Characterization of lung cancer radiosensitizers from genome-wide RNAi screens

描述（由申请人提供）：我作为放射肿瘤学家的职业目标是通过实验室启动的基础和转化研究改善肺癌预后，并在临床前模型中验证，为I/II期研究提供理论依据。目前，我将75%的精力投入到研究中，剩下的25%在诊所为胸部肿瘤患者进行咨询，治疗计划和随访。我的博士我与诺贝尔奖获得者Peter Agre博士的论文项目是水通道蛋白的结构-功能关系，我开发了用于纯化，重建和生物物理特性的技术，这些水通道蛋白在广泛的物种中。在我的研究即将结束时，我的父亲被诊断出患有肺癌，并在16个月后去世。我最终接受了放射肿瘤学的培训，加入了与疾病的斗争。因此，我的研究工作采取了不同的方向相比，我的博士学位，我在Alan D 'Andrea博士的指导下学习DNA修复生物学。我花了18个月的时间在他的实验室的霍尔曼研究路径住院期间，毕业后，我加入了达纳法伯癌症研究所的教师，继续我开始的研究。我获得了两个2011年美国放射肿瘤学会（ASTRO）青年教师研究培训奖之一。这是这个奖项的期望，我申请并获得K 08或同等的资金，以进一步我在癌症生物学研究的培训。我的目标是通过开发全基因组RNAi筛选鉴定的肺癌放射增敏剂来改善肿瘤控制。环境：考虑到我致力于癌症生物学研究的时间相对较短， 继续在艾伦·德安德烈亚博士的指导下工作他在DNA修复生物学和去泛素化酶方面的专业知识将对本提案的所有三个具体目标的实施非常宝贵。Kwok-Kin Wong博士开发了将在特定目标1和2中研究的肺癌基因工程小鼠模型（GEMM），他的共同指导也将是非常宝贵的，因为我在动物研究方面的经验相对较少，迄今为止还没有GEMM。该项目还利用了关键的部门资源，包括我们最近委托的小动物辐射研究平台（SARRP），用于CT引导的靶向小鼠放射治疗。职业发展计划的关键要素还包括哈佛公共卫生学院关于基因组数据操作和统计方法的课程，Countway医学图书馆的研讨会，以及哈佛Catalyst提供的关于资助写作和转化研究的资源。研究：放射治疗（RT）是治疗肺癌的一种重要方式。然而，大多数具有潜在可治愈疾病的患者存在局部晚期肿瘤。可以安全施用的RT的剂量是有限的，这是由于放射敏感结构（包括肺、脊髓、食管和心脏）的浸润或接近放射敏感结构，导致尽管进行了最佳治疗，但局部区域失败率仍在30%左右。为了确定可能提高治疗指数的放射增敏剂，我在2-3周内每天接受或不接受放射治疗的非小细胞肺癌（NSCLC）细胞系中进行了全基因组合并的shRNA筛选。基于最高命中率，我确定了NSCLC放射增敏的两种潜在策略。在具体目标1中，我假设蛋白酶体抑制可能通过抑制DNA双链断裂（DSB）修复来发挥作用。我计划探索的机制，蛋白酶体抑制作用的影响同源重组和非同源末端连接使用已建立的报告结构和检测。在具体目标2中，我假设USP 9 X去泛素化酶抑制增强细胞凋亡。我计划使用几种已建立的细胞凋亡测定法在NSCLC中评估这一点，并观察USP 9 X抑制对Bcl-2家族蛋白表达的影响。在目标1和2中，我将尝试用我们的SARRP在基因工程小鼠模型中证明放射增敏作用。在具体目标3中，我建议确定潜在的生物标志物，用于选择患者进行治疗， 同时放化疗和蛋白酶体和/或USP 9 X抑制剂，通过分析从用放射性蛋白酶体和/或USP 9 X抑制剂治疗的肺癌的多个GEMM获得的肿瘤。我还将分析医学上可用的、临床注释的患者数据集，以确定基因表达和其他改变与患者结局的关联。通过这样做，我希望为在I/II期试验中检测蛋白酶体和/或USP 9 X抑制提供理论依据，并为患者选择确定生物标志物。

DESCRIPTION (provided by applicant): My career goal as a radiation oncologist is to improve lung cancer outcomes through basic and translational research initiated in the laboratory and validated in pre-clinical models to provide rationale for Phase I/II studies. I am currently dedicating 75% effort to research, and the remaining 25% in the clinic performing consultations, treatment planning and follow-ups for thoracic oncology patients. My Ph.D. thesis project with Nobel laureate Dr. Peter Agre was on aquaporin structure-function relationships, and I developed techniques for the purification, reconstitution and biophysical characterization of these water channel proteins in a wide range of species. Toward the end of my studies, my father became diagnosed with lung cancer and died 16 months later. I ultimately sought training in radiation oncology to join the fight against the disease. As a result, my research efforts took different direction compared to my Ph.D., and I sought mentorship under Dr. Alan D'Andrea to study DNA repair biology. I spent 18 months during residency on the Holman Research Pathway in his laboratory, and after graduating I joined the faculty at the Dana-Farber Cancer Institute to continue the research I started. I received one of two 2011 American Society for Radiation Oncology (ASTRO) Junior Faculty Research Training Awards. It is the expectation of this award that I apply for and obtain K08 or equivalent funding to further my training in cancer biology research. My aim is to improve tumor control via development of lung cancer radiosensitizers identified by whole genome RNAi screens. ENVIRONMENT: Considering the relatively short time that I have dedicated to cancer biology research, I will benefit greatly from continued mentorship under Dr. Alan D'Andrea. His expertise in both DNA repair biology and deubiquitinating enzymes will be invaluable for the undertaking of all three Specific Aims of this proposal. Co-mentorship under Dr. Kwok-Kin Wong, who developed the genetically engineered mouse models (GEMMs) of lung cancer that will be studied in Specific Aims 1 and 2, will also be invaluable, as I have relatively less experience in animal studies and none to date in GEMMs. The project also makes use of key departmental resources, including our recently commissioned small animal radiation research platform (SARRP) for CT-guided targeted mouse radiotherapy. Key elements of the career development plan also include courses at the Harvard School of Public Health on genomic data manipulation and statistical methods, seminars at the Countway Library of Medicine, and resources offered by Harvard Catalyst on grant writing and translational research. RESEARCH: Radiation therapy (RT) is a critical modality in the treatment of lung cancer. The majority of patients with potentially curable disease, however, present with locally advanced tumors. The dose of RT that can be safely administered is limited due to infiltration of, or proximity to, radiosensitive structures including the lungs, spinal cord, esophagus and heart, resulting in locoregional failure rates of around 30% despite optimal treatment. To identify radiosensitizers that may improve the therapeutic index, I performed whole genome pooled shRNA screens in non-small cell lung cancer (NSCLC) cell lines treated with or without daily irradiation over a period of 2-3 weeks. Based on top hits, I identified two potential strategies for NSCLC radiosensitization. In Specific Aim 1, I hypothesize that proteasome inhibition may serve by inhibiting DNA double strand break (DSB) repair. I plan to explore the mechanism by which proteasome inhibition impacts homologous recombination and non-homologous end joining using established reporter constructs and assays. In Specific Aim 2, I hypothesize that USP9X deubiquitinating enzyme inhibition potentiates apoptosis. I plan to assess this in NSCLC using several established apoptosis assays, and to observe effects of USP9X inhibition on expression of Bcl-2 family proteins. In both Aims 1 and 2, I will attempt to demonstrate radiosensitization in genetically engineered mouse models with our SARRP. In Specific Aim 3, I propose to identify potential biomarkers for patient selection for treatment with concurrent chemoradiotherapy and proteasome and/or USP9X inhibitors, by profiling tumors obtained from multiple GEMMs of lung cancer treated with radiation ¿ proteasome and/or USP9X inhibitors. I will also analyze publically available, clinically annotated patient datasets t identify associations of gene expression and other alterations and patient outcomes. In so doing, I hope to provide rationale for testing proteasome and/or USP9X inhibition in Phase I/II trials, and to identify biomarkers for patient selection.