利用CRISPR/Cas9文库筛选技术探索急性髓系白血病细胞对高三尖杉酯碱的耐药机制

Acute myeloid leukemia (AML) accounts for 15%-20% of pediatric acute leukemia, and its prognosis is much worse than that of pediatric acute lymphoblastic leukemia. Drug resistance is the main cause for its relapse and refractoriness. Homoharringtonine (HRT) is a commonly used chemotherapeutic drug which was isolated from high tricus genus by Chinese researchers, and multiple HRT-based chemotherapy regimens have been established from long-term clinical practice to promote the prognosis of AML. Despite its wide clinical use, very little research has been done to explore HRT resistance in AML treatment. Revealing the underlying mechanisms in HRT resistance is of great significance in guiding its application in relapsed and refractory pediatric AML treatment. Our project intends to use CRISPR/Cas9 library and high-throughput sequencing strategy，combined with the results of multi-group sequencing of clinical initial remission cases and refractory/relapsed cases, to screen for genes and pathways that may play roles in HRT chemoresistance, and validate the functions of candidate genes and pathways in vitro models; furthermore to screen for targeted small molecule compounds which could reverse HRT resistance in vitro models and then test the effect in PDX mouse models, thus provide solutions for targeted treatment of relapsed and refractory pediatric AML.

急性髓系白血病（Acute myeloid leukemia，AML）约占儿童急性白血病的15%～20%，疗效远低于儿童急性淋巴细胞白血病。化疗药物耐药是儿童AML难治复发的主要原因。高三尖杉酯碱（Homoharringtonine, HRT）是由我国科研工作者从高三尖属植物中分离而来，并在大量临床实践中形成了以HRT为基础的化疗方案。然而，目前对于HRT的耐药机制尚不清楚，揭示其耐药机制对指导HRT在复发/难治的儿童AML的化疗应用具有重要的意义。本项目拟利用CRISPR/Cas9文库和高通量测序技术，结合临床初治缓解和难治/复发病例的多组学测序结果，筛选可引起HRT化疗耐药的基因和信号通路，并利用体外模型进行验证，以探索HRT在儿童AML化疗中的耐药机制，利用体内外模型筛选和验证可逆转HRT耐药的靶向小分子化合物，为靶向治疗难治/复发的儿童AML提供解决方案。

儿童急性髓系白血病(acute myeloid leukemia, AML)是一组造血系统的恶性克隆性疾病，约占儿童急性白细胞的15%~20%，5年EFS不足70%，近年来其疗效无显著改善。主要原因是由于耐药性导致的治疗不缓解或缓解后复发(30%)。高三尖杉酯碱（homoharringtonine, HHT）是我国特有的有效的化疗药物，安全性高，但有效率仅约60%。目前对HHT在AML治疗中发生耐药的机制研究不甚详尽。在本项目中，我们利用CRISPR/Cas9文库技术在AML细胞系中筛选出参与HHT耐药的候选基因并进行了体外验证，同时我们搜集和总结了22例采用包含HHT方案治疗的疗效并对遗传学异常进行分析，与文库发筛选出的候选耐药基因比对发现TP53变异为共同变异基因。利用目前的体系我们将继续探索TP53变异引起AML对HHT耐药的机制，优化TP53变异分层对预后及耐药性的影响，并筛选可逆转耐药性的小分子化疗物，以优化含HHT的方案在儿童AML中的应用，提高HHT在治疗儿童AML时的有效率，从而提高儿童AML的预后。