Dissecting hybrid epithelial-mesenchymal states in head and neck cancer

项目摘要 头颈部鳞状细胞癌（HNSCC）是癌症相关死亡的第六大原因， 大多数患者预后不佳，五年生存率不到40%。大多数死亡 可归因于转移和治疗失败，不幸的是，我们对这些途径的理解是， 不完整因此，没有针对这些生物过程的靶向治疗。最近我们 对HNSCC肿瘤进行了单细胞分析，揭示了一种新的转录途径--被称为杂交 上皮/间质状态（HEM）-作为侵袭和转移的关键驱动因素。这条路显然 与经典EMT途径不同，因为大多数关键EMT转录因子（TF）不表达， 除了Snail 2。这一途径对疾病病因学的重要性突出表现在以下事实： 肿瘤中HEM特征的存在比其他任何常见的治疗结果都更能预测治疗结果。 使用病理学或人口统计学因素。 HEM的主要驱动因子似乎是Snail 2转录因子，但关于Snail 2如何在HEM中起作用知之甚少。 指挥HEM。我们建议确定Snail 2的直接和间接靶点，并评估它们在以下方面的作用： 促进侵袭和转移。这将揭示HEM途径中促进肿瘤生长的蛋白质。 因此代表了合理设计治疗剂的靶点。因为HNSCC肿瘤是 高度异质性，我们预计批量基因组方法可能无法捕获被激活的Snail 2靶标， 或在肿瘤细胞的小亚群中被抑制;然而这些靶点仍然可能与病因学高度相关。 HNSCC的进展。因此，我们还将使用一种基于转座子的技术，称为“电话卡”， 具有单细胞分辨率，可以在不同的细胞中绘制Snail 2转录靶点的综合图谱。 混合上皮-间充质细胞状态（Aim 1）。完成这一目标将确定关键的职能目标 Snail 2的表达，以及我们提出的敲低、过表达和表型分析将直接决定 如果这些靶点的抑制（或激活）阻断了Snail 2对侵袭和转移的作用。到 作为对这种方法的补充，在目标2中，我们将研究Snail 2是否与其他TF合作， 然后学习这种合作性的功能后果。这一点很重要，因为尽管事实证明， 很难找到抑制TF-DNA相互作用的小分子，靶向TF之间的协同相互作用 正在成为一种可行的靶向致癌TF的策略。此外，虽然这些TF与Snail 2合作， 在某些位点，它们可能独立地或通过彼此相互作用激活其他HEM基因， 他们的目标将增加我们对这一途径的了解，并扩大可药用HEM目标的列表。 这些目标的完成将提供一个新的途径，在HNSCC中发挥关键作用的详细地图 并将确定合理的药物设计和靶向治疗的机会， HNSCC。

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.