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

项目摘要/摘要 PTEN是所有癌症类型中最常见的失活肿瘤抑制基因之一。损失的损失 PTEN激活PI3K/Akt，抑制Gsk3β，从而稳定Myc，促进肿瘤的发生。MYC 招募组蛋白乙酰转移酶以增加两个细胞中涉及的目标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-Gcn5乙酰转移酶 (SAGA)复合体优先乙酰化组蛋白H3赖氨酸9和组蛋白H4赖氨酸16激活基因 表情。一项对相互排斥的基因失活模式的泛癌症分析发现，以前 在PTEN缺陷性癌症中作为合成必需的未鉴定的长非编码RNA(LncRNA)。 初步研究表明，这种lncRNA抑制sagA介导组蛋白乙酰化，从而抑制Myc。 靶基因的反式激活。PTEN和lncRNA双基因敲除的SF-763胶质瘤细胞具有Myc通路 在野生型和单倍体中没有观察到的富集型，损害细胞活力和明显的非整倍体。 基因敲除的细胞系。我们假设，这种lncRNA抑制sagA介导的乙酰化，从而抑制Myc 促凋亡靶基因的反式激活和Myc驱动的内复制，从而促进癌症 生死存亡。这项提议将调查针对癌症研究较少的非编码基因组的可能性。 治疗，促进我们对组蛋白乙酰化在癌症基因组稳定性中的作用的了解(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来调节Myc的双重 在细胞增殖和凋亡中的作用。我们将验证lncRNA在不同癌症类型的细胞系中的表达 并在临床样本中分别验证本研究的泛癌相关性和翻译潜力。 Annexin V和caspase3/7检测将被用来评估Myc诱导细胞凋亡的假说。 还将使用BrdU掺入Colcemid受阻细胞来探索内复制。染色质分离 通过RNA纯化和染色质免疫沉淀测序，将展示lncRNA是如何 通过SAGA抑制组蛋白乙酰化。为了评估lncRNA在体内的作用，我们将从功能上验证其 推测小鼠同源基因并生成基因工程小鼠模型以表征其对肿瘤的影响 发展。将lncRNA基因敲除的等位基因培育成Qki；Pten；Trp53胶质母细胞瘤小鼠模型 评估在Pten缺失的背景下抑制LncRNA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，确保他能成功完成拟议的工作和 为他职业生涯的下一阶段做好准备。培训将在罗纳德·德皮尼奥博士在MD的实验室完成 安德森，在那里申请人将可以访问资源、设施，最重要的是，可以访问同事 这将培养他继续发展和成长为一名内科科学家。

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.