CRISPR/Cas9介导的全基因组敲除鉴定急性淋巴细胞白血病蒽环类耐药的相关基因

Acute lymphoblastic leukemia is a highly heterogeneous hematological malignancy, the prognosis in adult ALL patients are uniformly poor compared to children. Drug resistance is the major reason that leads to treatment failure. Previously by the support of National Natural Science Foundation of China, we have clarified two nuclear protein, NPM/B23 and NCL/C23, to be the novel potential targets in chemotherapy-resistant ALL. However, due to the complexity of the disease, one or two targets cannot improve the treatment response significantly, we need a better understanding of genome-scale reaction to chemotherapy. Anthracycline is a group of classical drugs in for ALL, which has been proved easily to induce chemotherapy resistance. We would like to screen for targets that lead to the resistance of Anthracycline using a genome-scale lentiviral single-guide RNA (sgRNA) library. By this pooled, loss-of-function CRISPR/Cas9 based screening, we will identify the resistance-origin candidates. To verify those candidates, the expression level in ALL patients and drug-resistant cell will be investigated. The functional study by single gene knockout will be done both in vitro and in vivo. From the screening and further validation, we will unmask the mechanism for the Anthracycline resistance and identify novel targets for relapse and refractory ALL. The goal of this study is to overcome refractoriness and improve ALL prognosis.

急性淋巴细胞白血病（ALL）是一种常见的血液肿瘤，成人疗效明显不如儿童，耐药是治疗失败的主要原因。前期项目组在国家基金支持下已取得较好结果，阐明了NPM/B23和NCL/C23在ALL耐药中的作用，但由于疾病的高度异质性，这些仅能解释耐药的部分问题，因此，进一步探寻ALL耐药机制对提高疗效具有紧迫性和必要性。为更全面了解ALL对蒽环类耐药的基因变化，本课题拟采用高通量基因编辑技术CRISPR/Cas9介导的全基因组敲除方法对ALL蒽环类耐药基因进行功能性筛选。通过全基因组测序分析，筛选出耐药细胞中被高度富集的基因，即为可能导致耐药的候选基因。检测不同阶段ALL临床样本中候选基因的表达水平，分析与ALL疗效及预后关系；通过细胞水平及动物体内实验等研究其生物学功能、涉及的信号通路及调控耐药的可能机制，将有助于探索新的ALL治疗靶点，对临床精确判断预后、精准治疗具有重要意义。这方面研究未见报道。

急性淋巴细胞白血病（ALL）是高度异质性、预后欠佳的的血液肿瘤，耐药是治疗失败的主要原因。项目组采用高通量基因编辑技术CRISPR/Cas9介导的全基因组敲除方法结合单细胞测序对ALL蒽环类耐药基因进行功能性筛选。通过不同的筛选条件，应用全基因组测序分析出在筛选条件下被高度富集的基因，获得了4个与增加药物敏感性有关的基因，6个可能导致耐药的基因，8个与细胞生存相关的基因，2个可能与预后有关的基因。在临床样本中检测上述基因的表达水平，分析与ALL疗效及预后关系。对这些潜在的靶向基因进行单基因的敲除和回复，通过细胞水平及动物体内实验等研究其生物学功能、涉及的信号通路及调控耐药的可能机制。对靶基因的进一步研究显示，PTPMT1通过调控氧化应激来影响ALL细胞在化疗药中的增殖能力，UHRF1通过甲基化修饰调控SFRP5的表达水平，SUMO2通过调控泛素化影响ALL对蒽环类化疗耐药水平。

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