Abstract 2339: RNAi discovery platform to identify novel genes that prevent immune surveillance in pancreatic ductal adenocarcinoma (PDAC)

Abstract

Abstract BACKGROUND: Being one of the most treatment-resistant cancer types, pancreatic ductal adenocarcinoma (PDAC) is characterized by its ability to escape immune surveillance by developing many immunological obstacles. These include a plethora of mechanisms that either dampen immune cell functionality, or foster tumor cell resistance towards immune attack. Immunotherapeutic strategies, such as immune checkpoint blockade, have proven clinical success in many cancer entities, but showed little clinical benefit in PDAC patients, emphasizing the need to identify more key players that could radically improve immunotherapy. AIM: We aim to systematically identify the whole arsenal of tumor-associated immune modulators by performing a high-throughput RNAi screen and subsequently validate novel therapeutic targets, whose blockade could potentially enhance anti-tumor immune response in PDAC patients. METHODS: We generated a luciferase-expressing PANC-1 cell line and knocked down 2514 genes using a siRNA library. Our library includes 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. RESULTS: We identified 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 genes, 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 different tumor entities. Among our candidates, 4 hits were chosen for further validation. We confirmed the expression of our selected candidates in several tumor cell lines and assessed the siRNA on-target effect using several non-overlapping siRNA sequences targeting the same hit. Transfection of PANC-1 with different siRNA sequences showed knock-down of the target gene as assessed via qPCR. Additionally we observed increased T-cell mediated killing as measured via luciferase-based killing assay and Chromium release assay. CONCLUSION: We set up a robust and systematic method to unravel novel key players of pancreatic cancer immune surveillance. Further functional validation of our candidate genes will prove their potential to be used as relevant therapeutic targets in the clinic. Citation Format: Antonio Sorrentino, Ayse Nur Menevse, Tillmann Michels, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Valentina Volpin, Sabrina Wagner, Rienk Offringa, Michael Boutros, Philipp Beckhove. RNAi discovery platform to identify novel genes that prevent immune surveillance in pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2339.