Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients

Abstract Background: Tregs trafficking is controlled by CXCR4. In Renal Cell Carcinoma (RCC), the new CXCR4 antagonist R54 was explored in peripheral blood Tregs isolated from primary RCC patients. Methods: PB-Tregs were isolated from 77 RCC patients and 38 healthy donors-(HD) and CFSE-Tregs suppression assay, IL-35 secretion and Nrp-1+Tregs frequency was conducted. PB-Tregs were analyzed for PTEN, CD25, TGF-β1, FOXP3, DNMT1 expression. PTEN-pAKT signaling was evaluated in the presence of R54 and/or triciribine (TCB), Akt inhibitor. TSDR (Treg-specific Demethylated Region) methylation analysis was conducted. Results: Ex vivo R54 impaired PB-RCC-Tregs function, reduced IL-35 release and Nrp-1+Tregs. As PTEN and CD25 expression significantly decreased in R54-PB-RCC-Tregs, PTEN signaling was evaluated. While CXCL12 recruited PTEN+CD25+Tregs cells, R54 significantly reduced it in PB-RCC-Tregs. Tregs activation through IL-2/PMA impaired pAKT+Tregs while R54 increased it. The Akt inhibitor, triciribine, prevented the pAKT+Tregs-R54 induction. As active Tregs present demethylated Treg-specific region (TSDR), a significant decrease in demethylation rate (DMR) of Foxp3-TSDR was observed in R54 treated PB-RCC-Tregs with significant reduction in DNMT1 and FOXP3 expression. Conclusion: R54 affects Tregs function in primary RCC patients targeting PTEN/PI3K/AKT pathway, reducing TSDR demethylation and thus Foxp3 and DNMT1 expression. CXCR4 Tregs targeting may improve the efficacy on RCC tumor microenvironment.