Characterization of lung cancer radiosensitizers from genome-wide RNAi screens

描述(申请人提供)：作为一名放射肿瘤学家，我的职业目标是通过在实验室启动并在临床前模型中得到验证的基础和转化性研究来改善肺癌结果，为I/II期研究提供理论基础。我目前将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。我与诺贝尔奖获得者Peter Agre博士的博士论文项目是水通道蛋白结构-功能关系，我开发了在广泛物种中纯化、重组和生物物理表征这些水通道蛋白的技术。在我的学习接近尾声时，我的父亲被诊断出患有肺癌，16个月后去世。我最终接受了放射肿瘤学方面的培训，加入了抗击这种疾病的斗争。因此，与我的博士学位相比，我的研究努力采取了不同的方向，我寻求艾伦·丹德里亚博士的指导，研究DNA修复生物学。我在霍尔曼研究之路的实验室里住了18个月，毕业后，我加入了达纳-法伯癌症研究所的教员，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)初级教师研究培训奖中的一个。我申请并获得K08或同等资助，以继续我在癌症生物学研究方面的培训，这是对这一奖项的期望。我的目标是通过开发全基因组RNAi筛查确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，我将从 在艾伦·丹德里亚博士的指导下继续指导。他在DNA修复生物学和去泛素酶方面的专业知识对于实现这项提案的所有三个具体目标将是无价的。黄国健博士开发了肺癌基因工程小鼠模型(GEMM)，将在特定的目标1和2中进行研究，他的共同指导也将是无价的，因为我在动物研究方面的经验相对较少，到目前为止还没有GEMM的经验。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引导的小鼠靶向放射治疗。职业发展计划的关键内容还包括哈佛公共卫生学院关于基因组数据处理和统计方法的课程、在Countway医学图书馆举行的研讨会以及哈佛大学促进会提供的关于赠款撰写和转化研究的资源。研究：放射治疗(RT)是肺癌治疗的关键手段。然而，大多数潜在可治愈疾病的患者都患有局部晚期肿瘤。由于肺、脊髓、食道和心脏等放射敏感结构的渗透或接近，可安全使用的RT剂量有限，导致局部失败率约为30%，尽管治疗是最佳的。为了确定可能改善治疗指数的放射增敏剂，我对接受或不接受每日照射的非小细胞肺癌(NSCLC)细胞株进行了全基因组池shRNA筛选，时间为2-3周。根据最高命中率，我确定了两种可能的非小细胞肺癌放射增敏策略。在特定的目标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。我计划利用已建立的报告结构和分析来探索蛋白酶体抑制影响同源重组和非同源末端连接的机制。在具体目标2中，我假设USP9X脱泛素酶抑制增强了细胞凋亡。我计划在NSCLC中使用几种已建立的细胞凋亡检测方法来评估这一点，并观察USP9X抑制对Bcl-2家族蛋白表达的影响。在目标1和目标2中，我将尝试用我们的SARRP在基因工程小鼠模型中演示放射增敏。在具体目标3中，我建议确定潜在的生物标记物，用于选择患者进行治疗 同步放化疗以及蛋白酶体和/或USP9X抑制剂，通过分析从肺癌的多个GEM中获得的肿瘤的概况，用蛋白酶体和/或USP9X抑制剂治疗肺癌。我还将分析公开的、有临床注释的患者数据集，以确定基因表达和其他变化与患者预后的关联。通过这样做，我希望为在I/II期试验中测试蛋白酶体和/或USP9X抑制提供理论基础，并识别用于患者选择的生物标记物。

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.