Altered Dependencies of Melanoma on Rewired Post-Transcriptional Regulation

项目摘要 黑色素瘤是所有皮肤癌中最致命的一种，其发病率正在迅速上升。最近FDA批准 作为转移性黑色素瘤治疗的支柱，靶向和免疫治疗受到以下因素的限制： 疗效受限、耐药性和严重毒性。因此，增进了对机制的了解， 推动黑色素瘤进展仍然是一个未满足的医疗需求，可以提供有效的手段， 用改进的靶向治疗策略预防肿瘤复发。 下一代测序技术的进步已经发现了广泛的香料异常 在大多数癌症中。尽管选择性剪接主要受RNA结合的调节， 蛋白质（RBP），用于多样化超过90%的人类蛋白质编码基因，新兴的研究 描述了这种转录后调节被劫持以支持致癌的新机制。 程序.最近的努力确定了肿瘤特异性的剪接体复合物和/或替代物的失调， 具有致癌或肿瘤抑制功能的同种型。在黑色素瘤中， 已经显示致癌驱动物促进转移和对BRAF抑制的抗性。尽管 令人信服的证据表明，选择性剪接有助于黑色素瘤的发病机制，一项全面的研究 介导这种效应的分子作用物的研究还没有进行。为了解决这个问题，我们的实验室分析了 在原发性和转移性黑素瘤患者样品以及对照中的基因表达和组蛋白标记中 黑素细胞在489例典型RBP中，原发性黑色素瘤17例，转移性黑色素瘤104例 相对于健康对照组，有52个RBP在 转移部位相对于疾病的原发部位。 为了研究黑色素瘤发病机制中对RBP的依赖性，我进行了CRISPR-Cas9 在人黑素瘤细胞系中的负选择筛选方法。由于RBPs对细胞和组织的影响很大， 通过RNA结合结构域（RBD）的特异性功能，设计了针对 所有489个典型RBP的RBD。使用这种负选择方法，我将hnRNPA 1鉴定为RBP 对体外黑色素瘤至关重要。HnRNP A1是一种广泛表达的蛋白质，是多种蛋白质家族的成员 异质性核糖核蛋白，广泛涉及RNA加工事件，如剪接， 稳定、核出口和翻译。HnRNP A1表达在黑色素瘤中上调， hnRNP A1的基因切除揭示其对黑色素瘤细胞存活至关重要。的 所提出的工作将描述黑色素瘤对hnRNPA 1的需求。这项工作将阐明 复杂的转录后调节是黑色素瘤发病机制的基础，并为黑色素瘤的发生提供了强有力的理论基础。 开发新的治疗策略，利用这些信息来改善患者的预后。

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.