Biochemical and Functional Characterization of the Mutagenic Cytidine Deaminase APOBEC3B in the contextof Squamous Cell Lung Carcinoma

 描述(申请人提供)：非小细胞肺癌(NSCLC)占所有肺癌的绝大多数，是当今与癌症相关的死亡的主要原因。尽管有这样的统计数据，但有效调节非小细胞肺癌驱动因素的靶向治疗药物的可用性令人惊讶地低。尤其对于非小细胞肺癌的一种亚型--鳞状细胞肺癌，到目前为止还没有成功的靶向治疗方法，因此，识别和验证驱动鳞状细胞肺癌的分子靶点是非常必要的。拟议的项目概述了对APOBEC3B的生化和基于细胞的研究，APOBEC3B是一种突变的胞苷脱氨酶，已知在包括SQCLC在内的多种癌症中过表达。该项目的目标是更好地描述APOBEC3B作为治疗SQCLC的潜在治疗靶点的特征。为了了解APOBEC3B致癌的分子机制，本研究旨在1)对APOBEC3B的催化、核酸结合和结构进行生化和生物物理研究，2)通过一系列基于细胞的实验验证其与SQCLC的相关性。在第一个目标中，利用重组表达和纯化的全长APOBEC3B，通过一系列的稳态酶分析、凝胶迁移率变化分析和X射线结晶学来了解APOBEC3B诱变活性的生化基础。在第二个目标中，将使用过表达和RNAi敲除方法测试一组SQCLC细胞系和正常肺癌细胞系，以验证在SQCLC背景下APOBEC3B表达水平和癌症发生之间的关联。综上所述，这些目标将在理解APOBEC3B诱导致癌的分子机制方面趋同，并将为发展SQCLC的靶向治疗策略提供新的见解。

 DESCRIPTION (provided by applicant): Non-small cell lung cancer (NSCLC) comprises the vast majority of all lung cancers, which is the leading cause of cancer-related deaths today. Despite this statistic, the availability of targeted therapeutics that effectively modulates the drivers of NSCLC is surprisingly low. Particularly for squamous cell lung carcinoma (SQCLC), a subtype of NSCLC, there are no successful targeted therapies to date, and thus, the identification and validation of molecular targets driving SQCLC are of high demand. The proposed project outlines the biochemical and cell-based investigation of APOBEC3B, a mutagenic cytidine deaminase known to be overexpressed in a wide array of cancers including SQCLC. The goal of this project is to better characterize APOBEC3B as a potential therapeutic target for SQCLC treatment. In understanding the molecular mechanisms of APOBEC3B-induced carcinogenesis, the proposed study aims to 1) biochemically and biophysically investigate APOBEC3B catalysis, nucleic acid binding, and structure, and 2) validate its association with SQCLC using a series of cell-based experiments. In the first aim, using recombinantly expressed and purified full-length APOBEC3B, a series of steady-state enzyme assays, electrophoretic mobility shift assays, and X-ray crystallography will be utilized to understand the biochemical basis of APOBEC3B mutagenic activity. In the second aim, a panel of SQCLC cell lines and normal lung cancer cell lines will be tested using overexpression and RNAi knockdown methodology to validate the association between APOBEC3B expression levels and carcinogenesis, in the context of SQCLC. Together, these aims will converge in understanding the molecular mechanisms of APOBEC3B-induced carcinogenesis and will provide novel insights into the development of targeted therapy strategies for SQCLC.