Smyd3-mediated immuno-modulation in HPV-negative head and neck squamous cell carcinoma mouse models.

SET and MYND-domain containing protein 3 (SMYD3) mediates epigenetic repression of type I IFN response genes in human papilloma virus (HPV)-negative HNSCC cells, and Smyd3 depletion using anti-sense oligonucleotides (ASOs) increases the sensitivity of syngeneic mouse oral carcinoma (MOC1) models to anti-PD-1 therapy. In this study, we interrogated the effects ASO-induced Smyd3 depletion in the cancer and immune cell populations, as well as the global changes in the tumor microenvironment (TME) of MOC1 tumors treated with Smyd3 ASOs and anti-PD-1. Single-cell RNA-seq of MOC1 tumors treated with Smyd3 ASOs revealed enrichment of type I IFN response pathways in cancer cells, a shift of CD8+ T-cells towards an activated/memory phenotype and a shift of neutrophils towards an anti-tumorigenic phenotype. While Smyd3 ASOs seemed to also promote a Treg and M2 macrophage phenotype, bulk RNA-seq of MOC1 tumors treated with Smyd3 ASOs and anti-PD-1 revealed enrichment of type I IFN response pathways and upregulation of CD8+ T-cell attracting chemokines, implying the induction of an inflamed TME. Non-responder MOC1 tumors to the Smyd3 ASO and anti-PD-1 combination revealed mechanisms of resistance derived from cancer cells, macrophages and CD8+ T-cells, including neutrophil enrichment through upregulation of Cxcl2, repression of Cxcl9 and defective antigen presentation. This study sheds light in the immunomodulatory functions of Smyd3 in an in vivo setting and provides insight into actionable mechanisms of resistance to improve the therapeutic efficacy of Smyd3 ASO and anti-PD-1 combination treatment.