promotes animal survival by reducing tumor cell viability(A) Primary ascites-derived tumor cell isolation scheme. (B) Representative western blots to highlight the primary tumor cell validation scheme and demonstrate that ARID1A loss and heightened P-AKT S473 is observed in primary Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells versus normal ovarian surface epithelial (OSE) cells. (C) Western blots of P-AKT S473 and total AKT levels in Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells grown in the presence 10, 1, 0.001, or 0 µM BKM120 or equivalent dilutions (v/v) of DMSO vehicle control for 48 hrs. (D) BKM120 treatment reduces tumor cell viability with a half-maximal inhibitory concentration (IC50) of 0.96 +/- 0.25 µM. Significant differences based on the average percent inhibition ±SD of three independent tumor cell lines treated with BKM120- versus vehicle-treated cells were calculated using a two-tailed Student’s t test (*p-value <0.05). (E,F) Administration of chow-fed BKM120 reduces PI3K pathway activity, as shown by reduced P-S6 Ser235/236 levels, and extends the Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R median survival latency to 11 weeks. BKM120 treatment commenced 4 weeks after AdCRE injection. P-S6 Ser235/236 IHC was performed on tumor histological sections from three independent BKM120 treated or untreated mice. (F) Untreated Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice succumb to cancer with a median (µ1/2) latency of period of 7.5 weeks. Statistical differences in survival (BKM120 chow-fed versus untreated) were calculated using a log rank test (p-values <0.05 were considered significant).

Fig 3: Coexistent ARID1A-PIK3CA mutations induce IL-6 expression in a cooperative manner(A) IL-6 mRNA expression in AdCRE-infected primary OSE cells carrying all allele combinations. (B) Normalized IL-6 ELISA measurements of AdCRE-infected primary OSE cells carrying all allele combinations. Significant differences based on the average normalized expression value ±SD among mutant allele combinations versus wildtype were calculated using a two-tailed Student’s t test. (C) Western blot showing ARID1A, P-AKT S473, total AKT, P-STAT3 Y705, and total STAT3 levels in AdCRE-infected primary OSE cells carrying all allele combinations. AdCRE-infected wildtype cells served as controls. (D) MTT cell viability assay on primary Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells treated with AdControl, AdCRE, or AdCRE supplemented with 5 µg mL-1 anti-IL-6 antibodies. (E) Treatment of normal (wild type) primary OSE cells with vehicle (v/v), 10 ng mL-1 IL-6, or 10 ng mL-1 IL-6 supplemented with 5 µg mL-1 anti-IL-6 antibodies. Significant differences based on the average MTT absorbance value ±SD between three independent replicates of AdControl- versus AdCRE-infected or vehicle versus IL-6-treated cells were calculated using a two-tailed Student’s t test. (F) Western blot of normal primary OSE cells showing dose-dependent increases in P-STAT3 Y705 levels following IL-6 treatment. Co-treatment with 5 µg mL-1 anti-IL-6 antibodies blocked P-STAT3 Y705 induction. (G) ARID1A occupancy at the IL6 locus was detected by chromatin immunoprecipitation (ChIP) using anti-ARID1A antibodies (denoted as IP) on crosslinked chromatin from AdControl- or AdCRE-infected Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R primary OSE cells. Non-specific isotype matched antibodies (denoted as NS) were used in control ChIPs. ARID1A occupancy at the IL6 promoter (site or primer pair B) is reduced in ARID1A depleted cells treated with AdCRE. Average percent ChIP input ±SD represent data from experiments performed using three primary OSE cell isolations. Significant differences were calculated using a two-tailed Student’s t test. (H) Proposed model of IL-6 regulation by ARID1A and PIK3CA mutations in OCCC. Only p-values <0.05 were considered significant.

Fig 4: A new Arid1a conditional allele to explore ARID1A tumor suppressor function in vivo(A) Targeting scheme to insert two loxP sites flanking exon 5 and 6 of mouse ARID1A. (B) Representative Southern blot showing properly targeted ES cell clones. Insertion of loxP sites generates 5.6 kb EcoRV and 8.5 kb ApaI genomic fragments. (C) PCR genotyping and quantitative, dual-color fluorescent western blot of ARID1A (red) and ßACTIN (green) protein expression in whole-embryo lysates from four Arid1afl/+ and four Arid1afl/+;Sox2Cre0/+ embryos. (D) Graph of quantitative western blot results depicting normalized protein levels. Significant differences based on the average normalized protein expression ±SD of four independent embryos were calculated using a two-tailed Student’s t test (p-values <0.05 were considered significant). (E,F) Representative LacZ-stained, control (uninjected) or intrabursal AdCRE-injected (Gt)Rosa26lacZ ovary showing patterns of CRE-mediated recombination in the ovarian surface epithelium. (F, inset) Histological section of LacZ-stained AdCRE-injected (Gt)Rosa26lacZ ovary. (G-I) Live-luminescence overlaid whole-mount images of an intrabursal AdCRE injected, (Gt)Rosa26luciferase female mouse. (I) Excised reproductive tract. Asterisks in H indicate wound staple.

Fig 5: Increased CRISPR-Cas9 editing efficiency of ARID1A at higher ribonucleoprotein concentration.(A) ARID1A editing efficiency in four porcine HCC cell lines following transfection with 25 nM ribonucleoprotein comprising Cas9 and gRNA#1. Genomic DNA was collected 2 days post-transfection and analyzed by targeted NGS. The bar graph depicts the percentages (%) of total reads that displayed indels at the gRNA target site occurring as a result of nonhomologous end joining. (B) Top 15 reads detected by targeted NGS analysis in the four porcine HCC lines, mapped to the reference sequence. The identity and frequency of indels in the four cell lines is consistent. The percentages of reads of each sequence are shown on the right. The location of the indel relative to the expected cleavage site is shown on the left, followed by a colon and the number of nucleotides inserted (i) or deleted (d). The asterisk denotes non-edited reads. Dashed line: predicted Cas9 cleavage position; red box: insertion; dash: deleted base.HCC: Hepatocellular carcinoma; NGS: Next-generation sequencing.