Fig. 5: Transcriptional profiling reveals a BTK-dependent signature of anti-apoptotic gene expression.

a–d RIVA, U2932 and OCI-LY3 cells were cultured in the presence or absence of ibrutinib for 6 h and subjected to RNA isolation; U2932 and RIVA cells were additionally orthotopically transplanted into six MISTRG mice per cell line and subjected to two weeks of continuous in vivo ibrutinib exposure (25 mg/kg, administered five times per week). Bone marrow cells were harvested from three ibrutinib-treated and three vehicle-treated control mice, and tumor cells were FACS-sorted to >95% purity prior to RNA isolation. In vitro and in vivo generated triplicate samples were subjected to bulk RNA sequencing. A heat map of 141 genes that are downregulated (log2 fold change <−0.5 and adjusted p value < 0.05) upon ibrutinib exposure in vitro in both RIVA and U2932 cells are shown for all 30 samples in a. Select genes are annotated (color code as in c). An UpSet plot of the intersections of overlapping differentially expressed, downregulated genes across the four indicated conditions is shown in b. The top bar chart indicates the number of downregulated genes shared between two, three, or all four conditions. Three select gene networks as identified by pathway analysis of the 141 commonly downregulated genes are shown in c, connecting the indicated genes assigned to each pathway (color-coded in yellow, blue and red as in a). The color of each dot indicates the average fold change in expression of the respective gene; its size indicates the number of genes from the list of 141 that are in the respective pathway. Whisker plots of the variance stabilized transformed expression of four select anti-apoptotic Bcl-2 family members (BCL2, MCL1, BCL2L1, and BCL2A1) are shown in d for all triplicate samples of eight conditions. e, f quantitative RT-PCR of the three indicated transcripts, as determined using an independently generated set of in vitro (6 h of exposure) and in vivo ibrutinib-exposed RIVA and U2932 cells. Each dot represents one sample; bars represent means + standard deviation. P-values were determined by Mann–Whitney test. g BCL2A1 gene expression of the indicated cell lines as determined by qRT-PCR; ABC-DLBCL cell lines are color-coded in red, and GCB-DLBCL cell lines in green; an unclassified cell line, RC-K8, is shown in black.