Abstract B39: Novel dinucleotides that activate STING signaling for immuno-oncology

Abstract

Abstract Background: Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. It is being recognized that induction of type I interferons (IFN) and interferon-stimulated genes (ISGs) in tumor cells and within the tumor microenvironment (TME) is essential for modulating the host-immune response and inducing apoptosis of tumor cells. Furthermore, the antigen-presenting cells within TME can cause induction of adaptive immune response, through priming of CD8+ T cells and tumor killing. Importantly, the DNA released from damaged cells and cancer cells can be sensed by cyclic GMP-AMP synthase (cGAS) leading to the synthesis of cyclic-GMP-AMP (2',3'-cGAMP), a second messenger that activates Stimulator of Interferon Genes (STING) pathway resulting in the production of type I IFN and ISGs. The cumulative effects of activation of innate and adaptive immune response can result in potent anti-cancer effects. Therefore, therapeutic agents that activate the cGAS-STING signaling pathway in tumor cells and TME are urgently needed. Herein, we describe the discovery of novel potent, first-in-class small molecules for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of cyclic dinucleotides bound to STING, a focused library of dinucleotide compounds was synthesized using phosphoramidite chemistry and evaluated for: (a) Induction of IFN signaling: The compounds were screened for the induction of Interferon regulatory factor (IRF), ISG54, and NF-κB using reporter assays. We used HEK293 cell line (SZ14) stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for screening and the active compounds were further characterized in THP1 cells and human primary PBMCs. The IRF, ISG54, and NF-κB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50s of the compounds were ascertained to identify active compounds, (b) Expression of IFN-β and IRF7 in THP1 cells: THP1 cells were treated with active compounds or controls for 22hrs. RNA was extracted and the expression of IFN-β, IRF7, was ascertained using semi-quantitative RT-PCR, (c) Induction of pathogen recognition receptors (PRRs) including RIG-I, MDA5, LGP2, and OAS-1 and ISG54: THP1 cells and PBMCs were treated with active compounds, 2',3'-cGAMP (control), or DMSO and the gene expression of different PRRs, ISGs, was determined by quantitative RT-PCR using ΔΔct method, (d) Induction of cGAS-STING signaling using reporter assays: HEK293 cells stably expressing ISG54 were transfected with plasmids encoding human cGAS (wild-type, or K384A, K400A, or K411A mutants) and treated with active compounds, poly (dA:dT) (positive control), or DMSO for 21 hrs. ISG54 induction was calculated as fold-change in luminescence compared to DMSO-treated controls. (e) Cytotoxicity assays: THP1 cells were treated with active compounds or DMSO control with Lipofectamine and cytotoxicity assessed using the CellTiter-Glo® Luminescent assays. Cytotoxicity was calculated from %-fold change in luminescence compared to DMSO-treated sample. Results: Through in vitro assays in conjunction with Structure Activity Relationship studies, we have identified potent compounds that activate cGAS-STING signaling pathway for induction of IRF, IFN, and NF-κB. These compounds also cause induction of expression of PRRs, including RIG-I, MDA5, LGP2, as well as, ISG54 and OAS-1. Conclusion: We have discovered potent, first-in-class agents that cause induction of IFN, NF-κB, ISGs, and PRRs. Further optimization and preclinical evaluation of the compounds for application in immuno-oncology is underway. Citation Format: Shenghua Zhou, Sreerupa Challa, Seetharamaiyer Padmanabhan, Anjaneyulu Sheri, Samantha Delaney, Geeta Meher, Dillon Cleary, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Novel dinucleotides that activate STING signaling for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B39.