Division of Cancer and Stem Cells, School of Medicine, Centre for Cancer Science, Biodiscovery Institute, University of Nottingham, Nottingham, NG2 7UH, UK.
Department of Basic Medical Science, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, 681 Samsen Road, Dusit, 10300, Bangkok, Thailand.
Cancer Immunol Immunother. 2023 Dec;72(12):4001-4014. doi: 10.1007/s00262-023-03534-z. Epub 2023 Nov 16.
Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain.
We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence.
The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab).
These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.
可变剪接的调控是癌症治疗的新方法。程序性细胞死亡受体 1（PD-1）是一种表达在免疫细胞上的免疫抑制受体，它与癌细胞表达的配体 PD-L1 和 PD-L2 结合，在肿瘤微环境中形成主要的免疫检查点途径。使用阻断抗体（nivolumab 和 pembrolizumab）靶向该途径是癌症免疫治疗的主要方法，恢复衰竭 T 细胞的功能。PD-1 可变剪接形成两种异构体，一种是跨膜信号受体（flPD1），通过与配体 PD-L1 结合介导 T 细胞死亡，另一种是可变剪接的、可溶性的、缺乏跨膜结构域的变体。
我们使用 PCR 和 Western blot 分析原发性外周血单核细胞（PBMCs）和 Jurkat T 细胞、IL-2 ELISA、流式细胞术、黑色素瘤和胆管细胞癌的共培养以及生物信息学分析和分子克隆来研究 PD1 剪接的机制及其后果。
PD-1 的可溶性形式，通过跳过外显子 3（∆Ex3PD1）产生，在 PBMCs 和 T 细胞中内源性表达，并防止癌细胞介导的 T 细胞抑制。SRSF1 的多个结合位点紧邻 PD-1 外显子 3 剪接位点。磷酸化模拟 SRSF1 的过表达导致 flPD1 的优先表达。通过 SRPK1 shRNA 敲低和选择性抑制剂 SPHINX31 抑制 SRSF1 磷酸化，导致剪接向 ∆Ex3PD1 转换。SPHINX31（相当于 pembrolizumab）逆转了胆管癌细胞对 T 细胞 IL-2 表达的抑制。
这些结果表明，通过靶向 SRPK1 从 flPD1 切换剪接决定为 ∆Ex3PD1 可能代表癌症中免疫检查点抑制的潜在新机制。
