BACKGROUND:Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined.METHODS:Using human induced pluripotent stem cells–derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity.RESULTS:Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector—ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD⁺ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell–derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro.CONCLUSIONS:Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

中文翻译：

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综合应激反应增强帕纳替尼引起的心脏毒性

背景：线粒体功能障碍是心脏收缩衰竭的主要驱动因素。然而，线粒体能量学和信号调节之间的相互影响仍然模糊。帕纳替尼是一种用于治疗慢性粒细胞白血病的酪氨酸激酶抑制剂，是最具心脏毒性的酪氨酸激酶抑制剂之一，会导致线粒体功能障碍。帕纳替尼诱导的线粒体功能障碍是否会触发综合应激反应（ISR）从而诱导帕纳替尼诱导的心脏毒性仍有待确定。 方法：使用人类诱导多能干细胞来源的心肌细胞和最近开发的帕纳替尼诱导的心脏毒性小鼠模型，我们进行了通过蛋白质组学分析、分子和生化分析来研究 ponatinib 诱导的线粒体应激与 ISR 之间的关系及其在促进 ponatinib 诱导的心脏毒性中的作用。结果：蛋白质组学分析显示 ponatinib 激活心脏细胞中的 ISR。我们将 GCN2（一般控制非去阻遏蛋白 2）确定为 eIF2α（真核翻译起始因子 2α）激酶，负责在接触 ponatinib 后传递线粒体应激信号以触发主要 ISR 效应子 ATF4（激活转录因子 4）。从机制上讲，ponatinib 治疗对 ATP 合酶活性产生抑制作用，并降低其表达水平，导致 ATP 缺乏。线粒体功能紊乱导致 ATP 缺乏，然后触发 GCN2 介导的 ISR 激活，而补充烟酰胺单核苷酸（一种 NAD ⁺前体）可以抵消这种作用。ATP 合酶的基因抑制也激活了 GCN2。有趣的是，我们发现 ATP 丰度的减少也促进了 ponatinib 与 GCN2 的直接结合，出乎意料地导致其激活，很可能是由于其结构的构象变化。重要的是，使用 ISR 抑制剂可以保护人诱导多能干细胞衍生的心肌细胞免受普纳替尼的侵害。接受 Ponatinib 治疗的小鼠还表现出心脏功能下降，这种影响在全身 ISRIB 给药后减弱。重要的是，ISRIB 不会影响 ponatinib 的体外抗肿瘤作用。结论：中和 ISR 过度激活可以预防或逆转 ponatinib 诱导的心脏毒性。ATP 产生受损会增强 GCN2 介导的 ISR 激活这一发现对各种心脏病具有广泛的影响。我们的结果还强调了尽管 ponatinib 作为 ATP 竞争性激酶抑制剂，但它在导致激酶靶标直接激活方面发挥着意想不到的作用。