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BACKGROUNDHead and neck squamous cell carcinoma not associated with HPV (HPV-unrelated HNSCC) is associated with a high rate of recurrence and poor survival.METHODSWe conducted a clinical trial in 14 patients with newly diagnosed HPV-unrelated HNSCC to evaluate the safety and efficacy of neoadjuvant bintrafusp alfa, a bifunctional fusion protein that blocks programmed death ligand 1 (PD-L1) and neutralizes TGF-β.RESULTSBintrafusp alfa was well tolerated, and no treatment-associated surgical delays or complications occurred. Objective pathologic responses (PRs) were observed, and 12 of the 14 (86%) patients were alive and disease free at 1 year. Alterations in Treg infiltration and spatial distribution relative to proliferating CD8+ T cells indicated a reversal of Treg immunosuppression in the primary tumor. Detection of neoepitope-specific tumor T cell responses, but not virus-specific responses, correlated with the development of a PR. Detection of neoepitope-specific responses and PRs in tumors was not correlated with genomic features or tumor antigenicity but was associated with reduced pretreatment myeloid cell tumor infiltration. These results indicate that dual PD-L1 and TGF-β blockade can safely enhance tumor antigen-specific immunity and highlight the feasibility of multimechanism neoadjuvant immunotherapy for patients with HPV-unrelated HNSCC.CONCLUSIONOur studies provide insight into the ability of neoadjuvant immunotherapy to induce polyclonal neoadjuvant-specific T cell responses in tumors and suggest that features of the tumor microenvironment, such as myeloid cell infiltration, may be a major determinant of enhanced antitumor immunity following such treatment.TRIAL REGISTRATIONClinicalTrials.gov NCT04247282.FUNDINGThis work was funded by the Center for Cancer Research, the NCI, and the Intramural Research Program of the NIDCD, NIH. Bintrafusp alfa was provided by the health care business of Merck KGaA (Darmstadt, Germany), through a Cooperative Research and Development Agreement with the NCI. Additional funding was provided by ImmunityBio through a Cooperative Research and Development Agreement with the NIDCD.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world, with Human Papilloma virus (HPV)-negative patients having a ~50% recurrence rate and poor median overall survival of ~13 months in the recurrent/metastatic setting. Pembrolizumab, a programmed-death-1 (PD-1) checkpoint inhibitor is active but with a response rate as low as 19%. Our preliminary work has identified SET and MYND Domain containing 3 (SMYD3) as a chromatin modifier that is significantly overexpressed in HPV-negative HNSCC tumor tissues compared to normal epithelium and is associated with low CD8+ T-cell infiltration in HPV-negative HNSCC. Furthermore, we have shown that knockdown of human SMYD3 upregulates the expression of multiple type I IFN response and antigen presentation machinery (APM) genes. This study aims to investigate whether Smyd3 depletion potentiates immune-mediated antitumor efficacy in anti-PD-1 resistant syngeneic mouse models of HPV-negative HNSCC (MOC1), as well as relevant mechanisms. SiRNA-mediated knockdown of Smyd3 in mouse HPV-negative MOC1 cells followed by RNA-seq revealed upregulation of multiple type I IFN response and APM genes in vitro. Multicolor flow cytometry of MOC1 tumors treated with control anti-sense oligonucleotides (ASOs) or Smyd3 ASOs showed that Smyd3 depletion in a syngeneic, heterotopic mouse model (C57BL/6) of flank MOC1 tumors induced intratumoral infiltration of CD8+ T-cells, as well as upregulation of H2-Kb (MHC class I) and mouse Pd-l1 in MOC1 cells. Combined treatment of Smyd3 ASOs with anti-PD-1 induced complete regressions of flank MOC1 tumors in 4/8 treated mice and significant tumor growth restriction in 2/8 mice, while 2/8 tumors “escaped” the treatment effect. Single-cell RNA seq of MOC1 tumors treated with control or Smyd3 ASOs is ongoing to evaluate the effects of Smyd3 depletion on MOC1 cancer cells and immune cell subsets of the tumor microenvironment and to identify potential mechanisms of resistance of Smyd3 ASO + anti-PD-1 combination treatment. Genome-wide mapping of Smyd3 and the repressive mark H4K20me3, which is known to be written by Smyd3, in MOC1 cells using CUT&RUN assays is also ongoing and aims to decipher whether Smyd3 directly binds to and regulates the expression of immune-related genes through H4K20me3. Ex-vivo cytotoxicity assays aim to evaluate whether CRISPR Smyd3 KO in MOC1 cells sensitizes cancer cells to TIL-mediated cytotoxicity. Smyd3 depletion induces an inflamed tumor microenvironment and may potentiate the immune-mediated antitumor efficacy of anti-PD-1 in syngeneic mouse models of HPV-negative HNSCC. This work may lay the biological rationale to combine SMYD3 inhibition with T-cell based immunotherapeutic approaches in HPV-negative HNSCC. Citation Format: Daniel E. Tsai, Yvette Robbins, Angel Huynh, Andrew Sinkoe, Cem Sievers, Madhavi Murali, Xiaolin Luo, Clint T. Allen, Vassiliki Saloura. Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6250.