Fig 2: OCA-B isoform proximity labeling identifies GATA3 as a consistently proximal protein. (A) Schematic showing the OCA-B isoform constructs and their mutations used to generate OCA-B-BirA fusion proteins and which OCA-B isoforms are expressed. OCA-B WT is capable of expressing OCA-B from both the canonical p34 start codon and the non-canonical p40 codon which gets myristoylated generating the p35 isoform. OCA-B p34 construct includes a p35 G to A mutation which prevents myristolation, resulting in only p34 and p40 isoform expression. OCA-B p35 includes two mutations, a p40 L to M mutation and a p34 M to S mutation, stopping p34 isoform expression and increasing p40 expression which gets myristoylated into the p35 isoform. Finally, OCA-B p40 contains three mutations, a p40 M to S, a p35 G to A, and a p34 M to S, which drives expression of the p40 isoform which is unable to be myristoylated. The expression chart shows isoform expression level (red line) for each construct. OCA-B-BirA isoform and BirA-only constructs were integrated into the SupT1 T cell line and then cells were supplemented with doxycycline and biotin to promote fusion protein expression and proximity biotinylation. Isolated protein was subjected to streptavidin pull-down to enrich biotinylated proteins and enriched proteins were analyzed by LC-MS/MS. (B) Immunoblot comparing OCA-B protein expression in primary mouse naïve, CD3 stimulated, and CD3/CD28 stimulated CD4+ T cells to the SupT1 T cell line. (C) Volcano plot depicting proteins identified by mass spectrometry positively and negatively enriched from wild-type OCA-B-BirA (n=3) transduced relative to BirA-only (n=3) transduced control SupT1 cells. Proteins significantly elevated with the wild-type OCA-B replicates are shown in red, and proteins significantly elevated in BirA-only replicates are shown in blue. (D) Venn diagram shown the number of proteins significantly elevated over the BirA-only control within each OCA-B isoform and any protein overlap between isoforms. (E) String map showing significantly enriched proteins compared to the BirA-only control from all OCA-B isoforms. Connecting line thickness represents the strength of the protein association. OCA-B (POU2AF1) is marked by a red box. (F) Coimmunoprecipitation assays evaluating protein-protein interactions between GATA3, Oct1, and OCA-B using SupT1 cell lysates. “WT” indicates normal parent SupT1 cells. “hOCA*” indicates SupT1 cells in which CRISPR was used to disrupt OCA-B, creating a significant truncation.

Fig 3: T cell OCA-B diminishes the Th2 immune response in vitro and in vivo. Splenic naïve CD4+ T cells were isolated from Ocabfl/fl (n=6) and Ocabfl/fl ;CD4-Cre (n=6) mice and differentiated in vitro into Th2 cells. (A) Representative flow cytometry plot showing the frequency of IFNγ and IL-4 expression by CD4+ Th2 differentiated cells isolated from Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice. (B) Quantification of the frequency of IL-4 expression by CD4+ Th2 differentiated cells isolated from Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice. (C) Quantification of the frequency of IL-13 expression by CD4+ Th2 differentiated cells isolated from Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice. (D) qPCR showing normalized gene expression of Il4, Il5, and Il13 in CD4+ Th2 differentiated cells isolated from Ocabfl/fl (n=4) and Ocabfl/fl ;CD4-Cre mice (n=4). (E) Schematic showing dosing routine for papain allergen challenge. 10 week-old male/female littermate Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice were subjected to saline or papain allergen challenge, and lungs were harvested on day 11 for flow cytometric and histological analysis (Ocabfl/fl papain n=5, Ocabfl/fl ;CD4-Cre papain n=7, Ocabfl/fl saline n=4, Ocabfl/fl ;CD4-Cre saline n=4). (F) Representative flow cytometry plot showing the frequency of CD4+ and CD8+ T cells isolated from the lungs of Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice treated with either saline or papain. (G) Quantification of the frequency of CD4+ T cells isolated from the lungs of Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice treated with either saline or papain. (H) Quantification of the frequency of CD8+ T cells isolated from the lungs of Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice treated with either saline or papain. (I) Representative H&E histopathological images of Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice after saline or papain allergen challenge. (J) Pathological scoring of lung capillary tissue from Ocabfl/fl and Ocabfl/fl ;CD4-Cre mice following saline or papain allergen challenge. All values represent mean ±SD. ns = p>0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Fig 4: POU2AF1-driven multiple myeloma is dependent on the mSWI/SNF complex(A) Scatterplot depicting gene dependency difference of all plasma cell myeloma versus other cancer types (left) and all B cell malignancies versus other cancer types (right) based on DepMap. The red circles indicate the top 5 essential genes among others.(B) Representative hematological cancer cell lines showing dose-response curves of AU-15330 at varying concentrations for five days. Sensitive cell lines are in red while relatively resistant cell lines are in blue. Data are presented as mean ± SD (n = 6).(C) ATAC-seq read-density heatmaps from MM1.S cells treated with DMSO or 1 μM AU-15330 for 4 h (n = 2 biological replicates).(D) Analysis of fold change and significance level for HOMER motifs that are enriched within sites dependent and independent of the mSWI/SNF complex after 4 h AU-15330 treatment in MM1.S cells (left panels) and NCI-H929 cells (right panels).(E) Scatterplot showing the dependency scores for IRF4/POU2AF1 in diffuse large B cell lymphoma (blue), multiple myeloma (red), and other cancer types based on DepMap dataset.(F) ChIP-seq read-density heat maps for POU2AF1 and IRF4 at the AU-15330-loss genomic sites in MM1.S cells after treatment with DMSO or AU-15330 (1 μM) for 6 h.(G) Volcano plot detailing proteins that interact with POU2AF1, as identified by POU2AF1 RIME analysis in MM1.S cells. mSWI/SNF components highlighted in orange (n = 3 biological replicates).(H) Co-immunoprecipitation (IP) of POU2AF1 or IRF4 in MM1.S cells followed by immunoblot for POU2AF1 and IRF4. This experiment was repeated independently twice.(I) GSEA plots illustrating genes regulated by IRF4. The plots use a gene signature ranked by fold change from AU-15330 treated NCI-H929 (top) and MM1.S (bottom) cells.(J) Combined ATAC-seq and ChIP-seq tracks for c-MYC locus in MM1.S cells with and without AU-15330 treatment.See also Figure S5.