AF1q调控CD44在慢性髓性白血病干细胞自我更新中的作用及机制研究

Leukemia stem cells (LSC) exist in the bone marrow of patients with chronic myelogenous leukemia (CML), even when they reach complete remission. LSC exhibits an inherent insensitivity to tyrosine kinase inhibitors and is the main reason for drug resistance and disease relapse.Accumulating evidence showed that CD44 expression was obviously increased in LSC which was essential for the maintenance of LSC self-renewal. However, the mechanisms of CD44 overexpression in LSC remain unclear. In our previous study, we found that a novel gene AFIq involved in leukemia progression can regulate the expression of CD44, which indicates that AFIq may play an important role in the self-renewal of LSC through regulating CD44. In this study,we intend to isolate LSC from CML patients and imatinib-resistant cell lines, use lentivirus to increase or decrease AF1q expression and investigate the effects of AFIq on the self-renewal and drug resistance of LSC. Indentify that AFIq can regulate CD44 in LSC and further demonstrate the mechanisms of AFIq in LSC. Then in NOD/SCID mice models, we will investigate the impact of AFIq on leukemia initiating ability of LSC and the possibility of eradicating LSC by targeting AFIq in vivo. Our study will of great significance for overcoming drug resistance and eradicating minimal residual disease in CML.

慢性髓性白血病（CML）缓解后患者体内残留少量白血病干细胞（LSC），其对酪氨酸激酶抑制剂不敏感，是CML耐药和复发的根源。研究发现LSC中CD44表达明显增高，对维持LSC自我更新至关重要，但CD44在LSC中表达增高的原因及调控机制目前尚不明确。我们前期研究发现一种与白血病发病密切相关的基因AF1q能调控CD44表达，提示AF1q可能通过调控CD44在LSC自我更新中发挥重要作用。本课题拟在前期工作基础上，分离纯化CML患者骨髓原代细胞和伊马替尼耐药细胞株中的LSC，慢病毒感染LSC上调或下调AF1q表达，研究AF1q对LSC自我更新及耐药性的影响；明确LSC中AF1q对CD44的调控作用，阐明AF1q的作用机制；利用NOD/SCID小鼠从体内研究AF1q对LSC致白血病能力的影响，探讨靶向AF1q清除LSC的可行性；为克服CML耐药及清除微小残留病变奠定理论基础。

随着酪氨酸激酶抑制剂在临床上的广泛应用和时间的推移，其耐药现象日益明显，导致CML复发率逐渐增高，因此探索CML耐药机制对克服耐药、延长生存具有重要的临床意义。研究显示，白血病干细胞的持续性存在是CML耐药和复发的根源。我们前期研究发现，一种与白血病发病密切相关的基因——AF1q，在CML患者中异常高表达，且上调AF1q可增加CD44的表达水平。本课题研究了AF1q在CML细胞和CD34+白血病干/祖细胞中的作用及机制，以期为CML的治疗提供新靶点。我们研究发现，AF1q在各期CML患者中均高表达，且CML CD34+白血病干/祖细胞中AF1q的表达明显高于CD34-细胞。在CML伊马替尼耐药细胞株K562/G01、CML原代细胞以及CD34+白血病干/祖细胞中，应用siRNA下调AF1q的表达后，细胞的增殖速度减慢，对伊马替尼的药物敏感性增高，细胞凋亡增加；同时在CML伊马替尼敏感细胞株K562中上调AF1q的表达后，细胞的增殖速度加快，对伊马替尼的药物敏感性降低，细胞凋亡减少。说明AF1q在CML细胞、CD34+白血病干/祖细胞中具有促进增殖、诱导耐药以及抵抗凋亡的作用。我们进一步研究发现，CD44在CML慢性期患者中高表达，且CD34+白血病干/祖细胞中的表达明显高于CD34-细胞，且CD44与AF1q的表达水平呈正相关；在CML原代细胞、CD34+白血病干/祖细胞和CML细胞系中改变AF1q的表达水平，CD44表达随之改变，证实AF1q能正向调控CD44的表达。我们进而在AF1q上调的K562细胞系中应用CD44中和抗体A3D8阻断其发挥生物学作用，结果显示细胞的增殖速度减慢，对伊马替尼的药物敏感性升高，细胞凋亡增加，明确了AF1q通过调控CD44发挥生物学功能。同时，NOD/SCID小鼠体内试验显示，AF1q可增强CML细胞在体内的定植能力，加快CML在小鼠体内的发生发展。目前，已有3篇受本课题资助的SCI论文发表，另有1篇论文正在投稿、资料整理过程中。