伴有STAT5B-RARA融合基因的急性早幼粒细胞白血病患者的耐药机制研究

Acute Promyelocytic Leukemia (APL) is a special subtype with unique clinical features in acute myeloid leukemia. APL is characterized by the generation of the PML-RARA fusion transcript as a result of a reciprocal chromosomal rearrangement, t(15;17) (q22;q21), with breakpoints within the PML gene and the RARA gene. Its presence predicts a near universal therapeutic response to two targeted agent,.all-trans retinoic acid (ATRA) and arsenic trioxide, making it one of the most successful models in leukemia treatment. However,in a small proportion of APL patients who lack the PML-RARA fusion, the RARA gene is reportedly fused with a number of alternative partner genes. These uncommonly RARA-related fusion gene mostly resistence to ATRA, especial STAT5B-RARA fusion gene. At presently, the mechanism of leukemogenesis and ATRA resistance of patients with STAT5B-RARA fusion gene remains largely unknown. In this study, we identified 4 patients harboring STAT5B-RARA fusion transcript by array CGH and RT-PCR analysis in 1188 patients with newly diagnosed APL. Interesting, patients with STATRB-RARA fusion gene displayed typical APL clinical, morphological and immunological features, wherase unresponsive to both ATRA and arsenic trioxide regimen in the clinical treatment, suggesting that they are different from the typical PML-RARA positive patients in pathogenesis and molecular mechanism. To date, the mechanism of pathogenesis and ATRA resistence are still unclear.We plan to determine the role of STAT5B-RARA and Polycomb group in leukemogenesis and ATRA resistence, and explore the value of the inhibitory drugs with JAK/STAT signal pathway in the treatment of this variant APL with STAT5B-RARA fusin gene by using multiple in vivo and in vitro techniques, such as liquid culture, colony formation assay, cell differentiation assay, ATRA-sensitive experiment, flow cytometry, gene transfection, co-immunoprecipitation chromatin immunoprecipitation and tumor formation in nude mice. This study will further reveal the pathogenesis of APL and provide theoretical and experimental evidences for seek new targets for this variant APL therapy.

PML-RARA融合基因是急性早幼粒细胞白血病(APL)的分子标志，靶向PML-RARA药物ATRA和砷剂的应用，使得APL长期生存率超过90%。部分APL患者可具有RARA与其他对手基因形成的变异型融合基因，且多对ATRA耐药，其发病机制和治疗策略是APL研究的难点。申请人前期对1188例APL患者进行了筛选，在形态和免疫表型显示典型APL特征、PML-RARA阴性的患者中，发现4例STAT5B-RARA阳性病例，且对ATRA和砷剂高度耐药。目前STAT5B-RARA的致病及耐药机制尚不明确。本课题拟应用细胞培养、集落形成、基因转染、CHIP和裸鼠成瘤等手段，阐述STAT5B-RARA的发病机制及PcG家族在ATRA耐药中的作用机理，并探讨JAK/STAT抑制剂在STAT5B-RARA阳性APL中的治疗价值。本课题对于阐明变异型APL的发病及耐药机制、寻找新的治疗靶点提供理论和实验依据。

急性早幼粒细胞白血病（APL）是常见的急性髓细胞白血病类型是，以形成PML-RARA融合基因为分子特征，靶向PML-RARA药物维甲酸（ATRA）和砷剂的广泛应用，使得APL长期生存率超过90%。但临床上仍有一小部分患者缺乏t(15;17)(q24;q21)/PML-RARA，通过本中心1215例APL的大样本筛选，共发现4例伴有STAT5B-RARA融合基因，另有2例为新发现的国内外尚未见报道的STAT3-RARA阳性病例。通过全基因组测序和RNA转录组测序，明确了STAT5B-RARA和STAT3-RARA两种融合基因在基因组水平上的断裂位置以及融合事件的发生。通过转染细胞的增殖分化实验，发现STAT5B-RARA转细胞株具有更强的生长增殖能力，并且在ATRA处理后，并不能明显降低其增殖能力。荧光素酶活性检测RARE启动子的活性，结果显示，STAT5B-RARa可以对RARa靶基因进行转录抑制，且在ATRA处理后，并不能减弱这种转录抑制作用，提示ATRA并不能逆转STAT5B-RARa融合蛋白通过抑制RARE启动子的作用引起的耐药。免疫共沉淀检测STAT5B-RARA与PcG家族BMI蛋白的相互作用，结果发现BMI蛋白可以与STAT5B-RARa蛋白存在直接相互作用关系，且这种作用ATRA处理后并不能解除，因此，这种异常结合可能是STAT5B-RARA对ATRA的耐药机制之一。同时，对新发现的STAT3-RARA，在研究分析其临床和实验室特征的基础上，通过免疫荧光、免疫印迹、免疫共沉淀等研究方法，进一步研究其致病机制。研究结果发现STAT3-RARA融合蛋白存在异常的细胞定位，可能是伴有STAT3-RARA融合基因的APL的致病机制之一。同时STAT3-RARA能上调磷酸化的AKT 和BCL2蛋白，而导致下游靶基因激酶活性的增强和抗凋亡能力的增强。此外，与其它RAR融合蛋白相同，STAT3-RARA也能自身结合并形成同源二聚体，以促进APL的发生。总之，通过本项目的研究，进一步明确了STAT5B-RARA和STAT3-RARA阳性APL患者的发病机理和维甲酸耐药机制，希望可以为该类预后不良的APL患者提供更有效的治疗指导策略提供帮助。