Abstract 1103: CDK4 copy number gain in BRAF V600E-mutated non-small cell lung cancers resistant to dabrafenib plus trametinib
Authors: Hirai Noriko , Sasaki Takaaki , Chiba Shinichi , Uno Yuji , Takei Hidehiro 
Published: July 2021

Background: Since the discovery of the oncogenic mutation B-Raf proto-oncogene, serine threonine kinase (BRAF) V600E, dabrafenib and trametinib (DT) combination therapies have been shown to have great anti-tumor activities against V600E-mutated non-small cell lung cancers (NSCLC). However, the resistant mechanisms for DT therapies are not fully understood. Single nucleotide mutation in KRAS, NRAS, MEK1, which locate in the universal growth pathway RAF-RAS-MEK-ERK, or frameshift stop of PTEN had been reported as acquired resistances previously. We had been aimed to search a new therapeutic target for clinically DT resistant, and have been performed comprehensive cancer drug screening by establishment of the clinically resistant patient's derived cell lines. In the process, we focused on a positive regulator of cell cycle cyclin dependent kinase 4 (CDK4) as a potential role in resistant acquisition.

Materials: Two patients' pleural effusion-derived NSCLC cell lines of BRAF V600E-mutant who were clinically resistant for DT therapies (LC-1, LC-6) were established and used for post-DT samples. The same patients' derived tissue samples collected at diagnosis (tLC-1, tLC-6) were used for pre-DT samples as comparison. Encompassing cancer drug screening were performed by MTS assay. Copy number variant (CNV) and single nucleotide variant (SNV) were analyzed by amplicon-captured next generation sequence (NGS). Each mRNA expression was quantified using TaqMan Array of human cancer drug panel. Protein expression were evaluated by western blotting and immunohistochemistry (IHC). As references for protein expression, we used patient's derived cell lines of BRAF K601E-mutant (LC-12), and BRAF V600E-mutant (OY) both of which were DT naive.

Results: Patient 1 was treated with DT for 3 months and progressed with pleural effusions. Patient 6 was treated with DT for 11 months and progressed with pleural effusions. In both LC-1 and LC-6, dabrafenib and trametinib showed resistance in each 1microM screening in vitro. Analysis of CNV and SNV pre- and post-treatment in each patient, copy number of CDK4 was significantly gained in the patient 1 after DT therapies. qPCR and western blotting revealed high expression of CDK4 in LC-1 while not remarkable in LC-6, LC-12 and OY. Protein expression assessed by IHC revealed that in pre- and post-DT sample, increased protein expression of CDK4, cyclinD1 phosphorylated-Rb were observed in LC-1, but remarkable change were not observed in the patient 6.

Conclusions: We highlight CDK4 copy number gain as a possible clinical resistant mechanism for DT therapies in BRAF V600E-mutated NSCLC. We are currently investigating how CDK4 contribute as the acquired resistance.

Citation Format: Noriko Hirai, Takaaki Sasaki, Shinichi Chiba, Yuji Uno, Hidehiro Takei. CDK4 copy number gain in BRAF V600E-mutated non-small cell lung cancers resistant to dabrafenib plus trametinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1103.