基于高通量CRISPR基因敲除筛选技术探讨肿瘤化疗耐药机制及对抗策略

Chemoresistance is the pivotal obstacle that restricts the effectiveness of chemotherapy in clinical practice for multiple types of cancer. However, it remains unclear that how chemoresistance develops genetically or epigenetically and how to effectively antagonize the resistance. Here， by focusing on chemoresistance in colon cancer, we propose to perform genome-wide CRISPR knockout screens in multiple colon cancer cell lines in order to systematically identify the genes that whose loss-of-function mutations can cause chemoresistance to three first-line cytotoxic drugs (fluorouracil, irinotecan and oxaliplatin). Novel molecular mechanism of chemoresistance will be deciphered by characterizing these drug resistance genes. To overcome the drug resistance caused by these gene mutations, we will perform a second round CRISPR screen on those resistance cells carrying specific gene mutations with a focused druggable gene library to identify target genes whose inhibition can specifically kill the corresponding chemoresistant cells. The target drugs corresponding to the target genes when available will be tested for the efficacy to overcome the corresponding chemoresistance. In addition to cell lines, we will also extend our investigations by incorporating in vivo mouse models and clinical colon cancer chemoresistant samples. The completion of this study will not only lead to better understanding of chemoresistance in cancer, but also provide novel genetic biomarkers for diagnosis and treatment strategies to overcome corresponding chemoresistance by way of precision medicine.

肿瘤化疗耐药一直是困扰肿瘤诊疗临床实践的重大问题，其背后的分子机制与遗传基础尚不十分清楚。本项目拟针对结肠癌的化疗耐药问题，通过全基因组范围的CRISPR基因敲除筛选技术在多株结肠癌细胞系中快速、高效的鉴定出与结肠癌一线化疗药物（氟尿嘧啶、伊立替康及奥沙利铂）耐药相关的基因，其突变或功能失活即能造成对特定化疗药物的耐药，继而深入探讨其耐药的分子机制。针对单基因突变或失活而形成的耐药细胞株，进一步利用特制的CRISPR基因敲除文库（druggable gene library，成药基因文库）开展新一轮的细胞生长或活力筛选，明确能够有效对抗相应耐药机制的基因靶点，进而探讨其对应的靶向药物对抗相应耐药机制的效果。在细胞、小鼠及临床样品等多维度水平上对上述发现进行验证。本项目一方面能够系统揭示肿瘤化疗耐药形成的分子机制，另一方面能够为肿瘤化疗耐药的临床诊断和治疗提供新的有效策略，实现精准诊疗。

肿瘤化疗耐药是肿瘤诊疗临床实践中遇到的常见问题，严重影响病人预后和临床治疗效果，目前尚未有系统的解决方案，其背后的分子机制和遗传基础也尚不明确。以CRISPR技术为基础的高通量遗传筛选系统的出现为探讨该问题提供了新的有效工具。本项目针对多种临床常见的肿瘤化疗药物，通过对包括结直肠癌细胞在内的多种癌细胞进行全基因组规模的CRISPR敲除筛选，系统地鉴定与化疗药物敏感性相关的基因驱动因素，探究化疗耐药现象背后的分子机制，并寻找对抗化疗耐药的基因靶点或靶向药物，为解决临床上的化疗耐药问题提供新策略。我们发现不同的肿瘤遗传背景及化疗药物对应着差异化的化疗耐药基因，化疗耐药基因的失活可通过多种不同的分子机制导致肿瘤细胞对特定化疗药物耐受。通过对结直肠癌奥沙利铂和伊立替康耐药细胞进行深入分析，我们揭示了相应化疗耐药的细胞学特征和基因表达谱。为进一步寻找对抗耐药的基因靶点，接下来，我们对奥沙利铂和伊立替康耐药细胞进行了第二轮CRISPR成药基因文库的筛选，鉴定出了一系列潜在的对抗耐药的基因靶点及潜在药物。我们利用细胞、小鼠及人类结直肠癌临床样品等模型，进一步证明某激酶基因失活或其特异性小分子抑制剂可以有效杀伤奥沙利铂耐药的结直肠癌细胞。本研究不仅为理解肿瘤化学耐药的分子基础提供了系统资源和新见解，还提出了对抗化疗耐药的潜在临床诊疗新策略。

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