Hyperactive downstream signaling in relapse CLL acquired PLCγ2 variants can be blocked by targeting SYK or LYN. (A) Calcium flux from 3 individual relapse CLL (R) bearing mutated PLCγ2 or 12 nonresistant CLL samples (NR) were measured, and 5E6 cells per well in 96-half well microplates were stimulated with 10 μg/ml α-IgM at 37°C for 15 minutes, and then measured by BD Calcium Assay Kit. The data represent AUC from 7 and 12 replicates in resistant and nonresistant samples, respectively; 1.0 μM ibrutinib was pretreated for 1 hour followed by washout. (B) The data represents calcium release by 3 individual relapse CLL (R) or the mean of 12 nonresistant samples (NR) stimulated with 10 μg/ml α-IgM in the presence of 0.5 μM GS-9973, 2.5 μM R406, or 0.1 μM dasatinib; 1.0 μM ibrutinib was pretreated for 1 hour followed by washout. ***P < .001. (C) The downstream phospho-protein activation was accessed in CLL cells from a single patient (patient #2 in Table 1) in the baseline or relapse setting. The detailed information of the 3 relapsed CLL patient samples analyzed here are listed in Table 1.