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

项目摘要和摘要 候选人：我是史蒂夫·利奇博士和奥马尔·阿卜杜勒-瓦哈布博士在纪念馆共同指导的博士后研究员 斯隆凯特琳癌症中心(MSKCC)。我有人口和分子流行病学方面的研究培训背景， 癌症易感性，癌症生物标记物的发现和验证，以及分子和细胞解剖方法 疾病中的信号通路。我目前的研究集中在突变型p53和异常选择性剪接在 胰腺导管腺癌(PDAC)。作为K99奖项的申请者，我的目标是寻求 我们希望为胰腺癌患者确定治疗机会的研究问题。我的长期目标 是成为一名独立的调查员，专注于揭示高度侵略性的基因转录基础 PDAC肿瘤由已知的驱动因素和未知的突变以及选择性剪接变异体的表达决定。至 为了达到这个目标，我的目标是扩大我对实验方法的熟悉和专业知识，这些方法是 研究战略中概述的实验计划，进一步发展研究的自主性，并招聘和培养人才 学生们。 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 生存结果最差的亚型表现为1)TP53突变和2)TP53表达改变 编码RNA加工机制的基因。这项提案的首要目标是确定p53是如何突变的 调节并依赖于特定的选择性mRNA剪接模式来促进、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R175H优先 促进多聚C序列盒外显子mRNAs的剪接，同时抑制外显子与多嘌呤的夹杂 序列。Poly-C序列导致富含脯氨酸的SH3结合域在A 小分子GTP酶的调节者，包括RAS和RHO。这些观察结果导致了我的假设p53R175H 改变驱动KRAS突变体Panin转化的GTPase调节器的RNA剪接，以促进PDAC 发病机制，并提出了一种潜在的细胞转化机制。此外，我们还发现小- 核心RNA剪接因子的分子抑制剂显著抑制肿瘤生长并延长PDAC的生存时间 表达p53R172H的小鼠，而不表达p53R172H的小鼠则不受影响。这表明p53R175H依赖于 通过控制AS来调节基因表达可能是治疗干预和发现的一个可行的机会 PDAC中其他p53突变的漏洞。 环境：MSKCC是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员所需的培训和知识。首先，我是 由大卫·M·鲁宾斯坦胰腺癌研究中心主任利奇博士指导， 这位研究人员因其在胰腺生物学分子机制方面的开创性工作而受到表彰 癌症、胰腺肿瘤和发展。此外，我还得到了大三学生阿卜杜勒-瓦哈布博士的共同指导 内科医生兼科学家，mRNA剪接调控专家，在鉴定分子方面做出了重要贡献 急性髓系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 我过渡到独立调查员的最佳环境和所需领域的培训 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.