Investigating Transcriptional Elongation and Nuclear RNA Surveillance in Melanoma

研究黑色素瘤的转录延伸和核 RNA 监视

• 批准号: 10215098
• 负责人: Megan Leigh Insco
• 金额: $ 5.95万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10215098
• 关键词: Affect; Aggressive behavior; Animal Model; Area; Award; Binding; Biological Assay; Boston; Cancer Biology; Cell Culture System; Cell Proliferation; Cells; Cellular Stress; Clinical; Collaborations; Complex; Cyclin-Dependent Kinases; Cyclins; Cytoplasm; Dana-Farber Cancer Institute; Data; Dissection; Dominant-Negative Mutation; Down-Regulation; Drug Targeting; Environment; Epigenetic Process; Failure; Family; Gene Expression; Genes; Genetic; Genetic Transcription; Grant; Growth; Heat shock proteins; Hematologist; Human; Image; Immune; Immunotherapy; In Vitro; Laboratories; Lead; Leadership; Learning; Lysosomes; Mainstreaming; Malignant Neoplasms; Measures; Melanoma Cell; Mentors; Metastatic Melanoma; Modeling; Molecular; Mutate; Mutation; Nuclear; Nuclear RNA; Oncogenic; Oncologist; Pathway interactions; Patients; Pediatric Hospitals; Peptides; Phenotype; Phosphorylation; Phosphotransferases; Phylogenetic Analysis; Pigments; Prognosis; Progressive Disease; Proteins; Proteomics; Publishing; RNA; Recurrence; Refractory; Reporter; Research; Research Personnel; Resistance; Scientist; Skin Cancer; Stress; System; Techniques; Testing; Therapeutic; Time; Training; Transcription Elongation; Translating; Translations; Work; Zebrafish; endoplasmic reticulum stress; exosome; in vivo; kinase inhibitor; medical schools; melanocyte; melanoma; member; mutant; premature; protein expression; skills; stress granule; transcriptome sequencing; transcriptomics; tumor; tumorigenesis

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.

项目摘要 转移性黑色素瘤是一种源自产生色素的黑素细胞的癌症，是最致命的皮肤类型 癌症。对当前疗法的难治性或耐药性的转移性黑色素瘤的预后较差。黑色素瘤 生长和治疗耐药性取决于基因表达失调，以及基因表达 黑色素瘤仍然了解不足。转录细胞周期蛋白依赖性激酶（CDK）是一个 直接调节转录子过程的系统发育不同的激酶群。我发现CDK13 在黑色素瘤中突变；这些突变是激酶死的，并通过干扰WT CDK13功能起作用 （主要负面）。 CDK13中的下调或突变与转移性预后不良有关 黑色素瘤患者，突变CDK13在斑马鱼模型中的表达加快了黑色素瘤的发作，并且 突变CDK13在人黑色素瘤细胞中的表达会导致细胞更加增殖。突变CDK13 未能磷酸化一种蛋白质，该蛋白质会激活过早截短的“垃圾” RNA的核降解 （ptrnas）。随后，PTRNA积累，导出到细胞质，并翻译成短 蛋白质。这项工作是在Biorxiv上新发表的。我最近还确定了两个重复的截断 核RNA监视成员：ZFC3H1和ZC3H18。在这里，我建议进一步研究机制 突变CDK13黑色素瘤中的肿瘤生成和核RNA监测。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）测试是否截断了蛋白质表达 足以概括斑马鱼黑色素瘤中的CDK13突变物表型。在AIM 2中，我将调查 是否在两个核监视复合构件（ZFC3H1和/或ZC3H18）中复发截断突变 引起更具侵略性的黑色素瘤，如果是这样，则使用蛋白质组学和3'影响核RNA监测如何影响 转录组学。这项工作有可能开设一个新的癌症生物学领域并导致治疗 不足核RNA监测的癌症的策略。 这项研究将在著名的血液学家/肿瘤学家Leonard Zon博士的实验室进行。 以及将斑马鱼开发为黑色素瘤主流模型生物的癌症生物学家 研究。在Zon博士的指导下和一个杰出的心理委员会的指导下，该委员会个人投资 我成为独立调查员的成长，该奖项将为发展技能提供必要的保护时间 研究黑色素瘤的核RNA监测作为独立研究者。按照此建议 委员会，我已经与史蒂夫·吉吉（Steve Gygi）博士建立了新的合作，以学习尖端的蛋白质组学 技术。我严格的培训计划将有助于我建立成功的科学和领导能力 当我过渡到独立时。达纳波士顿儿童医院的研究和临床环境 Farber癌症研究所和哈佛医学院是成功发展的理想环境 研究黑色素瘤中核RNA监测的独立科学家。