PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, with the majority of deaths attributable to tumor metastasis and failures in treatment. Because most cases of HNSCC result from tobacco and alcohol exposure, these tumors are highly heterogeneous, greatly complicating diagnosis, treatment, and investigations into the biology of this disease. We recently performed single cell RNA- sequencing (scRNA-seq) in HNSCC and identified a partial epithelial-to-mesenchymal (p-EMT) transcriptional program that is predictive of poor clinical outcomes including nodal metastasis and diminished survival (Puram et al., Cell). Understanding the regulatory factors that control the p-EMT program in HNSCC is of critical importance as targeting multiple genes in a complex pathway such as p-EMT is particularly challenging, yet has the potential to significantly improve HNSCC outcomes and treatment decision-making. RNA modification proteins, which can directly read/write nucleotide marks on RNA, have emerged as one exciting class of such regulatory proteins. Because these proteins modulate multiple RNA transcripts, targeting RNA modification proteins may disrupt expression of multiple disease-related genes and make resistance less likely to emerge. In HNSCC, we have found that METTL3, an RNA methyltransferase which catalyzes the N6-methyladenosine (m6A) modification, is a key regulator of the p-EMT program in HNSCC. In preliminary studies, we have discovered that perturbation of METTL3 in HNSCC disrupts p-EMT signaling and reduces invasion in vitro. However, the precise mechanisms by which METTL3 and other RNA modification proteins exert their function are poorly understood, but may depend on changes in transcript stability and/or translation. Thus, a better understanding of how METTL3 modulates p-EMT in HNSCC is likely to improve rational drug design and future small molecule- and biologically-based screens in search of effective epitranscriptomic therapeutics. We hypothesize that METTL3 antagonizes p-EMT in HNSCC by disrupting the translation of critical p-EMT target genes. To test which domains in METTL3 are essential for its function, we will first perform sophisticated structure-function analyses in HNSCC cell lines and patient-derived xenograft organoid (PDXOs) (Aim 1). We will determine which domains are required for m6A function using state-of-the-art mass spectrometry methods, while also mapping the binding partners of the critical domains. To determine if METTL3 controls the transcription or translation (or both) of p-EMT genes, we will utilize advanced biochemical techniques including meRIP-seq to study the stability of p-EMT RNA transcripts and polysome profiling and PAR-CLIP to investigate the effects of METTL3 on translation of p-EMT genes (Aim 2). These studies will provide indispensable insight into the mechanism by which METTL3 directs HNSCC tumorigenesis, specifically focusing on its reg...