Abstract LB412: BRAFp.G469E hotspot mutation promotes nasopharyngeal carcinoma tumorigenesis with potential druggability

Abstract

Abstract Nasopharyngeal Carcinoma (NPC) is a distinct type of Epstein-Barr Virus (EBV)-associated head & neck cancer. It is most prevalent in Southeast Asia with a high incidence rate of ~15/100,000. While mutations of the MAPK pathways are found in nearly one-fifth of head & neck squamous cell carcinoma (HNSCC) in Western countries (TCGA-HNSCC Firehose cohort, N=512), and some of which are druggable in HNSCC setting (e.g. MAPK1 and HRAS mutations), we sought to determine the cumulative mutational rate of this potentially druggable pathway in 164 micro-dissected NPC cases using recently published next-generation sequencing data (98 whole-exome and 63 whole-genome-sequenced cases). An overall rate of 9.32% cases of NPC harbored MAPK pathway mutations (16 mutations in 15 tumors). Four genes are recurrently mutated: 4 NRAS (all Q61R/L hotspot mutations), 3 HRAS (G12S, Q61R, S106L), 2 BRAF (R726H, Y647*), and 2 SHC4 (Q519R, A552T) mutations. Among which, both patients bearing BRAF mutations died of NPC, suggestive of the biological importance of BRAF mutations in NPC progression. Prompted by an independent clinical report on an advanced NPC patient with somatic co-mutations of BRAFp.G469E and KRASp.G12D being sensitive to the B-raf/C-raf inhibitor sorafenib, we first examined the biological functions and potential druggability of this particular BRAFp.G469E mutation in NPC, as it represents a hotspot mutation site across 20 cancer types (BRAFp.G469E/A/R/V/* found in 137/70655 pan-cancer samples, cBioPortal). Based on the NPC clinical report, we hypothesized that this BRAFp.G469E hotspot mutation could drive NPC tumorigenesis and might be potentially sensitive to the B-raf inhibitor sorafenib. By ectopic expression of BRAFp.G469E mutation, BRAF-wildtype (wt) and EGFP-control gene into a recently developed Epstein-Barr virus-positive nasopharyngeal carcinoma [EBV(+)NPC] cell line, NPC43, we determined the biological functions of this mutation in NPC. We showed that NPC43-BRAFp.G469E-expressing cells significantly drove a 4-fold increase in 3D tumoroid growth vs. BRAF-wt and EGFP-control cells on day 7 (flat-bottom low attachment plates). Further, wound healing assay demonstrated a significantly higher migration rate of NPC43-BRAFp.G469E cells achieving ~80% wound healing (at 96h) vs. 30% and 10% healing in BRAF-wt and EGFP-control cells. Lastly, our preliminary drug study showed that NPC43-BRAFp.G469E cells displayed higher sensitivity to low dose sorafenib (33.3nM and 100nM) than BRAF-wt and EGFP-control cells, achieving ~20% and 30% growth inhibition at 72h, vs. no growth inhibition in control cells. Thus, BRAFp.G469E hotspot mutation can drive NPC tumorigenesis, and may confer sensitivity to raf inhibitors such as sorafenib in NPC. Potential druggability of other BRAF mutations in NPC should be investigated. Acknowledgements: The study was supported by Research Impact Fund (R4015-19F), Research Grants Council, University Grants Committee, Hong Kong SAR during 6/29/20-9/7/21. Citation Format: Chun Ho Law, Zoey Wing Yee Liu, Franky Leung Chan, Tin Yu Samuel Law, Vivian W.Y. Lui. BRAFp.G469E hotspot mutation promotes nasopharyngeal carcinoma tumorigenesis with potential druggability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB412.