Two Transforming C-RAF Germ-Line Mutations Identified in Patients with Therapy-Related Acute Myeloid Leukemia

Abstract

Mutations leading to activation of the RAF-mitogen-activated protein kinase/extracellular signal-regulated (ERK) kinase (MEK)-ERK pathway are key events in the pathogenesis of human malignancies. In a screen of 82 acute myeloid leukemia (AML) samples, 45 (55%) showed activated ERK and thus were further analyzed for mutations in B-RAF and C-RAF. Two C-RAF germ-line mutations, S427G and I448V, were identified in patients with therapy-related AML in the absence of alterations in RAS and FLT3. Both exchanges were located within the kinase domain of C-RAF. In vitro and in vivo kinase assays revealed significantly increased activity for (S427G)C-RAF but not for (I448V)C-RAF. The involvement of the S427G C-RAF mutation in constitutive activation of ERK was further confirmed through demonstration of activating phosphorylations on C-RAF, MEK, and ERK in neoplastic cells, but not in nonneoplastic cells. Transformation and survival assays showed oncogenic and antiapoptotic properties for both mutations. Screening healthy individuals revealed a <1/400 frequency of these mutations and, in the case of I448V, inheritance was observed over three generations with another mutation carrier suffering from cancer. Taken together, these data are the first to relate C-RAF mutations to human malignancies. As both mutations are of germ-line origin, they might constitute a novel tumor-predisposing factor.

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

“…Our list of BCAR genes contains several genes (NRG1, FGF17, HRAS, RAF1, and the membrane receptors PDGFRA, PDGFRB, EGFR, and CSF1R), which are well documented with respect to their role in normal and malignant cell growth (Hunter 2000, Gschwind et al 2004). In addition, RAS, RAF, and PDGFRA are frequently mutated in different types of cancer (Bos 1989, Emuss et al 2005, Zebisch et al 2006, Brugge et al 2007. RAD21 is known to play a role in chromosome cohesion during cell cycle, homologous recombination-mediated double strand break repair, and apoptosis.…”

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia

“…Western blot analyses were performed as described 17,18 using the following antibodies: anti-RKIP (Upstate, Billerica, MA, USA), anti-b-actin, anti-FLAG M2 (both from Sigma-Aldrich, St Louis, MO, USA), anti-ERK1/2, anti-phospho-ERK1/2 (all from Cell Signaling Technology, Beverly, MA, USA) and anti-GAPDH (Ambion/Applied Biosystems, Foster City, CA, USA). Data generated with Affymetrix U133A GeneChips (Affymetrix, Santa Clara, CA, USA) have been used for analysis of RKIP and Snail mRNA expression.…”

“…13 --16 We previously described loss of RKIP in patients with therapyrelated AML and C-RAF germline mutations. 17,18 RKIP silencing was shown to be a somatic, leukemia-specific event and contributed to C-RAF driven malignant transformation. However, it is unknown whether loss of RKIP is restricted to this small subset of cases or whether it is of broader significance for myeloid leukemogenesis.…”

Isolation of a Novel Thioflavin S–Derived Compound That Inhibits BAG-1–Mediated Protein Interactions and Targets BRAF Inhibitor–Resistant Cell Lines

“…19) and 16% to 29% are V600K mutations (20,21). Because of the role of RAF kinases in promoting cancer cell growth, mainly through enforcing cell-cycle progression and enhancing cell survival (1,5,18,22,23), the RAS-RAF-MEK module has become a promising target for therapeutic intervention. This led to the development of small molecular weight inhibitors of RAF and MAP-ERK kinase (MEK; refs.…”