Eccdna-encoded LIPIN1 drives DGAT1-dependent lipid droplet biogenesis to promote Acute Myeloid Leukemia progression and chemoresistance

Abstract Introduction: Extrachromosomal circular DNA (eccDNA) has emerged as a critical player in cancer biology. Accumulating evidence indicates that eccDNA promotes tumor progression by inducing genomic instability, amplifying oncogenes, and regulating gene expression. However, its biological function and mechanistic role in acute myeloid leukemia (AML) remain unexplored. In this study, we investigated the functional significance and regulatory mechanisms of eccDNA in AML, aiming to identify potential therapeutic targets. Methods Bone marrow samples were collected from newly-diagnosed and chemo-resistant AML patients with informed consent. High-throughput eccDNA sequencing was performed to determine eccDNA length distribution. The presence of gene-containing eccDNAs was validated by PCR and fluorescence in situ hybridization (FISH), and synthetic eccDNA constructs were generated to investigate their functional roles in AML. In vivo, we evaluated the impact of eccDNA-encoded LIPIN1 (ecc-LIPIN1) on AML progression and chemoresistance, and developed a PEI/PC7A nanoparticle system for therapeutic intervention. Proteomic and transcriptomic analyses were performed to explore the downstream targets. Results High-throughput eccDNA sequencing revealed abundant eccDNAs (0.3-1 kb) across newly-diagnosed and chemo-resistant AML samples, mapped to diverse genomic regions. Analysis of the GEO dataset (GSE165430) identified genes upregulated in chemo-resistant AML patients. Integrating these data with eccDNA-derived genes highlighted ecc-LIPIN1 as a potential resistance driver. PCR and FISH confirmed the presence of eccDNA harboring LIPIN1 nucleotide fragments. Ecc-LIPIN1 levels were significantly elevated in AML samples, particularly in chemo-resistant cases, and associated with poor prognosis. A circular DNA containing LIPIN1 was synthesized based on sequencing data and transfected into AML cells, resulting in elevated LIPIN1 expression. Functional assays demonstrated that ecc-LIPIN1 promoted AML cell proliferation and chemoresistance. Given its metabolic role, metabolomic profiling assay revealed that ecc-LIPIN1 primarily regulated lipid metabolism. Fluorescence microscopy and flow cytometry confirmed that ecc-LIPIN1 enhanced lipid droplet formation, contributing to AML progression and chemo-resistance. Proteomic and transcriptomic analyses of ecc-LIPIN1-overexpressed AML cells identified DGAT1, a key enzyme in lipid metabolism, as a downstream effector. Silencing DGAT1 in ecc-LIPIN1-overexpressed AML cells markedly reduced lipid droplet accumulation, indicating that ecc-LIPIN1 drives lipid storage through DGAT1. Further analyses revealed that LIPIN1 also modulated endoplasmic reticulum (ER) stress pathways. In LIPIN1-overexpressed AML cells, ER-stress markers CHOP and ATF4 were markedly elevated. Silencing DGAT1 reversed ER-stress activation, suggesting that LIPIN1 promoted AML progression by regulating ER stress via DGAT1. To validate the oncogenic function of ecc-LIPIN1, we generated eccDNA constructs containing either wild-type or mutant LIPIN1 nucleotide sequences and transfected them into AML cells. The wild-type ecc-LIPIN1 markedly enhanced AML progression and chemo-resistance both in vitro and in vivo. To counteract this effect, we developed a PEI/PC7A/siLIPIN1 nanoparticle system. Treatment with these nanoparticles efficiently suppressed ecc-LIPIN1 expression, inhibited AML progression, and mitigated chemo-resistance. Conclusions This study provides preclinical evidence that eccDNA plays a critical role in AML progression and chemoresistance by encoding LIPIN1. Mechanistically, ecc-LIPIN1 promotes lipid droplet formation and ER stress via DGAT1, ultimately driving chemoresistance. These findings identify ecc-LIPIN1 as a novel oncogenic factor in AML and highlight its potential as a therapeutic target. Future research into eccDNA biogenesis in AML may uncover new strategies for combating drug resistance and disease progression. Keywords Extrachromosomal circular DNA; Acute Myeloid Leukemia; drug resistance; lipid metabolism; LIPIN1.