Dziubek Katarzyna, Faktor Jakub, Lokhande Kiran Bharat, Shrivastava Ashish, Papak Ines, Chrusciel Elzbieta, Pilch Magdalena, Hupp Theodore, Marek-Trzonkowska Natalia, Singh Ashutosh, Parys Maciej, Kote Sachin
International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland.
Department of Life Sciences, Translational Bioinformatics and Computational Genomics Research Lab, Shiv Nadar Institution of Eminence, Gautam Buddha Nagar, UP, India.
Cell Commun Signal. 2024 Dec 18;22(1):605. doi: 10.1186/s12964-024-01935-w.
The PD-1/PDL-1 immune checkpoint inhibitors revolutionized cancer treatment, yet osteosarcoma remains a therapeutic challenge. In some types of cancer, PD-1 receptor is not solely expressed by immune cells but also by cancer cells, acting either as a tumor suppressor or promoter. While well-characterized in immune cells, little is known about the role and interactome of the PD-1 pathway in cancer. We investigated PD-1 expression in human osteosarcoma cells and studied PD-1 protein-protein interactions in cancer. Using U2OS cells as a model, we confirmed PD-1 expression by western blotting and characterized its intracellular as well as surface localization through flow cytometry and immunofluorescence. High-throughput analysis of PD-1 interacting proteins was performed using a pull-down assay and quantitative mass spectrometry proteomic analysis. For validation and molecular modeling, we selected tyrosine kinase receptor AXL-a recently reported cancer therapeutic target. We confirmed the PD-1/AXL interaction by immunoblotting and proximity ligation assay (PLA). Molecular dynamics (MD) simulations uncovered binding affinities and domain-specific interactions between extracellular (ECD) and intracellular (ICD) domains of PD-1 and AXL. ECD complexes exhibited strong binding affinity, further increasing for the ICD complexes, emphasizing the role of ICDs in the interaction. PD-1 phosphorylation mutant variants (Y223F and Y248F) did not disrupt the interaction but displayed varying strengths and binding affinities. Using bemcentinib, a selective AXL inhibitor, we observed reduced binding affinity in the PD-1/AXL interaction, although it was not abrogated. To facilitate the future translation of this finding into clinical application, we sought to validate the interaction in canine osteosarcoma. Osteosarcoma spontaneously occurs at significantly higher frequency in dogs and shares close genetic and pathological similarities with humans. We confirmed endogenous expression of PD-1 and AXL in canine osteosarcoma cells, with PD-1/AXL interaction preserved in the dog cells. Also, the interacting residues remain conserved in both species, indicating an important biological function of the interaction. Our study shed light on the molecular basis of the PD-1/AXL interaction with the implication for its conservation across species, providing a foundation for future research aimed at improving immunotherapy strategies and developing novel therapeutic approaches.
PD-1/PDL-1免疫检查点抑制剂彻底改变了癌症治疗方式，但骨肉瘤仍然是一个治疗难题。在某些类型的癌症中，PD-1受体不仅由免疫细胞表达，癌细胞也会表达，其作用既可以是肿瘤抑制因子，也可以是肿瘤促进因子。虽然在免疫细胞中对其已有充分了解，但关于PD-1通路在癌症中的作用和相互作用组却知之甚少。我们研究了人骨肉瘤细胞中PD-1的表达，并研究了癌症中PD-1的蛋白质-蛋白质相互作用。以U2OS细胞为模型，我们通过蛋白质印迹法确认了PD-1的表达，并通过流式细胞术和免疫荧光对其细胞内以及表面定位进行了表征。使用下拉分析法和定量质谱蛋白质组学分析对PD-1相互作用蛋白进行了高通量分析。为了进行验证和分子建模，我们选择了酪氨酸激酶受体AXL——一种最近报道的癌症治疗靶点。我们通过免疫印迹法和邻近连接分析（PLA）证实了PD-1/AXL相互作用。分子动力学（MD）模拟揭示了PD-1和AXL的细胞外（ECD）和细胞内（ICD）结构域之间的结合亲和力和结构域特异性相互作用。ECD复合物表现出很强的结合亲和力，ICD复合物的结合亲和力进一步增加，强调了ICD在相互作用中的作用。PD-1磷酸化突变体变体（Y223F和Y248F）并未破坏相互作用，但显示出不同的强度和结合亲和力。使用选择性AXL抑制剂贝美替尼，我们观察到PD-1/AXL相互作用中的结合亲和力降低，尽管并未消除。为了促进这一发现未来转化为临床应用，我们试图在犬骨肉瘤中验证这种相互作用。骨肉瘤在犬类中自发发生的频率明显更高，并且与人类在基因和病理上有密切的相似性。我们证实了犬骨肉瘤细胞中PD-1和AXL的内源性表达，犬类细胞中保留了PD-1/AXL相互作用。此外，相互作用的残基在两个物种中都保持保守，表明这种相互作用具有重要的生物学功能。我们的研究揭示了PD-1/AXL相互作用的分子基础及其在物种间的保守性，为未来旨在改进免疫治疗策略和开发新治疗方法的研究奠定了基础。
