BRAF inhibition induces apoptosis in primary HCL (but not HCL-like cells), which is partially rescued by coculture with bone marrow stromal cells blunting MEK-ERK dephosphorylation. (A) Quantification of apoptosis (by flow cytometry for ANXA5) in leukemic cells from 12 HCL (circles) and 4 HCL-like (squares) patients (supplemental Table 5), treated in vitro for 48 to 96 hours with the indicated drugs in triplicate (average shown), except 2 HCL cases run in duplicate or in single. All HCL cases showed drug-induced reduction of living cells from 1 (vehicle; horizontal bar) down to a maximum of 0.156 (ie, 84.4% relative decrease; P < .05 in all patients analyzed in replicate; supplemental Table 5). Conversely, no HCL-like cases featured such a reduction (and rather displayed a paradoxical increase in some instances). (B) Apoptosis by vemurafenib vs dabrafenib in 4 HCL patients (including 2 relapsed after vemurafenib; denoted with R and not included in panel A), performed in triplicate (circles; horizontal line: average) except in patient HCL 10 (single replicate). In all cases, dabrafenib reduced living cells more (1.3- to 3.8-fold) than vemurafenib in a statistically significant manner (P < .05) for all 3 cases analyzed in triplicate. (C) Western blotting for phospho-ERK1/2 and phospho-MEK1/2 of hairy cell protein lysates from a representative HCL patient (HCL 22; supplemental Table 1) exposed in vitro to the indicated drugs for 6 hours, in the presence or absence of HS5 bone marrow stromal cells. (D) Drug-induced apoptosis in the absence of HS5 cells was reduced (1.2- to 2.3-fold for vemurafenib, 1.7- to 7.3-fold for dabrafenib; P < .05, not shown) upon coculture with HS5 cells in all 4 cases tested except HCL 23. However, in the presence of HS5 cells dabrafenib produced more apoptosis than vemurafenib in 2 of 3 cases (HCL 1R, 1.7-fold; HCL 23, 3.4-fold).