Investigating Transcriptional Elongation and Nuclear RNA Surveillance in Melanoma

项目摘要 转移性黑色素瘤是一种源于产生色素的黑素细胞的癌症，是最致命的皮肤类型 癌症。转移性黑色素瘤是难治性或抗药性的现有治疗方法，预后很差。黑色素瘤 生长和治疗耐药依赖于基因表达异常，而基因表达在 黑色素瘤仍然知之甚少。转录周期蛋白依赖性蛋白激酶(CDK)是一种 在系统发育上不同的一组直接调节转录子过程的激酶。我发现CDK13 在黑色素瘤中发生突变；这些突变是激酶死亡，并通过干扰WT CDK13功能发挥作用 (显性否定)。CDK13基因下调或突变与转移性肿瘤预后不良相关 黑色素瘤患者，斑马鱼模型中突变CDK13的表达加速黑色素瘤的发生 突变的CDK13在人黑色素瘤细胞中的表达使细胞更具增殖能力。突变体CDK13 不能磷酸化一种蛋白质，这种蛋白质可以激活过早截断的“垃圾”RNA的核降解 (PtRNAs)。随后，ptRNAs积累，被输出到细胞质，并被翻译成短的 蛋白质。这项工作是最近在BioRxiv上发表的。我最近还发现了两个经常性的截断 核RNA监测成员：ZFC3H1和ZC3H18。在此，我建议进一步研究其机制。 黑色素瘤中突变CDK13致癌和核RNA监测的研究。在Aim1中，我计划a)确定如何 蛋白质应激途径在CDK13突变细胞中受到影响，b)测试截短的蛋白质表达 足以概括斑马鱼黑色素瘤的CDK13突变表型。在《目标2》中，我将调查 两个核监视复合体成员(ZFC3H1和/或ZC3H18)是否存在反复截断突变 导致更具侵袭性的黑色素瘤，如果是这样的话，使用蛋白质组学和3‘ 转录学。这项工作有可能开辟一个新的癌症生物学领域，并导致治疗 针对核RNA监测不足的癌症的策略。 这项研究将在著名的血液学家/肿瘤学家伦纳德·宗博士的实验室进行 和癌症生物学家，他已经将斑马鱼培育成黑色素瘤的主流模式生物 研究。在Zon博士和一个杰出的指导委员会的指导下，个人投资于 我成长为一名独立的调查员，这个奖项将提供必要的保护时间来发展技能 作为独立的调查者研究黑色素瘤的核RNA监测。如此所建议的 委员会，我与Steve Gygi博士开展了一项新的合作，以学习尖端蛋白质组学 技巧。我严格的训练计划将帮助我建立成功所必需的科学和领导技能 当我过渡到独立的时候。达纳波士顿儿童医院的研究和临床环境 法伯癌症研究所和哈佛医学院是发展成为成功的理想环境 研究黑色素瘤核糖核糖核酸监测的独立科学家。

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.