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Altered RAS-BRAF-MAPK-ERK pathway in CLL
The overall survival of patients with mutations in RAS- BRAF-MAPK-ERK pathway genes was similar to that of patients without mutations in this pathway (Table 2). When mutations in TP53, ATM or BIRC3 were taken into account, the overall survival of patients with mutations in genes of the RAS-BRAF-MAPK-ERK pathway alone was similar to that of patients without adverse mutations (Figure 2D) [5-year overall survival of patients without mutations, 84% (95% CI: 78-92%); with mutations only in the RAS-BRAF-MAPK-ERK pathway, 80% (95% CI: 64-99%); with adverse mutations only, 66% (95% CI: 53- 79%); and with both abnormalities in RAS-BRAF-MAPK- ERK pathway genes and adverse mutations, 66% (95% CI: 45-100%), P=0.003]. Multivariate analysis including IGHV status, mutations in genes of the RAS-BRAF- MAPK-ERK pathway, and adverse mutations in a final model with 439 patients showed an independent impact on overall survival for IGHV status [HR 3.3 (95% CI: 1.9- 5.9), P<0.001] and adverse mutations [HR 1.7 (95% CI: 1.1-2.8), P=0.02].
Functional and gene expression analysis
To assess the functional impact of these genomic alter- ations on the RAS-BRAF-MAPK-ERK pathway, we ana- lyzed the phosphorylation status of ERK as a surrogate marker of activation of the pathway. Western blotting with an antibody that specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2 showed higher levels of endogenous ERK phosphoryla- tion (3.3- to 4.4-fold induction) in CLL cases with muta- tions in KITLG, BRAF, MAP2K2 and MAP2K1 genes com- pared to U-IGHV CLL cases with no alterations in the MAPK/ERK pathway (Figure 3A). The same results were obtained when analyzing the phosphorylated forms of ERK by flow cytometry, labeling cells with phospho (T202/Y204)-ERK1/2-phycoerythrin. Figure 3B shows that cases with mutations in genes of the RAS-BRAF-MAPK-
ERK pathway (PTPN11, BRAF, and MAP2K1 mutations) had higher basal levels of phosphorylated ERK than cases of U-IGHV CLL (5- to 10-fold).
To identify the differential biological characteristics of cells carrying mutations in the RAS-BRAF-MAPK-ERK pathway, we conducted a gene expression profiling study in CD19+ tumor CLL cells from 143 CLL cases, 17 of which carrying functional mutations according to PolyPhen-2, SIFT and CADD phred-like predictions. With the C2 Biocarta analysis, we detected 126 of 149 gene sets upregulated in the group carrying mutations in genes of the RAS-BRAF-MAPK-ERK pathway, including the Biocarta MAPK pathway (NES=1.90; P<0.001; FDR=0.013) (Online Supplementary Table S2 and Figure 3C). Similar results were obtained when carrying out a C2 KEGG analysis. We detected 104 of 178 gene sets upregulated in the group carrying mutations in genes of the RAS-BRAF-MAPK-ERK pathway, including the KEGG MAPK signaling pathway (NES=1.85; P<0.001; FDR=0.013) (Online Supplementary Table S3 and Figure 3D). Genes belonging to the Biocarta and KEGG MAPK pathways are listed in Online Supplementary Tables S4 and S5, respectively.
Response to MAPK pathway inhibitors
We next evaluated the effect of BRAF inhibitors (vemurafenib, a specific inhibitor of the BRAF V600E mutation, and dabrafenib, specific for BRAF V600E and V600K variants) in cells from 17 CLL cases, nine contain- ing mutations in genes of the RAS-BRAF-MAPK-ERK pathway (KITLG, PTPN11, KRAS, BRAF, MAPK1, MAP2K1 and MAP2K2) and eight U-IGHV CLL cases with no alterations in this pathway. Vemurafenib, at a dose of 2.5 mM, was not able to inhibit basal ERK phos- phorylation or after anti-IgM stimulation in mutated cases, while a slight effect was observed after treatment with 2.5 mM of dabrafenib. Furthermore, upregulation of
Figure 1. Brick-plot showing gene mutations, cytogenetic abnormalities and the type of RAS-BRAF-MAPK-ERK pathway mutations. Clonal mutations are labeled in dark blue, subclonal mutations in light blue, normal genes or chromosomal regions in light gray, and mutated/deleted genes or chromosomal regions in dark gray. Adverse alterations: TP53, ATM or BIRC3.
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