Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry–based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI–sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis.

致癌性间变性淋巴瘤激酶（ALK）是神经母细胞瘤中少数可药物治疗的靶标之一，对靶向ALK的酪氨酸激酶抑制剂（TKIs）的治疗耐药性是不可避免的临床挑战。因此，必须更好地了解由ALK驱动的致癌信号网络重新布线，以改善和指导未来的治疗方法。在这里，我们对使用三种临床相关的ALK TKI（克唑替尼，LDK378或lorlatinib）之一或实验性使用的ALK TKI（TAE684）处理的神经母细胞瘤细胞进行了基于质谱的蛋白质组学研究，以阐明异常的ALK信号通路。我们的综合近端蛋白质组学（IPP）策略包括多个信号层，例如ALK相互作用组，磷酸酪氨酸相互作用组，磷酸化蛋白质组和蛋白质组。我们确定了信号转导蛋白IRS2（胰岛素受体底物2）是主要的ALK靶点，并且是由致癌ALK驱动的神经母细胞瘤细胞中的ALK TKI敏感信号转导节点。TKI治疗减少了IRS2向ALK的募集，并减少了IRS2的酪氨酸磷酸化。此外，siRNA介导的ALK或IRS2耗竭减少了存活促进激酶Akt和下游靶标转录因子FoxO3的磷酸化，并降低了三种ALK驱动的神经母细胞瘤细胞系的生存能力。总而言之，我们的IPP分析通过揭示IRS2作为衔接蛋白，通过Akt-FoxO3信号传导轴将ALK与成神经细胞瘤细胞存活联系起来，从而提供了对癌基因ALK信号传导近端结构的深入了解。