Key Points• The V600E kinase-activating mutation of BRAF profoundly shapes the distinct identity of HCL among B-cell neoplasms.• Clinically available BRAF and MEK inhibitors exert potent antileukemic activity in patients' HCL cells in vitro and in vivo.Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and

Background Hairy cell leukemia (HCL) is a peripheral B-cell neoplasm with indolent clinical course and peculiar morphology and phenotype. Recently, we identified the BRAF-V600E kinase mutation as the genetic lesion underlying HCL and distinguishing it from other leukemias and lymphomas, including HCL-mimics such as splenic marginal zone lymphoma and HCL-variant. The BRAF-V600E mutation is known to be an oncogenic driver in melanoma and other solid tumors thorough constitutive phosphorylation of the kinase MEK (the immediate downstream target of BRAF), which in turns phosphorylates the kinase ERK. Thus, the BRAF-MEK-ERK pathway appears an ideal therapeutic target in HCL to be attacked by small-molecule BRAF inhibitors (such as PLX4720, Vemurafenib and Dabrafenib) and MEK inhibitors (such as Trametinib), which have already proven effective in clinical trials of BRAF-V600E-positive melanoma patients and (for Vemurafenib) in anecdotal HCL patients. However, a thorough analysis of the effects of these drugs in primary HCL cells has not been conducted so far. Aim To study in vitro the effects of BRAF and MEK inhibitors on the specific biochemical, morphological and anti-apoptotic features of HCL, using primary leukemic cells purified from HCL patients (as we previously showed that the putative “HCL” cell lines described in the literature do not carry the BRAF-V600E mutation and are, thus, unlikely to be of true HCL origin). Methods Using CD19-MACS, we purified primary hairy cells from the peripheral blood of 20 HCL patients and 12 HCL-like patients and treated these cells in vitro with different BRAF inhibitors (Vemurafenib, PLX4720 and Dabrafenib) or the MEK inhibitor Trametinib, at various concentrations (up to 1 µM) for different time periods (30 minutes to 72 hours), and monitored the phosphorylation status of MEK and ERK by Western blotting. Primary hairy cells were also followed, up to 5 days, for: i) the potential loss of their typical hairy projections (rich in F-actin), through phalloidin staining and confocal microscopy analysis (in 6 HCL and 5 HCL-like patients); ii) the potential reduction of viability, through the MTT or WST metabolic assays (in 12 HCL and 6 HCL-like patients); and iii) the potential induction of apoptosis, through double staining with AnnexinV/Propidium-Iodide and flow cytometry analysis (in 12 HCL and 4 HCL-like patients). Results Treatment with any BRAF inhibitors resulted in consistent, rapid, sustained and dose-dependent MEK and ERK dephosphorylation at all time points in all HCL cases, as opposed to vehicle-treated HCL cells and to inhibitor-treated HCL-like cells. Trametinib also produced a strong ERK dephosphorylation in HCL cells. These early biochemical events were followed, in a time frame of 2-3 days, by a consistent and statistically significant reduction of the hairy projections in still viable leukemic cells (AnnexinV-negative). After additional 1-2 days, we observed loss of viability (20% to 60% decrease relative to the drug vehichle – p value <0.05 in all instances) and induction of apoptosis (20% to 59% relative increase – p value <0.05 in all instances) specifically in leukemic cells of the vast majority of HCL patients as opposed to none of the HCL-like patients. Conclusion These results represent a comprehensive pre-clinical evidence of the significant anti-leukemic activity obtained through inhibition of the BRAF-MEK-ERK pathway in HCL. V.P. and A.S. equally contributed to this work. E.T. and B.F. equally contributed to this work. Disclosures: No relevant conflicts of interest to declare.