Project Summary/Abstract The long-term objectives of this application are to understand how epigenetic factors control the invasive growth and metastasis of head and neck squamous cell carcinoma (HNSCC) and to develop novel therapeutics for HNSCC. Patients with HNSCC, particularly human papillomavirus (HPV)-negative HNSCC, exhibit poor overall 5-year survival rates compared to breast and colorectal cancers. Therefore, novel effective therapies need to be developed for HNSCC patients. Emerging evidence suggests that histone methylation plays a critical role in activation of gene transcription in HNSCC by regulating chromatin accessibility. We have identified that the histone demethylase KDM4A as a key epigenetic factor, stimulated the transcription of the invasive genes to promote HNSCC invasion and metastasis by erasing repressive H3K9me3 marks. To further confirm that KDM4A is an important target for HNSCC treatment, we took advantage of the 4-nitroquinoline 1-oxide (4-NQO)- induced mouse model of HNSCC, which fully simulates HNSCC development and lymph node metastasis in a syngeneic tumor immune microenvironment. We found that the specific deletion of KDM4A significantly inhibited HNSCC invasive growth and lymph node metastasis. Our RNA-seq analysis found that KDM4A ablation significantly suppressed the gene expression associated with cell migration and epithelial mesenchymal transition in primary HNSCC, which confirmed our previous findings. Unexpectedly, a set of genes associated with immune response was robustly upregulated in primary HNSCC tissues from KDM4A knockout mice. Immunostaining revealed that the inhibition of KDM4A increased the infiltration of CD8+ T cells in HNSCC. Recently, immune checkpoint inhibitors targeting PD1/PD-L1 have achieved great success in several solid tumors including HNSCC. Although anti-PD1 therapy has been approved for treating recurrent or metastatic HNSCC, the objective response rate is less than 20%, indicating that HNSCC cells might be intrinsically resistant to checkpoint blockades. Thus, reversing the hypo-immunogenicity of HNSCC cells has become increasingly imperative to ongoing cancer immunotherapy. Given the critical role of KDM4A in chromatin accessibility and DNA replication, we hypothesize that targeting KDM4A not only inhibits the expression of invasive genes, but also potently activate tumor-intrinsic immunity and induce CD8+ T cell infiltration by inducing DNA replication stress. To test our hypothesis, we will propose the following specific aims: 1) Determine whether KDM4A ablation inhibits the expression of invasive genes and activates tumor cell-intrinsic immunity in HNSCC; 2) Explore targeting KDM4A impairs DNA replication and activates tumor cell-intrinsic immune responses by inducing replication stress; and 3) Determine whether targeting KDM4A can help to overcome HNSCC resistance to PD- 1 blockade therapy by recruiting and activating CD8+ T cells. The results from our studies might help ...