Fig 2: A schematic model of the mechanism underlying transcriptional regulation of SLC7A11 expression by SWI/SNF chromatin remodeling complexes. (a). A Schematic model of transcriptional regulation of the SLC7A11 gene in cooperation with cBAF, PBAF, and ncBAF complexes. cBAF (SMARCA4, ARID1A, SMARCB1) localizes to the promotor and enhancer regions of the SLC7A11 locus. PBAF (SMARCA4, PBRM1, SMARCB1) and ncBAF (SMARCA4, BRD9) localize to the promotor region of the SLC7A11 locus. When either SMARCA4, SMARCB1, or PBRM1 is deficient, the transcription of the SLC7A11 is attenuated by disrupting the coordinated transcriptional regulation among the cBAF, PBAF, and ncBAF complexes. Therefore, the absence of any of the complexes attenuates the transcription of SLC7A11. (b). A Schematic model of transcriptional regulation of the SLC7A11 gene and the balance between GSH and ROS in SWI/SNF-proficient cells. The BAF, PBAF, and ncBAF complexes act cooperatively to remodel the chromatin at the promotor and enhancer regions of the SLC7A11 locus, thereby facilitating transcription. This is followed by recruiting transcription factors such as NRF2 and RNA polymerases II (RNAPII). SLC7A11 incorporates Cystine (Cys-Cys) into the cells, and then Cys is produced in cells. Cys (cysteine) is associated with Glu (glutamate) and Gly (glycine), and Glutathione (GSH) is made in excess of the amount of ROS in the cell. At this time, even if a GSH inhibitor is administered, ROS can be suppressed, so the cell survives. (c). A Schematic model of transcriptional regulation of the SLC7A11 gene and the balance between GSH and ROS in SWI/SNF-deficient cells. Deficiency in either cBAF or PBAF suppresses the recruitment of transcription factors, leading to repressed expression of SLC7A11. This reduces cystine uptake, resulting in a shortage of intracellular cysteine. Because Glutathione (GSH) is synthesized from Cystine (Cys-Cys), Glu (glutamate), and Gly (glycine), a shortage of Cys (cysteine) leads to a reduction in GSH synthesis. Thus, SMARCA4-, SMARCB1- and PBRM1-deficient cells have low levels of GSH, making them vulnerable to ROS. Further suppressing GSH in SMARCA4-, SMARCB1- and PBRM1-deficient cells by treatment with eprenetapopt decreases basal levels of GSH to an even greater extent. This ultimately leads to an increase in intracellular ROS, which triggers apoptosis-mediated cell death.

Fig 3: cBAF- and PBAF-deficient cancer cells show low basal levels of GSH due to attenuated expression of SLC7A11. (a). Relative expression of SLC7A11 mRNA in SWI/SNF-proficient cell lines (H2122, H2228, PC9) and SMARCA4-deficient (H1703, H1819), SMARCB1-deficient (JMU-RTK-2, G402), or PBRM1-deficient (KMRC-1, RCC-MF) cell lines (relative to that in the H2122 cell line). Data are presented as SEM (the mean ± standard error of the mean), n = 3 independent experiments. (b). Basal levels of GSH in SWI/SNF-proficient cell lines (H2122, H2228, PC9) and SMARCA4-deficient (H1703, H1819), SMARCB1-deficient (JMU-RTK-2, G402), or PBRM1-deficient (RCC-MF, KMRC-1) cell lines (relative to that in the H2122 cell line). Data are presented as SEM, n = 3 independent experiments. For all experiments, p values were determined by an unpaired two-tailed Student’s t-test. **p < 0.01, ***p < 0.001.

Fig 4: Damaging Mutations in PBRM1 are associated with MCL1 dependency and sensitivity to MCL inhibition in ccRCC. (A) MCL-1 gene dependency analysis in ccRCC cell lines with damaging PBRM1 mutations compared to WT. Dependency scores (Chronos, 22Q2) were retrieved from DepMap (https://depmap.org) (B). PBRM1 mRNA levels in ccRCC cell lines inversely correlate with MCL1 dependency. *P<0.05, versus WT by t-test. (C) Waterfall plot showing half maximal inhibitory concentration (IC50) for PRT1419 in the ccRCC cell line panel. (D) Cell Titer-Glo (CTG) assay assessing inhibition of 3D spheroid growth in PBRM1-mut and WT ccRCC cell lines following treatment with MCL-1 inhibitor PRT1419 (72h). Values shown are cell viability calculated as percentage of vehicle DMSO control. (E) Caspase 3/7 activity assay showing induction of apoptosis following 4h treatment with PRT1419 in PBRM1-mut and WT lines. (F) CTG assay assessing inhibition of 3D cell growth by PRT1419 in 769-P (PBRM1 WT) cells following PBRM1 depletion via RNAi. (G) Caspase 3/7 activity assays assessing induction of apoptosis by PRT1419 in 769-P (PBRM1 WT) cells following PBRM1 depletion via RNAi. (H) Western blot validating PBRM1 expression following RNAi knockdown in 769-P cells (I) OSRC-2 (PBRM1 mutant ccRCC) cell line-derived xenograft (CDX) assessing anti-tumor activity of intravenously administered PRT1419 at 20 mg/kg. Animals were dosed once weekly with PRT1419. Data represented as mean ± SEM. N=8, * P<0.05 by Mann-Whitney U test. (J) Representative images of tumors from mice in both vehicle and PRT1419-treated groups at the end of the study.

Fig 5: SMARCA4/SMARCA2 deficiency and SS18–SSX fusion lead to upregulation of KREMEN2 gene expression. A, Expression of KREMEN2 mRNA in SWI/SNF-proficient, SMARCA4/SMARCA2-deficient, and SS18–SSX fusion cell lines (relative to that in DMS114 cells). Data are presented as the mean ± SEM; n = 3 independent experiments. B, Violin plots showing clustered expression of KREMEN2 mRNA in SWI/SNF-WT, SMARCB1-deficient, ARID1A-deficient, PBRM1-deficient, SMARCA4-deficient, SMARCA4/SMARCA2-deficient, and SS18–SSX fusion cell lines based on an analysis of mutations, gene CN, and gene expression data from DepMap (data version 23Q2). SWI/SNF-proficient (n = 1,238), SMARCB1-deficient (n = 19), ARID1A-deficient (n = 52), PBRM1-deficient (n = 23), SMARCA4-deficient (n = 34), SMARCA2-deficient (n = 27), SMARCA4/SMARCA2-deficient (n = 6), and SS18–SSX (n = 5) cell lines were analyzed, and data are presented as the mean ± SEM. n indicates the number of independent samples. C, Localization of signals generated by SMARCA4 and H3K27ac ChIP-seq, ATAC-seq, and RNA-seq around the KREMEN2 locus in SMARCA4/SMARCA2-deficient BIN-67 cells transduced with an SMARCA4 expression vector (+SMARCA4) and in BIN-67 cells without the SMARCA4 expression vector (−SMARCA4). D, Localization of signals generated by SS18, SMARCA4, RNAPII, and SUZ12 ChIP-seq, ATAC-seq, and RNA-seq around the KREMEN2 locus in SS18–SSX-fusion Aska-SS cells depleted of SS18–SSX by SSX-targeting shRNA (shSSX; SS18-WT) and in SS18–SSX fusion Aska-SS cells not depleted of SS18–SSX [i.e., transduced with NT shRNA (shNT; SS18–SSX)].