Abstract 2320: Dissecting the effects of Smyd3 depletion in the tumor microenvironment of HPV-negative head and neck squamous cell carcinoma mouse models

• Published in: Cancer research (Chicago, Ill.) Ill.), 2023-04, Vol.83 (7_Supplement), p.2320-2320
• Main Authors: Patel, Malhar, Tsai, Daniel, Abdelmaksoud, Abdalla, Kedei, Noemi, Hernandez, Maria, Karim, Baktiar, Saloura, Vassiliki
• Format: Article
• Language: English
• ISSN: 1538-7445; 1538-7445
• DOI: 10.1158/1538-7445.AM2023-2320

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 chemoki