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Development of a Cell Line Containing the Chimeric ETV6-NTRK3 Gene. The Search for Mutations of the Tyrosine Kinase
U A Boyarskikh1, T A Savostyanova1, I P Oscorbin1
1Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia.
Abstract:
The development, registration, and further use of entrectinib and larotrectinib for the treatment of tumors resulting from oncogenic stimulation of chimeric neurotrophin receptors (TRK) attracted much interest to the mechanisms of tumor cells resistance to TRK inhibitors during treatment. In the presented study, a cell line carrying the chimeric gene ETV6-NTRK3 (HFF-EN) was created on the basis of human fibroblasts. The transcription level of the chimeric ETV6-NTRK3 gene in HFF-EN was comparable to the transcription level of the household ACTB gene, the expression of the ETV6-NTRKA protein was confirmed by immunoblotting. A comparison of the dose-effect curves of fibroblasts and HFF-EN cells showed a ~38-fold increase in the sensitivity of HFF-EN to larotrectinib. To obtain a cell model of the resistance to larotrectinib in NTRK-dependent cancer, we used cell passages with a gradually increasing concentration of larotrectinib and obtained six resistant clones. p.G623E c.1868G>A mutation was found in five clones, and p.R582W c.1744C>T mutation, previously not described as a resistance mutation, was found in one clone showing significantly less resistance. These results can be further used for more complete understanding of the mechanisms of the resistance to TRK inhibitors and for the development of new drugs.
Insights
Researchers developed a new cell model to study resistance to TRK inhibitors like larotrectinib. They identified specific gene mutations, including a novel one, that contribute to treatment resistance in NTRK-driven cancers.
Area of Science:
- Oncology
- Molecular Biology
- Drug Resistance Mechanisms
Background:
- Entrectinib and larotrectinib target tumors with oncogenic NTRK fusions.
- Understanding resistance to TRK inhibitors is crucial for effective cancer treatment.
Purpose of the Study:
- To create a cell line model for studying resistance to TRK inhibitors.
- To identify genetic mutations associated with resistance to larotrectinib in NTRK-driven cancers.
Main Methods:
- Established a human fibroblast cell line (HFF-EN) with the ETV6-NTRK3 chimeric gene.
- Developed resistant cell clones by stepwise exposure to increasing larotrectinib concentrations.
- Utilized immunoblotting and sequencing to confirm gene expression and identify mutations.
Main Results:
- HFF-EN cells demonstrated significantly increased sensitivity to larotrectinib (~38-fold).
- Six larotrectinib-resistant clones were generated.
- Commonly identified mutation: p.G623E (ETV6-NTRK3).
- Novel mutation p.R582W (ETV6-NTRK3) found in one clone with lower resistance.
Conclusions:
- The study successfully created a valuable cell model for TRK inhibitor resistance research.
- Identified known and novel mutations contributing to resistance, aiding in understanding treatment failure.
- Findings support the development of next-generation TRK inhibitors and personalized treatment strategies.
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