Abstract 6170: SPHK2 modulates the tumor-intrinsic response to STING agonism in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) accounts for 10-15% of breast cancers and has a poorer prognosis than other breast cancer subtypes. TNBCs with robust immune infiltrates are known to have improved responses to chemotherapy, immunotherapy, and radiotherapy. Stimulator of interferon genes (STING) promotes both innate and adaptive anti-tumor immune cell infiltration through type I interferon production, and we and others have shown that its activation is associated with therapeutic sensitivity in TNBC. Repression of STING expression or activity are commonly observed in various cancer types, and this has been shown to attenuate responses to treatment. TNBCs overexpress sphingosine kinase 2 (SPHK2), which produces the bioactive lipid spingosine-1-phosphate (S1P). SPHK2-mediated S1P biosynthesis has been shown to suppress inflammatory responses in lung injury models via inhibition of STING. In this study, we sought to determine if SPHK2 attenuated STING activity in TNBC cells. We analyzed publicly available RNA-seq data in The Cancer Genome Atlas and found that TNBC patients with higher SPHK2 expression had significantly worse disease-free survival and overall survival. For in vitro studies, we stably overexpressed SPHK2 in two commonly studied TNBC cell lines with mid-range SPHK2 and STING protein expression: MDA-MB-231 and BT-549. STING was stimulated with the synthetic agonist diABZI, the endogenous agonist 2’3’-cGAMP, or with ionizing radiation. Phosphorylated interferon regulatory factor 3, a downstream event of STING activation, was decreased in SPHK2 overexpressing cells following diABZI stimulation. We measured type I interferon signaling by analyzing transcriptional activity of interferon-stimulated response elements (ISRE) using a luciferase reporter assay. SPHK2 overexpressing MDA-MB-231 and BT-549 cells had decreased transcriptional activity of ISRE compared to controls at baseline and after both diABZI and 2’3’-cGAMP treatment, suggesting that SPHK2 suppresses STING-dependent type I interferon production in TNBC. To test a therapeutically relevant model, cells were also exposed to photon radiation. Compared to control cells, SPHK2 overexpressing cells had decreased radiation induced ISRE activity. The FDA-approved SPHK2 inhibitor opaganib was able to augment diABZI-stimulated ISRE transcriptional activity in a dose-dependent manner. These data suggest that malignancy-associated SPHK2 overexpression may attenuate STING activation in TNBC, and that SPHK2 inhibition may be a therapeutically relevant and viable approach to augment anti-tumor immunity. The interactions between SPHK2 and the STING-interferon pathway warrants further study to develop potential therapeutic combinations to benefit patients with TNBC. Citation Format: Colette R. Worcester, Shane R. Stecklein. SPHK2 modulates the tumor-intrinsic response to STING agonism in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6170.