Fig 2: PBRM1 restoration into PBRM1-mutated RCC cells shifted coregulator content of the PAX8 hub to CoAs(A) The experimental approach.(B) Western blot and IP-western blot analyses of empty vector versus FLAG-PBRM1 transfected RCC (ACHN) cells. PAX8 versus IgG control IP was performed in lysates from empty vector versus PBRM1-FLAG transfected cells 48 h after transfection.(C) Heatmaps to indicate amounts of coregulators in the PAX8 interactome in empty vector versus FLAG-PBRM1 transfected cells. PAX8 was IP and proteins analyzed by LCMS/MS. Analyses were 48 h after transfection. A minimum Mascot ion score of 25 and peptide rank 1 were used for automatically accepting all peptide MS/MS spectra. STRING was used to construct the protein-protein interaction network with a STRONG score >0.4. Circle size indicates abundance of protein in the interactome. Detection, quantification, GO, and STRING analyses data are in Table S6.(D) Relative abundances of CoA and CoR complexes with empty vector versus PBRM1 transfection. The individual proteins constituting CoA and CoR are listed in (A). Median ± IQR. Values analyzed are tabulated in Table S6.

Fig 3: The PBRM1 protein interactome in kidney lineage cells(A) Endogenous PBRM1 was immunoprecipitated (IP) from human embryonic kidney cells (HEK293, left) and RCC cells (ACHN, right), and co-purified proteins were analyzed by LCMS/MS. Shown are identified coactivator (CoA) components and transcription factors. Circle size indicates abundance of protein in the interactome. A minimum Mascot ion score of 25 and peptide rank 1 were used for automatically accepting all peptide MS/MS spectra. The most enriched functional group was “ATPase chromatin remodeling complex” (p value Bonferroni-corrected 1.42 × 10−27). The STRING database system was used to construct the protein-protein interaction network with a parameter STRONG score >0.4. Detection, quantification, Gene Ontology (GO), and STRING data for these and other proteins are in Table S1. Heatmap shows quantification versus IP with immunoglobulin G (IgG) isotype control.(B) Heatmap summary of the data shown in (A).(C) Bi-directional IP-western blots in 293T and three RCC cell lines confirmed the interaction between PBRM1 and PAX8. The RCC cell lines used were ACHN, SKRC45, and SKRC29.

Fig 4: IRF9 and STAT2 expression significantly correlated with the expression of PBRM1, SETD2, or BAP1 in ccRCC tumors.(A) 20x IHC images of representative ccRCC foci stained with indicated antibodies. (B) Spearman correlation coefficient, rho, and p-value of nuclear (nuc) IRF9 or cytoplasmic (cyto) IHC signals with those of PBRM1, SETD2 or BAP1. The analysis was performed with either all the foci or the foci within each tumor stage or tumor grade. (C) Kaplan-Meier curves of patient overall survival based on the positive or negative IHC signal of nuclear IRF9 and (D) cytoplasmic STAT2. Statistical significance calculated using the log-rank test. (E) A schematic diagram showing how HIF and secondary tumor suppressors converge on ISGF3 to regulate tumor growth in ccRCC. HIF activates ISGF3 that suppresses tumor growth. PBRM1, KDM5C, SETD2 and BAP1 all support the function of ISGF3. Mutations in any of them relieve the tumor suppressive function of HIF2α.

Fig 5: Components of the PBRM1/PBAF CoA recruited by PAX8 are recurrently deleted/mutated in RCC, while druggable CoRs recruited by PAX8 are recurrently amplified(A) The analysis approach.(B) Copy numbers of genes for components of the PBAF CoA and for druggable CoR components recruited by PAX8. Gistic thresholded copy-number data from TCGA (n = 342).(C) The copy-number alterations (predominantly deletions) and inactivating mutations to genes for PBAF CoA components impact their expression accordingly. Gene expression in normal kidney (NKid) and RCCs stratified by recurrent deletions and mutations of CoA genes (RNA sequencing [RNA-seq] TCGA, NKid cortex n = 72, RCC n = 342). Median ± interquartile range (IQR). *p < 0.001, two-sided Mann-Whitney test.(D) The copy-number alterations to genes for druggable CoR components (predominantly gains/amplifications) impact their expression accordingly. Gene expression in NKid and RCCs stratified by recurrent gains/amplifications of CoR genes (RNA-seq TCGA, NKid cortex n = 72, RCC n = 342). Median ± IQR. *p < 0.001, two-sided Mann-Whitney test.