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Altered RAS-BRAF-MAPK-ERK pathway in CLL
ulation of the RAS-BRAF-MAPK-ERK signaling pathway.37 Mutations in this pathway seem to be mutually exclusive as only in one case were two different mutations observed simultaneously in the pathway. In this way, oncogene mutations that activate common downstream pathways often occur in a mutually exclusive fashion,38 as has been reported for BRAF and MAP2K1 in HCL-variant.34
The upregulation of genes of the MAPK pathway observed in the gene expression profiling analysis as well as the higher levels of phosphorylated ERK, a surrogate marker of MAPK pathway activation,39 in cases with mutations in genes of the RAS-BRAF-MAPK-ERK path- way suggested the activation of this pathway in this sub- group of patients. Importantly, no ERK phosphorylation was observed in unmutated cases. Overall, these results agree with those found in other cancers, in which it has been postulated that the activation of RAS-RAF-MEK-ERK signaling can occur through mutations in several genes in the pathway.40
Our data suggest that mutations in the RAS-BRAF- MAPK-ERK pathway are associated with adverse biologi- cal features such as U-IGHV, high expression of ZAP-70, CD38 and CD49d, abnormal values of lactate dehydroge- nase, and accumulation of three or more driver mutations. Importantly, mutated CLL cases had a 5-year TTFT similar to that of patients with adverse mutations (TP53, ATM or BIRC3), whereas patients carrying both types of muta- tions simultaneously had the worst 5-year TTFT, as reported by our group and others.7,9,22,33 In our series of patients, the impact of mutations in genes of the RAS- BRAF-MAPK-ERK pathway on TTFT was independent of that of IGHV status and mutations in TP53, ATM or BIRC3. However, mutations in genes of the RAS-BRAF- MAPK-ERK pathway did not affect overall survival. Recently it was reported that BRAF mutations were asso- ciated with adverse overall survival, whereas KRAS and NRAS mutations were not.24
Vemurafenib (in 2011) and dabrafenib (in 2013) were the first selective BRAF inhibitors clinically approved for the treatment of melanoma with BRAF mutations.30 MEK inhibitors have also shown efficacy in BRAF-mutant melanoma and in 2014 and 2015 the Food and Drug Administration approved the use of MEK inhibitors in combination with BRAF inhibitors as standard-of-care for BRAF-mutant advanced melanoma.41 With these com- pounds, clinical response rates of around 50% and increased survival have been reported in BRAF-mutant melanoma42 as well as in cases of HCL refractory to con- ventional therapy.43,44 However, the majority of responses are transient and resistance is often associated with a plethora of different mechanisms that allow tumor cells to bypass BRAF/MEK inhibition and restore ERK-dependent signaling.45 Our results showed that vemurafenib and dabrafenib were not able to decrease levels of ERK phos- phorylation significantly in mutated cases, although a slight effect was observed after dabrafenib treatment which could be an off-target effect. Accordingly, a differ- ent spectrum of efficacy against non-V600 BRAF mutants has been described for vemurafenib and dabrafenib.46 In contrast, activation of ERK was detected in unmutated CLL cases, potentially due to ERK activation by the B-cell receptor signaling complex as it has been described that
BRAF inhibitor-related ERK phosphorylation can be par- tially abrogated by blocking B-cell receptor signaling with SYK inhibitors.47
It has been postulated that cancer cells can dynamically rewire their signaling networks to restore ERK activity and override the actions of inhibitors that act upstream of ERK.48 We, therefore, consider ERK itself as one of the “best” nodes for effective disruption of ERK signaling. Our results demonstrated that ulixertinib (BVD-523), a potent and highly selective inhibitor of ERK1/2, was able to inhibit ERK phosphorylation in vitro in all CLL cases with mutations in genes of the RAS-BRAF-MAPK-ERK path- way. Ulixertinib has shown activity in BRAF- and RAS- mutant cell lines. Results of phase I studies in solid tumors have documented a safe and well-tolerated effect in patients who harbored BRAF-, NRAS- and MEK-mutant solid tumors, supporting the ongoing development of ulix- ertinib for patients with MAPK-activating alterations.49 Recently it was reported that CLL cells with trisomy 12 showed increased sensitivity to MEK and ERK inhibitors, pointing to an essential role for MEK/ERK signaling in CLL with trisomy 12.50
In conclusion, we showed that the RAS-BRAF-MAPK- ERK pathway is one of the cellular processes affected in CLL and identified novel CLL drivers. Patients with muta- tions in genes of the RAS-BRAF-MAPK-ERK pathway had adverse biological features and most of them required treatment. Furthermore, our results suggest that inhibition of ERK phosphorylation in this subgroup of mutated CLL patients can be achieved using new, specific ERK inhibitors that have recently entered clinical trials. Pharmacological inhibition of the RAS-BRAF-MAPK-ERK pathway may represent a therapeutic approach to improve responses in this subgroup of CLL patients.
Acknowledgments
This study was supported by the Ministerio de Economía y Competitividad, Grant n. SAF2015-67633-R ,and PI16/00420 which are part of Plan Nacional de I+D+I and are co-financed by the European Regional Development Fund (FEDER-“Una manera de hacer Europa”) and the CERCA program from Generalitat Catalunya. European Union’s Seventh Framework Programme for research, technological development and demon- stration under grant agreement n. 306240; Generalitat de Catalunya Suport Grups de Recerca AGAUR 2017-SGR- 1009, and Departament de Salut (SLT002-16-00350), Instituto de Salud Carlos III (ISCIII) International Cancer Genome Consortium for Chronic Lymphocytic Leukemia (ICGC-CLL Genome Project), and project PM15/00007, which is part of Plan Nacional de I+D+I and are co-financed by FEDER. NG is a recipient of a predoctoral fellowship from Agaur and EC is an Academia Researcher of the "Institució Catalana de Recerca i Estudis Avançats" (ICREA) of the Generalitat de Catalunya. This work was mainly developed at the Centre Esther Koplowitz (CEK), Barcelona, Spain. We are indebted to the Genomics core facility of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) for technical help. We are grateful to N Villahoz and MC Muro for their excellent work in the coordination of the CLL Spanish Consortium and also thank L Jimenez, S Cabezas, and A Giró for their excellent technical assistance. Finally, we are very grate- ful to all patients with CLL who participated in this study.
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