What question did this study set out to answer?

This research aims to investigate whether SPHK2 influences STING pathway activity in TNBC, potentially enhancing anti-tumor immunity.

April 5, 2026

Abstract 4323: The SPHK2 inhibitor opaganib potentiates tumor-intrinsic STING activation in triple-negative breast cancer in vitro

Key Points

This research aims to investigate whether SPHK2 influences STING pathway activity in TNBC, potentially enhancing anti-tumor immunity.
Evaluated effects of opaganib, an SPHK2 inhibitor, in TNBC cell lines (BT-549, HCC70, MDA-MB-231, MDA-MB-468).
Assessed STING activation using synthetic agonist diABZI and analyzed downstream signaling via western blot.
Conducted transcriptional activity analysis with ISRE luciferase reporter in BT-549 cells.
Examined mRNA expression of 770 immuno-oncology genes using NanoString technology.
Opaganib alone did not enhance STING activation, but increased activation when combined with diABZI.
Enhanced phosphorylation of STING pathway proteins (STING, IRF3, TBK1) observed with opaganib-diABZI combination.
Pre-treatment with exogenous S1P reduced STING activity, while opaganib followed by diABZI improved ISRE activity.
Upregulation of cytokine signaling and immune response pathways identified in opaganib-diABZI treated cells.

Abstract

Abstract Triple-negative breast cancer (TNBC) has the poorest prognosis of breast cancer subtypes. Patients with TNBCs that exhibit robust stromal tumor infiltrating lymphocytes and/or gene expression signatures indicative of immune activation have improved response rates to chemotherapy and immunotherapy. DNA damage induces anti-tumor immunity through the stimulator of interferon genes (STING) protein pathway, and STING activation has been associated with better response rates and improved prognosis in TNBC. However, TNBCs also have elevated levels of sphingosine-1-phosphate (S1P), an immunomodulatory biolipid with pleiotropic effects. S1P is produced by sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). SPHK1 has been shown to lead to cancer cell progression and metastasis, while the role of SPHK2 in TNBC is less well-studied. SPHK2-dependent S1P production has been shown to inhibit STING in an acute lung injury model. Whether tumor-intrinsic STING is inhibited by the SPHK2-S1P axis is not known. We hypothesized that SPHK2 modulates STING pathway activity, and that SPHK2-dependent S1P biogenesis could be targeted to augment anti-tumor immunity in TNBC. We evaluated opaganib, an SPHK2 small molecule inhibitor with FDA orphan drug designation, in in vitro TNBC models (BT-549, HCC70, MDA-MB-231, and MDA-MB-468). Opaganib alone did not increase STING activation. However, pre-treatment with opaganib potentiated the synthetic STING agonist diABZI in all cell models. Compared to diABZI alone, western blots of cells treated with the opaganib-diABZI combination had enhanced phosphorylation of STING pathway proteins, such as STING, IRF3, and TBK1. Using BT-549 cells with an interferon stimulated response element (ISRE) luciferase reporter system, we analyzed transcriptional activity downstream of the STING pathway and found that ISRE activity increased in a dose-dependent manner with diABZI-mediated STING stimulation. However, pre-treatment of BT-549 cells with exogenous S1P decreased the diABZI-mediated ISRE activity. Conversely, pre-treatment with opaganib followed by low-dose diABZI treatment potentiated the downstream STING-mediated effects. To further analyze cellular responses to SPHK2 inhibition and STING agonism, we evaluated mRNA expression of 770 immuno-oncology-related genes (NanoString PanCancer IO 360 panel for nCounter) in drug-treated cells. Upregulated pathways in cells treated with the opaganib-diABZI combination compared to cells treated with diABZI alone or opaganib alone included cytokine signaling in immune system and responses to cytokine stimulus. In summary, we show that malignancy-associated production of S1P blunts tumor-intrinsic STING activity and suggest that targeting the SPHK2-S1P axis with opaganib may augment anti-tumor immunity in TNBC. Citation Format: Colette R. Worcester, Amrita Mitra, Harsh B. Pathak, Shane R. Stecklein. The SPHK2 inhibitor opaganib potentiates tumor-intrinsic STING activation in triple-negative breast cancer in vitro abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4323.

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