Altered mRNA splicing dependent on mutant p53 identifies novel therapeutic vulnerability in pancreatic cancer

项目总结和摘要 候选人：我是一名博士后研究员，由史蒂夫·利奇博士和奥马尔·阿卜杜勒-瓦哈卜博士共同指导 斯隆凯特琳癌症中心（MSKCC）。我有人口和分子流行病学的研究训练背景， 癌症易感性，癌症生物标志物的发现和验证，以及分子和细胞方法来剖析 疾病的信号通路。我目前的研究主要集中在突变型p53和异常选择性剪接在 胰腺导管腺癌（PDAC）。作为K99奖的申请人，我的目标是寻求 研究问题，我们希望确定胰腺癌患者的治疗机会。我的长期目标 是成为一个独立的调查员，重点是揭示高度侵略性的转录组学基础。 PDAC肿瘤由众所周知的驱动因素和未探索的突变以及可变剪接变体的表达决定。到 为了达到这个目标，我的目标是扩大我的熟悉和专业知识与必要的实验方法， 在研究战略中概述实验计划，进一步发展研究自主权，并招募和培养人才 学生 研究：最近的研究根据基因表达谱确定了PDAC患者的分子亚型，其中 具有最差存活结果的亚型显示1）TP 53突变的富集，和2） 编码RNA加工机制的基因。这项提议的首要目标是确定突变的p53 调节并依赖于特定的mRNA剪接模式，以促进、启动和维持 PDAC。我的初步数据显示，癌蛋白p53最常见的突变形式，R175 H， 促进多聚C序列盒外显子mRNA的剪接，同时抑制外显子与多聚嘌呤的结合 序列的多聚C序列导致富含脯氨酸的SH 3结合结构域在蛋白质同种型中的表达。 一类小GTP酶的调节剂，包括Ras和Rho。这些观察结果导致我的假设是p53 R175 H 改变驱动KRAS突变PanIN转化的GT3调节因子的RNA剪接，以促进PDAC 发病机制，并提出了细胞转化的潜在机制。此外，我们发现，小- 核心RNA剪接因子的分子抑制剂显著降低了肿瘤生长，延长了PDAC的生存期。 表达p53 R172 H的小鼠，而在不存在p53 R172 H的情况下没有观察到效果。这表明p53 R175 H依赖于 通过控制AS来调节基因表达可能是一个可行的机会， PDAC中其他p53突变的脆弱性。 环境：MSKCC是一个理想的地方，我执行本提案中概述的研究，并获得 成为一所主要大学的独立教师所需的培训和知识。首先，我是 由大卫M.鲁宾斯坦胰腺癌研究中心和 研究人员承认他在胰腺癌生物学的分子机制方面的开创性工作， 癌症、胰腺肿瘤和发展。另外，我的导师是大三学生阿卜杜勒-瓦哈卜博士 mRNA剪接调控方面的医生兼科学家专家，通过鉴定分子 急性髓细胞白血病和骨髓增生异常综合征中异常剪接的机制我确信这是 最好的环境，让我过渡到成为一个独立的调查员和培训所需的领域， 成为RNA和PDAC生物学领域的专家。

PROJECT SUMMARY AND ABSTRACT CANDIDATE: I am a postdoctoral research fellow co-mentored by Drs. Steve Leach and Omar Abdel-Wahab at Memorial Sloan Kettering Cancer Center (MSKCC). I have research training background in population and molecular epidemiology, cancer predisposition, cancer biomarker discovery and validation, and molecular and cellular approaches to dissect signaling pathways in disease. My current research focuses on the role of mutant p53 and aberrant alternative splicing in pancreatic ductal adenocarcinoma (PDAC). My goal as an applicant of the K99 award is to pursue answers to the research questions where we hope to identify therapeutic opportunities for pancreatic cancer patients. My long-term goal is to become an independent investigator with the focus of uncovering the transcriptomic foundations of highly aggressive PDAC tumors dictated by well-known driver and unexplored mutations and expression of alternatively spliced variants. To reach this goal, my objectives are to broaden my familiarity and expertise with experimental approaches necessary for the experimental plan outlined in the research strategy, develop further autonomy in research, and recruit and train talented students. RESEARCH: Recent studies have identified PDAC patient molecular subtypes based on gene expression profiles, where the subtype with the worst survival outcome displays enrichment of 1) mutations in TP53, and 2) altered expression of genes encoding the RNA processing machinery. The overarching goal of this proposal is to determine how mutated p53 regulates and depends on specific patterns of alternative mRNA splicing for promotion, initiation, and maintenance of PDAC. My preliminary data shows that the most commonly mutated form of oncoprotein p53, R175H, preferentially promotes splicing of poly-C sequences cassette exon mRNAs, while repressing the inclusion of exons with poly-purine sequences. The poly-C sequences result in the expression of proline-rich, SH3 binding domains in protein isoforms of a class of regulators of small-GTPases, including Ras and Rho. These observations led to my hypothesis that p53R175H alters RNA splicing of GTPase regulators that drive transformation of KRAS-mutant PanIN, to promote PDAC pathogenesis, and presents a potential mechanism of cellular transformation. In addition, we have found that small- molecule inhibitors of the core RNA splicing factors significantly reduced tumor growth and extended the survival of PDAC mice expressing p53R172H, whereas no effect was seen in the absence p53R172H. This suggests that p53R175H’s dependence on regulating gene expression through control of AS may be a viable opportunity to therapeutically intervene and discover vulnerabilities for other p53 mutations in PDAC. ENVIRONMENT: MSKCC is an ideal place for me to perform the research outlined in this proposal and obtain the necessary training and knowledge to become an independent faculty member at a major university. First, I am being mentored by Dr. Leach, the director of the David M. Rubenstein Center for Pancreatic Cancer Research and an investigator recognized for his groundbreaking work on the molecular mechanisms regarding the biology of pancreatic cancer, pancreatic neoplasia and development. In addition, I’m being co-mentored by Dr. Abdel-Wahab, a junior physician-scientist expert in mRNA splicing regulation who has done important contributions by identifying molecular mechanisms of aberrant splicing in acute myeloid leukemia and myelodysplastic syndromes. I am convinced that this is the best environment for me to transition to becoming an independent investigator and training in areas required to become an expert in the fields of RNA and PDAC biology.