Abstract 3224: Copper homeostasis: A new player in anti-tumor immune response
Authors: Voli Florida , Lerra Luigi , Kimpton Kathleen , Saletta Federica , Shen Sylvie , Cirillo Giuseppe , Kavallaris Maria , Vittorio Orazio 
Published: August 2019

Abstract
Immunotherapy has shown great potential for treating aggressive cancers and it is becoming the fourth and newest pillar of cancer therapy complementing surgery, cytotoxic therapy, and radiotherapy. In particular, immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown extraordinary clinical efficacy in several types of cancer.
This is because tumor cells express molecules, such as the Programmed Death Ligand 1 (PD-L1), to prevent immune cells activity (immune-evasion). The immune checkpoint protein Programmed Death receptor 1 (PD-1) expressed by lymphocytes instructs T-cells not to attack any tumor cell expressing PD-L1. Several therapies anti PD-1/PD-L1 have been approved by FDA, but concerns have been raised about their long term efficacy and safety. Therefore, there is a need for better understanding of the biology and the mechanisms regulating PD-1/PD-L1 axis, to develop different approaches to target this pathway. Drugs modulating the transcriptional and post-transcriptional regulation of PD-L1 could represent new therapeutic strategies for increasing the efficacy and reducing side effects of the current anti PD-L1 antibodies.
Copper transporter 1 (CTR-1) and copper levels are elevated in tumors and the use of copper targeting agents is currently under intense investigations. It has been also reported that copper plays a major role in the immune-system, but its activity is unclear.
In this study we demonstrated that copper plays a key role in the expression of PD-L1 in cancer cells. Tissue microarrays from neuroblastoma (NB) and glioblastoma patients showed a significant correlation between CTR-1 and PD-L1 expression (p=0.00014 and p=0.012 respectively). In vitro experiments showed that downregulation of CTR-1 caused a decrease of intracellular copper which in turn led to a downregulation of PD-L1 expression in cancer cells. On the other hand, addition of copper into the media clearly induced PD-L1 upregulation. RNA-seq analysis revealed specific pathways and candidate genes associated with tumor copper homeostasis and PD-L1 expression. Consistently, Dextran-Catechin (DC) and TEPA, drugs reducing copper, were able to downregulate PD-L1 expression in tumors. In vivo studies showed that copper lowering drugs prolonged mice survival, and ex vivo immunohistochemistry staining confirmed the downregulation of CTR1 and PD-L1 expression. In addition, 24h and 48h of DC treatments showed an increase of tumor-infiltrating CD4+ and CD8+ lymphocytes and activated Natural Killer cells (NK) cells in NB immune-competent mouse model. In conclusion, there is a strong association between PD-L1 expression and intracellular copper levels. Copper dysregulating agents reduce PD-L1 in vitro and in vivo, highlighting the possibility to enhance tumor immune surveillance by targeting intracellular copper levels. This study shows the potential utility of copper targeting drugs to improve anti-cancer immunotherapies.
Citation Format: Florida Voli, Luigi Lerra, Kathleen Kimpton, Federica Saletta, Sylvie Shen, Giuseppe Cirillo, Maria Kavallaris, Orazio Vittorio. Copper homeostasis: A new player in anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3224.