KDM5 inhibition regulates KMT2D target genes and BCR signaling regulators. (A) Heatmap indicating normalized enrichments scores (NESs) of KMT2D and CREBBP signatures in KDM5-inh1–treated cells, following GSEA of RNA-seq profiles using a manually curated database of B-cell signatures. (B) Overlap between KDM5 inhibition–regulated regions in SU-DHL-6 and CREBBP²⁶  or KMT2D bound regions,^9,10  with observed/expected and FDR values for the overlaps indicated. (C) Deeptools³²  was used to calculate summary scores at the promoters (transcription start site [TSS] ± 500 bp) of genes in each cluster (Figure 3G) plus nonbivalent (H3K4me3+/H3K27me3−) and bivalent (H3K4me3/H3K27me3+) control promoters for ChIP-seq datasets of histone mark deposition (ENCODE/BLUEPRINT) and KMT2D,^9,10  CREBBP,²⁶  and EZH2/SUZH12³³  binding. The overall direction of change in RNA expression, following KDM5i or KMT2D loss, is indicated for each cluster in the first 2 columns. (D-E) Heatmaps indicating NES following GSEA of the Reactome database in RNA-seq profiles from KDM5-inh1–treated cells (D) and RNA-seq plus promoter H3K4me1 and H3K4me3 profiles from KDM5-inh1–treated WSU-DLCL2/WSU#22^−/+ cells (E). (F) Log₂FC values of B-cell signaling regulators in SU-DHL-6, OCI-LY-18, and HT cells treated with KDM5-inh1. (G-H) Levels of pERK1/2 (T202/Y204) and ERK1/2 were quantified by western blot in cell lines exposed to 1 μM KMD5-inh1 for 5 days or 1 μM entospletinib for 48 hours (G), with the quantification of 3 individual replicates (H), alongside levels of ERK1 Y204 measured by phosphoproteomics and MEK1 activity quantified by kinase substrate enrichment analysis.³⁴  (I) Activation of SYK was investigated by western blot analysis in SU-DHL-6, OCI-LY-18, and OCI-LY-7 cells treated with DMSO or KDM5-inh1 for 72 hours, followed by anti-IgM F(ab′)₂ antibody stimulation for 10 minutes or 1 hour.