FoxM1调控DNA损伤修复在慢性髓性白血病伊马替尼耐药中的作用及机制研究

Imatinib has been considered as first-line treatment of chronic myeloid leukemia (CML). However, resistance to imatinib severely impairs treatment efficiency and survival, thereafter remaining a challenge for CML treatment. Genetic mutations and over-amplification of oncogenic fusion gene Bcr-Abl are suggested to be the main reason for imatinib resistance. Accumulating evidence showed that Bcr-Abl was closely related to DNA damage repair, which may play an important role in the imatinib-resistance of CML. A novel transcription factor FoxM1 was demonstrated to regulate DNA damage repair, but whether FoxM1 was essential for the imatinib-resistance of CML remained unclear. Based on our previous studies, we speculated that FoxM1 could regulate DNA damage repair and affect Bcr-Abl kinase activity, which contributed to imatinib resistance. In the following study, we intend to detect the expression of FoxM1 in patients with CML and analyze its clinical significance; investigate the effect of FoxM1 up/down-regulation on the regulation of Bcr-Abl mutations and over-amplification as well as determine the role of FoxM1 in imatinib resistance; screen and identify FoxM1 downstream targets associated with DNA damage repair, which was involved in imatinib resistance, and further clarify the underlying mechanisms of FoxM1 in imatinib resistance; assess the feasibility of imatinib sensitivity enhancement by targeting FoxM1 in the NOD/SCID mouse models engrafted with CML cells. Our study will be of great importance for overcoming imatinib resistance in CML.

Bcr-Abl基因点突变或扩增是伊马替尼（IM）耐药产生的主要原因，严重制约慢性髓性白血病（CML）疗效提高。研究表明，DNA损伤修复对Bcr-Abl依赖的IM耐药至关重要。新近研究显示，转录因子FoxM1可调控DNA损伤修复，但是否参与IM耐药目前尚不明确。我们前期发现，FoxM1能调控DNA损伤修复，影响Bcr-Abl激酶活性，在IM耐药中起关键作用。本课题拟在前期工作基础上，检测CML患者骨髓中FoxM1表达，分析其与IM敏感性的关系；研究过表达和沉默FoxM1在调控Bcr-Abl点突变或扩增中的作用及对IM耐药的影响；利用芯片筛选并鉴定出参与IM耐药的FoxM1下游DNA损伤修复基因，阐明FoxM1调控IM耐药的新机制；利用NOD/SCID小鼠从体内探讨靶向FoxM1逆转IM耐药的可行性。本研究将从FoxM1这一新视点为揭示IM耐药机制奠定基础，为克服IM耐药提供靶向治疗新思路。

白血病干细胞（LSC）是慢性髓性白血病（CML）耐药复发的根源，BCR-ABL基因扩增或点突变是伊马替尼（IM）耐药的主要原因。新近研究显示，增殖特异性转录因子FoxM1参与肿瘤发生、发展，在维持肿瘤干细胞自我更新和耐药中发挥重要作用。亦有研究表明，DNA损伤修复对BCR-ABL依赖的IM耐药至关重要，且FoxM1分子可调控DNA损伤修复。然而，FoxM1是否参与CML耐药目前尚不明确，其能否影响LSC存活有待进一步研究。本课题成功分选出CML初诊患者骨髓CD34+白血病干/祖细胞，阐明该群LSC细胞对IM的耐药性。首次报道，上调miR-370可减弱LSC和K562/G01细胞的生存能力，并增强CML细胞对IM的敏感性及其诱导的细胞凋亡；K562细胞中下调miR-370能产生相反的效果。同时证实，CML细胞中miR-370对FoxM1的靶向调节作用，并发现下调FoxM1可抑制DNA损伤修复，减弱BCR-ABL激酶活性，促进IM诱导的LSC和K562/G01细胞凋亡，从而逆转IM耐药；上调FoxM1对K562细胞的作用相反。此外，我们应用全基因表达谱芯片筛选并验证出PARP是FoxM1通路介导IM耐药的关键DNA损伤修复基因。进一步研究显示下调PARP能够抑制DNA损伤修复，增强IM诱导的K562/G01细胞凋亡，从而减弱BCR-ABL依赖的IM耐药；且PARP表达上调能够中和FoxM1抑制诱导的IM敏感性和DNA损伤增强，说明FoxM1能调控DNA损伤修复基因PARP表达，促进IM耐药。临床标本检测发现，FoxM1分子在CML患者中存在异常激活，并在CD34+白血病干/祖细胞中高表达，且FoxM1表达与CML患者对IM的治疗反应关系密切。最后，我们还利用NOD/SCID小鼠从体内探讨靶向FoxM1逆转IM耐药的可行性。本研究揭示miR-370可通过下调其靶基因FoxM1，抑制PARP表达，调控DNA损伤修复，促进LSC的存活及BCR-ABL依赖的IM耐药。从FoxM1这一新视点，明确了miR-370-FoxM1-PARP调控轴在CML白血病干细胞及IM耐药中的重要地位。此原创性研究揭示靶向CML 干细胞miR-370-FoxM1-PARP通路有望成为逆转IM耐药治疗的新策略，为克服CML耐药及清除微小残留病提供靶向治疗的新思路，对白血病的临床治疗具有重大的参考价值。