背景:细胞功能取决于蛋白质运输的精心编排,在空间和时间上。这个过程的核心是逆行贩运,负责将蛋白质靶向细胞核。尽管它与许多疾病有关,胶质母细胞瘤(GBM)逆行贩运的意义尚不清楚.
方法:为了确定TMZ抗性的遗传驱动因素,我们进行了全面的CRISPR基因敲除筛选,揭示ADP-核糖基化因子4(ARF4),逆行贩运的监管机构,作为主要贡献者。
结果:抑制ARF4可显著增强GBM患者来源的异种移植(PDX)模型中的TMZ敏感性,导致提高生存率(p<0.01)在原发和复发系。我们还观察到TMZ暴露刺激ARF4介导的逆行贩运。不同ARF4水平GBM细胞的蛋白质组学分析揭示了该通路对EGFR信号的影响。在ARF4过表达和TMZ处理的细胞中观察到EGFR的核运输增加。此外,GBM患者组织的空间分辨RNA测序显示ARF4与关键核EGFR(nEGFR)下游靶标之间存在实质性相关性,比如MYC,STAT1和DNA-PK。DNA-PK活性降低,nEGFR信号下游的DNA修复蛋白,有助于TMZ抗性,在ARF4水平受抑制的细胞中观察到。值得注意的是,用DNA-PK抑制剂治疗,KU57788,在具有复发性PDX系的小鼠中导致延长的生存期(p<0.01),强调靶向蛋白质依赖于ARF4介导的逆行贩运的有希望的治疗意义。
结论:我们的研究结果表明,ARF4介导的逆行贩运有助于TMZ耐药性的发展,巩固这一途径作为克服GBM化学耐药性的可行策略。
BACKGROUND: Cellular functions hinge on the meticulous orchestration of protein transport, both spatially and temporally. Central to this process is retrograde trafficking, responsible for targeting proteins to the nucleus. Despite its link to many diseases, the implications of retrograde trafficking in glioblastoma (GBM) are still unclear.
METHODS: To identify genetic drivers of TMZ resistance, we conducted comprehensive CRISPR-knockout screening, revealing ADP-ribosylation factor 4 (ARF4), a regulator of retrograde trafficking, as a major contributor.
RESULTS: Suppressing ARF4 significantly enhanced TMZ sensitivity in GBM patient-derived xenograft (PDX) models, leading to improved survival rates (P < .01) in both primary and recurrent lines. We also observed that TMZ exposure stimulates ARF4-mediated retrograde trafficking. Proteomics analysis of GBM cells with varying levels of ARF4 unveiled the influence of this pathway on EGFR signaling, with increased nuclear trafficking of EGFR observed in cells with ARF4 overexpression and TMZ treatment. Additionally, spatially resolved RNA-sequencing of GBM patient tissues revealed substantial correlations between ARF4 and crucial nuclear EGFR (nEGFR) downstream targets, such as MYC, STAT1, and DNA-PK. Decreased activity of DNA-PK, a DNA repair protein downstream of nEGFR signaling that contributes to TMZ resistance, was observed in cells with suppressed ARF4 levels. Notably, treatment with DNA-PK inhibitor, KU-57788, in mice with a recurrent PDX line resulted in prolonged survival (P < .01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking.
CONCLUSIONS: Our findings demonstrate that ARF4-mediated retrograde trafficking contributes to the development of TMZ resistance, cementing this pathway as a viable strategy to overcome chemoresistance in GBM.