Combined BRAF and MEK inhibition counteracts the protective effect of bone marrow stromal cells against ERK dephosphorylation and HCL cell apoptosis induced by single BRAF or MEK inhibition. (A) Western blot analysis of phospho-ERK1/2 levels in hairy cells purified from patient HCL 24 (supplemental Table 1) and exposed in vitro to the indicated BRAF and MEK inhibitors alone or in combination, for 6 hours, in the presence of HS5 bone marrow stromal cells. After cotreatment with dabrafenib 50 nM and trametinib 1 nM, the phospho-ERK bands, relative to the total ERK bands as loading control, are weaker than after either drug alone (twofold and fivefold weaker, respectively, as quantified in the phospho-ERK/ERK ratio indicated below each lane and obtained by densitometry using the ImageJ software). A similar result was obtained in another HCL patient (no. 26, supplemental Table 1) (B) Flow cytometric quantification of living (ANXA5-negative) cells in primary blood leukemic cells purified from a HCL patient (HCL 25) and in vitro exposed in triplicate for 48 hours to the indicated drugs, in the presence or absence of HS5 stromal cells. In the absence of HS5 cells, trametinib 1 nM and dabrafenib 50 nM were able to induce significant (P < .05, supplemental Table 5) HCL cell apoptosis and their combination was not more effective than dabrafenib alone. Conversely, in the presence of HS5 cells drug-induced apoptosis was dampened but combined BRAF and MEK inhibition resulted in more apoptosis than BRAF or MEK inhibition alone, as quantified in the histograms of panel C (mean ± standard deviation [SD], relative to trametinib 1 nM). A similar result was obtained in another HCL patient (no. 26, supplemental Table 5).