ALKBH5 enhances efficiency of anti-PD-1/PD-L1 therapy by reducing Lnc-XIST/miRNA- 124-3p mediated FGL1 expression in bladder cancer

Abstract

Abstract Background Fibrinogen-like 1 (FGL1), the promising inhibitory immune checkpoint, has been proved to promote immune escape and abrogate the efficacy of immunotherapy in malignancy. However, knowledge on its dynamic expression and regulatory mechanism in course of cancer immunotherapy is limited in bladder cancer (BCa). The vital roles of N6‑methyladenosine (m6A) RNA methylation have been identified in multiple biological activities, including tumor immunity. However, the roles of m6A modification on FGL1 expression and anti-tumor immunity are unknown. Methods The associations of dynamic FGL1 expression with immunotherapy, tumor-infiltrated immune cells and prognosis in BCa patients were assessed in public datasets, vitro and vivo tumor models. Furthermore, a siRNAs kit targeting m6A related genes were utilized and identified that ALKBH5 regulated the expression of FGL1. Moreover, methylated RNA immunoprecipitation (Me‑RIP), RT‑qPCR and rescue experiments were performed to validate the molecular mechanism underlying ALKBH5/Lnc-XIST axis in FGL1 expression. And the luciferase report assays were carried out to identify the sponge of Lnc-XIST with miR-124-3p and interactions between miR-124-3p and FGL1 and PD-L1. The xenograft tumor mice models were constructed to verify the anti-tumor effects of single or combined ICIs in BCa with aberrant expressions of FGL1 and ALKBH5. Results In this study, we found that high expression level of FGL1 was associated with poor immunotherapy response and prognosis in BCa. Functionally, elevated FGL1 expression reducing tumor-infiltrated CD8+ T cells and abrogated anti-tumor immunity in an immunocompetent mouse model. Furthermore, ALKBH5 knockdown significantly promoted FGL1 expression via up-regulating Lnc-XIST expression in an m6A dependent manner. Lnc-XIST was found to act as a ceRNA by sponging miR-124-3p which reversed up-regulation of FGL1 induced by ALKBH5. Further analysis identified that PD-L1 was also a downstream target of miR-124-3p in BCa. In addition, co-blockade of FGL1/LAG3 and PD-L1/PD-1 axis motivated more effective antitumor immune response in BCa with low ALKBH5 expressions. Conclusion Our study suggested that ALKBH5 regulated FGL1 expression via Lnc-XIST/ miR-124-3p axis in an m6A dependent manner and dual blockade of FGL1/LAG3 and PD-L1/PD-1 axis could significantly inhibit tumor growth in BCa with low-ALKBH5 expressions. These results will provide implications for precise and efficient therapeutic strategies in the BCa immunotherapy.