Abstract LB-118: SR8541A is a potent inhibitor of ENPP1 and exhibits dendritic cell mediated antitumor activity

Purpose: In recent years, programs targeting the innate immune pathways alone and alongside the adaptive immune pathways have become increasingly popular. A main area of interest in innate immunity is the STING (STimulator of INterferon Genes) pathway. STING plays an integral role by activating type 1 interferons in response to detection of cytosolic nucleic acid by cGAS (Cyclic GMP-AMP Synthase). cGAS detects cytosolic DNA and converts it into 2′3′ cGAMP, which is the direct ligand of STING. Targeting the direct activation of this pathway using synthetic, non-hydrolyzable forms of this ligand has shown to be effective in preclinical efficacy models, but has failed to impress in phase 1 clinical trials. Another method of activating this pathway is to inhibit the direct negative regulator, ENPP1 (Ectonucleotide pyrophosphatase/phosphodiesterase 1). ENPP1 constitutively hydrolyzes 2′3′-cGAMP, suppressing the pathway both in the cancer cells and in the surrounding tumor microenvironment. Previously, we reported that SR-8314, a highly selective and potent ENPP1 inhibitor, induces the activation of the STING pathway via type I interferon response in both in vitro and in vivo models. In this study, we continue this research and present our nominated clinical candidate, SR-8541A, which shows improved physiochemical characteristics and increased immune response. Methods: Direct binding of SR-8541A to ENPP1 was evaluated using a thermal shift assay. Inhibition of ENPP1 enzymatic activity was shown using TMP or ATP as substrates in either cell free or cell based assays. RT-PCR, western blots, immune infiltration using peripheral blood mononuclear cells (PBMCs), and MesoScale Disovery (MSD) ELISA assays were performed to evaluate the effect of SR-8541A on the STING pathway. ENPP1 CRISPR knockout cells were generated to demonstrate on-target activity of SR-8541A. Pharmacokinetics, stability, and selectivity were completed with outside vendors. Results: SR-8541A exhibits strong binding to ENPP1 and shows inhibitory activity at an IC50 of 1.4 nM. STING pathway activation was confirmed by a significant increase in gene expression of IFNβ, ISG15 and CXCL10 in SR-8541A treated cells. An increase in secreted type 1 interferon and other cytokines was also observed in these treated cells. Using immune infiltration assays, we show that SR-8541A stimulates the migration and infiltration of immune cells (PBMC) into cancer spheroids. Importantly, depletion of dendritic cells results in loss of infiltration of remaining PBMCs into cancer spheroids. Additionally, natural killer (NK) cells do not infiltrate into SR-8541A treated cancer spheroids unless co-cultured with dendritic cells. ENPP1 CRISPR knockout cell models confirmed that the drug effect was dependent on the presence of ENPP1. SR-8541A was stable for >60 mins in liver microsomes and S9 fractions from various species. Rodent pharmacokinetics showed a strong oral bioavailability of ~50% and tumor efficacy studies are ongoing. Conclusions: In summary, we show that SR-8541A is a potent and selective inhibitor of ENPP1 that displayed strong immune response in 3D spheroid models. The results from these studies along with physiochemical properties and tumor model data will be presented. The promising physiochemical properties make it a strong candidate for clinical studies. Citation Format: ALEXIS S. WESTON, Trason G. Thode, Ryan Rodriguez del Villar, Srikanta Dana, Srinivas Kasibhatla, Mohan R. Kaadige, Sunil Sharma. SR8541A is a potent inhibitor of ENPP1 and exhibits dendritic cell mediated antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-118.