Altered Dependencies of Melanoma on Rewired Post-Transcriptional Regulation

项目总结 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。最近获得FDA批准 靶向和免疫疗法是转移性黑色素瘤的主要治疗方法，但受到以下因素的限制 疗效受限、抗药性和严重毒性。因此，提高了对机制的理解 推动黑色素瘤进展仍然是一种未得到满足的医疗需求，可以提供有效的治疗手段 通过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揭示了广泛存在的调味料异常 在大多数癌症中。尽管选择性剪接主要由RNA结合调控 新的研究表明，蛋白质(RBPs)用于使90%以上的人类蛋白质编码基因多样化 描述这种转录后调控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体和/或替代的去调控 具有致癌或肿瘤抑制功能的异构体。在黑色素瘤中，差异表达的mRNA在 致癌驱动因素已被证明可以促进转移和抵抗BRAF的抑制。尽管 一项全面的研究表明，选择性剪接有助于黑色素瘤的发病机制 调节这一效应的分子因子中的一部分还没有被执行。为了解决这个问题，我们的实验室分析了一些变化 在原发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。在489个典型的RBPs中，17个位于原发黑色素瘤，104个位于转移性黑色素瘤 与健康对照组相比显著上调，其中52个RBP显著上调 相对于原发部位的转移部位。 为了研究在黑色素瘤发病机制中对限制性商业惯例的依赖性，我进行了CRISPR-Cas9 人黑色素瘤细胞系的阴性选择筛选方法。由于限制性商业惯例对细胞和组织产生了高度的影响- 通过RNA结合域(RBD)、单引导(Sg)RNAs设计了针对 所有489个典型限制性商业惯例的RBDS。使用这种负选择方法，我将hnRNP A1识别为RBP 在体外对黑色素瘤是必不可少的。HnRNP A1，一种普遍表达的蛋白质，是多样化家族的成员 异质核糖核蛋白，广泛涉及RNA加工事件，如剪接， 稳定、核出口和翻译。HnRNP A1在黑色素瘤中的表达上调 黑素细胞和hnRNP A1的基因消融表明，它对黑色素瘤细胞的生存至关重要。这个 拟议的工作将表征黑色素瘤对hnRNP A1的要求。这项工作将阐明 复杂的转录后调控在黑色素瘤发病机制中的作用，并为 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.