PI3K inhibitor nonresponders show upregulation of ERK1/2 signaling. (A) Western blot analysis of peripheral blood mononuclear cells (PBMCs) from a PI3K responding (left) and nonresponding (right) patient pretreated with either vehicle or 1 μM idelalisib (Idela) or 1 μM CI-1040 (MEK1/2 inhibitor) for 1 hour and stimulated with anti-IgM for 15 minutes. The samples were profiled for pERK, total ERK, pAKT, total AKT, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Quantification of pAKT (B) and pERK (C) from 10 independent responders and 5 independent nonresponders. A different symbol is used for each patient to trace their response. The measurements were log transformed to satisfy statistical model assumptions. (D) Fold inhibition of pERK and pAKT with 1 μM idelalisib, in 10 responders (R) and 5 nonresponders (NR) compared with the vehicle-treated group. (E) CellTiter-Glo luminescent cell viability experiment at 48 hours for a patient who initially responded to idelalisib but later progressed on the drug. The patient’s PBMCs from responding and progressing time points were treated with the indicated doses of idelalisib, CI-1040 (MEK1/2 inhibitor), SCH772984 (ERK1/2 inhibitor), or a combination of idelalisib with either CI-1040 or SCH772984 at 1 μM and 5 μM concentrations. (F) Representative immunoblot of a sample from a patient with acquired resistance at responding and progressing time points. The sample was treated with either vehicle or 1 μM idelalisib or 1 μM CI-1040 for 1 hour and stimulated with anti-IgM for 15 minutes. The samples were profiled for pERK, total ERK, pAKT, total AKT, and GAPDH. Data are presented as mean ± standard error of the mean (SEM). ***P < .001; **P < .01; *P < .05 by mixed effects analysis of variance, Holm-Sidak test. CAL-101 (idela), idelalisib; DMSO, dimethyl sulfoxide; SCH, SCH772984 (a selective ERK1/2 inhibitor); w.r.t., with respect to.