Abstract 7655: The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines
Authors: Auletta Federica , Ieranò Caterina , Rea Giuseppina , Napolitano Maria , Galdiero Francesca , Bello Anna Maria , Tassone Pierfrancesco , Di Martino Maria Teresa , Scala Stefania 
Published: March 2024

Abstract
Purpose: Renal cell carcinoma (RCC) is one of the most common malignancies in the genitourinary system. Combinations of Immunocheckpoints inhibitors plus TKIs represents the actual standard of therapy for metastatic RCC patients, while mTOR inhibitors play a role in the second-line treatment of mRCC in combination with lenvatinib. Thus characterizing resistance mechanism to mTOR inhibitors is unmet need.
Experimental Design: A498 RAD-resistant renal cell carcinoma (RCC) cells were generated through increasing doses of RAD001 (RAD1-5-10 μM). Gene expression was assessed by qRT-PCR. pERK and transwell migration were evaluated in A498, RAD10 and RAD10shP. PBRM1sh was obtained in RAD10 with lentiviral particles while CXCR4 transfection in RAD10 was realized using pCMV6-AC-h-CXCR4-GFP. ChIP assay was assessed using Chromata ChIP kit.
Results: Gene expression analysis revealed reduction in CXCR4/CXCR7 expression in A498 RAD 1-5-10-resistant cells (RAD1-5-10) (CXCR4:32.8, 37, 40.7-fold and CXCR7:2.3, 2.3, 2-fold, respectively), while PBRM1 and CXCL12 were overexpressed as compared to A498 (PBRM1: 2.2, 2.34 and 3.32-fold, and CXCL12: 7.2-, 5.6-, and 10-fold, respectively). The axis CXCL12-CXCR4 was functionally inhibited in RAD10 as demonstrated by inhibition in CXCL12- pERK and migration. YY1, already described as CXCR4 and CXCR7 negative regulator, was upregulated in RAD10 cells. ChIP confirmed higher binding of YY1 on CXCR4 and CXCR7 promoters in RAD10 as compared to A498. To address CXCR4 role, human CXCR4 was transfected in RAD10 cells (RAD10 CXCR4+) and RAD001 cytotoxicity was evaluated; interestingly, RAD10 CXCR4+ entirely restored RAD001 sensitivity in RAD10 cells. PBRM1 silencing in RAD10 (RAD10shP) downregulated mTOR and 4EBP1 (1.54 and 3-fold, compared to RAD10). As described, CXCR4 decreased in RAD10 and PBRM1 silencing did not further affect it, while CXCR7 increased (20-fold) in RAD10shP, indicating that PBRM1 regulates CXCR7 expression rather than CXCR4 in RAD001 resistant cells. RAD10shP migrated more towards CXCL11 than CXCL12 according to the overexpression of CXCR7 in RAD10shP cells. YY1 expression in RAD10 was higher than WT (2.6-fold) and was reduced in RAD10shP compared to RAD10 (1.6-fold) displaying a possible role of PBRM1 in regulating CXCR7 in RAD001-resistant RCC through YY1. CXCR4/CXCL12 signaling in RAD10shP cells more actively induced pERK and pAKT following CXCL12 or CXCL11-mediated as compared to RAD10 demonstrating that PBRM1 silencing restored a sensitive-like phenotype; however, PBRM1 silencing partially restored RAD001 sensitivity in RAD10 (RAD10 were 48-fold more resistant than A498, RAD10shP were 8.8-fold more resistant than A498).
Conclusion: Taken together, these results demonstrate that both PBRM1 and CXCR4 are responsible for RAD001 resistance with PBRM1 regulating CXCR7 and mTOR shedding light on the new mechanistic roles of PBRM1 in RCC
Citation Format: Federica Auletta, Caterina Ieranò, Giuseppina Rea, Maria Napolitano, Francesca Galdiero, Anna Maria Bello, Pierfrancesco Tassone, Maria Teresa Di Martino, Stefania Scala. The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines [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 7655.