A multi-faceted approach to identifying K-Ras synthetic lethal relationships

 描述(申请人提供)：每年有超过20万美国人死于肺癌和结直肠癌。减少这些癌症的死亡频率无疑需要根据癌症中发生的特定突变量身定做个人的治疗。K-RAS癌蛋白的激活突变在肺癌和结直肠癌中很常见，与常规和靶向治疗的反应特别差有关。我们的首要目标是了解突变K-RAS致癌特性的潜在机制，以便开发有针对性的治疗策略。该项目包括三个阶段。在我们项目的第一阶段，我们将使用CRISPR技术来产生表达内源性突变K-RAS的肺癌和结直肠癌细胞的K-RAS野生型衍生物。然后，我们将综合表征与突变K-RAS缺失相关的细胞和分子表型，例如，通过将多路复用质谱学与计算建模相结合来识别突变K-RAS用于转化细胞的信号转导网络。这项研究还将分析野生型和突变型细胞的辐射反应，因为激活的K-RAS被认为对电离辐射具有抵抗力。在我们项目的第二阶段，我们将进行各种全基因组和有针对性的筛选，以寻找在体外和体内突变KRAS背景下被击倒或过度表达导致致命性的基因。这些研究将利用最先进的高通量筛选技术，包括我们在过去十年中完善的多西环素诱导shRNA和开放阅读框架。在项目的最后阶段，我们将确定K-RAS合成致死物在治疗上具有靶向性，然后在肺癌和结肠癌的基因工程小鼠模型上进行临床前研究。利用基因控制的小鼠和人类实验系统将使我们能够识别表达突变K-RAS的癌细胞真正选择性地需要的基因产物。最终，这项工作将对患K-RAS突变癌症的患者产生重大影响。

 DESCRIPTION (provided by applicant): More than 200,000 Americans die as a result of lung and colorectal cancer each year. Decreasing the frequency of deaths due to these cancers will undoubtedly require tailoring an individual's treatment to the specific mutations that have occurred in their cancer. Activating mutations in the K-Ras oncoprotein are common in lung and colorectal cancers and are associated with particularly poor response to both conventional and targeted therapies. Our overarching goal is to understand the mechanisms underlying the oncogenic properties of mutant K-Ras in order to develop targeted therapeutic strategies. This project includes three phases. In the first phase of our project, we will use CRISPR technology to generate K-Ras wild-type derivatives of lung and colorectal cancer cells expressing endogenous mutant K-Ras. We will then comprehensively characterize the cellular and molecular phenotypes associated with loss of mutant K-Ras, for example by combining multiplexed mass spectrometry with computational modeling to identify the signal transduction network utilized by mutant K-Ras to transform cells. This study will also include an analysis of radiation response in wild-type and mutant cells, as activated K-Ras is known to confer resistance to ionizing radiation. In the second phase of our project, we will perform a variety of genome-wide and targeted screens for genes that when knocked down or over-expressed cause lethality in the context of mutant KRas in vitro and in vivo. These studies will utilize stat-of-the-art high-throughput screening technologies, including doxycycline-inducible shRNAs and open reading frames, that we have perfected over the past decade. In the final phase of the project, we will identify K-Ras synthetic lethals that are therapeutically targetable and then perform preclinical studies in genetically engineered mouse models of lung and colon cancer. The utilization of genetically controlled mouse and human experimental systems will allow us to identify gene products that are truly selectively required in cancer cells expressing mutant K-Ras. In the end, this work will have a major impact for patients who develop K-Ras mutant cancer.