Fig. 5: Effects of ibrutinib in RAD51-mediated HR repair in CLL.

a Left panel: representative images and quantification of the number RAD51-positive cells 6 h after irradiation (2 Gy) in HG3^WT and HG3-del(11q) ATM^KO clones. Cells were pretreated for 24 h with 5 μM olaparib, 1 μM ibrutinib or the drug combination. Data are represented as the mean values ± SD of three independent experiments. Cells were scored RAD51 + when five or more foci were formed. At least 100 cells per experiment were counted. Right panel: volcano plots of transcripts changes comparing 1- (top) and 6-month (bottom) post-ibrutinib initiation vs. pretreated longitudinal samples in 14 CLL patients. RAD51 expression is significantly downregulated in samples after 1 month and 6 months of ibrutinib therapy. Log₂ of fold-changes (treatment vs. control) are shown in x axis and statistical significance (-log₁₀ of q value) is shown in y axis. RNA-seq data were previously generated in Landau et al. [23]. b Primary CLL cells were seeded in co-culture with HS-5 bone marrow stromal cells, 1.5 μg/mL CpG and 50 ng/mL IL-2 and treated with the indicated drugs and doses for 5 days. Normalized surviving fraction is expressed relative to untreated cells. Data are presented as the mean ± SD. c Upper panel displays a representation of the HR-reporter plasmid adapted from Seluanov et al. [43]. Lower-left panel represents the HR repair efficiency as calculated by dividing the number of GFP + cells of the totality of positive-transfected DsRed + cells. Data represent mean ± SD of three independent experiments. Right panel displays representative plots of the HR efficiency of HG3 treated with DMSO or ibrutinib (1 μM).