Analysis of Mutational Profile of Hypopharyngeal and Laryngeal Head and Neck Squamous Cell Carcinomas Identifies KMT2C as a Potential Tumor Suppressor

Abstract

Hypopharyngeal cancer is a poorly characterized type of head and neck squamous cell carcinoma (HNSCC) with bleak prognosis and only few studies focusing specifically on the genomic profile of this type of cancer. We performed molecular profiling of 48 HPV (Human Papilloma Virus)-negative tumor samples including 23 originating from the hypopharynx and 25 from the larynx using a targeted next-generation sequencing approach. Among genes previously described as significantly mutated, TP53, FAT1, NOTCH1, KMT2C, and CDKN2A were found to be most frequently mutated. We also found that more than three-quarters of our patients harbored candidate actionable or prognostic alterations in genes belonging to RTK/ERK/PI3K, cell-cycle, and DNA-damage repair pathways. Using previously published data we compared 67 hypopharyngeal cancers to 595 HNSCC from other sites and found no prominent differences in mutational frequency except for CASP8 and HRAS genes. Since we observed relatively frequent mutations of KTM2C (MLL3) in our dataset, we analyzed their role, in vitro, by generating a KMT2C-mutant hypopharyngeal cancer cell line FaDu with CRISPR-Cas9. We demonstrated that KMT2C loss-of-function mutations resulted in increased colony formation and proliferation, in concordance with previously published results. In summary, our results show that the mutational profile of hypopharyngeal cancers might be similar to the one observed for other head and neck cancers with respect to minor differences and includes multiple candidate actionable and prognostic genetic alterations. We also demonstrated, for the first time, that the KMT2C gene may play a role of tumor suppressor in HNSCC, which opens new possibilities in the search for new targeted treatment approaches.

KMT2 (MLL) family of methyltransferases in head and neck squamous cell carcinoma: A systematic review

“…Moreover, the co‐occurrence of KMT2D and KMT2C mutations has been reported by several studies, which may suggest an important epigenetic event in HNSCC carcinogenesis 23,25,30,33,37,40,46,48–51 . In a similar study that also utilized data from the cBioPortal, a significant trend toward the co‐occurrence of KMT2C and KMT2D mutations was observed in lung adenocarcinoma and squamous cell carcinoma (p‐value <0.001), and these mutations were associated with driver mutations in PIK3CA , PTEN , and ARID1A in various cancer types 13 .…”

“…The functional effects of such KMT2D inactivation were investigated in various tumors aiming to better understand its role. In this systematic review, we found a study that suppressed KMT2D in oral squamous cell carcinoma cell line using CRISPR‐Cas9, which led to decreased colony formation, sphere formation, cell migration, and invasion compared to control cells 40 . In contrast, Dauch et al showed that the loss of KMT2D in a cutaneous squamous cell carcinoma cell line was associated with increased proliferation, cell colony growth, migration, and accelerated progression through the cell cycle 87 .…”

“…Regarding the risk of bias evaluated by Joanna Briggs Institute Critical Appraisal Checklist for Studies Reporting Prevalence Data, 18 studies were classified as having a low risk of bias, 27,29,34,35,37–45,49–51,54,55 and the others 15 were classified as having a moderate risk of bias 23–26,28,30–33,36,46–48,52,53 . The summary of each study's risk of bias assessment is in Table S3.…”

“…The included studies were conducted in 16 different countries, that is Austria, 23 Brazil, 24 Canada, 25,26 China, 27,28 Denmark, 29,30 France, 31,32 Germany, 33 India, 34–36 Japan, 37 Malaysia, 38 Pakistan, 39 Poland, 40 Taiwan, 41,42 Thailand, 43 the Netherlands, 44 and the United States of America 45–55 . All of them were published in English between 2011 and 2022.…”

“…KMT2C mutations (3.53%) were identified by 14 23,25,30,33,36,37,39,40,46,48–51,55 out of 29 studies, which detected different mutations, such as single nucleotide variation, nonsense mutation, missense mutation, synonymous variation, and frameshift deletion. Curiously, one study showed that all KMT2C mutated cell lines also displayed CDKN2A promoter methylation 46 …”