Fig 2: SMARCA4 protein levels are higher in H3.3K27M compared to H3WT cells. (A) Immunoblots of patient-derived H3 WT (SF188, SJGBM2, and UMPED37), H3.3G34V (KNS42), or H3.3K27M (DIPG007, DIPGXIII*p, and SF7761) cell lines probed for SMARCA4, SMARCA2, PBRM1, ARID2, BRD7, and SMARCB1; western blot for HSP90 and Ponceau staining were used as loading control. (B) Immunoblots of DIPG007 cells with or without stable knockdown (KD) of H3-3A with two independent shRNAs (sh1 or sh2) probed for H3.3K27M, H3K27me3, and H3K27ac; total H3 was probed as loading control. (C) Heatmap of normalized protein abundance (Z-score) of key components of BAF and PBAF (PBRM1, ARID2, and BRD7) SWI/SNF complex in DIPG007 H3.3K27M with or without H3-3A KD acquired by untargeted proteomics (n = 3, each). (D) Immunoblots of DIPG007 cells with or without H3-3A KDs from B probed for SMARCA4, SMARCA2, PBRM1, ARID2, BRD7, and SMARCB1. HSP90 was probed as loading control. (E) Representative images of H3K27M and H3WT (2 cases each) tumors stained for SMARCA4 by immunohistochemistry. (Scale bar, 50 μM.) (F) Matlab-based quantification of SMARCA4 (Y axis, a.u.=arbitrary units determined by number of pixels × pixel intensity; three randomly selected regions/case) in H3WT (n = 5) and H3K27M (n = 6) DMGs. Data were analyzed by two-sided, two-tailed, nonpaired t test.

Fig 3: H3.3K27M cell lines are sensitive to SMARCA2, SMARCA4, and PBRM1 protein degradation using the PROTAC AU-15330. (A) Heatmap of normalized protein abundance (Z-score) of key SWI/SNF complex components in untreated DIPG007 cells (n = 3) or DIPG007 cells treated with AU-15330 PROTAC (n = 4, 1 µM for 24 h) or vehicle (Veh, DMSO, n = 3), acquired by untargeted proteomics. Data were analyzed by ANOVA; P values indicated are between Veh and AU-15330-treated cells. (B) Immunoblots of H3.3K27M mutant cell lines (SF7761, BT245, and DIPG007) treated with AU-15330 (1 µM for 24 h) or Veh and probed for SMARCA4, SMARCA2, PBRM1, and SMARCB1. HSP90 was probed as loading control. (C) Immunoblots of H3.3G34V mutant (KNS42) and H3WT (SJGBM2 and SF188) cell lines treated with AU-15330 (1 µM for 24 h) or Veh and probed for SMARCA4, SMARCA2, PBRM1, and SMARCB1. HSP90 was probed as loading control. (D) Cell viability (normalized to Veh, percentage, Y axis) of H3WT (SJGBM2 and SF188), H3.3G34V mutant (KNS42), and H3.3K27M mutant (SF7761, BT245, and DIPG007) cell lines on treatment with different concentrations of AU-15330 (log concentrations, X axis) for 5 d (n = 3 for each concentration/cell line). (E) Cell viability (normalized to Veh, percentage, Y axis) of DIPG007 cells with or without for H3-3A KD with two independent shRNAs (H3-3A sh1 or sh2) treated with different concentrations of AU-15330 (log concentrations, X axis) for 5 d (n = 3 for each concentration/cell line). (F) Half maximal inhibitory concentration (IC50) of AU-15330 (μM=Molar, Y axis) for H3.3K27M mutant (SF7761, BT245, and DIPG007), H3-3A KD sh1 and sh2 DIPG007, H3.3G34V mutant (KNS42), and H3WT (SJGBM2 and SF188) cells based on cell viability curves in (D) and (E).

Fig 4: Cell viability screening for ATRi in combination with ATMi, DNA-PKi, or PARPi in SWI/SNF-deficient LUAD cells.A LUAD cell lines harboring deficiency of SMARCA4 (A549 and H1299), ARID1A (H2172 and SK-LU-1), and PBRM1 (ABC1 and H226) were constitutively expressed the corresponding wild-type proteins via lentiviral cDNA expression system. Expression of SMARCA4, ARID1A, and PBRM1 proteins was analyzed by western blotting. β-Actin was detected as loading control. B Cell viability screening for tuvusertib (0, 3.9–1000 nM) in combination with lartesertib (0, 39.1–10000 nM), peposertib (0, 39.1–10000 nM), or niraparib (0, 39.1-10000 nM). Cells were treated with these combinations for 4 days, then a cell viability assay was performed. Using SynergyFinder plus, dose response and Bliss synergy maps in A549 cells are shown in Fig. 1B, and other cells are shown in Fig. S1E–G. These maps indicate the mean of three independent experiments. C Combination sensitivity score-Bliss synergy score plots of tuvusertib in combination with lartesertib, peposertib, or niraparib in the cell viability screening shown in Fig. 1B.