K. Hirai1, M. Lin1, S. Hoque1, M. Choi1, Y. Zhang1, G. V. Georgakis1, A. R. Sasson1, M. Gao1, J. Kim1 1Stony Brook University Medical Center,Stony Brook, NY, USA
Introduction: We have detected the immune checkpoints programmed cell death ligand 1 (PD-L1) and programmed cell death 1 (PD-1) on pancreatic cancer cells and tissues, even in the absence of immune cells. In this study we sought to determine the functional role of PD-L1/PD-1 in pancreatic cancer cells and tissues and to determine the cytotoxic efficacy when targeting the PD-L1/PD-1 axis with current FDA approved drugs.
Methods: To evaluate the function of immune checkpoints in pancreatic cancer, we used the established human pancreatic cancer cell lines AsPC-1, MIAPaCa-2, and PANC-1. With IRB approval and written informed consent, we established pancreatic adenocarcinoma patient derived organoids (PDO) from operative tissues. Initially, we exposed all pancreatic cancer cells to exogenous PD-L1 or PD-1 (1μg/mL) and examined the activation of the mitogen activated protein kinase pathway. To assess the specificity of the PD-L1-PD-1 interaction, we stably transfected PD-1 short hairpin RNA into PANC-1 cells and also used pembrolizumab, a clinically active anti-PD-1 monoclonal antibody. We utilized pancreatic cancer cells and PDOs to assess the cytotoxic efficacy of current FDA approved inhibitors of the PD-L1/PD-1 axis.
Results: Exposure of exogenously administered PD-L1 or PD-1 increased levels of ERK phosphorylation in all 3 pancreatic cancer cell lines. Using stably transfected PD-1 shRNA in PANC-1 cells, we observed that ERK phosphorylation was attenuated when cells were exposed to PD-L1. Additionally, pretreatment of the 3 pancreatic cancer cell lines to pembrolizumab (anti-PD-1) inhibited ERK phosphorylation. Altogether, these studies indicate that the specific PD-L1-PD-1 interaction results in activation of MAPK signaling. Using pancreatic cancer cells and successfully created PDOs, we sought to determine treatment efficacy using FDA approved immune checkpoint inhibitors [nivolumab, NIV (anti-PD-1); pembrolizumab, PEM (anti-PD-1); and atezolizumab, ATE (anti-PD-L1)]. We utilized daratumumab (DARA) as a negative monoclonal antibody control. In PDOs, we also assessed trametinib (TRAM), a small molecule MAPK inhibitor. We completed a cytotoxicity assay (CellTiter-Glo) in triplicate and observed considerable PDO cytotoxicity with NIV (34.1%), PEM (52.0%), and ATE (39.4%). However, greatest PDO death was observed when anti-PD1 drug (PEM) was combined with TRAM (83.2%).
Conclusion: The immune checkpoints PD-L1 and PD-1 are expressed on pancreatic cancer cells and activate oncogenic signaling pathways. Drug therapies combining immune checkpoint inhibitors with MAPK antagonists have tremendous potential for novel therapeutic advances in pancreatic cancer.
