Investigating cellular heterogeneity in lung cancer

描述(申请人提供)：非小细胞肺癌(NSCLC)是全球癌症相关死亡的主要原因。由致癌基因Kras表达驱动的肿瘤约占NSCLC亚型的25%，尤其是对于这些肿瘤，缺乏有效的化疗方法。标准化疗在这些肿瘤中失败的一个可能的解释是肿瘤内存在的细胞异质性。这项建议的目的是了解Kras驱动的肺腺癌的细胞异质性。具体地说，我将探索Wnt和R-Respondin/Lgr5家族信号通路作为旁分泌调节因子在肿瘤细胞去分化和“干性”中的作用。为了做到这一点，我将在Kras驱动的肺腺癌的复杂小鼠模型以及人类肺腺癌细胞系中使用一系列新的工具。我建议测试Wnt和R-Respondin/LGR通路在肺癌发生和发展中的作用，以及潜在的治疗靶点。为了做到这一点，我将使用Wnt合成的小分子抑制剂或双启动子慢病毒通过RNAi来沉默该途径的关键组件，包括Lgr5家族受体。此外，新的Wnt报告慢病毒或表达Lgr5或Lgr6驱动和他莫昔芬可激活的Creer重组酶的敲入等位基因将被用于进行谱系追踪实验，使我能够在经历自然肿瘤进展且不基于细胞系移植的肿瘤模型中跟踪或切除假定的肺腺癌干细胞的命运，这在大多数当前文献中是一个警告。使用Wnt-Response和Lgr5/6记者还将使我能够分离出可能的肺腺癌干细胞，用于基因表达谱分析，这可能会导致发现新的干细胞标记和可用药的途径。阐明调节癌症细胞(分化)状态的分子机制将为绘制肿瘤细胞图景提供新的标记；其中一些将被证明是药物干预的有用靶点，最终将改善这种基本上难以治愈的疾病的治疗结果。因此，我觉得这项提议与国家癌症研究所的使命完全一致。在这份申请中，我还提出了一个广泛的培训计划，旨在促进我过渡到一个独立的首席调查员职位。麻省理工学院贾克斯实验室和周边地区的研究环境为科学讨论、合作和培训提供了无与伦比的机会。我目前指导一名本科生和一名技术助理，他们直接与我一起做与我的研究有关的实验。这是一次令人难以置信的经历，它将赋予我管理独立实验室的许多必要技能。麻省理工学院、博德研究所和哈佛医学院的科学界提供了无数的课程、研讨会和研讨会，这些课程、研讨会和研讨会将继续促进我的科学发展。我有幸组建了一支由导师(杰克斯博士和温伯格博士)和顾问(克里弗斯博士和斯卡登博士)组成的梦之队，他们将在K99/R00奖的整个期间为我提供必要的指导和支持。重要的是，我的主要导师杰克斯博士将允许我带着我目前和计划中的所有研究作为我未来研究计划的基础。我在研究控制(肿瘤)血管生成和淋巴管生成的细胞-细胞相互作用方面的经验，以及过去两年在杰克实验室学习新的方法和概念，形成了在这一申请中提出的研究。我打算启动一个独立的研究计划，利用这些强大的体内系统。我已经证明了我的独立性，在我们实验室以前没有研究过的领域创建了一个项目，并以研究金和小额项目赠款的形式获得了独立资金。这一点，再加上我开发的大量试剂，为我提供了完成拟议计划所需的动力。从长远来看，我相信这些实验将为我的研究计划提供坚实的基础。我期待着指导那些和我一样热爱癌症研究的学生和博士后。

DESCRIPTION (provided by applicant): Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death globally. Tumors driven by expression of oncogenic Kras account for approximately 25% of NSCLC subtypes and, for these tumors in particular, effective chemotherapies are lacking. One possible explanation for the failure of standard chemotherapies in these tumors is the cellular heterogeneity that exisists within tumors. The goal of this proposal is to understand the cellular heterogeneity in Kras-driven lung adenocarcinoma. Specifically, I will explore the role of the Wnt and R-spondin/Lgr5 family signaling pathways as a paracrine regulators of cellular de-differentiation and "stemness" in the tumors. To do so, I will employ a series of novel tools in a sophisticated mouse model of Kras- driven lung adenocarcinoma as well as in human lung adenocarcinoma cell lines. I propose to test the role of the Wnt and R-spondin/Lgr pathways in lung tumor initiation and progression, as well as potential targets of therapy. To do this, I will use small molecule inhibitors of Wnt synthesis or dual-promoter lentiviruses to silence key components of the pathway, including Lgr5 family receptors, using RNAi. Furthermore, novel Wnt reporter lentiviruses or knock-in alleles expressing Lgr5 or Lgr6 driven and tamoxifen-activatable CreER recombinase will be used to perform lineage-tracing experiments that allow me to track the fate of or ablate the putative lung adenocarcinoma stem cells in a tumor model that undergoes natural tumor progression and is not based on cell line transplantation, a caveat in most of the current literature. Use of Wnt-responsive and Lgr5/6 reporters will also enable me to isolate the putative lung adenocarcinoma stem cells for gene expression profiling, which may lead to the discovery of novel stem cell markers and druggable pathways. Elucidating the molecular mechanisms that regulate cell (differentiation) states in cancer will provide novel markers for mapping the cellula landscape of tumors; some will prove to be useful targets for pharmacological intervention, which will eventually improve treatment outcomes in this largely intractable disease. Thus, I feel that this proposal is fully aligned with the mission of the National Cancer Institute. In this application I also propose an extensive training program that is designed to facilitate my transition to an independent Principal Investigator position. The research environment in the Jacks Laboratory, MIT, and the surrounding area offers unparalleled opportunities for scientific discussion, collaboration and training. I currently supervise an undergraduate student and a technical assistant that work directly with me on experiments pertaining to my research. This is an incredible experience that will endow me with many of the necessary skills to manage an independent laboratory. The scientific community at MIT, the Broad Institute, and Harvard Medical School offers countless courses, seminars and workshops that will continue to foster my scientific development. I have been fortunate in having been able to assemble a dream team of Mentors (Dr. Jacks and Dr. Weinberg) and Consultants (Dr. Clevers and Dr. Scadden), who will provide me with the necessary guidance and support throughout the entire duration of the K99/R00 Award. Importantly, my Primary Mentor Dr. Jacks will allow me to take all of my current and proposed research with me to serve as the foundation of my future research program. The research proposed within this application has been shaped by my experiences in studying cell-cell interactions controlling (tumor) angiogenesis and lymphangiogenesis, as well as by the past 2 years in the Jacks Laboratory learning novel methodology and concepts. I intend to start an independent research program that will capitalize on these powerful in vivo systems. I have already demonstrated my independence by creating a project in a field not previously studied in our lab as well as by obtaining independent funding in the form of Fellowships and small project grants. This, in combination with the large number of reagents that I have developed, provides me with the momentum needed to complete the proposed program. For the long-term, I am confident that these experiments will provide a solid foundation on which my research program can be built upon. I look forward to mentoring students and postdocs that share my passion for cancer research.