Abstract 4196: Alt-NHEJ inhibition leads to cGAS/STING-dependent immunogenic cell death in multiple myeloma

Abstract Background: Alternative Non Homologous End Joining (Alt-NHEJ) DNA repair is considered a major player in cancer genomic instability. Here, we investigated cGAS-STING pathway as crucial node in the interplay between Alt-NHEJ repair and immune response, in the aim to discover novel therapeutic vulnerability in Multiple Myeloma (MM). Methods: In silico analyses were performed by consulting public MM dataset CoMMpass. Anti-proliferative activity was evaluated by CellTiter-Glo, while flow cytometry analysis was used to determine the apoptotic process, cell cycle, phagocytosis, micronuclei detection, Calreticulin and T-cell activation markers. Protein expressions were detected by western blot of whole or fractioned protein extracts. Results: We first examined the prognostic role of a cGAS-STING signature described by Sokac et al, by interrogating public MM database. 767 MM patients were selected from CoMMpass dataset and divided into 4 groups according to cGAS-STING signature groups (CSG 1-4). Interestingly, 130 patients were classified into CSG2 group with non functional cGAS-STING pathway, 105 into CSG3 with active cGAS-STING and 532 patients into CSG4 group with abnormal activation of cGAS-STING pathway; no patients where classified as CSG1 with inactive cGAS-STING. Importantly, CSG3-4 groups showed significantly reduced PFS and OS as compared to CSG2, supporting the idea that cGAS-STING has a pivotal role in the progression of MM. Indeed, Gene Set Enrichment Analysis (GSEA) of CSG3 versus CSG2 revealed differential involvement of several pathways such as DNA repair, TNFA signaling, oxidative phosphorylation and MYC targets. In addition, gene expression analysis showed a higher mRNA expression DNA Ligase 3 (LIG3) and PARP1 in CSG3 and CSG4 group as compared to CSG2. On this basis, we evaluated the activity of Alt-NHEJ inhibitors Talazoparib (PARP1-inhibitor) and L67 (LIG3 inhibitor) on MM cell lines, focusing in their capability to modulate cGAS-STING pathway. We first detected a significant reduction of cell proliferation and the induction of apoptosis following Talazoparib and L67 treatment, which in turn induced DNA damage response and cell cycle blockade, and finally cGAS-STING pathway activation. Finally, by performing co-culture experiments with healthy donor’s peripheral blood mononuclear cells (PBMCs), we demonstrated the increase of i) CD80 and CD83 maturation markers on dendritic cells, ii) CD69 and CD25 activation markers on T cells and iii) phagocytosis of tumor cells. Importantly, these results were confirmed also in HR-defective breast and ovarian cancer cell lines, further suggesting the onset of an immunogenic cascade upon Alt-NHEJ inhibition. Conclusion: Taken together, our findings indicate that Alt-NHEJ inhibitors are potential immune-stimulating agents for MM with hyperactivation of cGAS-STING pathway, coherently with our working hypothesis. Citation Format: Giada Juli, Domenica Ronchetti, Sara Squillacioti, Nicoletta Polerà, Anna Maienza, Michelangelo Vocaturo, Emanuela Altomare, Katia Grillone, Maria Gaetano, Stefania Signorelli, Antonio Galvano, Niccolò Bolli, Pietrosandro Tagliaferri, Pierfrancesco Tassone, Daniele Caracciolo. Alt-NHEJ inhibition leads to cGAS/STING-dependent immunogenic cell death in multiple myeloma [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 4196.