Investigating Mechanisms of Cancer Redox Homeostasis with Metabolic CRISPR-Based Screens

项目摘要和摘要 NRSA个人奖学金的广泛目标有两个：1)促进 使应聘者成为一名有效的内科科学家的基本技能，以及2)调查 基于CRISPR的筛查发现癌症氧化还原动态平衡的机制。这位候选人和他的 门拓为实现这些目标设计了具体的培训计划。该计划包括一项严格的 为事业成功奠定基础的研究组成部分。这项建议与肺有关 腺癌与氧化应激的调节。肺癌仍然是癌症死亡的首要原因 在男人和女人中都有。最近新陈代谢药物的成功，如复合体I抑制剂，已经 重新燃起了针对癌症新陈代谢的兴趣。然而，这些药物中的许多都有不可接受的一面 效果。对癌症代谢重新编程有更深入的了解是发展所必需的 有效、安全、代谢靶向的癌症治疗方法。 最近的研究发现，十字花科蔬菜的饮食消费量，如西兰花，是 与降低患癌症的风险有关。它们的化学防护性归因于异硫氰酸酯 如异硫氰酸苯乙酯(PEITC)。尽管它知道PEITC扰乱了微妙的癌症氧化还原 平衡，这种情况发生的机制尚不清楚。候选人有初步数据，使用 代谢聚焦CRISPR筛选显示PEITC诱导肺所需的KDM5C 用PEITC诱导腺癌细胞死亡和SCD1合成致死。这项提案的重点是验证 这些基因敲击并检测KDM5C和SCD1调节细胞抗氧化剂的机制 回应。这项提案的长期目标是确定治疗新陈代谢的目标，这些目标可能导致更多 肺癌患者的有效和更安全的治疗。 在具体目标1中，候选人将确定PEITC诱导的肺是否需要KDM5C 腺癌细胞通过抑制抗氧化剂基因死亡。KDM5C对组蛋白的调控分析 修饰及其对基因表达的下游影响将用于确定在多大程度上 KDM5C是细胞抗氧化反应的负调节因子。在具体目标2中，候选人将确定 抑制SCD1与PEITC诱导肺合成致死的分子机制 腺癌细胞死亡。脂质组学将量化SCD1调节细胞脂质水平的程度。 内质网和线粒体应激的测量将为深入了解SCD1的机制提供帮助。 用PEITC合成致死物质。拟议的研究将确定并因果验证以前未知的 肿瘤细胞氧化还原动态平衡的机制及为设计有效的药物组合提供依据 以此为目标的治疗方法。

Project Summary and Abstract The broad objectives of this NRSA Individual Fellowship are two-fold: 1) to facilitate the development of essential skills that will allow the candidate to become an effective physician-scientist, and 2) to investigate mechanisms of cancer redox homeostasis uncovered by CRISPR-based screens. The candidate and his mentor have designed a specific training plan to achieve these objectives. The plan includes a rigorous research component that lays the foundation for a successful career. This proposal concerns lung adenocarcinoma and regulation of oxidative stress. Lung cancer remains the leading cause of cancer death among both men and women. The recent success of metabolic drugs, such as complex I inhibitors, have revitalized interest in targeting cancer metabolism. However, many of these drugs have unacceptable side effects. A more thorough understanding of cancer metabolic reprogramming is necessary for the development of effective and safer, metabolically targeted cancer therapies. Recent studies have uncovered that the dietary consumption of cruciferous vegetables, such as broccoli, is associated with a decreased risk of cancer. Their chemoprotective properties are attributed to isothiocyanates such as phenethyl isothiocyanate (PEITC). Although it known that PEITC disrupts the delicate cancer redox balance, the mechanism by which this occurs is unknown. The candidate has preliminary data utilizing a metabolism focused CRISPR-based screen revealing KDM5C as required for PEITC-induced lung adenocarcinoma cell death and SCD1 as synthetic lethal with PEITC. The focus of this proposal is to validate these gene hits and examine the mechanisms by which KDM5C and SCD1 regulate the cellular antioxidant response. The long-term goal of this proposal is to identify therapeutic metabolic targets that may lead to more effective and safer therapies for patients with lung cancer. In Specific Aim 1, the candidate will determine whether KDM5C is required for PEITC-induced lung adenocarcinoma cell death by repressing antioxidant genes. Analysis of KDM5C regulation of histone modifications and their downstream effects on gene expression will be used to determine the extent to which KDM5C is a negative regulator of cellular antioxidant response. In Specific Aim 2, the candidate will determine the molecular mechanism by which inhibition of SCD1 is synthetic lethal with PEITC to induce lung adenocarcinoma cell death. Lipidomics will quantify the extent to which SCD1 regulates cellular lipid levels. Measurements of ER and mitochondrial stress will provide insight into the mechanism by which SCD1 is synthetic lethal with PEITC. The proposed studies will identify and causally validate previously unknown mechanisms of cellular redox homeostasis in cancer and provide evidence to devise effective combinatorial therapies to target this balance.