新研究表明c-kit/PDGFRA基因二次突变等基因组不稳定性是导致胃肠道间质瘤(GIST)酪氨酸激酶抑制剂继发耐药(TSR)的分子基础，并明确DNA损伤反应(DDR)是维护基因组稳定的基石。前期，我们在TSR临床标本中检测到缺氧诱导因子-1a(HIF-1a)高表达，并证实其能促进TSR克隆形成、减少细胞凋亡、增加基因突变频率；重要的是，HIF-1a及其下游分子FOXO3a抑制GIST细胞DDR功能执行蛋白磷酸化。据此我们提出TSR新机制：缺氧环境下，HIF-1a上调FOXO3a表达、抑制DDR损伤激活和修复功能执行，诱发GIST基因组不稳定，进而形成TSR。为验证该机制，本项目将逐步分析HIF-1a对FOXO3a、FOXO3a对DDR功能执行的作用机制；并通过TSR体内外模型，明确通路关键基因对TSR的调控作用。通过此研究，力图阐明TSR形成机制，为临床开展GIST新诊疗方案提供依据。

Genomic instability, such as secondary mutations of c-kit/PDGFRA gene abnormal amplification, has been found to be the key driver for secondary resistance of tyrosine kinase inhibitor (TSR). Furthermore, more and more studies have confirmed that the inhibition of DNA damage response (DDR) is related to the development of genomic instability and indefinite cell proliferation. In our preliminary study, we found the increased expression of hypoxia-inducible factor-1a (HIF-1a) in TSR GIST samples, and HIF-1a could increase TSR clone and gene mutation frequency and decrease apoptosis. Importantly, HIF-1a and its downstream molecular FOXO3a could decrease the phosphorylation of several DDR related protein. Accordingly, we hypothesized a new mechanism for TSR: HIF-1a, through up-regulating the expression of FOXO3a, inhibits the function of activation and repair of DDR, thus resulting in genomic instability and TSR. To test this hypothesis, we will take GIST cell line as the carrier, and explore, step by step, the transcriptional regulation mechanism of HIF-1a on FOXO3a，and the protein and protein interaction between FOXO3a and DDR related proteins; additionally, by using in vitro and in vivo model, we will investigate the role of HIF-1a/FOXO3a/DDR pathway in the formation of TSR. Through this research, we will try to clarify the mechamisms of TSR and supply the experimental data for GIST therapy.