NSD1 Mutations by HPV Status in Head and Neck Cancer: Differences in Survival and Response to DNA-damaging Agents.

Compared to HPV-negative head and neck squamous cell carcinomas (HNSCCs), HPV-positive HNSCCs are associated with a favorable prognosis in part due to their improved treatment sensitivity. Inactivating mutations in NSD1 were shown to be a favorable prognostic biomarker in laryngeal cancers. Here, we characterize NSD1 mutations from the expanded The Cancer Genome Atlas (TCGA) HNSCC cohort (n = 522) and examine their prognostic implications based on HPV status of the tumor. We also begin to examine if NSD1 regulates response to platinum-based drugs and other DNA-damaging agents. TCGA HNSCC samples were segregated by HPV and NSD1 mutations using cBioPortal and patient survival was determined. Pathogenicity of mutations was predicted using UMD-Predictor. NSD1-depleted cell lines were established by transfection with control or shRNAs against NSD1, followed by puromycin selection, and confirmed by qRT-PCR. Cell sensitivity to DNA damaging agents was assessed using short-term proliferation and long-term clonogenic survival assays. Among 457 HPV(−) tumors, 13% contained alterations in the NSD1 gene. The majority (61.3%) of NSD1 gene alterations in HPV(−) specimens were truncating mutations within or before the enzymatic SET domain. The remaining alterations included homozygous gene deletions (6.7%), missense point mutations (30.7%) and inframe deletions (1.3%). UMD-Predictor categorized 18 of 23 missense point mutations as pathogenic. For HPV(+) HNSCC (n = 65), 6 NSD1 mutations, comprised of two truncating (33%) and 4 missense point (66%) mutations, were identified. Three of the 4 missense point mutations were predicted to be pathogenic or probably pathogenic by UMD-Predictor. Kaplan-Meier survival analysis determined significantly improved survival of HPV(−) HNSCC patients whose tumors harbored NSD1 gene alterations, as compared to patients with wild-type NSD1 tumors. Interestingly, the survival effect of NSD1 mutations observed in HPV-negative HNSCC was reversed in HPV(+) tumors. Proliferation and clonogenic survival of two HPV(−) cell lines stably expressing control or NSD1 shRNAs showed that NSD1-depleted cells were more sensitive to cisplatin and carboplatin, but not to other DNA damaging drugs. Genetic alterations in NSD1 hold potential as novel prognostic biomarkers in HPV(−) head and neck cancers. NSD1 mutations in HPV(+) cancers may also play a prognostic role, although this effect must be examined in a larger cohort. NSD1 downregulation results in improved sensitivity to cisplatin and carboplatin, but not to other DNA-damaging agents, in epithelial cells. Increased sensitivity to platinum-based chemotherapy agents associated with NSD1 depletion may contribute to improved survival in HPV(−) HNSCCs. Further studies are needed to determine mechanisms through which NSD1 protects HPV(−) HNSCC cells from platinum-based therapy, as well as confirmation of NSD1 effect in HPV(+) HNSCC.