MEC1 cells overexpressing MAP2K1 mutations show constitutive ERK phosphorylation. (A) Graphical representation of MAP2K1 mutations identified in this PI3K resistance cohort and a relapsed refractory CLL cohort. (B) Representative immunoblot of MEC1 cells overexpressing WT and mutant MAP2K1, both unstimulated and with anti-IgM stimulation for 15 minutes. The labels on the top indicate the construct used: GFP only, WT MAP2K1, and MAP2K1 mutants (E203K, F53L, and Q56P). The cells were profiled for pERK, pAKT, and HA-tag. (C) Quantification of pERK from MEC1^WT and MEC1^MUT cells, unstimulated and after anti-IgM stimulation (n = 7 experiments). (D) Quantification of pAKT from MEC1^WT and MEC1^MUT cells, unstimulated and after anti-IgM stimulation (n = 7 experiments), showed no significant differences. (E) Representative immunoblot of MEC1 cells overexpressing WT or MAP2K1 mutants after in vitro treatment with either vehicle or 1 μM idelalisib for 1 hour and stimulation with anti-IgM for 15 minutes. (F) Quantification of pERK from MEC1^WT and MEC1^MUT cells after 1 μM idelalisib treatment in vitro (n = 10 experiments). (G) Quantification of pAKT from MEC1^WT and MEC1^MUT cells after 1 μM idelalisib treatment in vitro (n = 10 experiments). (F-G) The measurements were log transformed to satisfy statistical model assumptions. Data represented as mean ± SEM. ****P < .0005; ***P < .001; **P < .01; *P < .05.