Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and is involved in polyamine transportation. We identified SLC3A2 as a potential Anaplastic Lymphoma Kinase (ALK) interacting partner in a BioID-proximity labeling screen in neuroblastoma (NB) cells. In this work we show that endogenous SLC3A2 and ALK interact in NB cells and that this SLC3A2:ALK interaction was abrogated upon treatment with the ALK inhibitor lorlatinib. We show here that loss of ALK activity leads to decreased SLC3A2 expression and reduced SLC3A2 protein stability in a panel of NB cell lines, while stimulation of ALK with ALKAL2 ligand resulted in increased SLC3A2 protein levels. We further identified MARCH11, an E3 ligase, as a regulator of SLC3A2 ubiquitination downstream of ALK. Further, knockdown of SLC3A2 resulted in inhibition of NB cell growth. To investigate the therapeutic potential of SLC3A2 targeting, we performed monotreatment of NB cells with AMXT-1501 (a polyamine transport inhibitor), which showed only moderate effects in NB cells. In contrast, a combination lorlatinib/AMXT-1501 treatment resulted in synergistic inhibition of cell growth in ALK-driven NB cell lines. Taken together, our results identify a novel role for the ALK receptor tyrosine kinase (RTK), working in concert with the MARCH11 E3 ligase, in regulating SLC3A2 protein stability and function in NB cells. The synergistic effect of combined ALK and polyamine transport inhibition shows that ALK/MARCH11/SLC3A2 regulation of amino acid transport is important for oncogenic growth and survival in NB cells.

溶质载体家族 3，成员 2（SLC3A2 或 4F2hc）是一种多功能糖蛋白，可介导整合素依赖性信号传导，充当氨基酸转运蛋白的运输伴侣，并参与多胺运输。我们在神经母细胞瘤 (NB) 细胞的 BioID 邻近标记筛选中将 SLC3A2 鉴定为潜在的间变性淋巴瘤激酶 (ALK) 相互作用伙伴。在这项工作中，我们证明内源性 SLC3A2 和 ALK 在 NB 细胞中相互作用，并且这种 SLC3A2:ALK 相互作用在用 ALK 抑制剂 lorlatinib 治疗后被消除。我们在此表明​​，在一组 NB 细胞系中，ALK 活性的丧失会导致 SLC3A2 表达减少并降低 SLC3A2 蛋白稳定性，而用 ALKAL2 配体刺激 ALK 会导致 SLC3A2 蛋白水平增加。我们进一步确定了 MARCH11（一种 E3 连接酶）作为 ALK 下游 SLC3A2 泛素化的调节因子。此外，SLC3A2 的敲低导致 NB 细胞生长受到抑制。为了研究 SLC3A2 靶向的治疗潜力，我们用 AMXT-1501（一种多胺转运抑制剂）对 NB 细胞进行单一治疗，但对 NB 细胞仅显示出中等效果。相比之下，lorlatinib/AMXT-1501 联合治疗可协同抑制 ALK 驱动的 NB 细胞系的细胞生长。综上所述，我们的结果确定了 ALK 受体酪氨酸激酶 (RTK) 与 MARCH11 E3 连接酶协同作用，在调节 NB 细胞中 SLC3A2 蛋白稳定性和功能方面的新作用。 ALK 和多胺转运抑制联合的协同效应表明 ALK/MARCH11/SLC3A2 对氨基酸转运的调节对于 NB 细胞中致癌生长和存活很重要。