放射治疗减少/延缓T790M介导的EGFR-TKI获得性耐药的分子机制研究

Acquired resistance is an important problem which limits the application of EGFR-TKI in advanced non-small cell lung cancer, how to reduce, delay or even reverse TKI resistance has become a pressing problem of the day. In clinic, EGFR mutation at T790M accounts for more than 50% of acquired resistance to TKI and T790M-mediated disease progression is more likely to occur in lung/pleura and lymph nodes than in more distant sites, indicating an opportunity for delivering thoracic radiotherapy. More supporting evidence showed integration of radiotherapy during TKI treatment prolonged progression-free survival in patients that respond to TKI; in pre-clinic, EGFR-TKI resistant cells with T790M exhibited enhanced sensitivity to radiation, suggesting administrating radiotherapy in relative early stage may help reduce or delay the resistance by decreasing the proportion of tumor cells carrying T790M or susceptible to acquired T790M. To gain insight about the promise of radiotherapy in reduction and delay of T790M-mediated EGFR TKI resistance, we aim to observe the emerging time of T790M and its proportion during chronic exposure to gefitinib in TKI-sensitivity cell lines, and to evaluate the anti-tumor effect of early radiation combined with gefitinib in nude mice xenograft model with different proportion of T790M. The success of this study will contribute to the molecular mechanisms of T790M-mediated EGFR TKI resistance, but also provide a new therapeutic strategy for patients in advanced non-small cell lung cancer to aid expansion of the effectiveness of TKI treatment through radiotherapy.

获得性耐药限制了晚期肺癌患者应用EGFR-TKI的价值，如何减少、延缓或逆转TKI耐药是目前临床亟待解决的难题。临床50％以上TKI耐药要归因于T790M突变，这些患者多表现为胸部局限性病灶进展，提示放疗干预有参与价值。临床资料显示早期放疗可以延长TKI治疗有效患者的疾病无进展生存时间；基础研究表明携带T790M突变的耐药细胞株对放疗敏感性高，因此我们推测放疗有可能杀死肿瘤组织中少量的携带有T790M突变的耐药细胞或易于发生T790M获得性突变的细胞，从而延缓或减少TKI 耐药的发生。本研究拟通过观察不同放疗剂量及不同时间点放疗后，T790M突变在EGFR-TKI敏感细胞株持续gefitinib诱导过程中出现的时间分布和在肿瘤细胞群中所占比例特征，评估放疗干预对携带不同比例T790M突变耐药小鼠肿瘤的控制效果，从而探讨放疗减少或延缓T790M突变介导的TKI获得性耐药的分子机制。

获得性耐药限制了晚期肺癌患者应用 EGFR-TKI 的价值，临床50％以上TKI耐药要归因于T790M突变，这些患者多表现为胸部局限性病灶进展，提示放疗干预有参与价值。临床资料显示早期放疗可以延长TKI治疗有效患者的疾病无进展生存时间；基础研究表明携带T790M突变的耐药细胞株对放疗敏感性高，因此我们推测放疗有可能杀死肿瘤组织中少量的携带有T790M突变的耐药细胞或易于发生T790M获得性突变的细胞，从而延缓或减少TKI耐药的发生。本研究通过观察不同放疗剂量及不同时间点放疗后，T790M突变在EGFR-TKI敏感突变细胞株持续gefitinib诱导过程中出现的时间分布和在肿瘤细胞群中所占比例特征，评估放疗干预对携带不同比例T790M突变耐药小鼠肿瘤的控制效果，从而探讨放疗减少或延缓T790M突变介导的TKI获得性耐药的分子机制。本研究首先通过gefitinib逐步加量建立了肺癌TKI耐药细胞株，在单纯TKI持续诱导和联合放疗监测T790M突变水平，发现放疗干预可以减少T790M突变的丰度，并且T790M突变细胞具有更高的放疗敏感性。接着以不同比例T790M突变细胞建立皮下肿瘤小鼠模型给予放疗处理，用于模拟TKI耐药后肿瘤进展的不同阶段（T790M突变比例随进展上升），在TKI使用的同时联用放射治疗证实了放疗对TKI耐药的延缓/减少作用。课题进一步采用microRNA和LncRNA芯片比较子母细胞株，发现miR-1243和LncRNA MIR31HG差异最显著的基因之一。敲除miR-1243后放疗敏感性增加，流式显示与凋亡增加有关，后续蛋白验证发现可能与下游PEG10蛋白有关。本课题体内外实验证实了放疗减少延缓T790M突变丰度，为临床TKI治疗早期应用放疗提供了理论依据，为晚期非小细胞肺癌患者提供了新策略，最大化了TKI的治疗效果，具有较好的社会经济效益。本项目初步探索了T790M突变细胞放疗敏感性增加的机制，丰富了TKI获得性耐药的分子机制理论，并为后续进一步研究提供了方向。