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.

项目总结 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，我们对这些路径的理解 侵袭和转移的基础是不完整的，但我们最近的单个细胞发现了一些线索。 测序分析显示，许多HNSCC肿瘤含有既不是完全上皮的细胞 也不是完全间充质，但处于一种独特的、混合的上皮-间充质(混合-EM)状态。他的存在 与吸烟史相比，杂交-EM细胞的存在更能预测不良反应，并且功能研究证实 混合-EM是侵袭和转移的关键驱动因素。综上所述，这些观察结果证明混合EM是一种 HNSCC进展中的中枢通路。然而，杂交-EM标记蛋白是不可下药的，所以它仍然 在治疗上以这种状态为目标具有挑战性。像混合-EM这样的细胞状态受多种因素的调节 机制，但特别是超级增强剂，已被确定为维持细胞特性的关键。 因此，我们使用BET抑制剂JQ1靶向HNSCC中的超级增强剂，并观察到 HNSCC侵袭~2倍，并抑制了杂交瘤的侵袭。这一结果是令人鼓舞的，但效果大小是 由于JQ1采取的是间接和非具体的行动模式，这一点并不严重。我们假设如果我们能澄清 在HNSCC中，受超级增强子调控的基因，我们可以识别更直接的分子途径 并与侵袭和转移有关，并发现更多有效的靶点。我们最初的实验 发现超级增强剂调节混合-EM细胞中的胆固醇生物合成基因，并且它们的敲除 (KD)有力地破坏了杂交-EM表型，KD的侵袭率分别减少了2000倍和8000倍 两个甲氧戊酸合成基因。重要的是，我们体内许多胆固醇基因的蛋白质产物 可被商业上可获得的小分子所抑制。事实上，我们发现他汀类药物抑制了 胆固醇的生物合成，有效地抑制入侵(&gt；减少10倍)。这些数据为我们的 原创假设。我们现在建议使用CRISPRi系统地研究胆固醇的作用 用复杂的体外和体内模型研究HNSCC侵袭过程中的代谢(目标1)。在目标2中，我们将 评估9种小分子胆固醇生物合成抑制剂在细胞系、患者衍生的有机化合物(PDO)、 以及体内的临床前模型。最后，在目标3中，我们将确定超级增强剂的完整监管网络 控制杂交-EM，特别是集中在JQ1抑制的Brd2-4上。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 而不是直接侵袭和转移的胆固醇生物合成。总之，这些实验将提供 对超级增强剂如何监管混合新兴市场的关键见解，同时探索通过瞄准 利用FDA批准的现有药物，我们可以通过超级增强子调节胆固醇生物合成途径 绕过漫长的药物开发过程，迅速进入人体试验。