Altered Dependencies of Melanoma on Rewired Post-Transcriptional Regulation

黑色素瘤对转录后调控的依赖性改变

• 批准号: 10062797
• 负责人: Kathryn Grace Hockemeyer
• 金额: $ 5.05万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10062797
• 关键词: Ablation; Address; Adhesions; Alternative Splicing; Automobile Driving; BRAF gene; Biochemical; Biological Assay; Biological Markers; Biology; CRISPR/Cas technology; Cancer Patient; Cell Line; Cell Survival; Cells; Clinical; Code; Complex; Data; Dependence; Development; Disease; Disease Progression; Down-Regulation; Drug resistance; Early Diagnosis; Event; FDA approved; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomics; Growth; Guide RNA; Histologic; Histones; Human; Immunotherapy; In Vitro; Incidence; Introns; Knock-out; Light; Maintenance; Malignant Neoplasms; Mediating; Medical; Melanogenesis; Melanoma Cell; Messenger RNA; Metastatic Melanoma; Metastatic to; Molecular; Mutation; Neoplasm Metastasis; Nuclear Export; Oncogenic; Pathogenesis; Patient-Focused Outcomes; Patients; Post-Transcriptional Regulation; Process; Protein Isoforms; Proteins; RNA Binding; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; Recurrence; Relapse; Resistance; Ribonucleoproteins; Role; Sampling; Signaling Protein; Site; Skin Cancer; Specimen; Spices; Spliceosomes; Technology; Tissues; Toxic effect; Transcript; Translations; Treatment Efficacy; Work; cancer cell; design; driver mutation; fitness; hnRNP A1; improved; in vitro Assay; in vivo; insight; mRNA Precursor; melanocyte; melanoma; member; new therapeutic target; next generation sequencing; novel; novel therapeutics; outcome forecast; prevent; prognostic; programs; protein expression; public health relevance; replication factor C; screening; side effect; support network; targeted treatment; therapeutic target; therapy resistant; transcription factor; transcriptome; transcriptome sequencing; tumor

PROJECT SUMMARY The incidence of melanoma, the deadliest of all skin cancers, is rapidly increasing. Recent FDA-approved targeted and immune therapies, the mainstay of treatment for metastatic melanoma, are constrained by restricted efficacy, drug resistance, and severe toxicities. Thus, improved understanding of the mechanisms driving melanoma progression remains an unmet medical need that could provide effective means of preventing tumor recurrence with improved on-target therapeutic strategies. Advances in next-generation sequencing technologies have uncovered widespread spicing aberrancies across the majority of cancers. Although alternative splicing, predominantly regulated by RNA-binding proteins (RBPs), serves to diversify more than 90% of human protein-coding genes, emerging studies describe novel mechanisms by which this post-transcriptional regulation is hijacked to support oncogenic programs. Recent efforts identify tumor-specific deregulation of the spliceosome complex and/or alternative isoforms with oncogenic or tumor suppressive function. In melanoma, differential mRNA processing in oncogenic drivers has been shown to promote metastasis and resistance to BRAF inhibition. Despite compelling evidence that alternative splicing contributes to melanoma pathogenesis, a comprehensive study of the molecular actors mediating this effect has not been performed. To address this, our lab profiled changes in gene expression and histone marks in primary and metastatic melanoma patient samples, as well as control melanocytes. Of 489 canonical RBPs, 17 RBPs in primary melanoma and 104 RBPs in metastatic melanoma were significantly upregulated relative to healthy controls, with 52 RBPs were significantly upregulated in metastatic sites relative to the primary site of disease. To investigate dependencies on RBPs in melanoma pathogenesis, I have performed a CRISPR-Cas9 negative selection screening approach in human melanoma cell lines. As RBPs exert highly cell- and tissue- specific function through RNA-binding domains (RBDs), single-guide(sg)RNAs were designed against the RBDs of all 489 canonical RBPs. Using this negative-selection approach, I identified hnRNP A1 as an RBP essential to melanoma in vitro. HnRNP A1, a ubiquitously expressed protein and member of the diverse family of heterogeneous ribonucleoproteins, is broadly implicated in RNA processing events, such as splicing, stability, nuclear export, and translation. HnRNP A1 expression was upregulated in melanoma relative to melanocytes, and genetic ablation of hnRNP A1 revealed it to be critical to melanoma cell survival. The proposed work will characterize the requirement for hnRNP A1 in melanoma. This work will elucidate the complex post-transcriptional regulation underlying melanoma pathogenesis and provide a strong rationale for developing novel therapeutic strategies, leveraging this information to improve patient outcomes.

项目概要 黑色素瘤是所有皮肤癌中最致命的一种，其发病率正在迅速增加。最近 FDA 批准 靶向和免疫疗法是转移性黑色素瘤治疗的主要方法，但受到以下因素的限制： 疗效有限、耐药性和严重毒性。因此，加深了对机制的理解 驱动黑色素瘤进展仍然是一个未满足的医疗需求，可以提供有效的方法 通过改进的靶向治疗策略来预防肿瘤复发。 下一代测序技术的进步发现了广泛的香料异常 跨越大多数癌症。尽管选择性剪接主要受 RNA 结合调节 新兴研究表明，蛋白质（RBP）使 90% 以上的人类蛋白质编码基因多样化 描述了这种转录后调控被劫持以支持致癌的新机制 程序。最近的工作确定了剪接体复合物和/或替代物的肿瘤特异性失调 具有致癌或肿瘤抑制功能的亚型。在黑色素瘤中，差异 mRNA 加工 致癌驱动因素已被证明可以促进转移和对 BRAF 抑制的抵抗。尽管 一项综合研究，有令人信服的证据表明选择性剪接有助于黑色素瘤发病机制 介导这种效应的分子演员的研究尚未进行。为了解决这个问题，我们的实验室进行了更改 原发性和转移性黑色素瘤患者样本以及对照中的基因表达和组蛋白标记 黑素细胞。 489 个典型 RBP 中，17 个原发性黑色素瘤中的 RBP 和 104 个转移性黑色素瘤中的 RBP 相对于健康对照显着上调，其中 52 个 RBP 显着上调 相对于原发疾病部位的转移部位。 为了研究黑色素瘤发病机制中 RBP 的依赖性，我进行了 CRISPR-Cas9 人黑色素瘤细胞系的阴性选择筛选方法。由于 RBP 具有高度的细胞和组织作用 通过 RNA 结合域 (RBD) 实现特定功能，单引导 (sg)RNA 是针对 所有 489 个规范 RBP 的 RBD。使用这种负选择方法，我将 hnRNP A1 鉴定为 RBP 在体外对黑色素瘤至关重要。 HnRNP A1，一种普遍表达的蛋白质，是多样化家族的成员 异源核糖核蛋白，广泛涉及 RNA 加工事件，例如剪接、 稳定、核出口和翻译。相对于黑色素瘤，HnRNP A1 表达上调 黑素细胞和 hnRNP A1 的基因消融表明它对于黑素瘤细胞的存活至关重要。这 拟议的工作将描述黑色素瘤中对 hnRNP A1 的需求。这项工作将阐明 黑色素瘤发病机制中复杂的转录后调控，并为 开发新的治疗策略，利用这些信息来改善患者的治疗结果。