Functional Networks for Persister Cell Sensitivities

项目摘要 在这项提案中，我们的目标是表征和识别持久细胞状态的机制。具体地说， 我们已经发现癌持续细胞对铁凋亡特异性和有效敏感，这是一种新的 发现了非凋亡细胞死亡程序，其中涉及脂质氢过氧化物的毒性积累。 铁中毒性死亡可以通过GPX 4的化学或遗传抑制来诱导，GPX 4是人类的主要代谢酶。 清除脂质过氧化物的抗氧化剂。我们已经发现，药物幼稚的亲本癌细胞和 未转化的人上皮细胞对铁凋亡不敏感，并提出阐明 持续细胞对铁凋亡敏感性的分子基础。这将通过蜂窝和 分子方法，包括高通量遗传相互作用筛选。 在目标1中，我们计划评估我们在其他乳腺癌中观察到的乳腺癌持续细胞的一般性。 癌症类型包括黑色素瘤、卵巢癌和肺癌。我们将确定为什么 坚持者对铁凋亡是有效和特异性敏感的，而他们的亲本癌细胞是 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（例如谷胱甘肽和铁），GPX4抑制后的差异过氧化脂质，ROS 信号通路和其他机制分析。这些实验将提供一个框架， 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GPX 4在以下方面的作用： 在癌细胞异种移植物、PDX和同基因中持续细胞存活和获得性耐药性 免疫活性小鼠乳腺癌和黑色素瘤模型的建立 in vivo.通过在体内诱导存留细胞中的铁凋亡来预防肿瘤复发将是一个有意义的方法。 这一结果将促进进一步开展针对GPX4的临床干预工作。 我们还将集中精力确定持续状态背后的遗传相互作用及其 对铁下垂敏感。在目标3中，我们将在现有努力的基础上开发一个平台， 进行持续基因相互作用图谱（EMAPs）。这将帮助我们确定 在我们的筛选结果之间，希望能识别基因和小分子之间的协同作用。 此外，它还可以帮助我们发现与可药物化基因的新的协同相互作用。我们已经 完成了一个试点水平的EMAP分析，以建立可行性，在这里，我们建议扩大 范围到整个基因组。癌症持续细胞的潜在机制尚未被阐明。 这项提案将确定基因和疾病的相关性。我们的成就 将产生第一个持久性细胞状态遗传相互作用图谱，并为识别多种疗法铺平道路 翻译到诊所。

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.