LSD1 inhibition remodels the tumor microenvironment to enhance anti-PD1 immunotherapy in HNSCC

Background: The histone demethylase LSD1 (KDM1A), an epigenetic regulator implicated in tumor progression and immune suppression. Programmed cell death protein-1 (PD-1/CD279) marks exhausted CD8+ T cells and binds PD-L1 (CD274) in the tumor epithelium. Anti-PD-1 therapy has limited success in head and neck squamous cell carcinoma (HNSCC). We hypothesized that LSD1 inhibitor (SP2509), due to its specific mechanisms, shows superior anti-cancer activity in combination with anti-PD1 therapy. Methods: We assessed SP2509's impact on the tumor microenvironment (TME) in a syngeneic 4MOSC1 oral squamous cell carcinoma (OSCC) model and 4NQO-induced progressive OSCC model. Anti-PD-1 monotherapy and anti-PD-1-SP2509 combination therapy were tested in a 4NQO model. Multiple methods like immunostaining, qRT-PCR, flowcytometry were employed to test the hypothesis. Ovalbumin overexpression assay followed by flowcytometry was used to determine the antigen presentation in every groups. ChIP-qPCR was used to determine the H3K4 and H3K9 methylation change status on HLA-A, HLA-B and PD-L1 gene locus. Public RNA-seq data (GSE153383) were analyzed to examine immune cell responses to anti-PD-1 and compared with our findings. Results: SP2509 increased immune cell infiltration, including CD8 + T cells, and reduced the frequency of PD-L1 + epithelial tumor cells in both 4MOSC1 and 4NQO models. Anti-PD-1 monotherapy expanded CD8 + T cells but did not alter PD-L1 + epithelial cells. Each treatment alone enhanced CD8+ T-cell IFN-gamma production, consistent with the GSE153383 analysis showing elevated T-cell infiltration and IFN-gamma in anti-PD-1 treated samples. Furthermore, we observed a significant increase in antigen presentation in anti-PD-1 and SP2509 combination group with ovalbumin assay. The combination regimen produced greater immune infiltration, particularly CD8+ T-cells with higher IFN-gamma levels, a significant reduction in PD-L1+ epithelial cells, and more pronounced tumor regression than anti-PD-1 alone was observed in vivo . ChIP-qPCR results shows increase in H3K4me2 in HLA-A and HLA-B gene locus while H3K9me2 increase in PD-L1 gene locus was observed after SP2509 treatment. We also observed DC activation in SP2509 alona and in combination groups and absent in only anti-PD1 treatment group. Conclusions: Targeting LSD1 with SP2509 enhances the anti-PD-1 efficacy in HNSCC by modulating the TME, augmenting CD8⁺ T-cell-mediated antitumor immunity and antigen presentation via MHC class I activation, and reducing tumor PD-L1 expression, ultimately leading to a reduction in HNSCC growth.