Abstract 2546: The immune checkpoint PD-L1 alters choline kinase expression and metabolism in triple negative breast cancer cells

Introduction Cancer immunotherapy is designed to activate the immune system against cancer cells. This field has been revitalized with the discovery of immune checkpoints such as programmed cell death protein-1 (PD-1) and its ligand (PD-L1). PD-L1 has been exploited for immune therapy with varying success depending on the tumor type, with several studies pointing towards cancer metabolism as one of the possible causes of this variability. One of the most common tumor associated metabolic alteration is increased expression of choline kinase-α (Chk-α), characterized by increased phosphocholine (PC) and total choline-containing compounds. Since the presence of PC on proteins has been related to escape from immune surveillance, here, we investigated the relationship between Chk-α, PC and PD-L1. Methods Experiments were performed using triple negative breast MDA-MB-231 cells transiently transfected with small interfering RNA (siRNA) against Chk-α or PD-L1 following standard protocols. Total RNA was isolated, complementary cDNA synthesised and quantitative real-time PCR performed using IQ SYBR Green supermix and gene specific primers. For high resolution 1H Magnetic Resonance Spectroscopy (MRS), cell extracts were obtained using a dual-phase extraction method. 1H MRS was recorded on a Bruker Biospin Avance-III 750 MHz spectrometer. Integrals of the metabolites of interest were determined and normalized to the TSP reference and the number of cells. Metabolites were estimated from at least three experimental samples. Statistical significance was evaluated using the Student t-test. Results and Discussion We observed that silencing Chk-α resulted in a significant increase of PD-L1 expression and silencing PD-L1 resulted in a significant increase of Chk-α expression. Consistent with the molecular changes, a significant increase of PC was observed in cells transfected with PD-L1 siRNA. Metabolic changes, however, extended well beyond the choline containing compounds. We identified changes in amino acids, organic acids, nucleotides and other compounds. Furthermore, we found that PD-L1 downregulation profoundly altered the lipid profile of MDA-MB-231 cells. Metabolic characterization showed that cells with downregulated Chk-α levels shifted towards a more immunosuppressive profile through metabolic reprogramming, increasing the production of metabolites that have been linked to decreased effectiveness of T-cells. Our data also suggest that low levels of PD-L1 can skew c