Fig 2: Context-dependent cellular phenotypes by genetic loss of individual EMT regulators.(A) Violin plot showing the distribution of the dependency for proliferation of all CCLE NSCLC cell lines for individual genes within the indicated families. The plot shows the phenotypic dependency calculated by the DepMap project. CRISPR (Avana) CERES score and RNAi DEMETER2 score are displayed in the left and right panels, respectively. Selected genes are labeled within their families. ANOVA, analysis of variance. (B) Bar plot of control and mutant A549-MGT#1 cell proliferation in parallel longitudinal analysis. Statistics: Significant by t test and Holm-Sidak post hoc test (P < 0.05; n = 4), BRD2, EPC1, ARID1A, and ACVR1 KOs versus control. (C) Bar plot of control and mutant A549-MGT#1 ± GSK126 cell colony formation assay. Statistics: Significant by t test and Holm-Sidak post hoc test (P < 0.05; n = 3) in DMSO group: ARID2, ARID1A, DOT1L, and ACVR1 KOs; GSK126: EPC1, ARID1A, BRD2, DOT1L, KMT2A, and ACVR1 KOs. (D) Left: Line plot of parallel longitudinal high-content wound healing analysis of A549-MGT#1 cells with the indicated genotypes under homeostatic conditions. Each dot represents the mean in each time point. Statistics: Two-way ANOVA and Dunnet post hoc test (n = 4). Asterisks denote significance for the indicated comparison. Antagonistic regulators of EMT and motility in A549 cells are shown to the right. (E) Left: Schematic representation of three-dimensional (3D) invasion assay. Right: Migration depth of DRAQ5-stained nuclei for each time point and clone normalized to time point T = 0 hours from high-content imaging. Statistical analysis for time point 24 hours shows corrected multiple t test (*P < 0.05; ***P < 0.001; n = 4). (F) FACS analysis (left) and quantification (right) of MGT#1 expression in lung and brain tumor cells with the indicated genotypes.

Fig 3: Frequent ARID2 mutations associated with protein loss.(a) Box representation of the mutated patients for the most significantly mutated genes according to OncodriveFML in the lung cancer cohort generated with Maftools 49. Each box in the central matrix represents an independent patient. Colored boxes represent mutated patients for the corresponding gene in a color code indicating the type of mutation. (b) Representation of the significance analysis of the functional impact of the mutations found in each gene performed by OncodriveFML. Genes in read showed a q-value < 0.1 after multi-test correction. (c) Visual representation of the location of all identified ARID2 mutations in our discovery and validation lung cancer cohort in relation to the functional protein domains. (d) Representative images of ARID2 immunohistochemistry experiments in two ARID2-mutated (right) and two ARID2-wildtype (left) lung adenocarcinomas.

Fig 4: ARID2 deficiency leads to altered gene expression programs linked to invasion(A) Association of ranked DEGs (heatmap) in SKmel147 ARID2 KO versus WT cells with their associated ATAC increased or decreased peaks (bottom), within a TAD and within ± 500 kb of the TSS. Replicates are shown (R1, R2). TEAD4/FOSL2 status is shown above and called if at least one of the gene-associated ATAC changing peaks is bound by FOSL2 and/or TEAD4.(B) Biological process gene ontology of genes in (A).(C) Heatmap of significantly upregulated genes (Z scores) from (B) of the pathway “response to TGFB.” TEAD4/FOSL2 status is shown on the left.(D) Heatmap of upregulated genes (Z scores) associated with regions with increased chromatin accessibility within a TAD overlapped with the “invasive melanoma signature” from Verfaillie et al. (2015).(E) UCSC genome browser (hg19) snapshot displaying a putative enhancer (highlighted in yellow) with increased accessibility and SS18/FOSL2 binding in ARID2 KO lines with the promoter of the associated upregulated gene TGFBR2 shown.(F) WCE immunoblots of SKmel147 ARID2 WT (parental and NTC) and KO (KO1.4 and KO3.20) lines probed for TGFBR2, phospho-SMAD3, and SMAD3. GAPDH was used as a loading control (n = 2). See also Figure S5 and Tables S1, S2, S3, S5, and S6.

Fig 5: ARID2 depletion leads to melanoma metastasis in vivo(A) Schematic of the chicken CAM xenograft assay and regions of the chicken embryo where cells disseminate to, including the lower CAM and the liver.(B) FACS (fluorescence-activated cell sorting) quantification of SKmel147 ARID2 WT (NTC) and KO (KO1.4) cells of the primary tumor (n = 12 and n = 11, respectively), lower CAM (n = 19 and n = 16, respectively), and liver (n = 14 and n = 10, respectively) of the CAM xenografts 7 days post-injection. Nineteen independent replicates are aggregated, line represents the median. Welch’s t test. Primary tumor, p = 0.8580; lower CAM, p = 0.0019; liver, p = 0.0156. *p < 0.05; **p < 0.01; ns, p > 0.05.(C) Representative images of SKmel147 ARID2 WT (NTC) and KO (KO1.4) GFP+ cells that disseminated to the lower CAM.(D) Quantification of SKmel147 ARID2 WT (NTC, n = 5) and KO (KO1.4, n = 5) metastatic colonies in the lung 3 weeks post-tail-vein injection detected by fluorescence imaging. Line represents the median. Welch’s t test. p = 0.029. *p < 0.05.(E) Fluorescence images of lungs from mice with SKmel147 NTC and KO1.4 cells injected into the lateral tail vein.(F) Quantification of 4L ARID2 WT (NTC, n = 5) and KO (KO1.17, n = 5; KO5.11, n = 5) metastatic colonies in the lung 5 weeks post-tail-vein injection detected by fluorescence imaging. Line represents the median. Welch’s t test. NTC versus KO1.17, p = 0.0421; NTC versus KO.511, p = 0.0334. *p < 0.05.(G) Model of the mechanisms by which ARID2 loss regulates chromatin dynamics across the three types of SWI/SNF-bound regions, and gene expression pathways affected by PBAF loss. See also Figure S6.