Abstract 1680: The immune checkpoint metabolome and its relationship with choline metabolism

Abstract

Abstract Introduction: Immune checkpoint inhibition has emerged as an exciting treatment option for several cancers. New insights into the role of immune checkpoints in cellular metabolism can be used to optimize their effectiveness in treatment. Programmed cell death-1 ligand (PD-L1) is an immune checkpoint overexpressed in cancers that has been successfully exploited for immune therapy. Here we investigated the relationship between the aberrant choline metabolism observed in most cancers and PD-L1 expression in triple-negative human MDA-MB-231 breast cancer cells, and characterized the metabolic effects of PD-L1 downregulation. Methods: Experiments were performed with MDA-MB-231 cells transiently transfected with small interfering RNA (siRNA) against either luciferase (control siRNA), choline kinase-α (Chk-α), or PD-L1, following standard protocols. Total RNA was isolated, complementary cDNA synthesized, and quantitative real-time PCR performed using IQ SYBR Green supermix and gene-specific primers. Cell extracts were obtained using a dual-phase extraction method and analyzed by high resolution 1H magnetic resonance spectroscopy. 3 to 6 independent experiments were performed for each group. Results: Silencing of Chk-α resulted in a significant increase of PD-L1 mRNA expression and silencing of PD-L1 resulted in a significant increase of Chk-α mRNA expression. These increases were not observed when both PD-L1 and Chk-α were silenced. Transfection with control siRNA did not alter Chk-α mRNA, but induced a small increase of PD-L1 mRNA compared to untreated cells. Transfection of cells with Chk-α and PD-L1 siRNA resulted in a significant decreases of Chk-α and PD-L1 mRNA. Consistent with the mRNA results, a significant increase of phosphocholine (PC) was observed in spectra obtained from cells transfected with PD-L1 siRNA compared to cells transfected with control siRNA, confirming that Chk-α increased in these cells. A significant increase of glutamate, glutathione, alanine and lactate was also observed in cells transfected with PD-L1 siRNA. As expected, cells transfected with a combination of Chk-α and PD-L1 siRNA showed a significant decrease of PC due to downregulation of Chk-α. Discussion: Our data have identified, for the first time, the inverse association between PD-L1 and Chk-α expression following downregulation. These data suggest that treatments that decrease Chk-α and PC could result in cancer cells escaping immune surveillance through increased expression of PD-L1. On the other hand, these cells may become more susceptible to checkpoint inhibitors such as anti-PD-L1 or anti-PD-1 antibodies. PD-L1 downregulation also significantly increased glutathione that acts as a major antioxidant. Our ongoing studies are characterizing the effects of anti-PD-L1 antibody treatment on Chk-α expression. Supported by NIH R35CA209960 and R01CA82337, and the Martin Escudero Foundation and Emerson Foundation. Citation Format: Jesus Pacheco-Torres, Marie-France Penet, Yelena Mironchik, Sridhar Nimmagadda, Balaji Krishnamachary, Zaver M. Bhujwalla. The immune checkpoint metabolome and its relationship with choline metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1680.