Identification and therapeutic application of miRNA-drivers in lung cancer

描述(由申请人提供)：拟议的工作重点是确定癌症中新的miRNA驱动因素，并利用这一知识和其他知识开发基于miRNA的疗法。这项“独立之路”奖项申请包括候选人Andrea Kasinski博士转变为独立研究员的有指导的职业发展计划，以及附带的研究计划，该计划描述了拟议的发现可增强KRAS驱动的肺腺癌的miRNAs的实验，并探索重新表达肿瘤抑制miRNAs的新治疗策略，其中包括高通量筛选以确定可以恢复miRNA加工的小分子，以及用于使肿瘤对传统化疗增敏的组合miRNA疗法。候选人卡辛斯基博士是耶鲁大学分子、细胞和发育生物学系弗兰克·斯拉克博士实验室的博士后研究员。导致她在埃默里大学获得遗传学和分子生物学研究生学位的工作是在傅海安博士的药理系实验室进行的，重点是针对治疗开发的细胞生存信号。在FU实验室，卡辛斯基博士执行了两个非常不同的项目，基于开发靶向疗法的最终目标，这两个项目是相互关联的：在一个项目中，卡辛斯基博士利用生化分析确定IKK-1是EF24的直接靶点，EF24是一种姜黄素的类似物，最终进入临床试验。在第二个项目中，使用了一系列的遗传学和分子技术来评估致癌的14-3-3家族成员的转录调控，希望这一知识可能会引领后续的研究，以阻断14-3-3的表达。指导和职业发展计划将补充她的背景，包括遗传学、分子生物学、整体动物研究和小分子筛选，并在每个领域以及本项目涉及的特定领域：小鼠生物学、癌症方面进行培训和指导。 生物学、细胞培养技术和高通量药物筛选。卡辛斯基博士的目标是成为跨学科生物科学、癌症生物学或学术、私立或政府机构类似部门的教员，在那里她可以研究癌症中miRNA的生物学，并致力于推进基于miRNA的疗法。这项关于miRNAs参与癌症的研究需要进行创新和选择性的筛查，卡辛斯基博士正在积极寻求这一点，首先是在细胞培养方面，并将进入动物模型。这项在软琼脂上进行的测试是为了筛选能够特异性地导致正常人类肺支气管上皮细胞转化的miRNAs。从这项测试中鉴定出的miRNAs将在细胞培养中进一步评估，并最终在体内进行评估。拟议中的涉及miRNA疗法的研究有两个方面。第一种考虑了LET-7和LIN-28的相互作用。卡辛斯基博士正与埃默里大学化学生物学和药物筛选中心联席主任傅海安博士积极合作，进行高通量筛选(HTS)，以确定这种蛋白质-RNA相互作用的抑制剂。这种HTS的HITS将通过几个独立的研究途径对细胞渗透性、动力学和体内治疗潜力进行评估。最后，基于Kasinski博士目前的发现，即miR-34和let-7代表了非小细胞肺癌的有效治疗选择，她将结合目前在细胞培养、异种移植和Kras；p53双重突变中使用的化疗和靶向治疗药物来评估这些miRNA疗法。这项工作是新颖的，及时的，对人类健康和生存具有明确和重大的影响。识别miRNAs和靶向治疗以干扰癌症中发生的miRNA失衡，对于癌症和miRNA生物学领域具有非常重要的价值，并具有直接的临床应用价值。

DESCRIPTION (provided by applicant): The proposed work focuses on identifying novel miRNA drivers in cancer and utilizes this and other knowledge to develop miRNA-based therapeutics. This "Pathway to Independence" award application includes a mentored career development plan for the transition of the candidate, Dr. Andrea Kasinski, into an independent investigator, as well an accompanying research plan describing the proposed experiments on discovering miRNAs that can potentiate KRAS-driven lung adenocarcinoma and exploring novel therapeutic strategies for re-expressing tumor-suppressive miRNAs, which includes a high-throughput screen to identify a small molecule that can restore miRNA processing and combinatorial miRNA therapies for sensitizing tumors to conventional chemotherapies. The candidate, Dr. Kasinski, is a postdoctoral fellow at Yale, in the lab of Dr. Frank Slack in the Department of Molecular, Cellular, and Developmental Biology. The work leading to her graduate degree in Genetics and Molecular Biology at Emory University was conducted in the lab of Dr. Haian Fu in the Department of Pharmacology and focused on targeting cell survival signaling for therapeutic development. In the Fu lab, Dr. Kasinski performed two very distinct projects that were interrelated based on the ultimate goal of developing targeted therapeutics: in one project Dr. Kasinski utilized biochemical assays to identified IKK-¿ as a direct target of EF24, an analogue of curcumin that ultimately made its way into clinical trials. For the second project a series of genetic and molecular techniques were used to evaluate the transcriptional regulation of an oncogenic 14-3-3 family member, with the hope that this knowledge might spearhead subsequent studies to block 14-3-3 expression. The mentoring and career development plan will supplement her background, which is evenly split between genetic, molecular biology, whole animal studies and small molecule screening, with training and instruction in each, and in the particular areas that this project involves: murine biology, cancer biology, cell culture technique and high-throughput drug screening. Dr. Kasinski's goal is to become a faculty member in an interdisciplinary biosciences, cancer biology, or similar department at an academic, private, or government facility, in which she can research the biology of miRNAs in cancer and work to advance miRNA-based therapies. This research on miRNAs involvement in cancer requires that an innovative and selective screen be performed which Dr. Kasinski is actively pursuing first in cell culture and will advance into animal models. This assay in soft agar is selecting for miRNAs that can specifically cause normal human lung bronchial epithelial cells to become transformed. MiRNAs identified from this assay will be evaluated further in cell culture and ultimately in vivo. The proposed study involving miRNA therapeutics is two fold. The first takes into account the interaction of let-7 and LIN-28. Dr. Kasinski is in an active collaboration with Dr. Haian Fu, Co-Director of the Chemical Biology and Drug Screening Center at Emory University, to perform a high throughput screen (HTS) to identify inhibitors of this protein-RNA interaction. Hits from this HTS will be evaluated for cell permeability, kinetics and in vivo therapeutic potential, by several independent avenues of investigation. Finally building on Dr. Kasinski's current findings that miR-34 and let-7 represent valid therapeutic options for non-small cell lung cancer, she will evaluate these miRNA therapies in combination with currently used chemotherapies and targeted-therapeutics in cell culture, xenografts and in the Kras;p53 double mutant. This work is novel, timely and has clear and significant implications for human health and survival. The identification of miRNAs and targeted-therapies to perturb miRNA imbalance that occurs in cancer is invaluable to the fields of cancer and miRNA biology, and has direct clinical application.