Functional dissection of BTK-dependent transcriptional programs. (A) Messenger RNAs (mRNAs) identified as significantly downregulated from the RNA sequencing analysis were submitted to Ingenuity pathway analysis for mapping of related upstream transcriptional regulators, which are shown in their respective networks. Known protein-protein interactions were included, as revealed by STRING database analysis. (B) KG-1or (C) MV4-11 cells were subjected to retroviral transduction with IRES-EGFP vectors inducing stable expression of EGFP, caSTAT5 and EGFP, caIKK and EGFP, or MYC and EGFP. The transduced cell batches were left untreated (left panel) or were treated with ibrutinib at a final concentration of 500 nM for up to 6 days (middle panels) or were treated with shRNAs specific for BTK (right panels). After confirmation of successful knockdown of BTK, cells were cultured for up to 6 days. The proportion of EGFP-positive cells was analyzed by flow cytometry. Wild-type cells were used as a negative control in all flow cytometric measurements (data not shown). (D-E) The functionally relevant and highly enriched transcription factors MYC, STAT5, E2F1, and NF-κB (square nodes) and their differentially regulated target genes (circular nodes) were used for STRING protein-protein interaction analysis (gray lines). The transcription factor to target gene relationships are indicated by black arrows. Target genes known to be involved in the regulation of the cell cycle or of apoptosis are shown with a red border. (F-G) Regulation of mRNA levels upon ibrutinib treatment were validated by quantitative polymerase chain reaction. Results are shown for 4 independent experiments (mean ± SD).