Fig 2: A947 is selectively efficacious in SMARCA4-mutant NSCLC xenograft models.a Tumor concentration (upper graphic) and pharmacodynamic biomarker responses (lower graphic) monitored over a 2 week period in HCC515 xenograft tumors following a single-dose, intravenous (i.v.) administration of A947 (40 mg/kg). Tumor levels of SMARCA2 protein were quantified by ImageLab from Western blots and normalized to a loading control protein (ß-actin). Tumor levels of the mRNA transcripts, KRT80 and PLAU, were quantified by Taqman. Data are presented relative to levels in untreated tumors and represented as mean ± s.d from n = 5 animals per timepoint. b, c Tumor volume in mice harboring either SMARCA4-mutant HCC515 (b) or HCC2302 (c) xenografts following administration of A947 (40 mg/kg, i.v.) or vehicle control. Data are presented as mean ± s.e.m. (n = 10 mice/group). Statistical significance was assessed by a two-sided, unpaired Student t test. p = 0.0002; p = 0.0049 (c). d Pharmacodynamic biomarker levels tumors collected at end of study from animals treated in (b and c) (SMARCA4 protein: HCC515-vehicle (n = 8), HCC515-A947 (n = 10), HCC2302-vehicle (n = 10), HCC2302-A947 (n = 9); KRT80 transcript: HCC515-vehicle (n = 9), HCC515-A947 (n = 9), HCC2302-vehicle (n = 10), HCC2302-A947 (n = 8). Mice received a final dose of A947 24 h prior to tumor collection. SMARCA2 protein and KRT80 mRNA levels were quantified as in (a) and presented relative to levels in the respective vehicle-treated tumors. e Log-normalized counts per million reads (CPM) of SMARCA2 target genes determined by RNAseq and presented relative to levels in vehicle-treated tumors. A consensus set of SMARCA2-regulated genes (n = 412) were defined based upon exhibiting acute (24 h) and sustained (96 h) suppression following A947 treatment of both models in vitro, as further described in the methods section. Boxes span from quartile 1 (Q1; 25th perentile) to quartile 3 (Q3; 75th percentile), with medians (50th percentile) represented by center lines. Whiskers span ± 1.5 times the interquartile range (Q3–Q1) and outliers are represented as dots. f Tumor volume in mice harboring SMARCA4 wild-type Calu-6 xenografts following administration of A947 (40 mg/kg, i.v.) or vehicle control. Data are presented as mean ± s.e.m. (n = 10 mice/group). Potential significance was assessed by a two-sided, unpaired Student t test. n.s. nonsignificant. g Levels of SMARCA2, SMARCA4 and PBRM1 protein in Calu-6 tumors (n = 10 for each group) were quantified by ImageLab from Western blots and normalized to a loading protein control (ß-actin). Calu-6 tumors were collected at the end of study from mice treated in (f) and mice received a final dose of A947 24 h prior to tumor collection. Source data are provided as a Source Data file.

Fig 3: The TISIDB database shows that PBRM1 mutation is significantly associated with activated CD4 (A) and activated CD8 T cells (B) (Wilcoxon Test).

Fig 4: PBRM1 loss induces resistance to ICB.a Early treatment with anti-PD-1 blockade. PD-1 antibodies were administrated at day 3, day 6, and day 9 after tumor inoculation. First dose was 400 µg/mouse, and the following two doses were 200 µg/mouse. b Delayed treatment with anti-PD-1 blockade. Anti-PD-1 antibody (200 µg/mouse) was administrated every third day once the tumors reached 100-200 mm3. Treatment schemas, In vivo tumor growth rates and survival rates of mice are shown. Two-way ANOVA and Log-rank (Mantel-Cox) analyses were performed with GraphPad Prism 7.03. Data in the graphs are means ± SEM. UnTX, untreated control. ns, P > 0.05, *P < 0.05, and **P < 0.001. c Patient response in the IMmotion150 cohort following treatment with either atezolizumab (Atezo) or atezolizumab in combination with bevacizumab (Atezo + Bev). Mono, PBRM1 mutation only, Dual, PBRM1 mutation in combination with a BAP1 or SETD2 mutation; WT, wild type; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. Cochran–Mantel–Haenszel test. d Comparison of patient response rate, defined as either a complete or partial response, in the IMmotion150 cohort. Cochran–Mantel–Haenszel test. e Overall survival of RCC patients treated with ICB from the MSKCC IMPACT cohort stratified by PBRM1 status. Log-rank test. f Overall survival of ccRCC patients from TCGA stratified by PBRM1 mutation status. Log-rank test. g Model for PBRM1 mediated IFNγ-STAT1 signaling and tumor immune microenvironment modulating. PBRM1 ensures IFNγ-induced STAT1 activity and autonomous expression of downstream genes involved in T-cell recruitment (e.g., CXCL9). Infiltrating, activated T cells in turn produce more IFNγ which stimulates tumor cells to secrete immunostimulatory chemokines and cytokines, and in parallel upregulate checkpoint pathways on T cells and tumors cells. Thus, the immunogenic TME of PBRM1 proficient tumors is primed to respond to ICB. On the other hand, PBRM1 loss reduces IFNγ-STAT1 signaling and downstream T cell attracting factors, which prevents T cell infiltration and IFNγ secretion. Such a non-immunogenic TME of PBRM1 mutated tumors blunts ICB response. Source data are provided as a Source Data file.

Fig 5: Bromodomain 4 of PBRM1 is required for recognition of acetylated K382 on p53.a Schematic depiction of the functional domains of PBRM1 and truncated constructs. WT: wild-type, BAH: bromo-adjacent homology domain, HMG: high-mobility group domain. b, c HEK293T cells were transfected with vectors, Flag-WT PBRM1 and Flag-PBRM1 truncated constructs (b), and each individual or all the PBRM1 bromodomains (BD1–6, c). After immunoprecipitation with Flag-M2 beads, the inputs and eluates were analyzed by immunoblots. d Schematic of key amino acid residues of binding pockets in PBRM1 bromodomains 4 and 5. The critical YN residues are underlined and mutations are red. e–g H1299 PBRM1 KO cells were transfected with Flag-PBRM1 bromodomains 4 and 5 (Flag-BD45) containing mutations (BD4* or BD5*) that abolish acetyl-lysine recognition in each domain (e), Flag-BD45 containing tumor-derived mutations (f) or full-length PBRM1 containing the BD4* mutation (g). Lysates were incubated with biotinylated p53 peptides with lysine acetylation at the indicated sites. The peptides were pulled down with streptavidin beads and the associated proteins were immunoblotted. h Vectors, Myc-p53, HA-p300, and Flag-PBRM1 with or without the BD4* mutation were transfected into HEK293T PBRM1 knockout cells. The lysates were subjected to anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were analyzed in immunoblots. Source data are provided as a Source Data file.