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N. Giménez et al.
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Figure 4. Effect of RAS-BRAF-MAPK-ERK inhibitors in cases of RAS-BRAF-MAPK-ERK-mutated and unmutated IGHV chronic lymphocytic leukemia. (A) Cells from 17 cases of chronic lymphocytic leukemia (CLL), nine containing mutations in the RAS-BRAF-MAPK-ERK pathway (KITLG, PTPN11, KRAS, BRAF, MAPK1, MAP2K1, MAP2K2) and eight with unmutated IGHV genes (U-IGHV) with no alterations in genes of the RAS-BRAF-MAPK-ERK pathway were treated with vemurafenib 2.5 mM or dabrafenib 2.5 mM. p-ERK levels were analyzed by flow cytometry after 1.5 h of treatment and expressed relative to untreated cells (Ct) at basal levels (unstimu- lated) or after stimulation with anti-IgM (stimulated) (*P<0.05). Histograms showing anti-IgM stimulation of ERK (T202/Y204) phosphorylation with and without vemurafenib or dabrafenib (2.5 mM) treatment in representative CLL cases (U-IGHV CLL and case 17 with a BRAF mutation). (B) Cell viability after treatment for 24 and 48 h with vemurafenib or dabrafenib at doses of 0.5-5 mM. Bars represent the mean ± standard error of the mean (SEM) of all samples analyzed (n=9 in the group of CLL cases with mutations in genes of the RAS-BRAF-MAPK-ERK pathway and n=8 in the unmutated CLL group) (*P<0.05; **P<0.01). (C) p-ERK levels after treatment with 0.1 or 2.5 mM ulixertinib (UT) relative to untreated (Ct) samples analyzed by flow cytometry at basal levels (unstimulated) or after stimulation with anti-IgM (stimulated). Bars represent the mean ± SEM of six samples analyzed in each group, six with mutations in genes of the RAS-BRAF-MAPK-ERK pathway (KITLG, PTPN11, BRAF, MAP2K1, MAP2K2, and MAPK1) and six U-IGHV CLL cases. Histograms showing anti-IgM stimulation of ERK (T202/Y204) phosphorylation and its inhibition by 100 nM and 2.5 mM ulixertinib (UT) in representative CLL cases (U-IGHV: CLL and case 15: BRAF mutation). Each patient is represented by a dif- ferent color depending on the RAS-BRAF-MAPK-ERK mutational status and the mutation position relative to BRAF.
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haematologica | 2019; 104(3)