Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.

间变性淋巴瘤激酶 (ALK) 是一种受体酪氨酸激酶 (RTK)，在大约 10% 的小儿神经母细胞瘤 (NB) 中发生突变。为了阐明 ALK 驱动的信号传导过程，我们采用了基于 BioID 的体内邻近标记以识别与 ALK 细胞内相互作用的分子。在存在和不存在 ALK 酪氨酸激酶抑制剂 (TKI) lorlatinib 的情况下，使用 ALKAL 配体生成和刺激表达诱导型 ALK-BirA* 融合蛋白的 NB 衍生 SK-N-AS 和 SK-N-BE(2) 细胞。LC/MS-MS 分析鉴定了多种蛋白质，包括 PEAK1 和 SHP2，它们在 NB 细胞中被验证为 ALK 相互作用物。对 ALK-SHP2 相互作用的进一步分析证实，ALK-SHP2 相互作用以及 SHP2-Y542 磷酸化依赖于 ALK 激活。使用 SHP2 抑制剂 SHP099 和 RMC-4550 可抑制 ALK 驱动的 NB 细胞中的细胞生长。此外，我们注意到 ALK 和 SHP2 联合抑制的强烈协同作用，这是 ALK 驱动的 NB 细胞所特有的，