Functional interrogation of epigenetic vulnerabilities in KRAS-mutant pancreatic cancer

项目摘要/摘要 胰腺导管腺癌(PDAC)是一种破坏性疾病，目前是导致胰腺癌的第四大原因。 美国与癌症相关的死亡人数。KRAS在大多数PDAC中发生突变，是主要的 导致这种疾病的致癌因素。不幸的是，试图开发针对突变的RAS蛋白的药物 已经不成功，并且单剂抑制突变KRAS下游的效应通路，如 作为RAF-MEK-ERK或PI3K通路，到目前为止也被证明无效。有一种严重的未得到满足的需求 新的治疗策略。这项提案的首要目标是利用功能遗传学方法 识别KRAS突变的PDAC中的新脆弱性和治疗策略。我们优化了CRISPR- 人PDAC细胞系CAS9基因组水平的负选择筛选。鉴于对RAS的抑制- 丝裂原活化蛋白激酶(MAPK)信号通路将是结合的重要骨架 PDAC的治疗方法，我们将基因组规模的CRISPR-Cas9筛查与小分子相结合 抑制MEK1/2或ERK1/2激酶以识别新的合成致死靶点 依赖中存在MAPK抑制。这些靶标包括许多表观遗传调节因子，如 MEN1/MLL1和PRC2复合体的成员，以及许多转录因子。此外，我们的 PDAC细胞系破坏后表观遗传学和转录谱综合法的初步研究 对KRAS信号的研究表明，KRAS介导了导致不同细胞状态的表观遗传重新编程 具有潜在的可针对性漏洞。这项建议建立在这些初步数据的基础上，并有具体的重点 1)MEN1和MLL1作为合成致死靶点的遗传学和药理学验证 MAPK抑制，2)表观遗传、转录和功能遗传图谱的综合分析 了解PDAC中因MAPK途径抑制而暴露的关键漏洞。功能性 对这些靶点的验证和机制理解可能会导致新的联合治疗策略 PDAC患者。安德鲁·阿吉雷博士的导师是威廉·哈恩博士，他是一位内科科学家和 功能癌症遗传学，并将受益于由马修博士组成的咨询委员会 迈耶森、拉梅什·希夫达萨尼博士和布莱恩·沃尔平博士将共同提供指导， 在癌症生物学、表观遗传学和胰腺癌转化研究方面的合作和专业知识。Dr。 Aguirre还制定了一项为期5年的培训计划，该计划将利用 DFCI和哈佛医学院，包括授课课程、科学会议和专业课程 发展机会，这将帮助他实现他的科学和职业目标，开发一个 独立的胰腺癌研究实验室。

Project Summary / Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease and currently the fourth-leading cause of cancer-related death in the United States. KRAS is mutated in the majority of PDAC and is the major oncogenic driver of this disease. Unfortunately, attempts to develop drugs that target mutant RAS proteins have been unsuccessful, and single agent inhibition of effector pathways downstream of mutant KRAS, such as the RAF-MEK-ERK or PI3K pathways, has also proven ineffective to date. There is a critical unmet need for novel therapeutic strategies. The overarching goal of this proposal is to utilize functional genetic approaches to identify novel vulnerabilities and therapeutic strategies in KRAS-mutant PDAC. We have optimized CRISPR- Cas9 genome-scale negative-selection screening in human PDAC cell lines. Given that inhibition of the RAS- mitogen-activated-protein-kinase (MAPK) signaling cascade will be an important backbone for combination therapy approaches in PDAC, we have combined genome-scale CRISPR-Cas9 screening with small molecule inhibition the MEK1/2 or ERK1/2 kinases to identify novel synthetic lethal targets that demonstrate greater dependency in the presence of MAPK inhibition. These targets include a many epigenetic regulators, such as members of the MEN1/MLL1 and PRC2 complexes, as well as numerous transcription factors. Additionally, our preliminary studies using integrative epigenetic and transcriptional profiling of PDAC cell lines upon disruption of KRAS signaling suggest that KRAS mediates epigenetic reprogramming that leads to a distinct cell state with potentially targetable vulnerabilities. This proposal builds on these preliminary data with a specific focus on: 1) genetic and pharmacologic validation of MEN1 and MLL1 as synthetic lethal targets in combination with MAPK-inhibition, 2) integrative analysis of epigenetic, transcriptional and functional genetic profiling to understand the key vulnerabilities unveiled in response to MAPK pathway inhibition in PDAC. Functional validation and mechanistic understanding of these targets may lead to novel combination therapy strategies in PDAC patients. Dr. Andrew Aguirre is mentored by Dr. William Hahn, a physician-scientist and expert in functional cancer genetics, and will also benefit from an advisory committee comprised of Dr. Matthew Meyerson, Dr. Ramesh Shivdasani and Dr. Brian Wolpin, who will collectively provide mentorship, collaboration and expertise in cancer biology, epigenetics and pancreatic cancer translational research. Dr. Aguirre has also formulated a 5-year training plan that will leverage the outstanding resources available at DFCI and Harvard Medical School, including didactic coursework, scientific meetings and professional development opportunities that will assist him in achieving his scientific and career goals of developing an independent pancreatic cancer research laboratory.