跨损伤DNA合成在伊玛替尼获得性耐药过程中的可能作用机制探讨

Chronic myelogenous leukemia (CML) is characterized by the presence of the t (9; 22) Philadelphia chromosome (Ph) translocation, which harbors the bcr-abl fusion gene encoding a chimeric BCR-ABL protein with a constitutive tyrosine kinase activity. The invention of the tyrosine inhibitor imatinib, which targets BCR-ABL kinase activity, has revolutionized drug therapy of chronic myelogenous leukemia. While most of the responses to Imatinib are sensitive and stable, resistance to treatment after an initial response is common in more advanced phases of the disease. The mechanisms underlying the acquired persistence of minimal residual disease are unknown. Translesion DNA synthesis (TLS) is a DNA damage tolerance pathway utilized to overcome replication blocks when unrepaired DNA lesions stall the replication machinery. TLS aids in DNA damage tolerance, introducing mutations and mediating chemoresistance. In the work, we find imatinib can upregulate TLS activities in CML cells, suggesting that TLS may mediate acquired imatinib resistance. In this project, we will focus on the detail mechanisms of TLS function in acquired resistance. We believe our results will provide new tools by targeting TLS pathway to overcome Imatinib resistance and improve cancer therapy.

慢性粒细胞性白血病主要是由t（9；22）号染色体易位产生bcr-abl融合基因（即费氏染色体）而导致的，其编码的癌蛋白BCR-ABL具有持续的酪氨酸激酶活性。靶向BCR-ABL激酶活性的抑制剂伊玛替尼的问世对慢粒的药物治疗具有革命性的意义，但问题在于后续的治疗中有不少耐药现象。目前对这种获得性耐药的机制尚不清楚。跨损伤DNA合成（Translesion DNA Synthesis，TLS）是细胞内DNA损伤耐受的重要修复机制，能在损伤位点对面插入碱基，使得DNA复制得以继续。TLS有助于DNA损伤的修复，但也可能引入突变，且在介导化疗耐药中发挥作用。我们前期结果发现，伊玛替尼能在CML细胞中引起TLS活化，提示TLS可能参与伊玛替尼的获得性耐药过程。本项目将重点研究TLS聚合酶在伊玛替尼耐药中的作用机制，旨在为克服耐药和癌症预防及治疗提供有力的理论参考。

伊玛替尼（Imatinib，IM）耐药是慢性粒细胞白血病治疗中待解决的关键问题之一。明确IM耐药机制及克服伊玛替尼耐药已成为医学界共同关注的问题。本项目主要对跨损伤DNA合成信号通路在IM耐药中的机制进行了探讨。通过免疫印迹实验，我们检测到IM引起了PCNA泛素化水平增加，通过免疫荧光实验，我们发现IM能够诱导磷酸化RPA的招募，这些结果表明IM引起了跨损伤DNA合成信号通路的活化。通过对DNA损伤标志物γH2AX检测发现，IM能够引起γH2AX增加，诱导DNA损伤。在K562细胞中干扰REV1，能够一定程度增加IM敏感性。这些结果说明跨损伤DNA合成信号通路确实参与了IM耐药的过程。为了进一步明确DNA损伤在IM耐药中的作用，我们尝试寻找DNA损伤诱导剂，检测其能否克服IM耐药。通过高通量筛选，我们发现斑螯素能有效的DNA损伤，进一步实验发现斑螯素能抑制IM耐药细胞生长和增殖，引起IM敏感和耐药株Mitosis周期抑制和细胞死亡。这些结果说明靶向DNA损伤能够克服IM耐药，是研发抗IM耐药的重要研究方向。