CHK1新型抑制剂抗急性白血病敏感标志物的发现与作用机制研究

Acute leukemia (AL) is a highly malignant blood tumor. Flt3 mutated AL accounting for approximately 30%, remains a difficult clinical problem for portending poor prognosis and relapse. Before, we found the clues that cell lines carrying FLT3 mutantion are sensitive to CHK1 inhibitors via bioinformatics analysis, and then the novel CHK1 inhibitor PY-34 was further evaluated in cellular and animal levels. Here we are proposing that AL Cells with FLT3 mutations are sensitive to CHK1 selective inhibitor used as single agents. The CRISPR / CAS9 gene editing technology will be used to identify the role of CHK1 gene in the sensitive process of FLT3 mutant cells to PY-34. And more other clinical FLT3 mutations will be tested to refine and consolidate the features of FLT3 mutation AL sensitive to CHK1 inhibitor. On the other hand, the genome-scale CRISPR-Cas9 knockout screening library will be used to identify the potent hit genes that are responsible for the molecular sensitive mechanisms of FLT3 mutant cells to PY-34; and FLT3 mutant cells resistant to PY-34 will be established, omics method will be used to identify the potential resistance mechanisms. Then the key signal molecule and network between CHK1 and FLT3 mutations will be refined and solid by combing the sensitive and resistance mechnims. All the work would lay the foundation for the CHK1 inhibitor personalized drug against AL research.

急性白血病（AL）恶性程度极高，预后差、易复发，是临床治疗难题，尤以占比近30%的FLT3基因突变型为甚。前期生物信息分析发现：FLT3突变细胞对CHK1抑制剂敏感。我们利用新型CHK1抑制剂PY-34在细胞与动物水平验证后，提出FLT3突变AL对CHK1抑制剂单用即敏感的设想。拟利用CRISPR/CAS9基因编辑技术研究CHK1抑制在FLT3突变细胞对PY-34敏感中的作用，并开展其它临床常见FLT3突变型对PY-34的敏感性考察，细化和夯实FLT3突变AL的敏感特征。同时，利用CRISPR/CAS9人全基因组KO库开展FLT3突变细胞对PY-34药物敏感的分子机制研究，并建立FLT3突变细胞的PY-34耐药株，通过组学方法发现潜在耐药机制。二者研究结果相结合，将建立较为精细与扎实的CHK1与FLT3突变间的关键分子信号网络，为推动CHK1抑制剂抗AL个性化药物研究奠定坚实基础。

近二十年来国内外多个CHK1抑制剂进入临床试验，但部分CHK1抑制剂由于疗效不佳而终止临床试验。所以挖掘CHK1抑制剂的敏感肿瘤类型、敏感机制及其预测和疗效生物标志物对CHK1抑制剂的药物研发极为重要和关键。.本课题系统完成了PY34抗血液肿瘤活性与机制、预测药效生物标志物以及敏感人群生物标志物等三方面的研究工作：发现包括PY34在内的多种CHK1抑制剂对血液肿瘤更为敏感，其单用即可对多种血液肿瘤无论是细胞水平还是整体动物水平均显示良好活性；通过转录组学系统分析发现无论是细胞株还是病人原代细胞均表现出PY34诱导的c-myc下调与细胞对其敏感性呈现良好相关性，表明c-myc基因下调有可能作为预测CHK1抑制剂的药效生物标志物；利用GDSC数据库挖掘结合细胞以及整体动物水平的实验验证发现，FLT3基因突变可能是CHK1抑制剂的敏感人群特征。.以上三方面的工作层层递进，PY34抗肿瘤活性评价结果显示血液肿瘤对CHK1抑制剂相对敏感；而c-myc基因转录下调以及FLT3基因突变为CHK1抑制剂个性化临床实验方案设计中的预测药效生物标志物及敏感人群生物标志物选择提供数据支持。这些数据强有力的支持了PY34的临床试验申请成功，为临床实验病人遴选提供了一种可能策略。