Role of RNA methylation in chemoresistant cancer cells

RNA 甲基化在化疗耐药癌细胞中的作用

• 批准号: 9896260
• 负责人: Shobha Vasudevan
• 金额: $ 23.19万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-15 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896260
• 关键词: 3' Untranslated Regions; ATM Signaling Pathway; Acute Myelocytic Leukemia; Adenosine; Affect; Affinity Chromatography; Antibodies; Biological Assay; Blast Cell; Blood; Cancer Etiology; Cancer Relapse; Cell Cycle; Cell Line; Cell Survival; Cells; Chemicals; Clinical; Co-Immunoprecipitations; Complex; Coupled; Data; Disease remission; Gene Expression; Genes; Genetic Transcription; Goals; Heterogeneity; Immunoprecipitation; Lead; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Methods; Methylation; Methyltransferase; Modification; N(6)-ribosyladenine; Normal Cell; Outcome; Patients; Population; Positioning Attribute; Post-Transcriptional Regulation; Proteome; Proteomics; RNA; RNA methylation; RNA-Binding Proteins; Reader; Recurrence; Regulation; Resistance; Role; Sampling; Signal Transduction; Site; Testing; Therapeutic; Translating; Translations; acute myeloid leukemia cell; base; biological heterogeneity; cancer cell; cancer stem cell; chemotherapy; crosslink; cytokine; immune resistance; in vivo; in vivo Model; inhibitor/antagonist; leukemic stem cell; mRNA Expression; new therapeutic target; protein degradation; targeted treatment; therapeutic biomarker; transcriptome; tumor

Summary Role of RNA methylation in chemoresistant cancer cells Goal Based on our data, we propose that transiently quiescent populations in acute myeloid leukemia (AML) are maintained in part by RNA methylation, permitting synthesis of survival and tumor promoting regulators necessary for chemoresistance and subsequent AML persistence. The primary objective is to characterize the role of RNA methylation in regulating gene expression in resistant AML, which contributes to chemosurvival. Significance AML is a serious malignancy that displays clinical resistance due to heterogeneity. Resistant AML cells include quiescent (G0) cells that are transiently arrested states, and thus resist clinical therapy that targets only cycling cells; these cells re-enter proliferation to cause AML persistence. A primary issue is that resistance regulators remain to be uncovered. A second need is to identify markers to detect resistant cells. Such cells show distinct gene expression that permits chemosurvival (PNAS 2014, Mol. Cell 2016, Biorxiv/ 418715). Canonical translation is decreased; yet specific mRNAs are expressed by unknown mechanisms. Regulation of specific gene expression in resistant AML needs to be uncovered & targeted to curtail AML. Premise Profiling in resistant cells revealed expression of factors that affect cell survival. Expressed mRNAs in G0 resistant cells have extended 3? untranslated regions (UTRs) to include regulatory sites, such as for RNA methylation. Methylation at N6 position of adenosines (m6A) are unique marks on RNAs that regulate gene expression via RNA binding proteins called readers, to control distinct cell states. Role of m6A in resistant AML remains to be uncovered. We find the m6A methyltransferase increases due to therapy induced signals. Our data reveal that m6A is needed for specific gene expression & resistance. We developed inhibitors to block therapy induced signals & antisense to block unique m6A sites on extended 3’UTRs, which reduce resistance. These data suggest that m6A regulates specific gene expression to enable therapy survival. Characterization of mRNAs that are modified & expressed in resistant cells, in vivo & in patient samples by m6A, their readers, their role in resistance, & of inhibitors that block m6A, will provide markers & therapeutics to curb resistance. Method Aim I will characterize m6A readers that regulate known modified mRNAs, by in vivo crosslinking coupled RNA affinity purification in resistant AML cells & in vivo models. Chemical inhibitors to therapy induced signaling that promotes m6A and antisense to block mapped m6A sites will be tested to curb resistance. Aim II will globally identify m6A targets that contribute to resistance in cell lines & in vivo models, using m6A antibody & methyltransferase immunoprecipitation. Role of readers & m6A mRNAs in resistant cells & in vivo is tested by depletions coupled with chemosurvival assays; their expression will be verified in resistant patient samples. Outcomes mRNAs expressed by m6A, their readers, their role in resistance in AML cells & in vivo, & inhibitors of m6A to curb resistance will be identified. Their expression & efficacy in patient samples will be uncovered.

RNA 甲基化在化疗耐药癌细胞中的作用总结 目标 根据我们的数据，我们建议急性髓系白血病 (AML) 中的短暂静止人群 部分由 RNA 甲基化维持，允许合成生存和肿瘤促进调节因子 化疗耐药性和随后的 AML 持续性所必需的。主要目标是表征 RNA 甲基化在调节耐药 AML 基因表达中的作用，有助于化疗存活。 意义 AML 是一种严重的恶性肿瘤，由于异质性而表现出临床耐药性。抵抗的 AML 细胞包括暂时停滞状态的静止 (G0) 细胞，因此会抵抗临床治疗 仅针对循环细胞；这些细胞重新进入增殖状态，导致 AML 持续存在。一个主要问题是 电阻调节器仍有待发现。第二个需要是识别标记物来检测耐药细胞。 这些细胞表现出独特的基因表达，允许化学存活（PNAS 2014，Mol. Cell 2016，Biorxiv/ 418715）。规范翻译减少；然而特定的 mRNA 是通过未知的机制表达的。 需要发现耐药性 AML 中特定基因表达的调控并有针对性地遏制 AML。 耐药细胞的前提分析揭示了影响细胞存活的因子的表达。表达的 mRNA 在 G0耐药细胞已延长3？非翻译区 (UTR) 包括调控位点，例如 RNA 甲基化。腺苷 (m6A) N6 位点的甲基化是调节基因的 RNA 上的独特标记 通过称为阅读器的 RNA 结合蛋白进行表达，以控制不同的细胞状态。 m6A 在耐药 AML 中的作用 仍有待揭开。我们发现 m6A 甲基转移酶由于治疗诱导信号而增加。我们的 数据显示，特定基因表达和抗性需要 m6A。我们开发了抑制剂来阻止 治疗诱导信号和反义来阻断扩展 3'UTR 上的独特 m6A 位点，从而降低耐药性。 这些数据表明 m6A 调节特定基因表达以使治疗存活。表征 m6A 及其读者在体内和患者样本中在耐药细胞中修饰和表达的 mRNA， 它们在耐药性中的作用以及阻断 m6A 的抑制剂将为抑制耐药性提供标记物和治疗方法。 方法目标 I 将通过体内交联来表征调节已知修饰 mRNA 的 m6A 阅读器 耐药 AML 细胞和体内模型中的耦合 RNA 亲和纯化。化学抑制剂诱导治疗 将测试促进 m6A 的信号传导和阻断映射的 m6A 位点的反义信号以抑制耐药性。目标二 将使用 m6A 抗体在全球范围内识别有助于细胞系和体内模型耐药性的 m6A 靶点 和甲基转移酶免疫沉淀。测试阅读器和 m6A mRNA 在耐药细胞和体内的作用 通过消耗与化学存活测定相结合；它们的表达将在耐药患者样本中得到验证。 m6A 表达的 mRNA 的结果、它们的阅读器、它们在 AML 细胞和体内耐药性中的作用以及抑制剂 m6A 抑制阻力的作用将被确定。它们在患者样本中的表达和功效将被揭示。