Elucidating KRAS-specific vulnerabilities in pancreatic cancer

项目总结/摘要 尽管在其他癌症的治疗方面取得了重大进展，但胰腺导管腺癌的治疗 （PDAC）在过去几十年中仅略有改善。因此，胰腺癌仍然存在 在美国癌症死亡的第四大原因和发病率和死亡率的主要原因 国际吧原癌基因KRAS的突变是PDAC的标志，发生在>90%的病例中。研究 在动物模型和细胞系中的研究表明，突变的KRAS对于启动和 维持PDAC，使KRAS成为有吸引力的治疗靶点。不幸的是，直接针对 KRAS已经被突变蛋白的几个独特特征所挫败，使得KRAS对 约会此外，目前抑制推定的下游信号传导途径的策略在很大程度上是 不成功。考虑到KRAS突变不仅在PDAC中而且在肺癌中的高患病率， 结肠癌，靶向KRAS突变癌症的新方法的开发仍然是一个显著的未满足的需求。 拟议研究的长期目标是提高我们对KRAS生物学的理解， 在PDAC和其他KRAS突变癌症中开发新的治疗策略。我们的初步研究 部分或完全抑制突变型KRAS可诱导基因表达的显著和可逆的变化， 表达，细胞信号传导和药物敏感性，表明KRAS促进特定的细胞状态，可能 适合于靶向抑制。在这个提议中，我们利用了新的PDAC细胞系，其中KRAS已经被 通过CRISPR/Cas介导的基因组编辑失活，慢病毒cDNA文库允许表达 不同的KRAS突变变体阵列，以及基于CRISPR的全基因组筛选文库， KRAS相关的功能途径，并阐明与KRAS突变的合成致死相互作用。 具体来说，我们建议重新表达不同的KRAS突变变体，以确定变体特异性效应子 通过转录和生化分析，提供了前所未有的KRAS突变评估， 癌细胞的变异。与此同时，我们计划阐明细胞中独特的遗传依赖性， 使用基于CRISPR的筛选缺乏KRAS功能。总之，这些方法可能会揭示新的 KRAS突变细胞中的靶向途径和蛋白质，我们将在体外其他细胞系中验证 以及体内移植和自体模型。 拟议的研究策略是一个广泛的培训计划的一部分，旨在促进我的过渡到 成为基础和转化癌症生物学的独立研究者。主要的实验将是 在麻省理工学院科赫综合癌症研究所的杰克斯实验室进行， 泰勒杰克博士的指导下，在KRAS生物学和癌症小鼠模型的国际领导者。共- 临床和转化科学的导师将由Charles Fuchs博士提供，他是一位领先的临床 丹娜-法伯癌症研究所和哈佛癌症中心的研究员和GI孢子主任 (DF/HCC）。科学界，包括DF/HCC、麻省理工学院科赫研究所和哈佛医学院 学校为科学讨论和合作提供了一个良好的环境。另送上 我组建了一个科学顾问委员会，由威廉·哈恩博士、马修·范德海登博士和亚历克博士组成 Kimmelman，他们在基础和转化研究方面拥有丰富的信息和专业知识。在 除了在哈佛和麻省理工学院社区的研讨会上介绍我的工作外，我还 我通过哈佛的催化剂计划提出了一套全面的教学课程，以提高我的 理解转化研究，重点是目标识别，临床前验证和T1 翻译. 作为一名医学肿瘤学家和医生科学家，我致力于改善癌症患者的护理， 重点关注胰腺癌和其他胃肠道恶性肿瘤。作为杰克斯实验室的博士后， 我领导了几个项目的开发，构建了我将在实验中使用的新颖工具和试剂。 提出的实验。这些研究的结果将构成开发一个独立资助的 实验室虽然我已经成功地获得奖学金，以支持我的初步研究， K 08奖将为继续指导研究和向独立过渡提供关键资金。

PROJECT SUMMARY/ABSTRACT Despite significant therapeutic advances in other cancers, treatment of pancreatic ductal adenocarcinoma (PDAC) has improved only modestly over the past several decades. Consequently, pancreatic cancer remains the fourth leading cause of cancer death in the United States and a major cause of morbidity and mortality worldwide. Mutation of the proto-oncogene KRAS is a hallmark of PDAC, occurring in >90% of cases. Studies in animal models and cell lines have suggested that mutant KRAS is important for both the initiation and maintenance of PDAC, making KRAS an attractive target for therapy. Unfortunately, efforts to directly target KRAS have been thwarted by several unique features of the mutant protein, making KRAS “undruggable” to date. Furthermore, current strategies to inhibit putative downstream signaling pathways have been largely unsuccessful. Given the high prevalence of KRAS mutations in not only PDAC but also cancers of the lung and colon, the development of new approaches to target KRAS mutant cancers remains a significant unmet need. The long-term goal of the proposed research is to improve our understanding of KRAS biology to facilitate the development of novel therapeutic strategies in PDAC and other KRAS mutant cancers. Our preliminary studies revealed that partial or complete inhibition of mutant KRAS induced marked and reversible changes in gene expression, cell signaling, and drug sensitivity, suggesting that KRAS fosters a specific cell state, potentially amenable to targeted inhibition. In this proposal, we leverage novel PDAC cell lines in which KRAS has been inactivated by CRISPR/Cas-mediated genome editing, a lentiviral cDNA library permitting expression of a diverse array of KRAS mutant variants, and CRISPR-based genome-wide screening libraries to interrogate key KRAS-relevant functional pathways and elucidate synthetic lethal interactions with KRAS mutation. Specifically, we propose to re-express different KRAS mutant variants to identify variant-specific effectors through transcriptional and biochemical profiling, offering an unprecedented evaluation of KRAS mutant variants in cancer cells. In parallel, we plan to elucidate unique genetic dependencies in cells harboring or lacking KRAS function using CRISPR-based screening. Together, these approaches are likely to reveal novel targetable pathways and proteins in KRAS mutant cells, which we will validate in additional cell lines in vitro and transplant and autochthonous models in vivo. The proposed research strategy is part of an extensive training program designed to facilitate my transition to becoming an independent investigator in basic and translational cancer biology. The main experiments will be conducted in the Jacks laboratory at the Koch Institute for Integrative Cancer Research at MIT under the guidance of Dr. Tyler Jacks, an international leader in KRAS biology and mouse models of cancer. Co- mentorship on clinical and translational science will be provided by Dr. Charles Fuchs, a leading clinical researcher and Director of the GI SPORE at the Dana-Farber Cancer Institute and Harvard Cancer Center (DF/HCC). The scientific community, encompassing the DF/HCC, Koch Institute at MIT, and Harvard Medical School, offers an outstanding environment for scientific discussion and collaboration. In addition, I have assembled a scientific advisory committee, comprised of Drs. William Hahn, Matthew Vander Heiden, and Alec Kimmelman, who collectively have a wealth of information and expertise in basic and translational research. In addition to presenting my work locally at seminars within the Harvard and MIT community, I have also proposed a comprehensive set of didactic coursework through the Harvard Catalyst program to enhance my understanding of translational research with a focus on target identification, preclinical validation, and T1 translation. As a medical oncologist and physician-scientist, I am committed to improving the care of cancer patients, with a focus on pancreatic cancer and other gastrointestinal malignancies. As a postdoctoral fellow in the Jacks lab, I have led the development of several projects, building novel tools and reagents that I will utilize in the proposed experiments. Results from these studies will form the basis for developing an independently-funded laboratory. While I have been successful in acquiring fellowship funding to support my preliminary studies, the K08 award will provide critical funding for continued mentored research and transition to independence.