Abstract 4109: Functional profiling of 17,000 open reading frames identifies regulators of PDL1 expression in HNSCC

Abstract

Abstract The interaction of Programmed Death Ligand 1 (PD-L1) with the PD-1 receptor serves as an immune checkpoint, dampening anti-tumor immunity. While immunotherapies targeting this pathway (Pembrolizumab and Nivolumab), were approved for use in recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) in 2016, overall response rates are low and few biomarkers have been identified to differentiate responders. We expect a more detailed understanding of PD-L1 checkpoint regulation in HNSCC to provide insight toward addressing these deficits. Thus, we developed a high- throughput screening protocol to discover signals modulating PD-L1 expression. We have employed a lentiviral open reading frame (ORF) library for overexpression of over 14,000 different genes in a pooled format. UM-SCC-49 cells transduced with this library were stained for cell surface PD-L1 and sorted by flow cytometry to select cells with enhanced PD-L1 expression. Sequencing of the selected sub-population was performed to determine ORFs enriched over an unsorted pool. To validate a role for individual target genes in modulating PD-L1 expression, monoclonal populations were expanded from the sorted pool and evaluated for PD-L1 expression. We confirmed increased PD-L1 expression in 7 of these cell lines over wild-type and LacZ overexpressing controls via western blotting and flow cytometry. Furthermore, we selected 5 ORFs of interest overrepresented in the sorted pool for further validation using stably expressed V5 tagged constructs. Target gene expression was confirmed by western blot, and enhanced cell surface PD-L1 expression was validated via flow cytometry for 4/5 cell lines. Thus, we provide evidence for successful identification of signals regulating PD-L1 using a novel high-throughput overexpression screen. Future experiments will aim to clarify the mechanism of PD-L1 induction in response to the signals identified here, and may ultimately provide a more thorough understanding of genetic factors modulating immunosuppression and response to immunotherapy in HNSCC patients. Citation Format: Jacqueline Mann, Samantha Devenport, Aditi Kulkarni, Rebecca Hoesli, Susan Foltin, Nicole Michmerhuizen, Alexey Nesvizhskii, Chad Brenner. Functional profiling of 17,000 open reading frames identifies regulators of PDL1 expression in HNSCC [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 4109.