JQ5 and JQ1 impair GCB cells through distinct transcriptomic signatures. All results from analysis of sorted GCBs collected ∼49 days posttransplant in cGVHD/BO B6→B10.BR model. Four samples in each group are used for analysis; samples were chosen to be most representative of each treatment condition by pulmonary function test results. (A) Principal component analysis (PCA) of top 4 samples in each treatment condition, calculated with top 500 most variable genes. Treatment condition variation along the top 2 PCAs explains ∼48% of variance within the dataset. All groups cluster independently. (B-C) Volcano plot of differentially expressed genes in either (A) JQ1- or (B) JQ5-treated samples against vehicle-treated (cGVHD) samples. Differentially expressed genes called as having an adjusted value of P <.05, and a log₂ fold change greater than 0.15. Twenty-four genes were differentially increased and 16 were reduced with JQ1, and 8 genes were differentially increased and 19 were reduced with JQ5. (D) Euler plots of overlapping differentially expressed genes in each of the 4 treatment conditions. Left plot is upregulated genes, whereas right is downregulated genes. (E) Gene set enrichment analysis (GSEA) network mapping of MsigDB hallmark gene sets in BM-only vs cGVHD comparison. Red nodes were increased with cGVHD vs BM-only samples, blue nodes were decreased. (F-G) Individual enrichment barcode plots for (F, left to right) MTORC1 signaling, Myc targets V1, oxidative phosphorylation; (G; left to right) allograft rejection, IL2-STAT5 signaling, and inflammatory response hallmark gene sets. On all plots, genes enriched in the treatment condition are on the left, whereas genes enriched in cGVHD are on the right. Gene sets in panel E JQ5 and JQ1 impacted enrichment differently, gene sets in panel F sets were enriched comparably. (H) Heatmap of high variance genes in major GSEA nodes described in panel D. Colors are blue for BM only, red for cGVHD, purple for JQ5 treated, and green for JQ1 treated (A,C,F-G).