IRF4 requires ARID1A to establish plasma cell identity in multiple myeloma

Abstract

Multiple myeloma (MM) is an incurable plasma cell malignancy that exploits transcriptional networks driven by IRF4. We employ a multi-omics approach to discover IRF4 vulnerabilities, integrating functional genomics screening, spatial proteomics, and global chromatin mapping. ARID1A, a member of the SWI/SNF chromatin remodeling complex, is required for IRF4 expression and functionally associates with IRF4 protein on chromatin. Deleting Arid1a in activated murine B cells disrupts IRF4-dependent transcriptional networks and blocks plasma cell differentiation. Targeting SWI/SNF activity leads to rapid loss of IRF4-target gene expression and quenches global amplification of oncogenic gene expression by MYC, resulting in profound toxicity to MM cells. Notably, MM patients with aggressive disease bear the signature of SWI/SNF activity, and SMARCA2/4 inhibitors remain effective in immunomodulatory drug (IMiD)-resistant MM cells. Moreover, combinations of SWI/SNF and MEK inhibitors demonstrate synergistic toxicity to MM cells, providing a promising strategy for relapsed/refractory disease.

Keywords: ARID1A; CRISPR; IRF4; MYC; SWI/SNF; multiple myeloma; plasma cell; proteogenomics; proteomics; transcription.

Figure 1.. ARID1A promotes IRF4 expression in multiple myeloma.

A) Multi-omics screening strategy to define MM-specific vulnerabilities. B) Ranked list of average CRISPR screen scores (CSS) of 22 MM cell lines. C) Average IRF4 expression score from KMS12PE, SKMM1, and H1112 IRF4-GFP knock-in cells. Left panel shows top hits. D) IRF4-BioID2 enrichment over empty vector in SKMM1 cells (FDR-corrected 2-way ANOVA, n=3). E) Gene ontology cellular component hypergeometric analysis of IRF4-BioID2 hits with an average Log2FC≥0.8 and P<0.05. F) Venn diagram of hits obtained from CRISPR essentiality screens (CSS≤-1.25), IRF4-GFP knock-in CRISPR screens (CSS≤-1.25), and IRF4-BioID2 (Log2FC≥1.0). G) Heatmap of Log2FC enrichment of SWI/SNF members in IRF4-BioID2 experiments. H) Scatter plot of average Log2FC BioID2-IRF4 enrichment from Fig. 1G (x-axis) vs. the corresponding average CSS values from Fig. 1B (y-axis). I) Western blot analysis of IRF4, ARID1A and GAPDH in MM cell lines 3 days after transduction with control shRNA (NT) or ARID1A targeting shRNAs (n=2). J) Proximity ligation assay (PLA) (red) of IRF4 and SWI/SNF family members in NCI-H929 and SKMM1 cells. DAPI (blue), wheat germ agglutinin (WGA; green). Scale bar is 10 μm, n=3. K) Representative images of ARID1A-IRF4 PLA in bone marrow aspirates from MM patients with PLA (red), CD138 (white) and DAPI (blue). Scale bar is 10 μm. L) Chromatin occupancy (CUT&RUN) of IRF4, ARID1A, SMARCA4, and SMARCB1 among IRF4 peaks in NCI-H929 and SKMM1. M) Venn diagram of IRF4 and ARID1A peaks in NCI-H929 and SKMM1. N) Transcription factor motif enrichment of ARID1A peaks (P<0.01 vs. isotype) in NCI-H929 and SKMM1. O) ARID1A mutation frequency in TCGA and MMRF-CoMMpass. See also Figure S1 and Tables S1–4.

Figure 2.. ARID1A is required for plasma cell development.

A) FACS gating of PCs from spleen and Peyer’s patches (PP). B) % live PCs in spleen and PP in Aicda^cre/+; Arid1a^flox/flox mice (red, n=8 and n=7, respectively) versus littermates (gray, n=10). Line represents the mean and comparisons between groups were performed using a two-tailed unpaired t-test. C) FACS gating of GC B cells from spleen and PP. D) % live GC B cells in spleen and PP in Aicda^cre/+; Arid1a^flox/flox mice (red, n=8 and n=7, respectively) versus littermates (gray, n=10). Line represents the mean and comparisons between groups were performed using a two-tailed unpaired t-test. E) FACS gating of PCs from LP. F) % live PCs in LP in Aicda^cre/+; Arid1a^flox/flox mice (red, n=4) versus littermates (gray, n=4). Line represents the mean and comparisons between groups were performed using a two-tailed unpaired t-test. G) Representative images of IgA⁺ PCs (green) in LP. DAPI (blue) and EPCAM (white), scale bar =100 μm. H) % live PCs in BM in Aicda^cre/+; Arid1a^flox/flox mice (red, n=7), versus littermates (gray, n=6). Line represents the mean and comparisons between groups were performed using a two-tailed unpaired t-test. I) CUT&RUN tracks of ARID1A in naïve B cells, GC B cells, and PCs. J) ARID1A CUT&RUN in FACS-purified B cell subsets (columns) among enriched (P<0.01 vs isotype antibody) ARID1A peaks in the same subsets (rows). K) Top 3 motifs from transcription factor motif analysis among the B cell subset specific ARID1A peaks shown in Fig. 2J. L) LOLA of ARID1A CUT&RUN peaks in naïve B cells, GC B cells, and PCs versus public chromatin profiling datasets in murine cells (n=786). A lower max TF rank indicates stronger overlap. See also Figure S2.

Figure 3.. SMARCA2/4 inhibition targets IRF4 and its underlying transcriptional network.

A) Heatmap of identified ATAC-seq peaks in SKMM1, NCI-H929, and a MM patient sample treated with DMSO or 1 μM AU-15330. B) LOLA of downregulated ATAC-seq peaks (P<0.0001) from Fig 3A. Peak locations queried against public human chromatin profiling datasets (n=689) and inverse max ranks are plotted against −Log10 P-value. C) GREAT analysis of the top downregulated ATAC-seq peaks (P<0.0001) from Fig. 3A (ranking based on FDR-corrected binomial P-value). D) Differential ATAC-seq peak downregulation among gene locations co-occupied by IRF4 CUT&RUN peaks (IRF4 genes) or not (non-IRF4 genes). Box plots represent median and 25–75% of data, whiskers incorporate 5–95% of data, and outliers are displayed as dots. P-values were determined by Mann Whitney U test. E) IRF4 chromatin occupancy among significantly (P<0.05) and non-significantly downregulated ATAC-seq peaks from Fig. 3A. F) Representative ARID1A, IRF4 (CUT&RUN), and ATAC-seq tracks in SKMM1 cells following treatment with DMSO or 1 μM AU-15330. G) Volcano plot of differentially expressed genes by RNA-seq in SKMM1 cells 24 hours following DMSO or 1 μM AU-15330 (FDR-corrected Wald test, n=2). H) Overlap of genes significantly downregulated by RNA-seq and ATAC-seq in SKMM1 and NCI-H929. I-J) Heatmap (I) and J) transcription factor motif analysis of the 264 overlapping gene regions. K) Top 25 genes in the AU-15330-down signature. L) Kaplan-Meier plot of disease-specific survival in MMRF CoMMpass trial divided into quartiles based on the expression of the AU-15330 down signature. Significance determined by a two-sided likelihood-ratio test based on a Cox proportional hazard model with the AU-15330 down signature treated as a continuous variable. (M-Q) Average AU-15330 down signature among CoMMpass samples for M) indicated MM molecular subsets (P-values from one way ANOVA versus all samples), N) 1q21 tetraploidy, O) TP53 copy number, P) disease type (GSE13591 ), or Q) in new diagnoses versus first relapse. Lines indicate mean expression values, comparisons between two groups (N-Q) were performed by Mann-Whitney U test. See also Figure S3 and Table S5.

Figure 4.. SMARCA2/4 inhibition is toxic to MM by targeting IRF4 and MYC.

A) Dose-response of AU-15330 and FHD-286 in MM lines 7 days after treatment (mean±SD, n=3). B) Apoptosis in primary MM cells 72h after treatment. C) Intracellular IRF4 staining (MFI relative to DMSO control) 48h after treatment. D) CSS values of SWI/SNF members in 22 MM cell lines. Lines indicate median CSS. E) Ranked SWI/SNF dependency among disease lineages (DepMap). Heatmap indicates average CHRONOS scores for SWI/SNF sub-complexes in each lineage; Ranking is based on average dependency rank of all three sub-complexes. F) FHD-286 IC₅₀ values obtained 7 days after treatment in MM (n=13), GCB-DLBCL (n=15), ABC-DLBCL (n=3), and adenocarcinoma cell lines (n=5). Red indicates ARID1A mutation/loss. Lines indicate mean IC₅₀ (n=3). G) Essential MM genes associated with transcription factor motifs under SMARCA4 CUT&RUN peaks. H) Western Blot analysis in NCI-H929 and SKMM1 24h after treatment (n=2). I) Venn diagram of SMARCA4, IRF4, and MYC peaks (CUT&RUN) in NCI-H929 and SKMM1. J) GREAT analysis of shared IRF4-MYC-SMARCA4 and MYC-SMARCA4 CUT&RUN peaks (ranking based on FDR-corrected binomial P-value). K) LOLA of IRF4-MYC-SMARCA4 versus MYC-SMARCA4 CUT&RUN peaks. L) Distribution of IRF4-MYC-SMARCA4 (pink) and MYC-SMARCA4 (blue) peaks relative to transcription start sites. M) Chromatin occupancy of MYC and H3K4me3 (CUT&RUN) among significantly (P<0.05) and non-significantly downregulated ATAC-seq peaks in NCI-H929 and SKMM1 4h after AU-15330 treatment. N) Tumor volume for MM.1S cells treated with vehicle (grey), 1.5mg/kg FHD-286 (orange), or 3 mg/kg FHD-286 (red) (n=5 per group). Error bars represent SEM of tumor size. See also Figure S4.

Figure 5.. SMARCA2/4 inhibitors effectively target IRF4 in lenalidomide-resistant cells.

A) Dose-response curves in lenalidomide sensitive and resistant variants of MM.1S and NCI-H929 72h after treatment (mean±SD, n=3). B) Western Blot analysis in NCI-H929 lenalidomide sensitive and resistant variants 24h after treatment (n=2). C) PLA between IRF4 and IKZF1 in lenalidomide sensitive and resistant NCI-H929 cells 24h after treatment with DMSO (0.1%), AU-15330 (1 μM), or lenalidomide (10 μM). Scale bar is 10 μm, (n=3). D) Corresponding median PLA scores between IRF4 and IKZF1. Box plots represent median and 25–75% of data, whiskers incorporate 5–95% of data, and outliers are displayed as dots. One-way ANOVA with Dunnet’s post test. E) PLA between IRF4 and ARID1A in lenalidomide sensitive and resistant NCI-H929 cells 24h after treatment with DMSO (0.1%), AU-15330 (1 μM), or lenalidomide (10 μM). Scale bar is 10 μm, (n=3). F) Corresponding median PLA scores between IRF4 and ARID1A. Box plots represent median and 25–75% of data, whiskers incorporate 5–95% of data, and outliers are displayed as dots. One-way ANOVA with Dunnet’s post test. H) Synergy heatmaps from XG7, INA6, and KMS12PE 72h after treatment with FHD-286 and lenalidomide at the indicated concentrations. See also Figure S5.

Figure 6.. FHD-286 synergizes with RAS pathway inhibitors.

A) Heatmap of the FHD-286 drug interaction landscape in SKMM1 and XG7. Drug synergy is ranked by average Excess HSA. B) MEK inhibitor enrichment plot from the Drug Set Enrichment Analysis (DSEA) of the FHD-286 vs MIPE6.0 screen. C) Response matrix for the FHD-286 trametinib combination in SKMM1. D) Excess HSA matrix for the FHD-286 trametinib combination in SKMM1. E) Dose-response curves of RAS-dependent MM cell lines treated with FHD-286 alone or in combination with trametinib at the indicated concentrations for 72h (mean±SD, n=3). F) MM cell apoptosis 48h after treatment (mean±SD, n=3). G) Tumor volume for MM.1S cells treated with vehicle (grey), 1 mg/kg trametinib (blue), 1.5mg/kg FHD-286 (red), or the combination (pink) (n=5 per group). Error bars represent SEM of tumor size. See also Figure S6.