Functional Networks for Persister Cell Sensitivities

项目摘要 在这项提案中，我们的目标是描述和识别持久细胞状态的机制。具体来说， 我们已经发现，癌症永存细胞对一种新的铁性上睑下垂特异而有效地敏感。 发现了非凋亡性细胞死亡计划，其中包括有毒的过氧化脂质积聚。 化学或遗传抑制Gpx4可以导致铁眼症死亡，Gpx4是人类最早的 清除过氧化脂质的抗氧化剂。我们发现药物幼稚的亲本癌细胞和 未转化的人上皮细胞对铁性下垂不敏感，并建议阐明 外周细胞对铁性下垂敏感的分子基础。这将通过蜂窝和 包括高通量遗传相互作用筛选在内的分子方法。 在目标1中，我们计划评估我们在其他组织中观察到的乳腺癌周围细胞的普遍性。 癌症类型包括黑色素瘤、卵巢癌和肺癌。我们将确定原因的机制 持久者对铁下垂非常敏感，而他们的亲代癌细胞对铁下垂敏感。 麻木不仁。这将通过测量抗氧化剂和促氧化剂细胞代谢物来实现。 和辅因子(如谷胱甘肽和铁)，不同的过氧化脂质对Gpx4的抑制，ROS 信号通路和其他机制分析。这些实验将提供一个框架，以 目标3中描述的遗传相互作用研究。在目标2中，我们还将确定Gpx4在 异种癌细胞、PDX和同种异体肿瘤细胞存活和获得性耐药 免疫活性小鼠乳腺癌和黑色素瘤模型的建立 在活体内。通过在体内诱导周围细胞铁性下垂来预防肿瘤复发将是一项重要的 结果促进了针对Gpx4的临床干预的进一步工作。 我们还将致力于确定持久状态及其背后的遗传相互作用。 对铁性下垂敏感。在目标3中，我们将在现有努力的基础上，为 传导持续型基因互作图谱(EMAPS)这将帮助我们确定这一关系 在我们的屏幕点击率之间，希望确定基因和小分子之间的协同效应。 此外，它还可能帮助我们发现与可用药基因的新的协同作用。我们已经这么做了 完成了试行级EMAP分析以确定可行性，在此我们建议扩展 范围扩大到整个基因组。癌症永存细胞的潜在机制尚未得到证实。 彻底探索，这项建议将确定基因和疾病的相关性。我们的成就 将产生第一个持久细胞状态遗传相互作用图，并为识别多种疗法铺平道路 转送到诊所。

Project Summary In this proposal we aim to characterize and identify mechanisms for the persister cell state. Specifically, we have found that cancer persister cells are specifically and potently sensitive to ferroptosis, a newly discovered non-apoptotic cell death program which involves toxic buildup of lipid hydroperoxides. Ferroptotic death can be induced by chemical or genetic inhibition of GPX4, the primary human antioxidant which scavenges lipid peroxides. We have found that drug naïve parental cancer cells and nontransformed human epithelial cells are insensitive to ferroptosis and propose to elucidate the molecular basis for persister cell sensitivity to ferroptosis. This will be accomplished via cellular and molecular approaches including high throughput genetic interaction screens. In Aim 1, we plan to evaluate the generality of our observations in breast cancer persister cells in other cancer types including melanoma, ovarian and lung cancer. We will determine mechanisms for why persisters are potently and specifically sensitive to ferroptosis, while their parental cancer cells are insensitive. This will be accomplished by measurement of anti- and pro-oxidant cellular metabolites and cofactors (e.g. glutathione and iron), differentially peroxidated lipids upon GPX4 inhibition, ROS signaling pathways and other mechanistic analyses. These experiments will provide a framework for genetic interaction studies described in Aim 3. In Aim 2, we will also establish the role for GPX4 in persister cell survival and acquired drug resistance in cancer cell xenografts, PDXs and syngeneic immunocompetent mouse models of breast cancer and melanoma by inducing ferroptosis in persisters in vivo. Preventing tumor recurrence by inducing ferroptosis in persister cells in vivo will be a significant result to promote further work towards clinical intervention targeting GPX4. We will also focus on our efforts to identify the genetic interactions behind the persister state and its sensitivity to ferroptosis. In Aim 3 we will build on our existing efforts to develop a platform for conducting persister gene interaction maps (EMAPs). This is will help us identify the relationship between our screen hits, with the hope of identifying synergies among the genes and small molecules. In addition it may help us discover new synergistic interactions with druggable genes. We have already accomplished a pilot level EMAP analysis to establish feasibility, and here we propose to expand the scope to the whole genome. The mechanisms underlying cancer persister cells have not been thoroughly explored and this proposal will identify genes and disease relevance. Our accomplishments will yield the first persister cell state genetic interaction map and pave the way to identify polytherapies for translation to the clinic.