Abstract B064: Uncovering the lineage state specific regulation of STING in neuroblastoma
Authors: Shapiro Matthew , Mishra Pamela , Narch Alaa , Dessau Jessica , Dang Chi V. , Wolpaw Adam 
Published: September 2024

Study Aim: This study aims to leverage our investigation of the transcriptional regulation of cGAS-STING signaling to identify the mechanisms that lead to differences in inflammatory signaling and immunogenicity between neuroblastoma lineage states.Neuroblastomas cells can exist in two distinct lineage states - a chemosensitive adrenergic state and a chemoresistant mesenchymal state. We previously found that the mesenchymal state has higher expression of pattern recognition receptors, including STING (stimulator of interferon genes), which transduces signals from the cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase). We hypothesize that identifying modifiers of STING signaling will uncover drivers of the lineage state differences in inflammatory signaling. Methods: We examined cGAS and STING expression at the RNA and protein level in cell lines, patient-derived xenografts, and patient tumors. We also assessed CpG island and histone methylation and how they differed based on lineage state.Through immunoblots and methylation specific PCR we assessed the restoration of cGAS-STING pathway specific signaling through the use of DNMT inhibitors and a DOX inducible system state switch system. We performed a genome wide flow-based CRISPR screen, using an IRF3-responsive GFP reporter, to assess genes involved in STING expression and function. Results: We found that STING is universally expressed in mesenchymal, but not adrenergic cell lines, while cGAS is nearly universally silenced in all neuroblastoma cell lines. Moreover, we found that neuroblastoma cell lines have extensive methylation at the cGAS promoter CpG islands.Using methylation-specific PCR, we showed that the DNMT inhibitor decitabine reduced CpG island methylation and rescued cGAS expression, which restored the immunogenic functionality of the cGAS-STING pathway measured through expression of pIRF3 and pSTAT1.Furthermore, through the use of DOX inducible expression of PRRX1, we showed that STING expression was restored when cells switch from the adrenergic to the mesenchymal state.Finally, we performed a genome wide CRISPR screen using an IRF3 reporter to identify targets responsible for STING expression in the mesenchymal state. This screen identified known components of the cGAS-STING signaling pathway as well as chromatin modifiers and transcription factors not previously associated with cGAS-STING signaling. Conclusions: The cGAS-STING pathway is inactivated in neuroblastoma cell lines due to silencing of cGAS, but can be restored in the mesenchymal state due to its expression of STING.When cGAS functionality is restored, downstream immunogenic signaling returns.We have identified known and novel genes required for cGAS-STING signaling in mesenchymal neuroblastoma cells. Further validation and mechanistic work on these genes is ongoing, including understanding how they alter inflammatory signaling more broadly and how to use this information to improve immunotherapeutic responses.

Citation Format: Matthew Shapiro, Pamela Mishra, Alaa Narch, Jessica Dessau, Chi V. Dang, Adam Wolpaw. Uncovering the lineage state specific regulation of STING in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B064.