Fig 2: Reduced cyclin D1 in SMARCA4-deficient non-small cell lung cancer (NSCLC) cells causessensitivities to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. a, b SMARCA4-deficient NSCLC cell lines express reduced cyclin D1 levels. Western blot analysis for the indicated proteins (a) and CCND1 messenger RNA (mRNA) expression (b) of a panel of NSCLC cell lines. HSP90 was used as a loading control. Relative CCND1 mRNA expression (relative to GAPDH) was measured by real-time quantitative reverse transcription PCR (RT-qPCR). A4: SMARCA4, A4/2: SMARCA4/2, Pro: proficient, Def: deficient, K: KRAS mutation. Empty triangles indicate RB-deficient cell lines. Turquoise color indicates cell lines with KRAS mutation. Error bars: mean ± standard deviation (s.d.) of biological replicates (n = 3); two-tailed t test, *p < 0.05. c, d SMARCA4-deficient NSCLC cells are highly sensitive to palbociclib treatment, similar to KRAS mutation cells. c Half-maximal inhibitory concentration (IC50) of palbociclib in the above cell line panel was determined by measuring cell viability using CellTiter-Blue assay. Error bars: mean ± s.d. of biological replicates (n = 4); two-tailed t test, *p < 0.05, **p < 0.01. d Colony formation assays of the representative cell lines. Cells were cultured in the absence or presence of palbociclib at the indicated concentrations for 10–14 days. For each cell line, all dishes were fixed at the same time. e, f Palbociclib treatment in SMARCA4-deficient NSCLC cells induces strong G1 cell cycle arrest. H1299 (e) and H1703 (f) cells treated with palbociclib for 24 h were fixed, stained with propidium iodide and analyzed by flow cytometry using the Guava easyCyte HT System. g, h Ectopic expression of cyclin D1 confers drug resistance to palbociclib in H1299 (g) and H1703 (h) cells. Upper, colony formation assays; lower, immunoblot of cells with stable ectopic expression of GFP or CCND1 and treated with palbociclib (H1299, 300 nM; H1703, 33 nM). i, j Cyclin D1 knockdown sensitizes HCC827 (i) and PC9 (j) cells to palbociclib. Upper, colony formation assays in the absence or presence of 300 nM palbociclib; lower, immunoblot of cells expressing pLKO control or short hairpin RNAs (shRNAs) targeting CCND1

Fig 3: Palbociclib is effective against SMARCA4-deficient non-small cell lung cancer (NSCLC) tumor growth in vivo. Palbociclib inhibits tumor growth in xenograft models of H1299 (a, b, e, f) and H1703 (c, d, g, h). a, c Tumor size from day 0 of treatment in H1299 (a, n = 4 per group) and H1703 (c, n = 8 for vehicle, n = 7 for palbociclib; 150 mg kg−1) models. Error bars represent mean ± standard error of mean (s.e.m.); two-way analysis of variance (ANOVA), ****p < 0.0001. b, d Final tumor weight measured after surgery in H1299 (b) and H1703 (d) models. Two-tailed t-test, **p < 0.01, ****p < 0.0001. e–h Palbociclib treatment resulted in suppression of RB phosphorylation, Ki67 expression and mitotic index in xenograft tumors of the trial endpoints. Representative images of Immunohistochemistry (IHC) (p-RB, Ki67) and hematoxylin and eosin (H&E) analysis of H1299 (e) and H1703 (g) xenograft tumor tissues. Bar 50 µm; black arrows point to mitotic active cells as examples. f, h Quantifications of p-RB, Ki67 and mitotic count of H1299 (f, n = 3) and H1703 (h, n = 4). Two-tailed t-test, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 4: Depletion of SMARCA4 decreases chromatin accessibility at a subset of AR-binding sites as assessed by ATAC-seq.a Chromatin accessibility sites as revealed by ATAC-seq in SMARCA4-depleted and control VCaP cells shown as heatmap of normalized tag counts together with SMARCA4 and AR binding. siSMARCA4-affected chromatin accessibility sites magnified and sorted by the pre-hormone (DHT) accessibility on the right. NC, sites not changed by siSMARCA4. b Boxplots of site accessibility changes by SMARCA4 depletion compared to control under vehicle or DHT exposure. c Boxplots of pre-hormone accessibility sites divided into pre-accessible sites that are open already under vehicle conditions, whereas de novo sites open under DHT exposure. d Motif analysis of NC and siSMARCA4-affected sites. e Binding of AR, FOXA1, ERG or HOXB13 on the sites in panel a in the presence and absence of androgen as indicated. Significant changes in accessibility are shown by asterisks ***<0.001, calculated with One-way ANOVA with Bonferroni post hoc test.

Fig 5: Purification of MITF-associated complexes.(A) Western blot of 501Mel cell lines stably expressing Flag-HA-tagged-MITF (F-H-MITF). (B) The immunoprecipitated material from the soluble nuclear extract (SNE) was separated by SDS PAGE and stained with silver nitrate. F-H-MITF is indicated along with * that designates a contaminating protein seen in the control immunoprecipitations. Lane M corresponds to a molecular mass marker indicated in kDa. (C) Immunoblot detection of HERC2, BRG1, USP7, USP11, XRCC5, and XRCC6 in the MITF-associated complexes. (D) Summary of proteins and complexes interacting with MITF. Shown are the proteins found specifically in the immunopurifications of F-H-MITF classified according to their function and organisation into known complexes.DOI: http://dx.doi.org/10.7554/eLife.06857.003