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Altered RAS-BRAF-MAPK-ERK pathway in CLL
Introduction
The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous.1,2 The mutational status of the immunoglobulin heavy-chain variable-region genes (IGHV) and deletions/mutations of 11q/ATM/BIRC3 and 17p/TP53 are important determinants of the clinical outcome of patients with CLL.3–6 Whole genome sequenc- ing and whole exome sequencing have identified recur- rent acquired mutations in the coding and non-coding regions of several genes. A few of them are mutated with moderate/low frequencies (11-15%), whereas the majori- ty are mutated at much lower frequencies (2-5%).7–10 This mutational landscape highlights the patients’ heterogene- ity. Several of the mutations, including some with a low incidence, have been reported to be associated with par- ticular clinical features and disease evolution.9,11–13
BRAF is a member of the serine-threonine kinase RAF family, comprising RAF-1/CRAF, ARAF, and BRAF. In nor- mal cells, BRAF functions as a mitotic signal transporter in the RAS/RAF/mitogen-extracellular signal-regulated kinase 1/2 (MEK1/2)/ extracellular signal-regulated kinase 1/2 (ERK1/2)/mitogen activated protein kinase (MAPK) pathway. This pathway plays a pivotal role in regulating embryogenesis, cell proliferation, differentiation, migra- tion, and survival.14 In the last decade, a high frequency of BRAF point mutations has been identified in melanoma and other human cancers.15,16 BRAF mutations are also a characteristic of hairy cell leukemia (HCL), being detected in 95% to 100% of patients with this type of leukemia.17,18 The most common BRAF mutation leads to the substitu- tion of a valine for glutamic acid at amino acid 600 (V600E) in the kinase domain of the protein. This substi- tution mimics the phosphorylation of the activation loop, thereby leading to its constitutive activation and phospho- rylation of MEK1 and MEK2, which in turn phosphorylate and activate the effector kinases ERK1 and ERK2.19 ERK proteins target numerous substrates, such as protein kinases, transcription factors, and cytoskeletal or nuclear proteins. Moreover, they are able to affect protein func- tions either by phosphorylating proteins in the cytoplasm or by translocating them into the nucleus where they acti- vate transcription factors that regulate proliferation- and cell survival-associated genes.20
BRAF mutations have been recurrently reported in CLL patients with a frequency of approximately 3%;21–24 most of these mutations cluster within or near the activation loop. Recently, novel CLL drivers (NRAS, KRAS, NRAS and MAP2K1) of the RAS-BRAF-MAPK-ERK pathway have also been described.9-24 However, the impact of BRAF mutations and other mutations in the RAS-BRAF-MAPK- ERK pathway in CLL is not well established.
We analyzed the clinical and biological characteristics and the impact of mutations in genes of the RAS-BRAF- MAPK-ERK pathway in CLL patients, the functional implications of these mutations and the in vitro response to different MAPK inhibitors.
Methods
Patients
Four hundred fifty-two patients (276 males/176 females) diag- nosed with CLL according to the World Health Organization cri- teria25 and included in the International Cancer Genome
Consortium for CLL (ICGC-CLL)7 were analyzed. All patients gave informed consent to inclusion in this study, according to the guidelines of the ICGC-CLL project and the local ethics commit- tees. The study was conducted in accordance with the Declaration of Helsinki.
Primary chronic lymphocytic leukemia cells
CLL cells were isolated, cryopreserved and stored in the Hematopathology collection registered at the Biobank (Hospital Clínic-IDIBAPS; R121004-094) (Online Supplementary Methods). Functional studies were done in all patients with mutations in genes of the RAS-BRAF-MAPK-ERK pathway for whom cryopre- served material was available.
Mutational analysis
Whole exome sequencing or whole genome sequencing was performed in 452 CLL patients. DNA from purified CLL cells (>95% tumor cells) was obtained before administration of any treatment, as described elsewhere.7 The median interval between diagnosis and sample analysis was 36 months (range, 0-300 months). Mutations in genes of the RAS-BRAF-MAPK-ERK path- way according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (KITLG, KIT, SOS2, PTPN11, GNB1, KRAS, NRAS, BRAF, MAP2K1, MAP2K2 and MAPK1) were selected for further analysis. Clonal mutations were considered when the vari- ant allele frequency (VAF) was ≥0.40 and subclonal when the VAF was <0.40. PolyPhen-2, SIFT and CADD algorithms were used for in silico prediction of the pathogenicity of the mutations. Coding mutations were considered pathogenic if they were reported as such by at least two algorithms (probably damaging by PolyPhen- 2 and/or damaging by SIFT and/or with a phred-like score >20 by CADD).
Gene expression analysis
The gene expression profile of 143 purified CLL samples with unmutated IGHV genes (U-IGHV) from the CLL-ICGC project7 was analyzed using the Gene Set Enrichment Analysis (GSEA) package version 2.0. Enrichment of the MAPK gene signature was investigated using the C2 Biocarta and C2 KEGG collection ver- sion 6.1 as reported in the Online Supplementary Methods. Gene sets with a P≤0.05, a false discovery rate (FDR) q-value ≤10% and a normalized enrichment score (NES) ≥1.5 were considered to be significantly enriched in the group with mutations in the RAS- BRAF-MAPK-ERK pathway.
Western blot analysis
Whole-cell protein extracts were obtained from CLL cells and peripheral blood mononuclear cells from healthy donors and western blot was performed with antibodies against phosphory- lated-T202/Y204 ERK 1/2 and total ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Online Supplementary Methods).
Analysis of viability
Vemurafenib, dabrafenib, and ulixertinib (BVD-523) were pur- chased from Selleckchem (Houston, TX, USA). Primary CLL cells were incubated for 24 or 48 h with the indicated doses of the drugs and then stained and analyzed as reported in the Online Supplementary Methods.
B-cell receptor stimulation and quantification of phosphorylated ERK by flow cytometry
B-cell receptors were stimulated by incubating CLL cells with 10 mg/mL of anti-IgM (Southern Biotech, Birmingham, AL, USA) and cells were stained for phospho (T202 and Y204)-ERK1/2-phyco-
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