TRDMT1 methyltransferase gene knockout attenuates STING-based cell death signaling during self-extracellular RNA-mediated response in drug-induced senescent osteosarcoma cells
Under stress conditions, endogenous biomolecules such as nucleic acids or proteins can be released from damaged cells and considered as damage-associated molecular patterns (DAMPs) activating innate immune system and context-dependent responses. In the present study, self-extracellular RNA was obtained from dying (RNA D) and senescent (RNA S) cellular models of osteosarcoma (OS), characterized by NGS, and tested against proliferating and non-proliferating (etoposide-indued senescent) OS cells (U-2 OS, SaOS-2, MG-63, 143B). RNA D and RNA S induced apoptosis, nitrooxidative stress, nucleic acid sensing pathways and cytokine production, and RNA m5C methyltransferase-based responses (TRDMT1 and NSUN2) in proliferating OS cells. In drug-induced senescent OS cells, TRDMT1 gene knockout (KO) prevented STING activation, related proinflammatory response, and cell death. Furthermore, IFNß binding RNA partners were identified, namely NSUN2, NSUN5, NSUN6, CDKN1A, MYC, and RAD51 transcripts and these interactions were compromised in TRDMT1 KO cells and upon RNA D and RNA S treatment. TRDMT1 KO also resulted in replication stress in OS cells that was potentiated by RNA D and RNA S stimulation and associated with elevated levels of APOBEC3A and APOBEC3G, members of the cytidine deaminase protein family. In conclusion, we showed that TRDMT1 KO restricted STING-based immune and cell death response to RNA D and RNA S in nonproliferating drug resistant OS cells that might have potential therapeutic implications. Overall design: Four cellular models of OS in vitro were used, namely U-2 OS, SaOS-2, MG-63, and 143B cells. OS cells were treated with etoposide, DNA damaging chemotherapeutic agent, at both death and senescence-inducting concentrations and RNA released to cell culture medium, RNA D and RNA S, respectively, was characterized using NGS. Proliferating and/or non-proliferating (drug-induced senescent) OS cells were then stimulated with RNA D and RNA S to reveal RNA-associated responses.