Genetic and pharmacological inhibition of SRC-family kinases block multiple oncogenic signals downstream BCR. (A) BCR signaling representation. The SRC family kinases LYN, FYN, and BLK transmit the signal to multiple effectors including SYK-BTK, CD19, and GSK3β. (B) Quantification of cell viability in SU-DHL-2 cells with dual or triple knockout of LYN, FYN, and BLK, based on the percentage of fluorescent cells (GFP, RFP, tagRFP657). Dual LYN-FYN (RFP, GFP-positive) cells were used as control. (C) Percentage of cells sensitive to the indicated SRC inhibitors based on COSMIC dataset. (D) Scatter plot representing the IC50s (the dotted line indicates the 50% inhibitory concentration (IC₅₀) threshold = 20 μM) for masitinib treatment in 923 cells lines (gray). The colored points represent the DLBCL cell lines. In green are the cell lines sensitive to masitinib (24 cell lines) with IC₅₀ lower than the threshold, and in red are the cell lines resistant to masitinib (5 cell lines), with IC₅₀ higher than the threshold. (E) Percentage of viable cells in the indicated ABC and GCB lymphoma cell lines, treated with DMSO or with masitinib at 2.5, 5, or 10 μM for 72 hours. Each cell line was analyzed in triplicate, and data are shown as a graph corresponding to the mean ± SD. (F) Scatter plot showing the expression of masitinib target genes (fpkm, fragments per kilobase million) vs their reported dissociation constant (K_d) for masitinib. (G,I,K) Quantification of fluorescence signals (MFIs) of BTK (G), CD19 (I), and GSK3β (K) phosphorylation assessed by phosphoflow cytometry for patients with DLBCL treated with DMSO or ibrutinib (0.5 μM) or masitinib (5 μM) for 6 hours and stimulated with H₂O₂ for 3 minutes. The bar plots correspond to the mean of normalized MFI ± SD of 3 replicates, and data were normalized on stained, unstimulated cells. Significant changes between stimulated cells (orange bars) and stimulated cells treated with ibrutinib (blue bars) or masitinib (green bars) are labeled with an asterisk (uncorrected P ≤ .05). Unlabeled bars indicate not statistically significant changes. (H,J,L) Quantification of BTK (H), CD19 (J), and GSK3β (L) phosphorylation fluorescence signals assessed by phospho-flow cytometry for indicated cell lines treated with DMSO or ibrutinib (0.5 μM) or masitinib (5 μM) for 6 hours and stimulated with H₂O₂ for 3 minutes. The bar plots correspond to the mean of normalized MFI ± SD of 3 replicates, normalized on stained, unstimulated controls. Significant changes between stimulated cells (orange bars) and stimulated cells treated with ibrutinib (blue bars) or masitinib (green bars) are labeled with an asterisk (P ≤ .05). Unlabeled bars indicate not statistically significant changes. (M) MYC expression in the indicated ibrutinib-resistant cell lines treated with DMSO or masitinib (5 μM) for 24 or 48 hours. Each cell line was analyzed in 3 biological replicates, and data are shown as a bar graph corresponding to the mean ± SD. P values were calculated using 2-tailed Student t test. Significant changes between DMSO-treated and masitinib-treated cells are labeled with **P ≤ .01; ****P ≤ .0001. (N) Western blot analysis of MYC in SU-DHL-2 cells ibrutinib-resistant treated with DMSO or masitinib (5 μM) for 24 or 48 hours.