Constitutive activation of the RAS-RAF-MEK-ERK pathway by the BRAF-V600E mutation in HCL. (A) The RAS-RAF-MEK-ERK signaling pathway is physiologically triggered when a ligand binds to its surface receptor tyrosine kinase (RTK). This event, in turn, activates RAS and then BRAF (and CRAF, not shown). BRAF-CRAF heterodimers phosphorylate MEK (MEK1 and MEK2), which in turn phosphorylate ERK (ERK1 and ERK2). The BRAF-V600E mutation renders BRAF constitutively active independently from upstream signals and from heterodimerization with CRAF, thereby deregulating signaling activity. Active ERK enters the nucleus, where it triggers a transcriptional response (for example, through AP1 transcription factors). Such transcriptional response favors cell survival, proliferation, growth, and motility (as well as neoplastic transformation when deregulated by the BRAF-V600E mutation), and comprises genes that are generally induced by the RAF-MEK-ERK signaling pathway as well as genes induced by this cascade in the specific cellular context of HCL, including some of its diagnostic makers and the whole expression signature specific of this leukemia in comparison with normal mature B cells and other peripheral B-cell neoplasms.²² (B) HCL cells (brown) express the BRAF-V600E mutant protein (Ventana immunostaining with VE1 monoclonal antibody; immunoperoxidase and hematoxylin counterstain; ×400). (C) HCL cells express phospho-ERK (red) in the cytoplasm and the nucleus (anti–alkaline phosphatase immunostaining and hematoxylin counterstain; ×400).