Dissecting hybrid epithelial-mesenchymal states in head and neck cancer

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PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, and most patients have poor outcomes, with a five-year survival rate that is less than 40%. The majority of deaths are attributable to metastasis and treatment failure, and unfortunately, our understanding of these pathways is incomplete. Consequently, there are no targeted therapies against these biologic processes. Recently, we performed a single cell analysis of HNSCC tumors that revealed a new transcriptional pathway – dubbed a hybrid epithelial/mesenchymal state (HEM) – as a critical driver of invasion and metastasis. This pathway is clearly distinct from the classical EMT pathway, as most key EMT transcription factors (TFs) were not expressed, with the exception of Snail2. The importance of this pathway to disease etiology was highlighted by the fact that the presence of the HEM signature in tumors was more predictive of treatment outcome than any other commonly used pathologic or demographic factor. The main driver of HEM appears to be the Snail2 transcription factor, but little is known about how Snail2 orchestrates HEM. We propose to determine the direct and indirect targets of Snail2 and assess their roles in promoting invasion and metastasis. This will reveal the proteins in the HEM pathway that promote tumor progression and thus represent targets for the rational design of therapeutics. Because HNSCC tumors are highly heterogeneous, we expect that bulk genomic approaches may not capture Snail2 targets that are activated or repressed in small subpopulations of tumor cells; yet such targets may still be highly relevant to the etiology of HNSCC progression. Therefore, we will also use a transposon-based technology known as “calling cards” that has single-cell resolution to make a comprehensive map of the transcriptional targets of Snail2 across distinct hybrid epithelial-mesenchymal cell states (Aim 1). Completion of this aim will identify the key functional targets of Snail2 and the knockdown, overexpression, and phenotyping assays that we propose will directly determine if the inhibition (or activation) of these targets blocks the effect of Snail2 on invasion and metastasis. To complement this approach, in Aim 2, we will investigate whether Snail2 acts cooperatively with other TFs and then learn the functional consequences of such cooperativity. This is important because while it has proven difficult to find small molecules that inhibit TF-DNA interactions, targeting cooperative interactions between TFs is emerging as a viable strategy for targeting oncogenic TFs. Furthermore, while these TFs cooperate with Snail2 at some loci, they likely activate other HEM genes independently or by interacting with one another, so identifying their targets will increase our knowledge of this pathway and expand the list of druggable HEM targets. Completion of these aims will provide a detailed map of a new pathway that plays a critical role in HNSCC metastasis and invasion and will identify opportunities for rational drug design and targeted therapeutics against HNSCC.