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 在侵袭和转移的基础上的基因是不完整的，但我们最近的单细胞研究发现了一些线索。 测序分析显示，许多HNSCC肿瘤含有既不是完全上皮细胞， 也不完全是间充质的，而是处于独特的上皮-间充质混合（混合-EM）状态。存在 杂交EM细胞的表达比吸烟史更能预测不良反应，功能研究建立了 杂交-EM作为侵袭和转移的关键驱动因素。总之，这些观察将混合EM确立为 HNSCC进展的中枢通路。然而，杂交EM标记蛋白是不可药物化的，所以它仍然存在。 很难治疗这种状态像混合EM这样的细胞状态是由多种 机制，但特别是超级增强子，已被确定为维持细胞身份所必需的。 因此，我们使用BET抑制剂JQ 1靶向HNSCC中的超级增强子，并观察到HNSCC中的增强子减少。 HNSCC侵袭约2倍，并抑制混合EM。这一结果是令人鼓舞的，但效果大小是 由于JQ 1的间接和非特异性作用模式，因此不明显。我们假设如果我们能阐明 在HNSCC中，通过超级增强子调控的基因，我们可以确定更直接的分子途径， 并特别涉及侵袭和转移，并发现更有效的靶点。我们最初的实验 发现超级增强子调节混合EM细胞中的胆固醇生物合成基因， (KD)稳健地破坏杂交EM表型，在KD时侵袭减少>2000倍和>8000倍 两个甲羟戊酸合成基因重要的是，我们体内的许多胆固醇基因的蛋白质产物， 已鉴定的可通过市售小分子抑制。事实上，我们发现，抑制 胆固醇生物合成，有效地抑制侵袭（>10倍减少）。这些数据为我们提供了有力的支持。 原始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（目的1）。在目标2中，我们将 评价细胞系、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子的完整调控网络 控制混合EM，特别是关注被JQ 1抑制的Brd 2 -4。对于这些实验，我们 将使用单细胞方法来解释肿瘤的异质性，从而发现其他途径 除了胆固醇的生物合成外，它还直接影响肿瘤的侵袭和转移。这些实验将提供 对超级增强子如何调节混合EM的重要见解，同时探索通过靶向 超级增强子调节胆固醇生物合成途径使用现有FDA批准的药物，我们可以 绕过漫长的药物开发过程，迅速进入人体试验。