Dissecting the effects of Smyd3 depletion in the tumor microenvironment of HPV-negative head and neck squamous cell carcinoma mouse models

Abstract Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) patients have a poor prognosis and most patients with metastatic disease are resistant to pembrolizumab. We recently showed that depletion of SET and MYND domain protein 3 (SMYD3), a protein methyltransferase, induces upregulation of multiple type I IFN response genes in HPV-negative HNSCC human and mouse cells in vitro. Systemic treatment of flank HPV-negative mouse oral carcinoma 1 (MOC1) tumors with Smyd3 anti-sense oligonucleotides (ASOs) induced influx of CD8+ T-cells, and combined Smyd3 ASOs and anti-PD-1 treatment induced tumor growth restraint, with 75% of tumors cured or reduced and 25% of tumors escaping. Here, we aim to investigate the changes in the tumor microenvironment (TME) of MOC1 tumors treated with Smyd3 ASOs and in combination with anti-PD-1, and to elucidate mechanisms of resistance. MOC1 tumors treated with control or Smyd3 ASOs (n=3 per group) were harvested and single-cell RNA-seq was conducted. GeoMx, a high-plex spatial proteomic assay, was utilized on FFPE sections of MOC1 tumors treated with control or Smyd3 ASOs with or without anti-PD-1 (n=2 per group). To decipher mechanisms of resistance, single-cell RNA-seq of MOC1 tumors that responded or not to combination Smyd3 ASOs and anti-PD-1 was conducted. 26926 cells from six samples were available for single-cell RNA-seq analysis of control versus Smyd3 ASO treated tumors. UMAP analysis identified distinct immune-, stroma- and cancer-cell populations. Comparative analysis of the single-cell Smyd3 mRNA expression revealed downregulation of Smyd3 mRNA in CD8+ and CD4+ T-cells, B-cells, NK-cells and cancer cells, but not in myeloid cells, endothelial cells and fibroblasts, implying differential uptake of Smyd3 ASOs in the TME. CD8+ T-cells in the Smyd3 ASO treated tumors exhibited higher expression levels of Gzma and Gzmb, indicating enhanced cytotoxic activity. Colony stimulating factor 1 (Csf1), which is important for the differentiation of macrophages, was upregulated in CD8+ T-cells of Smyd3 ASO treated tumors. Single-cell RNA-seq of a non-responder versus a responder MOC1 tumor yielded 9170 and 11410 cells respectively, with more than a two-fold increase in the ratio of neutrophils and a two-fold decrease for CD8+ T-cells comparing the non-responder to responder tumor. Accordingly, cancer cells, CD8+ T-cells and macrophages of the non-responder tumor exhibited higher expression of Cxcl2, a chemokine that attracts PMNs. Differential gene expression analysis in all cell subsets in control versus Smyd3 ASO and responder versus non-responder tumors is ongoing. This work will elucidate the TME changes induced by Smyd3 ASOs in MOC1 tumors, and will reveal potential mechanisms of resistance to the Smyd3 ASOs and anti-PD-1 combination, enabling translation of this therapeutic approach in HPV-negative HNSCC. Citation Format: Malhar Patel, Daniel Tsai, Abdalla Abdelmaksoud, Noemi Kedei, Maria Hernandez, Baktiar Karim, Vassiliki Saloura. Dissecting the effects of Smyd3 depletion in the tumor microenvironment of HPV-negative head and neck squamous cell carcinoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2320.