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Altered RAS-BRAF-MAPK-ERK pathway in CLL
Discussion
CLL is characterized by a heterogeneous mutational landscape, with the presence of certain mutations being associated with progression of the disease and refractori- ness to immuno-chemotherapy, which lead to a poor out- come.6,13,28 Recently, it has been proposed that the MAPK– ERK pathway could be one of the cellular processes affect- ed in CLL through mutations in novel CLL drivers such as NRAS, KRAS, BRAF, PTPN11 and MAP2K1.9,24 The RAS- BRAF-MAPK-ERK pathway plays a central role not only in regulating normal cellular processes involved in prolifera- tion, growth, and differentiation, but also in oncogenesis,29 and it is an important key dysregulated pathway in cancer.30
In our series, we observed mutations in genes belonging to the RAS-BRAF-MAPK-ERK pathway in 5% of CLL patients, a frequency similar to that already described.13 When we evaluated each mutation specifically, BRAF mutations were detected in 2% of our CLL series, as pre- viously reported.9,21 BRAF mutations did not involve the canonical hotspot (V600E) seen in other malignancies,17 which leads to constitutive activation of BRAF, but rather were clustered around the activation segment of the
A
kinase domain.9,23 Mutations in these positions confer vari- able but increased signaling and have oncogenic capacity.31 Mutations in exon 15 of BRAF have been associated with refractoriness to fludarabine22 although they do not seem to be selected during progression to refractory CLL.21 Furthermore, the frequency of BRAF V600E mutations is higher in Richter syndrome than in untransformed CLL32, and this mutation could be acquired during the evolution of CLL. Recently, our group reported that the mere detec- tion of a BRAF mutation, even at a very low frequency, had a prognostic impact on TTFT.33 However, given the low frequency of mutations observed in CLL patients, larger series of patients are needed to corroborate these observations.
Mutations in genes upstream and downstream of BRAF were observed in 64% (16/25) of cases. MAP2K1 muta- tions have already been described in HCL-variant and con- ventional HCL with rearranged IGHV4-34,34 Langerhans cell histiocytosis,35 and pediatric-type follicular lym- phoma.36 This mutation, similar to those of BRAF, leads to activation of the downstream target, ERK.36 Moreover, we found mutations in additional genes of this pathway, such as MAP2K2, which encodes MEK2, and PTPN11, which encodes SHP-2. Both these proteins participate in the reg-
B
CD
Figure 3. Activation of the RAS-BRAF-MAPK-ERK pathway. (A) Basal phosphorylated (p)-ERK and ERK levels analyzed by western blot in cases of chronic lymphocytic leukemia (CLL) with mutations in genes of the RAS-BRAF-MAPK-ERK pathway (case 1: KITLG mutation, case 16: BRAF mutation, case 27: MAP2K2 mutation and case 25: MAP2K1 mutation), in unmutated IGHV (U-IGHV) CLL and in peripheral blood mononuclear cells (PBMC). α- tubulin was used as a loading control. p-ERK/ERK levels were quantified relative to the U-IGHV case. (B) Basal p-ERK levels were analyzed by flow cytometry in CLL cases with mutations in genes of the RAS-BRAF-MAPK-ERK pathway (case 6: PTPN11 mutation, case 15: BRAF mutation and case 25: MAP2K1 mutation). Expression levels are relative to those in U-IGHV CLL. (C) Gene set enrichment analysis (GSEA) plots of the Biocarta- MAPK and KEGG MAPK signaling pathway gene sets regarding mutational status in genes of the RAS-BRAF-MAPK-ERK pathway in U-IGHV cases. The enrichment plot contains profiles of the run- ning enrichment scores (ES) and positions of gene set members on the rank ordered list in GSEA (126 unmutated and 17 mutated CLL cases). NES, normalized enrichment score. FDR, false discov- ery rate.
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