Project Summary Checkpoint blockade targeting the Programmed cell Death (PD-1) / PD-Ligand1 pathway has proven effective in a range of cancers by releasing a restraint on cytotoxic T lymphocytes (CTLs). However, a concern has been that this very mechanism might predispose to cardiovascular and autoimmune diseases. Anecdotal evidence of a cardiovascular hazard has emerged and abundant data point to exacerbation of atherosclerosis in mice consequent to Pd-1 deletion. Recently, the generation of prostaglandin (PG) E2 by the sequential action of the cyclooxygenase (COX)-2 and microsomal PGE synthase enzymes, acting via its E prostanoid (EP) receptors, EP2 and EP4, has been shown also to promote lymphocyte exhaustion. This raises the possibility of a combinatorial approach to cancer with nonsteroidal anti-inflammatory drugs (NSAIDs) and checkpoint inhibitors. However, COX-2 inhibition alone confers a cardiovascular risk and may exacerbate one consequent to checkpoint blockade. Here, we address this possibility, first seeking to build on our preliminary data in mouse and human cells that there appears to be a bidirectional regulatory interaction between the PD-1 and PG pathways. Using pharmacological and genetic approaches, we will determine whether modulation of PGE2 alters lymphocyte phenotype and function and how regulation of PD-1 may influence the PGE2 biosynthetic response pathway both in mouse and human cells. We will use atherogenesis in the mouse as a surrogate for cardiovascular risk in humans (as it proved to be for NSAIDs and as it correlates to date with checkpoint inhibitors) and determine if deletion of Cox-2 accelerates and exacerbates the immuno- inflammatory atherosclerotic phenotype consequent to Pd-1 deletion. We will then determine whether alternative approaches to PGE2 suppression (deletion of the microsomal PGE Synthase [mPGES] – 1; EP blockade or inhibition of either enzyme restricted to myeloid cells) might limit or avoid the acceleration of atherosclerosis consequent to deletion of Pd-1. These studies will combine differential perturbations of the PG pathway in cells obtained from melanoma patients receiving PD-1 blockade, novel mouse models, state of the art immunophenotyping, single cell transcriptomics and mass-spectrometry based lipidomic substrate imaging and product analysis defining atherosclerotic lesions to understand a potentially serious risk of combining NSAIDs with checkpoint inhibitors. We shall also explore novel alternative approaches to suppressing PGE2 that might conserve the anti-cancer efficacy and minimize the cardiovascular risk of combinatorial therapy.