功能性筛选的慢性粒细胞白血病抗药抑癌基因下游非依赖BCR-ABL新药靶点的发现与验证及机理研究

BCR-ABL induced Chronic Myelogenous Leukemia (CML) is a malignant disorder of stem cells in which BCR-ABL targets HSCs and creates Leukemic stem cells (LSCs) that maintain the leukemia and are a source of treatment resistance. Tyrosine kinase inhibitors (TKI), such as imatinib, induce a complete hematologic and cytogenetic remission in most CML patients, but cannot completely eradicate BCR-ABL-expressing cells. In vitro, in vivo, and clinical evidence indicate that LSCs evade BCR-ABL kinase inhibitors and provide the source of CML resistance and progression. Because CML patients diagnosed in China are much younger than CML patients in western countries, long-term treatment of CML with TKI provides significantly increased chance for the resistance of LSCs to TKI and CML to relapse. We have previously established CML resistant cell lines with shRNA lentivirus library screening under the selection of Imatinib. We discovered 28 new leukemia suppressor genes (LSG). We identified PRKD2 as a novel therapeutic target, and we used its chemical inhibitor CRT0066101 to reduce CML cell proliferation and increase apoptosis. With the same strategy, we plan to validate these LSGs in a CML humanized mouse model. By taking advantages of biochemistry, molecular biology analytical methods, together with the next generation RNA-Sequencing and protein mass spectrum technology, we will identify even more therapeutic targets, define their signaling pathways and reveal their regulatory mechanism for the generation of TKI resistance. We will also evaluate the therapeutic effects of inhibitors of these target genes in the treatment of CML. Thus, exploration to the molecular pathways and regulatory mechanism of LSCs for their TKI resistance will provide novel insights into the therapy treating CML with TKI resistance and eventually eradicating LSCs to cure CML.

慢性粒细胞白血病（CML）是由病变的白血病干细胞增殖导致的髓性粒细胞的积累。靶向药物酪氨酸激酶抑制剂能够有效抑制CML，但无法杀死白血病干细胞并根治CML。前期我们用RNA干扰慢病毒文库和CRISPR基因组编辑技术建立CML抗药细胞株，筛选出6个抗药抑癌基因（LSG），结合信号通路等方法确定PRKD2作为新的非依赖BCR-ABL的新药靶点。本项目拟用CML小鼠模型验证LSG对抑制CML的发展和产生抗药性的机理和信号途径；结合RNA测序、信号通路和生物信息学等技术，再发现一个非依赖BCR-ABL的靶点，并在抗TKI的CML细胞系和白血病患者的白血病干细胞、CML人源化小鼠模型中检验PRKD2等新靶点药物治疗CML的效果，分析这些新药靶点调控白血病细胞产生抗药性的机理和信号途径。深入探究白血病干细胞的分子调控和抗药机制，将为解决其抗药性、开发新药、治愈CML提供新的理论依据和更有效的治疗方法。

该项目自实施以来，项目负责人积极组织相关研究人员对该项目进行了研究，按计划进行了实验，以前期工作筛选的6个CML白血病抗药抑癌基因（LSG）为基础，验证6个LSG对抑制CML的体内发展和产生抗药性的作用机制,结果说明PKCH下游的丝氨酸/苏氨酸蛋白质激酶PRKD2可能是非依赖BCR-ABL的新药靶点。此结果揭示CML干细胞通过NF-κB/PRKD通路介导，产生了不依赖于BCR-ABL的伊马替尼耐药性，这一先前未知的耐药机制。选择蛋白激酶D（PRKD）抑制剂CRT0066101和伊马替尼联合用药或组合用药，可克服CML对酪氨酸激酶抑制剂耐药性，这以联合用药技术方案可应用于对酪氨酸激酶抑制剂耐药的CML的靶向治疗。本实验通过PRKD抑制剂与IM两者在慢性粒细胞白血病小鼠模型中的联合治疗确认了其有效性。结果发现伊马替尼和PRKD2抑制剂CRT0066101（简称CRT）联合用药实验可有效抑制慢性耐药粒细胞白血病。. 另外，我们也期望从传统中药中发现抗慢粒白血病新药，我们用紫草素提取物乙酰紫草素处理K562慢粒白血病细胞，结果表明24h何48h处理的IC50分别为2.03 μM 和1.13 μM；乙酸紫草素以剂量依赖方式诱导K562细胞凋亡，被阻滞于S期,可有效降低Bcr-Abl蛋白激酶的表达，可抑制NF-κB通路的IκBα的磷酸化以及P65从细胞质向细胞核的转移, 从而抑制了K562细胞的NF-κB通路，表以乙酰紫草素可能通过抑制K562的酪氨酸激酶活性和NF-κB通路来诱导K562细胞凋亡，有望成为增强伊马替尼的酪氨酸激酶靶向药物。我们还研究了独蒜兰（中药山慈菇）提取物对白血病细胞的诱导凋亡作用。结果显示独蒜兰乙酸乙酯萃取物能显著抑制慢粒白血病细胞K562、HL-60和THP-1的细胞增殖，并通过触发内源性线粒体凋亡通路诱导了细胞凋亡。