MIR4726EccDNA drives bortezomib resistance in multiple myeloma by enhancing MIR4726-5p/NXF1/NKIRAS2 axis dependent autophagy

Background: Despite many new drugs, multiple myeloma (MM) remains an incurable plasma cell malignancy, and drug resistance is a long-standing topic in this field. Characterized by efficient transcription without being limited by the double helix structure and promoter, extrachromosomal circular DNA (EccDNA) has been proven to be widely involved in Cancer development and drug resistance.

Methods: We performed circle-seq and mRNA-seq on samples from three MM patients at the time of complete response and relapse to screen EccDNA candidate molecules. Outward PCR and Sanger Sequencing were used to identify EccDNA molecules. RT‒qPCR and WB were performed to detect gene expression levels. Fluorescence in situ hybridization (FISH) was carried out to detect the deletion of chromosome 17p (del (17p)). Transmission electron microscopy (TEM) was conducted to observe autophagosomes. Luciferase reporter assays were performed to validate the binding of MicroRNAs to target genes. Cell viability assays and Apoptosis assays were employed to assess drug resistance. Xenograft tumor mouse models were established for in vivo experiments. Immunohistochemistry (IHC) was used to detect protein expression levels.

Results: We successfully identified an EccDNA molecule (EccDNA^{chr17:38719676-38719812}) in one relapsed MM patient with del(17p) and named it MIR4726^EccDNA. We demonstrated that the overexpression of MIR4726^EccDNA in MM cells can increase bortezomib resistance. We further confirmed that the precursor miRNA carried by MIR4726^EccDNA can be efficiently transcribed in MM cells and that MIR4726^EccDNA drives bortezomib resistance via the MIR4726-5p/NXF1/NKIRAS2 axis. We further revealed that downregulation of NFKB inhibitor interacting Ras like 2 (NKIRAS2) activated the NF-κB pathway and increased Autophagy. Moreover, we established a xenograft model of human MM via subcutaneous inoculation. We administered intra-tumoral injection of AgoMIR4726-5p and intraperitoneal injection of bortezomib and found that AgoMIR4726-5p promoted tumor progression and partially drove bortezomib resistance.

Conclusions: In summary, our findings indicate that artificially synthesized MIR4726^EccDNA is functional in cells and that MIR4726^EccDNA enhances tumor progression and partially mediates drug resistance by enhancing MIR4726-5p/NXF1/NKIRAS2 axis dependent Autophagy.

Autophagy; Bortezomib; Extrachromosomal circular DNA (EccDNA); MIR4726-5p; Multiple myeloma; NKIRAS2; NXF1.