Abstract 245: Identification of novel immune checkpoints as potential therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) using RNAi screening
Authors: Antonio Sorrentino, Tillmann Michels, Ayse Nur Menevse, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Rienk Offringa, Michael Boutros, Philipp Beckhove
Journal: Cancer Research (2015)

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of pancreatic cancers and constitutes the fourth leading cause of cancer related death worldwide. In contrast to other malignancies, PDAC is highly resistant to chemotherapy and radiotherapy. Additionally, few immunotherapies are currently available because this malignancy was thought to be poorly immunogenic. Recent studies have shown that infiltration of immune cells in biopsies of PDAC patients correlates with improved clinical outcome. Nevertheless, tumor cells can elude the immune system through several inhibitory mechanisms including the expression of immune checkpoints. These are a plethora of molecules that can either boost or dampen the T-cell receptor (TCR) signaling. Some of these molecules, such as PD-L1, have been successfully used as therapeutic targets of novel anticancer drugs. AIM: We hypothesize that many immune checkpoint molecules on tumor cells remain undiscovered and we performed a high-throughput RNAi screen to unravel the whole arsenal of immune modulators. METHODS: We generated a luciferase-expressing PANC-1 cell line and knocked down 2514 genes using a siRNA library. Our library included G-protein coupled receptors, protein kinases and 1117 surface proteins. We co-cultured HLA-A201+ matched tumor infiltrating lymphocytes (TILs) derived from a PDAC patient with the transfected tumor cells. We then measured the remaining luciferase intensity of the tumor cells as an estimation of TIL-mediated cytotoxicity. In order to exclude genes whose knock-down affected cell viability per se, we cultivated tumor cells with the siRNA library in the absence of TILs. Data were analyzed with the cellHTS2 R package. RESULTS: Our screen revealed 155 candidate genes whose knock-down enhances TIL-mediated killing more efficiently than PD-L1 down-regulation. 35% of these genes are surface molecules and are most likely to directly mediate tumor immune evasion. Beside novel undescribed immune checkpoints, our list contains well characterized immune modulators, supporting the reliability of our approach. Of note 13 of our hits were also found in a related melanoma screen and might play a role in the regulation of immune surveillance of many solid tumors. Among our candidates, TONI1 was one of the most prominent hits. So far, we confirmed the role of TONI1 in inhibiting TIL-mediated killing both in chromium release and luciferase based kill assays. Additionally we detected increased T-cell activity upon TONI1 down-regulation, as measured with interferon-γ ELISPOT and TNF-α ELISA. Since the presented work is considered for patent protection, some gene targets are masked. CONCLUSION: We set up a robust and systematic method to identify novel immune checkpoints for pancreatic cancer. Further functional validation of our candidate genes will prove their use as therapeutic targets. Citation Format: Antonio Sorrentino, Tillmann Michels, Ayse Nur Menevse, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Rienk Offringa, Michael Boutros, Philipp Beckhove. Identification of novel immune checkpoints as potential therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) using RNAi screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 245. doi:10.1158/1538-7445.AM2015-245