Abstract 702: A novel small molecule inhibitor of ENPP1 promotes T and NK cell activation and enhances anti-tumor efficacy in combination with immune checkpoint blockade therapy
Authors: Yoon Pyoung Oh , Kim Juhyeon , Lee A-Ram , Son Eunji , Lee Young cheol , Jung Eunmi , Kim HyeongRae , Lee YeonHee , Park SoEun , Lee Dae-Yon , Chung Chul-Woong 
Published: April 2023

Abstract
Background: The cGAS-STING signaling pathway has led to significant anti-cancer innate immune responses in cancer immunotherapy. 2’3’-cGAMP binds to STING and promotes the production of various pro-inflammatory cytokines such as type I interferon, which changes cold tumors to hot tumors through modulating tumor microenvironment. Therefore, activation of STING pathway has been considered as an important target for cancer immunotherapy. Ecto-Nucleotide Pyrophosphatase/Phosphodiesterases 1 (ENPP1), highly expressed membrane-bound enzyme in cancer cells, modulates cGAMP levels by hydrolyzing 2’,3’-cGAMP to alter the inflammatory mileu. In this study, we show that targeting ENPP1 is a promising strategy for STING regulation in cancer immunotherapy through indirect activation of the STING pathway.
Methods: The ENPP1 inhibitory activities were measured by ENPP1 enzyme assay using both pNP-TMP and cGAMP as substrates. Cell-based activities were assessed by measuring IFN-beta release using THP-1 dual reporter assay. To validate biological functions, we evaluated cancer-cell killing effects of immune cells in co-culture spheroid system and T cell proliferation assay. The in vivo anti-tumor efficacy of our compound was assessed by monitoring tumor growth in CT-26 syngeneic mouse model.
Results: LCB33 ENPP1 inhibitor shows excellent ENPP1 inhibitory activity at an IC50 of 0.9pM and 1nM in enzyme assay using pNP-TMP and cGAMP as substrates, respectively. Our compound showed extensive ENPP1 selectivity in the PDE and kinase panel assays and favorable in vivo pharmacokinetic properties. The results from THP-1 dual reporter assay demonstrated the potency and efficacy of our compound which induce STING-mediated type I IFN release. In co-culture spheroids, our compounds induced immune cell penetration into spheroids, which indicates that immune-cell mediated tumor cell death via ENPP1 inhibition. Furthermore, LCB33 compound stimulated cytokine production without affecting to the proliferation of human T cells. Finally, in the CT-26 colorectal syngeneic mouse model, oral administration at 5 mg/kg demonstrated a TGI of 39% as monotherapy and a TGI of 72% in combination with anti-PD-L1, which describes our compound enhance anti-cancer effect of immune checkpoint blockade.
Conclusions: Taken together, we report a novel and potent small molecule ENPP1 inhibitor in this study. Our ENPP1 inhibitor showed immune-cell mediated anti-cancer effects without affecting T cell proliferation. Additionally, we confirmed that our compounds showed synergistic anti-cancer effect with immune checkpoint blocker, anti-PD-L1, in colon-cancer mouse model. Further, we are going to perform Pharmacodynamic analysis in tumor infiltrating lymphocytes and tumor associated macrophages to verify immune-modulatory function of our ENPP1 inhibitors.
Citation Format: Pyoung Oh Yoon, Juhyeon Kim, A-Ram Lee, Eunji Son, Young cheol Lee, Eunmi Jung, HyeongRae Kim, YeonHee Lee, SoEun Park, Dae-Yon Lee, Chul-Woong Chung. A novel small molecule inhibitor of ENPP1 promotes T and NK cell activation and enhances anti-tumor efficacy in combination with immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 702.