Inhibition of GCN2 Reveals Synergy with Cell-Cycle Regulation and Proteostasis

The integrated stress response is a signaling network comprising four branches, each sensing different cellular stressors, converging on the phosphorylation of eIF2α to downregulate global translation and initiate recovery. One of these branches includes GCN2, which senses cellular amino acid insufficiency and participates in maintaining amino acid homeostasis. Previous studies have shown that GCN2 is a viable cancer target when amino acid stress is induced by inhibiting an ad-ditional target. In this light, we screened myriad drugs for their potential to synergize with the GCN2 inhibitor TAP20. The drug sensitivity of six cancer cell lines to a panel of 25 compounds was assessed. Each compound was then combined with TAP20 at concentrations below their IC50, and the impact on cell growth was evaluated. The strongly synergistic combinations were further characterized using synergy analyses and matrix-dependent invasion assays. Inhibitors of proteostasis and the MEK–ERK pathway, as well as the pan-CDK inhibitors, flavopiridol, and seliciclib, were potently synergistic with TAP20 in two cell lines. Among their common CDK targets was CDK7, which was more selectively targeted by THZ-1 and synergized with TAP20. Moreover, these combinations were partially synergistic when assessed using matrix-dependent invasion assays. However, TAP20 alone was sufficient to restrict invasion at concentrations well below its growth-inhibitory IC50. We conclude that GCN2 inhibition can be further explored in vivo as a cancer target.整合应激反应（integrated stress response）是一套由四个分支组成的信号网络，每个分支均可感知不同的细胞应激原，最终汇聚于eIF2α的磷酸化过程，以下调全局翻译并启动细胞修复程序。其中一个分支包含GCN2，该蛋白可感知细胞内氨基酸匮乏，并参与维持氨基酸稳态。既往研究表明，当通过抑制另一靶点诱导氨基酸应激时，GCN2是可行的癌症治疗靶点。基于此，我们对众多药物与GCN2抑制剂TAP20的协同潜力开展了筛选。我们评估了6种癌细胞系对25种化合物组成的化合物库的药物敏感性。随后，我们将每种化合物以低于其IC50的浓度与TAP20联合给药，并评估其对细胞增殖的影响。对具有强协同效应的联合用药方案，我们进一步通过协同效应分析及基质依赖性侵袭实验进行了表征。蛋白稳态（proteostasis）抑制剂、MEK–ERK通路抑制剂，以及泛CDK抑制剂夫拉平度（flavopiridol）和塞利西利（seliciclib），在两种癌细胞系中与TAP20展现出强效协同作用。这些化合物的共同CDK靶点包括CDK7，而THZ-1可更特异性地靶向CDK7，且其与TAP20亦存在协同效应。此外，通过基质依赖性侵袭实验评估发现，上述联合用药方案仅呈现部分协同效应。但在远低于其抑制细胞生长的IC50浓度下，单独使用TAP20即可有效限制细胞侵袭。综上，GCN2抑制作为癌症治疗靶点，可在体内开展进一步的探索研究。