Zapata Dongo Richard Junior, Fontana Diletta, Mologni Luca, Faya Castillo Juan Enrique, Infante Varillas Stefany Fiorella
Department of Basic Sciences, Bioethics and Human Life, Faculty of Human Medicine, University of Piura, Miraflores, Lima, Perú.
Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy.
PLoS One. 2025 Jan 21;20(1):e0308747. doi: 10.1371/journal.pone.0308747. eCollection 2025.
The anaplastic lymphoma kinase (ALK) oncoprotein plays a crucial role in non-small cell lung cancer (NSCLC) by activating signaling pathways involved in cell proliferation and survival through constitutive phosphorylation. While first-line crizotinib can regulate phosphorylation, mutations in the ALK gene can lead to resistance against ALK inhibitors (ALKi) such as ceritinib and alectinib. On the other hand, overexpression of BCL2, a protein involved in cell death regulation, has been observed in NSCLC and is considered a potential therapeutic target. In this study, we propose to inhibit BCL2 as a secondary therapeutic target in EML4-ALK cell models to overcome resistance caused by ALK mutations. Four Ba/F3 EML4-ALK cell models (WT, C1156Y, L1196M, and G1202R) generated by site-directed mutagenesis exhibited varying levels of BCL2 expression. Both the WT and G1202R models showed overexpression of BCL2, while C1156Y and L1196M models approached baseline levels. We treated these cells with ABT-199, a selective BCL2 inhibitor, and found that models with high BCL2 expression exhibited resistance, while those with lower expression showed sensitivity to BCL2 inhibition. In addition, our analysis using bioinformatics indicated that ABT-199 not only targets BCL2 but also binds to the active site of all ALK mutants, it was contrasted by in vitro ALK kinase activity inhibition by ABT-199 (5.5 μM). This interaction was further supported by a significant decrease of ALK phosphorylation in single and combination treatment with 300nM ABT-199. Finally, when ABT-199 was combined with ALKi, we observed a wide range of synergistic effects in the WT and G1202R cell models, while the C1156Y and L1196M models showed limited synergy. In conclusion, our findings indicate that BCL2 targeting with ABT-199, in combination with ALKi, can significantly reduce tumor cell survival in Ba/F3 EML4-ALK cell models.
间变性淋巴瘤激酶（ALK）癌蛋白通过组成性磷酸化激活参与细胞增殖和存活的信号通路，在非小细胞肺癌（NSCLC）中发挥关键作用。虽然一线克唑替尼可以调节磷酸化，但ALK基因的突变可导致对色瑞替尼和阿来替尼等ALK抑制剂（ALKi）产生耐药性。另一方面，在NSCLC中观察到参与细胞死亡调节的蛋白质BCL2的过表达，其被认为是一个潜在的治疗靶点。在本研究中，我们提议在EML4-ALK细胞模型中抑制BCL2作为次要治疗靶点，以克服由ALK突变引起的耐药性。通过定点诱变产生的四种Ba/F3 EML4-ALK细胞模型（野生型、C1156Y、L1196M和G1202R）表现出不同水平的BCL2表达。野生型和G1202R模型均显示BCL2过表达，而C1156Y和L1196M模型接近基线水平。我们用选择性BCL2抑制剂ABT-199处理这些细胞，发现BCL2高表达的模型表现出耐药性，而那些表达较低的模型对BCL2抑制敏感。此外，我们使用生物信息学的分析表明，ABT-199不仅靶向BCL2，还与所有ALK突变体的活性位点结合，这与ABT-199（5.5μM）对体外ALK激酶活性的抑制形成对比。在300nM ABT-199的单药和联合治疗中，ALK磷酸化的显著降低进一步支持了这种相互作用。最后，当ABT-199与ALKi联合使用时，我们在野生型和G1202R细胞模型中观察到广泛的协同效应，而C1156Y和L1196M模型显示出有限的协同作用。总之，我们的研究结果表明，用ABT-199靶向BCL2并与ALKi联合使用，可以显著降低Ba/F3 EML4-ALK细胞模型中的肿瘤细胞存活率。
Zotero 插件
