AML patient and healthy donor T-cell proliferation response to treatment with trametinib in vitro differs. Mononuclear cells isolated from peripheral blood or bone marrow samples from patients or healthy donors were stained with CellTrace Violet and treated in vitro with anti-CD3 and/or trametinib followed by a 5-day incubation period. (A) Representative flow cytometry plots of proliferation from a healthy donor gated on live CD3⁺ cells with and without treatment with trametinib. (B) Summary of the percentage of dividing T cells for healthy donors (n = 4) plated with mononuclear cells after treatment with trametinib. (C) Summary of the percentage of purified healthy donor (n = 3) T cells after treatment with trametinib. (D) Report of the percentage of dividing T cells from primary patient samples (n = 8) after stimulation and treatment with trametinib grouped according to presence or absence of an identified RAS or RAF mutation. (E) Mean ± SD percentage of dividing T cells after stimulation with anti-CD3 and treatment with trametinib 10 nM separated by the presence of either an RAS or RAF mutation or the absence of both. (F) T-cell proliferation of a patient sample after anti-CD3 stimulation and treatment with anti–PD-1 and/or trametinib 10 or 100 nM. (G) Western blot analysis of p-ERK and total ERK from viably frozen AML patient samples with and without an RAS (R) mutation. (B-C) Displaying Tukey pairwise comparisons between treatment conditions. (E) Two-sided Student t test with unequal variances. (B-C,E) Statistically significant results displayed as: *P < .05; **P < .01; ***P < .001.