Epigenetic silencing by SMYD3 represses tumor intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck [RNA-seq]

Cancers often display immune escape, but the mechanisms and potential for reversibility are incompletely understood. Epigenetic dysregulation has been implicated in the immune escape of various cancer types. We have identified the epigenetic modifier SET and MYND-domain containing protein 3 (SMYD3) as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab, a programmed-death-1 (PD-1) targeting antibody. SMYD3 depletion increased the sensitivity of HNSCC cancer cells to IFN-β, resulting in upregulation of type I IFN response and antigen presentation machinery genes. We found that SMYD3 binds to and regulates the transcription of Ubiquitin-Like PHD And RING Finger Domain-Containing Protein 1 (UHRF1), a key epigenetic reader of trimethylated lysine 9 on histone H3 (H3K9me3), which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1 and silences their expression. SMYD3 further maintains the repression of immune-related genes through the deposition of H4K20me3 within the promoters and/or gene body regions of these genes. In an anti-PD-1 immune checkpoint resistant syngeneic mouse model of HPV-negative HNSCC, Smyd3 depletion induced influx of CD8+ T-cells, upregulated PD-L1 and MHC class I molecules, and increased sensitivity to anti-PD-1 therapy. SMYD3 overexpression was associated with decreased CD8 T-cell infiltration in tumor samples from patients with HPV-negative HNSCC and poor response to neoadjuvant pembrolizumab. Overall, these data highlight a previously unreported function of SMYD3 as a master epigenetic regulator of anti-tumor immune response in HPV-negative HNSCC and provide a rationale for translational approaches combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in this devastating disease. For samples VS265-VS279, HN-6 (parental control cells) and 5-3 cells (SMYD3 KO cell line) were grown and exposed to human IFN-β for 24h prior to cell collection. For samples VS7-VS14 and VS748-VS753, HN-6 cells were transfected with control siRNA or siSMYD3 or siUHRF1 for 72h and exposed to human IFN-β for 24h prior to cell collection. For samples VS212-VS217, HN-6 cells were exposed to PBS or SMYD3 targeting ASOs (ASO 081) for 72h and exposed to human IFN-β for 24h prior to cell collection. Two to three biological replicates were generated per condition. RNA was extracted using the Direct-zol RNA miniprep kit (Zymo Research).