Exploration of long non-coding RNAs as synthetic essential targets in Pten-deficient cancers

项目概要/摘要 PTEN是所有癌症类型中最常失活的肿瘤抑制基因之一。的损失 PTEN激活PI 3 K/AKT，PI 3 K/AKT抑制GSK 3 β，从而稳定Myc并促进肿瘤发生。Myc 招募组蛋白乙酰转移酶，以增加参与两种细胞的靶基因的染色质可及性， 增殖和凋亡。在这些组蛋白乙酰转移酶中，Spt-Ada-Gcn 5乙酰转移酶 （佐贺）复合物优先乙酰化组蛋白H3赖氨酸9和组蛋白H4赖氨酸16以激活基因 表情一项对互斥基因失活模式的泛癌分析发现， 未表征的长非编码RNA（lncRNA）在PTEN缺陷型癌症的背景下是合成必需的。 初步研究表明，这种lncRNA抑制SAGA介导的组蛋白乙酰化，从而抑制Myc 靶基因的反式激活。PTEN和lncRNA双敲除的SF-763胶质瘤细胞显示Myc通路 富集，受损的细胞活力，和明显的非整倍性，这在野生型或单倍性中没有观察到。 敲除细胞系。我们假设这种lncRNA抑制SAGA介导的乙酰化作用， 促凋亡靶基因的反式激活和Myc驱动的核内复制，从而促进癌症 生存这项提案将调查针对研究不足的癌症非编码基因组的潜力 治疗，推进我们对组蛋白乙酰化对癌症基因组稳定性的作用的认识（广泛靶向 使用遗传毒性药物和放射治疗），并描述了一种新的机制，调节Myc的双重 在增殖和凋亡中起作用。我们将验证各种癌症类型的细胞系中lncRNA的表达 和临床样品中，以分别验证本研究的泛癌相关性和转化潜力。 将使用膜联蛋白V和半胱天冬酶3/7测定来评估Myc驱动的细胞凋亡的假设。 还将使用BrdU掺入秋水仙胺阻滞的细胞中来探测核内复制。染色质分离 通过RNA纯化和染色质免疫沉淀测序将被用来证明如何lncRNA 通过佐贺抑制组蛋白乙酰化。为了评估lncRNA在体内的作用，我们将在功能上验证其在体内的作用。 推定的小鼠同源物，并产生基因工程小鼠模型，以表征其对肿瘤的作用 发展lncRNA敲除等位基因将繁殖到Qki;Pten; Trp 53胶质母细胞瘤小鼠模型中， 评估在Pten缺失的情况下lncRNA抑制的效果。培训计划将解决以下方面的差距： 申请人的研究和临床能力，确保他能够成功完成拟议的工作， 为他职业生涯的下一阶段做准备培训将在MD的罗纳德德皮尼奥博士的实验室完成 安德森，申请人将有机会获得资源，设施，最重要的是，同事 这将培养他继续发展和成长为一名医生科学家。

Project Summary/Abstract PTEN is one of the most frequently inactivated tumor suppressor genes across all cancer types. The loss of PTEN activates PI3K/AKT, which inhibits GSK3β, thereby stabilizing Myc and contributing to oncogenesis. Myc recruits histone acetyltransferases to increase chromatin accessibility of target genes involved in both cell proliferation and apoptosis. Among these histone acetyltransferases, the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex preferentially acetylates histone H3 lysine 9 and histone H4 lysine 16 to activate gene expression. A pan-cancer analysis of mutually exclusive gene inactivation patterns identified a previously uncharacterized long non-coding RNA (lncRNA) as synthetic essential in the context of PTEN deficient cancer. Preliminary studies suggest that this lncRNA inhibits SAGA-mediated histone acetylation, thereby inhibiting Myc transactivation of target genes. PTEN and lncRNA double-knockout SF-763 glioma cells showed Myc pathway enrichment, impaired cell viability, and pronounced aneuploidy, which was not observed in wild-type or single knockout cell lines. We hypothesize that inhibition of SAGA-mediated acetylation by this lncRNA inhibits Myc transactivation of pro-apoptotic target genes and Myc-driven endoreduplication, thereby promoting cancer survival. This proposal will investigate the potential of targeting the poorly studied non-coding genome for cancer treatment, advance our knowledge of the role of histone acetylation on cancer genomic stability (widely targeted using genotoxic drugs and radiotherapy), and describe a novel mechanism for the regulation of Myc's dual functions in proliferation and apoptosis. We will verify lncRNA expression in cell lines from various cancer types and in clinical samples to validate the pan-cancer relevance and translational potential of this study, respectively. Annexin V and caspase 3/7 assays will be used to assess the hypothesis of Myc-driven apoptosis. Endoreduplication will also be probed using BrdU incorporation into colcemid-arrested cells. Chromatin isolation by RNA purification and chromatin immunoprecipitation sequencing will be used to demonstrate how the lncRNA inhibits histone acetylation by SAGA. To assess the role of the lncRNA in vivo, we will functionally validate its putative mouse homolog and generate a genetically engineered mouse model to characterize its effects on tumor development. The lncRNA knockout allele will be bred into a Qki;Pten;Trp53 glioblastoma mouse model to assess the effects of lncRNA suppression in the context of Pten deletion. The training plan will address gaps in the applicant's research and clinical abilities, ensuring that he can successfully complete the proposed work and preparing him for the next stage of his career. The training will be completed in Dr. Ronald DePinho's lab at MD Anderson, where the applicant will have access to the resources, facilities, and, most importantly, colleagues that will nurture his continuing development and growth into a physician-scientist.