胸段食管鳞癌术前新辅助化疗有效率约2/3，耐药是导致术前治疗失败的主因，其机制未完全明确。有研究提示NOTCH1表达异常可致食管癌化疗耐药，然而关于NOTCH1基因错义突变致耐药的研究，尚未见报道。我们前期研究发现耐药患者中NOTCH1错义突变检出率显著增加，且集中在配体结合区。通过PyMOL点突变三维结构分析发现NOTCH1p.E450K错义突变受体与配体DLL4结合更紧密；通过CRISPR-Cas9建立NOTCH1p.E450K细胞株，发现点突变细胞发生耐药等表型改变；HES1转录、表达增加，NOTCH1等信号通路激活。因此提出假设NOTCH1p.E450K突变导致NOTCH1信号通路激活进而耐药。本课题将从结构生物学的角度验证该假说并通过细胞分子生物学手段证实NOTCH1p.E450K为食管鳞癌新辅助化疗耐药突变，明确其分子机制。为逆转耐药提供新思路，为寻找靶点研发新药提供理论基础。

Two thirds of esophageal squamous cell carcinoma have response to neoadjuvant chemotherapy. Chemoresistance is the main reason for the failure of preoperative therapy. The mechanism remains unclear. Previous studies have suggested that aberrant expression of NOTCH1 might lead to chemoresistance in esophageal cancer. However, the drug resistance caused by missense mutation of NOTCH1 gene has not been reported. Our previous study have found the detection rate of NOTCH1 missense mutation was significantly increased in chemoresistance patients. Most of the missense mutation were detected in the ligand-binding region. The analysis by PyMOL of the three-dimensional structure of NOTCH1 p.E450K receptor has shown it was more tightly bounding to the ligand DLL4. The NOTCH1 p.E450K cell line was established by CRISPR/Cas9 system, and it was found that the missense mutant cells had chemoresistance and other phenotypic changes. The transcription and expression of HES1 were increased. The NOTCH1 and other signaling pathways were activated. Therefore, it was hypothesized that NOTCH1 p.E450K missense mutation led to activation of the NOTCH1 signaling pathway and chemoresistance. This project will verify the hypothesis by means of Structural Biology. This project will prove that NOTCH1 p.E450K is a drug resistant mutation of neoadjuvant chemotherapy in esophageal squamous cell carcinoma by means of Cell and Molecular Biology. We will try to clarify its molecular mechanism. It will provide new ideas for reversal chemoresistance and provide a theoretical basis for finding new drugs of target therapy.