Deciphering epigenetically-regulated pathways to improve targeted therapy for invasion and metastasis in head and neck cancer

破译表观遗传调控途径以改善头颈癌侵袭和转移的靶向治疗

• 批准号: 10650527
• 负责人: Robi D Mitra
• 金额: $ 60.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10650527
• 关键词: Anabolism; Binding; Bromodomains and extra-terminal domain inhibitor; CRISPR interference; Cancer Etiology; Cell Line; Cells; Cholesterol; Cholesterol Homeostasis; Cisplatin; Clinical Trials; Data; Disease; Drug Kinetics; Drug Targeting; Enhancers; Epigenetic Process; Epithelium; FDA approved; Fatty Acids; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Human; Hybrids; In Vitro; Invaded; Lead; Learning; Length; Longevity; Malignant Neoplasms; Mass Spectrum Analysis; Mesenchymal; Metabolic Pathway; Metabolism; Modeling; Molecular; Morbidity - disease rate; Neoplasm Metastasis; Oncology; Operative Surgical Procedures; Organoids; Outcome; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Pre-Clinical Model; Process; Prognosis; Proteins; Radiation; Radiation therapy; Recurrence; Role; Smoking History; Technology; Testing; Treatment Failure; adverse outcome; attributable mortality; candidate selection; chemotherapy; cholesterol biosynthesis; drug development; drug testing; experimental study; gene network; gene synthesis; genome-wide analysis; improved; in vivo; in vivo Model; inhibitor; insight; knock-down; lymph nodes; metabolomics; mevalonate; mortality; neoplastic cell; oxidation; prevent; protein biomarkers; response; single cell analysis; single cell sequencing; small molecule; small molecule inhibitor; targeted treatment; therapeutic target; transcriptome sequencing; treatment response; tumor; tumor heterogeneity; tumor xenograft

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, with most deaths attributable to metastasis and treatment failure. Unfortunately, our understanding of the pathways that underlie invasion and metastasis is incomplete, but some hints were uncovered by our recent single cell sequencing analyses which revealed that many HNSCC tumors contained cells that were neither fully epithelial nor fully mesenchymal, but were in a unique, hybrid epithelial-mesenchymal (hybrid-EM) state. The presence of hybrid-EM cells was more predictive of poor response than smoking history, and functional studies established hybrid-EM as a key driver of invasion and metastasis. Together, these observations establish hybrid-EM as a central pathway in HNSCC progression. However, hybrid-EM marker proteins are not druggable, so it remains challenging to therapeutically target this state. Cell states like hybrid-EM are regulated by a variety of mechanisms, but super-enhancers, in particular, have been identified as essential for maintaining cell identity. We therefore targeted super-enhancers in HNSCC using the BET inhibitor JQ1 and observed a reduction in HNSCC invasion of ~2-fold and a suppression of hybrid-EM. This result was encouraging, but effect size was modest due to the indirect and non-specific mode of action of JQ1. We hypothesized that if we could elucidate genes regulated by super-enhancers in HNSCC, we could identify molecular pathways that are more directly and specifically involved with invasion and metastasis and uncover more potent targets. Our initial experiments found that super-enhancers regulate cholesterol biosynthesis genes in hybrid-EM cells and that their knockdown (KD) robustly disrupts hybrid-EM phenotypes, with a >2000-fold and >8000-fold reduction in invasion upon KD of two mevalonate synthesis genes. Importantly, the protein products of many of the cholesterol genes we identified can be inhibited by commercially available small molecules. Indeed, we found that statins, which inhibit cholesterol biosynthesis, potently inhibit invasion (>10-fold reduction). These data provide strong support for our original hypothesis. We now propose to use CRISPRi to systematically investigate the role of cholesterol metabolism in invasion in HNSCC using sophisticated in vitro and in vivo models (Aim 1). In Aim 2, we will evaluate 9 small molecule inhibitors of cholesterol biosynthesis in cell lines, patient-derived organoids (PDOs), and in vivo pre-clinical models. Finally, in Aim 3, we will identify the full regulatory network of super-enhancers that control hybrid-EM, specifically focusing on Brd2-4 which are inhibited by JQ1. For these experiments, we will use single cell approaches to account for tumor heterogeneity and thereby uncover additional pathways beyond cholesterol biosynthesis that direct invasion and metastasis. Together, these experiments will provide critical insights into how super-enhancers regulate hybrid-EM, while exploring the possibility that by targeting the super-enhancer-regulated cholesterol biosynthesis pathway using existing FDA-approved drugs, we could circumvent a length drug development process and move quickly into human trials.

项目概要 头颈鳞状细胞癌 (HNSCC) 是癌症相关死亡的第六大原因， 大多数死亡归因于转移和治疗失败。不幸的是，我们对路径的理解 侵袭和转移的基础尚不完整，但我们最近的单细胞揭示了一些线索 测序分析表明，许多 HNSCC 肿瘤含有既不完全上皮的细胞 也不完全是间充质的，而是处于一种独特的混合上皮-间充质（hybrid-EM）状态。存在感 杂交 EM 细胞的数量比吸烟史更能预测不良反应，并且功能研究已建立 混合电磁作为侵袭和转移的关键驱动因素。总之，这些观察结果将混合电磁学确立为一种 HNSCC 进展的中心途径。然而，混合 EM 标记蛋白不可药物化，因此它仍然存在 治疗上针对这种状态具有挑战性。像混合EM这样的细胞状态受到多种因素的调节 机制，但特别是超级增强子，已被认为对于维持细胞身份至关重要。 因此，我们使用 BET 抑制剂 JQ1 靶向 HNSCC 的超级增强子，并观察到 HNSCC 侵袭约 2 倍并抑制混合 EM。这个结果令人鼓舞，但效果大小 由于 JQ1 的作用方式是间接和非特异性，因此影响不大。我们假设如果我们能够阐明 HNSCC 中受超级增强子调节的基因，我们可以识别更直接的分子途径 并特别涉及侵袭和转移并发现更有效的靶标。我们的初步实验 发现超级增强子调节杂交EM细胞中的胆固醇生物合成基因，并且它们的敲低 (KD) 强力破坏杂交 EM 表型，KD 后入侵减少 >2000 倍和 >8000 倍 两个甲羟戊酸合成基因。重要的是，我们发现许多胆固醇基因的蛋白质产物 已确定可以通过市售小分子来抑制。事实上，我们发现他汀类药物可以抑制 胆固醇生物合成，有效抑制入侵（减少>10倍）。这些数据为我们的 原来的假设。我们现在建议使用 CRISPRi 系统地研究胆固醇的作用 使用复杂的体外和体内模型研究 HNSCC 侵袭中的代谢（目标 1）。在目标 2 中，我们将 评估细胞系、患者源性类器官 (PDO) 中胆固醇生物合成的 9 种小分子抑制剂， 和体内临床前模型。最后，在目标 3 中，我们将确定超级增强剂的完整监管网络 控制混合EM，特别关注被JQ1抑制的Brd2-4。对于这些实验，我们 将使用单细胞方法来解释肿瘤异质性，从而揭示其他途径 超越直接侵袭和转移的胆固醇生物合成。这些实验将共同提供 对超级增强子如何调节混合电磁的关键见解，同时探索通过靶向 使用 FDA 批准的现有药物的超级增强子调节的胆固醇生物合成途径，我们可以 绕过漫长的药物开发过程并快速进入人体试验。