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Updated: May 7, 2026
Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
Published on: July 17, 2019
Resistance to vemurafenib resulting from a novel mutation in the BRAFV600E kinase domain
Timothy R Wagenaar1, Leyuan Ma, Benjamin Roscoe
1Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
Abstract:
Resistance to the BRAF inhibitor vemurafenib poses a significant problem for the treatment of BRAFV600E-positive melanomas. It is therefore critical to prospectively identify all vemurafenib resistance mechanisms prior to their emergence in the clinic. The vemurafenib resistance mechanisms described to date do not result from secondary mutations within BRAFV600E. To search for possible mutations within BRAFV600E that can confer drug resistance, we developed a systematic experimental approach involving targeted saturation mutagenesis, selection of drug-resistant variants, and deep sequencing. We identified a single nucleotide substitution (T1514A, encoding L505H) that greatly increased drug resistance in cultured cells and mouse xenografts. The kinase activity of BRAFV600E/L505H was higher than that of BRAFV600E, resulting in cross-resistance to a MEK inhibitor. However, BRAFV600E/L505H was less resistant to several other BRAF inhibitors whose binding sites were further from L505 than that of PLX4720. Our results identify a novel vemurafenib-resistant mutant and provide insights into the treatment for melanomas bearing this mutation.
Insights
A novel BRAFV600E mutation (L505H) confers resistance to vemurafenib in melanoma. This finding is crucial for developing new treatment strategies against BRAF-mutant melanomas.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Vemurafenib resistance is a major challenge in treating BRAFV600E-positive melanomas.
- Existing resistance mechanisms do not involve secondary mutations in BRAFV600E.
- Identifying novel resistance mutations is critical for effective melanoma treatment.
Purpose of the Study:
- To systematically identify BRAFV600E mutations conferring vemurafenib resistance.
- To characterize the functional impact of novel BRAF mutations on drug sensitivity.
- To inform future therapeutic strategies for BRAF-inhibitor resistant melanomas.
Main Methods:
- Targeted saturation mutagenesis of BRAFV600E.
- Selection of vemurafenib-resistant cell lines and mouse xenografts.
- Deep sequencing to identify resistance-conferring mutations.
Main Results:
- A single nucleotide substitution (T1514A) encoding L505H was identified.
- BRAFV600E/L505H exhibited increased kinase activity and conferred significant vemurafenib resistance.
- This mutant showed cross-resistance to a MEK inhibitor but varied sensitivity to other BRAF inhibitors.
Conclusions:
- A novel BRAFV600E resistance mutation, L505H, has been discovered.
- The L505H mutation impacts BRAF kinase activity and drug resistance profiles.
- Understanding this mutation provides insights for treating resistant melanomas.
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