基于AKT/TPR-FGFR1通路在8p11骨髓增殖综合征中的作用及机制研究

The molecular hallmarks of 8p11 myeloproliferative syndrome (EMS) are the fusion of fibroblast growth factor receptor1 (FGFR1) and various partner genes, which result in the creation of chimeric protein with constitutive activation of the FGFR1 tyrosine kinase. However, treatment with FGFR inhibitor alone was not sufficient to prevent EMS. In our previous study, we characterized and cloned a novel fusion gene named TPR-FGFR1 from a patient of EMS with t(1;8)(q25;p11.2), which is formed from a chromosomal translocation between the long arm of chromosome 1 and the short arm of chromsome 8. Further analysis revealed that increasing levels of TPR-FGFR1 were strongly associated with the progression of EMS. Its pathogenicity was preliminarily confirmed through in vivo experiments. Moreover, in vitro results showed that this gene can activate its downstream signaling pathways and promote mouse pre-B cell proliferation. However, the activation of FGFR downstream signaling molecule, AKT, was not affected by FGFR inhibitor. Thus, we hypothesize that AKT pathway might play important role in the development and progression of EMS. In order to verify our hypothesis, we will perform series experiments in the TPR-FGFR1 fusion gene transduced IL-3 dependent rodent Ba/F3 cells and murine Lin-Sca-1+c-Kit+ progenitor cells, as well as bone marrow transduction/transplantation mouse models, through genetic and pharmacological approaches. In addition, we will test whether combining FGFR and AKT inhibitors significantly suppress tumorigenesis in transduction/transplantation mouse models. We propose that the present studies will uncover the molecular etiology of EMS induced by the TPR-FGFR1 fusion gene and provide new insights for developing a therapeutic strategy that is tailored to treat this devastating disease.

8p11骨髓增殖综合征(EMS)分子特征为成纤维细胞生长因子受体1(FGFR1)与伙伴基因融合并伴FGFR1激酶组成性激活，但FGFR抑制剂治疗效果不佳。申请人前期在伴t(1;8)(q25;p11.2)EMS患者鉴定和克隆出新融合基因TPR-FGFR1，并发现其水平增加与EMS进展关系密切，通过体内实验初步证实其致病性，体外研究显示TPR-FGFR1可激活下游信号通路并促进鼠前B细胞增殖，但使用FGFR抑制剂对FGFR1下游AKT分子无明显抑制作用。据此提出假说：AKT通路在伴TPR-FGFR1的EMS发病中起重要作用，是潜在治疗靶点。为验证假说，我们拟采用鼠Ba/F3细胞系、造血干细胞和骨髓移植模型，运用逆转录病毒转染、Western Blot等技术手段，从细胞、分子、组织及动物整体水平全面探讨AKT通路在伴TPR-FGFR1的EMS发病中的功能及作用机制，为EMS临床治疗提供新思路。

本课题组在国际首次鉴定并克隆出8p11骨髓增殖综合征（EMS）的第13种融合基因TPR-FGFR1，并从细胞水平证实其致病能力，发现使用酪氨酸激酶抑制剂（TKIs）如AZD4547可部分抑制转染TPR-FGFR1的BaF3细胞增殖，且可以完全抑制FGFR1磷酸化，但对FGFR1下游AKT分子的活化却无明显抑制作用，提示AKT通路活化并非由FGFR1激活。本项目探讨AKT信号通路在含TPR-FGFR1的EMS中的作用。qRT-PCR和Western Blot结果表明，在稳转TPR-FGFR1的Ba/F3中，PTEN在mRNA和蛋白水平均较未转染组下降，AKT及下游分子的蛋白磷酸化水平增加。将AKTsiRNA稳转至含TPR-FGFR1的Ba/F3细胞，CCK-8、流式、集落形成和Western Blot结果表明:敲除AKT或使用FGFR1抑制剂的含TPR-FGFR1的Ba/F3细胞增殖受抑、凋亡增加。AKT敲除组抑制AKT下游蛋白活化，而使用与未使用FGFR1抑制剂组AKT及其下游蛋白的磷酸化水平无明显差异。将AKT shRNA稳转至含TPR-FGFR1的Balb/c小鼠造血干细胞，再移植入Balb/c小鼠体内，结果证明敲除AKT后，含TPR-FGFR1的Balb/c小鼠存活时间较未敲除组延长、组织中髓系细胞减少、外周血髓系抗原表达减少。分别单独或联合给予TKI(NVP-BGJ398)、AKT抑制剂(GSK2141795)处理稳转TPR-FGFR1 的Ba/F3细胞，结果表明单独使用NVP-BGJ398或GSK2141795可抑制含TPR-FGFR1的BaF3细胞增殖，促进其凋亡，联合使用则可明显增强这一效应。制作含TPR-FGFR1的Balb/c小鼠模型，待移植小鼠外周血测出TPR-FGFR1，分别单独或联合给予NVP-BGJ398、GSK2141795治疗小鼠两周，发现单独使用NVP-BGJ398或GSK2141795治疗含TPR/FGFR1的Balb/c小鼠，均可延长小鼠的生存期，联合治疗延长更为显著。此外，课题组拓展研究了FGFR1相关DDX21蛋白在结肠癌增殖中的作用。本项目发现AKT信号通路在含TPR-FGFR1的EMS中存在异常激活，同时使用TKI和AKT抑制剂可显著抑制肿瘤细胞的活性、延长含TPR-FGFR1的小鼠生存期，为EMS的治疗提供了新的选择。