Investigating Transcriptional Elongation and Nuclear RNA Surveillance in Melanoma

项目摘要 转移性黑色素瘤是一种由产生色素的黑色素细胞衍生的癌症，是最致命的皮肤类型 癌对当前疗法难治或耐药的转移性黑素瘤具有不良预后。黑素瘤 生长和治疗抗性依赖于失调的基因表达， 黑色素瘤仍然知之甚少。转录细胞周期蛋白依赖性激酶（CDKs）是一种 直接调节转录子过程的遗传学上不同的激酶组。我发现CDK 13 在黑色素瘤中发生突变;这些突变是激酶死亡的，通过干扰WT CDK 13功能发挥作用 （显性否定）。CDK 13的下调或突变与转移性肺癌的预后不良相关 在黑色素瘤患者中，突变型CDK 13在斑马鱼模型中的表达加速黑色素瘤发作， 突变CDK 13在人黑素瘤细胞中的表达导致细胞更具增殖性。突变型CDK 13 未能磷酸化一种蛋白质，该蛋白质激活过早截短的“垃圾”RNA的核降解 （ptRNAs）。随后，ptRNA积累，被输出到细胞质，并被翻译成短的 proteins.这项工作最近发表在BioRxiv上。我最近还发现了两个经常性的截断 核RNA监视成员：ZFC 3 H1和ZC 3 H18。在此，我建议进一步研究其机制 在黑色素瘤中的MUSC-CDK 13肿瘤发生和核RNA监测。在目标1中，我计划a）确定如何 蛋白应激途径在CDK 13突变细胞中受到影响，和B）测试截短的蛋白表达是否 足以概括斑马鱼黑色素瘤中的CDK 13突变表型。在目标2中，我将研究 是否在两个核监视复合体成员（ZFC 3 H1和/或ZC 3 H18）中的复发性截短突变 导致更具侵袭性的黑色素瘤，如果是这样，核RNA监视如何使用蛋白质组学和3' 转录组学这项工作有可能打开一个新的癌症生物学领域，并导致治疗 针对缺乏核RNA监测的癌症的策略。 这项研究将在著名血液学家/肿瘤学家伦纳德·宗博士的实验室进行 癌症生物学家，他将斑马鱼发展成为黑色素瘤的主流模式生物 research.在Zon博士和一个特殊的指导委员会的指导下， 我成长为一个独立的调查员，这个奖项将提供必要的保护时间来发展技能 作为一名独立的研究者研究黑色素瘤的核RNA监测。根据这一建议， 委员会，我已经开发了一个新的合作与博士史蒂夫Gygi为了学习尖端的蛋白质组学 技术.我严格的培训计划将帮助我建立成功所必需的科学和领导技能 当我过渡到独立。波士顿儿童医院的研究和临床环境，达纳 法伯癌症研究所和哈佛医学院是发展成为一个成功的 研究黑色素瘤核RNA监测的独立科学家。

Project Summary Metastatic melanoma, a cancer derived from pigment-producing melanocytes, is the deadliest type of skin cancer. Metastatic melanoma that is refractory or resistant to current therapies has a poor prognosis. Melanoma growth and therapy resistance is dependent on dysregulated gene expression, and gene expression in melanoma remains poorly understood. The transcriptional cyclin dependent kinases (CDKs) are a phylogenetically distinct group of kinases that directly regulate transcriptional subprocesses. I found that CDK13 is mutated in melanoma; these mutations are kinase-dead and act by interfering with WT CDK13 function (dominant negative). Downregulation or mutation in CDK13 is associated with poor prognosis in metastatic melanoma patients, expression of mutant CDK13 in a zebrafish model expedites melanoma onset, and expression of mutant CDK13 in human melanoma cells causes the cells to be more proliferative. Mutant CDK13 fails to phosphorylate a protein that activates nuclear degradation of prematurely truncated ‘junk’ RNAs (ptRNAs). Subsequently, ptRNAs accumulate, are exported to the cytoplasm, and are translated into short proteins. This work is newly published on BioRxiv. I have also recently identified recurrent truncations in two nuclear RNA surveillance members: ZFC3H1 and ZC3H18. Here, I propose to further investigate the mechanism of mutant-CDK13 oncogenesis and nuclear RNA surveillance in melanoma. In Aim1, I plan to a) determine how protein stress pathways are affected in CDK13-mutant cells and b) to test whether truncated protein expression is sufficient to recapitulate the CDK13-mutant phenotype in zebrafish melanoma. In Aim 2, I will investigate whether recurrent truncating mutations in two nuclear surveillance complex members (ZFC3H1 and/or ZC3H18) cause more aggressive melanoma and if so, how nuclear RNA surveillance is impacted using proteomics and 3’ transcriptomics. This work has the potential to open a new cancer biology field and to lead to therapeutic strategies for cancers with deficient nuclear RNA surveillance. This research will be conducted in the laboratory of Dr. Leonard Zon, a renowned hematologist/oncologist and cancer biologist who has developed the zebrafish into a mainstream model organism for melanoma research. Under the guidance of Dr. Zon and an exceptional mentoring committee, that is personally invested in my growth into an independent investigator, this award will provide the necessary protected time to develop skills to study nuclear RNA surveillance in melanoma as an independent investigator. As recommended by this committee, I have developed a new collaboration with Dr. Steve Gygi in order to learn cutting-edge proteomic techniques. My rigorous training plan will help me build scientific and leadership skills necessary to succeed when I transition to independence. The research and clinical environment at Boston Children’s Hospital, Dana Farber Cancer Institute, and Harvard Medical School is the ideal environment to develop into a successful independent scientist who investigates nuclear RNA surveillance in melanoma.