Abstract

Aim: Acute myeloid leukemia (AML) remains a highly aggressive hematologic malignancy with limited therapeutic options. This study aimed to elucidate how reticulocalbin-1 (RCN1) regulates mitochondrial DNA (mtDNA)-mediated innate immune signaling in AML and to evaluate the therapeutic potential of extracellular vesicle (EV)-mediated small interfering RNA (siRNA) delivery targeting RCN1. Methods: Stable RCN1 knockdown was achieved in AML cells using lentiviral delivery of short hairpin RNA. Mitochondrial transcription factor A (TFAM) expression, mtDNA leakage, and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-type I interferon pathway were analyzed by immunoblotting and quantitative polymerase chain reaction. Protein interactions were assessed by co-immunoprecipitation, proximity ligation assay, and immunofluorescence. TFAM rescue experiments were performed to evaluate its contribution to the downstream effects of RCN1 knockdown. For translational evaluation, EVs were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and marker protein detection, followed by loading with siRNA targeting RCN1. Therapeutic efficacy and safety were assessed in an AML xenograft mouse model. Results: RCN1 knockdown reduced TFAM expression, leading to cytoplasmic mtDNA accumulation and activation of the cGAS-STING-type I interferon signaling pathway. Restoration of TFAM expression attenuated mtDNA leakage and downstream signaling. EV-delivered siRCN1 effectively suppressed tumor growth in vivo without detectable toxicity. Conclusion: RCN1 silencing destabilizes mtDNA integrity and activates innate immune signaling in AML. EV-based delivery of siRCN1 may represent a promising and potentially safe therapeutic strategy for AML.