Mutations in signal transduction components, BCR signaling, and sensitivity to BTK inhibition. (A) Nonsynonymous mutations predicted to have a significant effect on the protein by at least 2 software programs were considered “relevant mutations.” Sixteen leukemic MCL samples were subject to RNA sequencing. A set of 137 genes representing the BCR and NF-κB (canonical and alternative) pathways (supplemental Table 4) was tested for point mutations using the following filters: minimum number of reads: 8; minimum percent of mutated reads: 20; duplicate reads excluded; synonymous mutations and mutations listed in the SNP137 database excluded. Samples are displayed according to their ascending BCR signature score and divided in 2 groups: (1) BCR-low, n = 9; and (2) BCR-high, n = 7, based on the BCR score mean. Allele frequency is median-centered and scaled as indicated. Each column represents a patient sample and each row represents a gene. (B) The BCR signature score, and the levels of p-p65 and p-SYK (expressed as log2 of the percent positive cells among CD5⁺/CD19⁺-gated cells) were median-centered and displayed according to the color scale. Each column represents a patient’s sample. Leukemic samples are arranged according to their BCR signature score. Suspension cells obtained from LN are shown on the far right for comparison (LNT). (C) Change in p-p65 MFI of 4 samples after exposure to ibrutinib, tested by flow cytometry. Each data point represents the mean of duplicates from 2 separate experiments. (D) MTS assay of samples as in (C) tested in triplicate against increasing concentrations of ibrutinib, the MALT1 inhibitor MI-2, and the IKK inhibitor PS1145 for 48 hours.