Abstract 2463: Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients
Authors: Rea Giuseppina , Santagata Sara , Bello Anna Maria , Capiluongo Anna , Napolitano Maria , Desicato Sonia , Fragale Alessandra , D'Alterio Crescenzo , Trotta Anna Maria , Ieranò Caterina , Portella Luigi , Napoli Marilena Di , Maro Salvatore Di , Feroce Florinda , Azzaro Rosa , Gabriele Lucia , Longo Nicola , Pignata Sandro , Perdonà Sisto , Scala Stefania 
Published: March 2024

Abstract
Purpose: In Renal Cell Carcinoma (RCC) a crucial role is played by tumor microenvironment (TME). The high presence of T regulatory cells (Tregs) in TME cause a suppressive environment that promotes the tumor growth and its treatments failure. Tregs trafficking is controlled by CXCR4. In RCC, the new CXCR4 antagonist R54 was explored in peripheral blood Tregs isolated from primary RCC patients.
Experimental Design: To investigate the mechanism by which the new CXCR4 antagonist R54 impairs Tregs activity, peripheral blood (PB) Tregs were explored on primary RCC patients. 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.
Result: 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. As consequence of TSDR modulation, the expression of Tregs regulating genes was evaluated with significant reduction in DNMT1 and FOXP3 expression. Taken together, our findings demonstrated that R54 causes an impairment of peripheral Tregs functionality in primary RCC patients through regulation of PTEN/PI3K/AKT pathway, resulting in impaired Tregs activity.
Conclusion: The CXCR4 antagonist R54 affects Tregs function in primary RCC patients through regulation of PTEN/PI3K/AKT pathway, reduction in TSDR demethylation and Foxp3 and DNMT1 expression. CXCR4 targeting is thus a strategy to inhibit Tregs activity contributing to the CXCR4 function in RCC tumor microenvironment.
Citation Format: Giuseppina Rea, Sara Santagata, Anna Maria Bello, Anna Capiluongo, Maria Napolitano, Sonia Desicato, Alessandra Fragale, Crescenzo D'Alterio, Anna Maria Trotta, Caterina Ieranò, Luigi Portella, Marilena Di Napoli, Salvatore Di Maro, Florinda Feroce, Rosa Azzaro, Lucia Gabriele, Nicola Longo, Sandro Pignata, Sisto Perdonà, Stefania Scala. Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2463.