Abstract
Acute myeloid leukemia (AML) is a clonal hematopoietic stem/progenitor cell disorder driven by aberrant proliferation, and undergoes extensive metabolic reprogramming to sustain rapid growth. While Azin1 is known to modulate human malignancies through polyamine-dependent metabolic pathways. However, the non-polyamine-mediated mechanisms through which Azin1 regulates AML pathogenesis remain largely undefined.
Firstly, we used Vav-iCre; Azin1fl/fl mouse model to explore the function of Azin1 in hematopoiesis. We observed that there was no significant impact on steady stable hematopoiesis when Azin1 deletion. However, under stress conditions—including competitive repopulation assays, 5-fluorouracil (5-FU) challenge, and irradiation—Azin1 deficiency markedly impaired hematopoietic reconstitution potential and reduced HSC self-renewal capacity. In addition, we established MLL-AF9(MA9) mouse model and found that Azin1 depletion suppressing MA9 cells proliferation, impairing leukemia stem cell (LSC) function, and prolonging survival.
To elucidate Azin1 downstream reprogram metabolism mechanisms in AML, we integrated RNA-sequencing and non-targeted metabolomics in Azin1 knockout(KO) MA9 and knockdown (KD) THP-1 cells. Metabolomics results revealed significant enrichment of lipids and unsaturated fatty acids (UFAs), while transcriptomics highlighted dysregulation of long-chain fatty acid biosynthesis and UFA metabolism. RNA-sequencing performed key enzymes SCD1, FADS3 and BTG3, critical for fatty acid synthesis, were downregulated in Azin1 KO MA9 cells and verified in vitro. Integrative analysis in Azin1 KO MA9 and KD THP-1 cells identified oleic acid (OA) as a top differentially reduced metabolite. Metabolic tracing with 13C6 confirmed reduced acetyl-CoA and OA in Azin1 KO MA9 cells, despite unchanged TCA cycle metabolites. Functionally, exogenous OA supplementation partially rescued the L-GMP frequency in Azin1 KO MA9 cells, supporting our hypothesis that Azin1 regulated L-GMP frequency to influence cell proliferation in AML through UFA metabolism.
To delineate the mechanism of lipid metabolic dysregulation in Azin1 KO AML cells, we employed mass spectrometry-based interaction screening. This revealed a novel interaction between Azin1 and trans-2-enoyl-CoA reductase (TECR), a key enzyme in fatty acid elongation, which was subsequently validated by Co-immunoprecipitation (Co-IP) in vitro. To verify the function of Azin1/TECR interaction, cycloheximide (CHX) chase assays was performed, which demonstrated that TECR protein stability was significantly enhanced in Azin1-overexpressing THP-1 cells. Conversely, KD TECR MA9 and THP-1 cells markedly inhibited proliferation and prolonged survival in vivo. Furthermore, to identify downstream effectors, RNA-sequencing of Azin1 KO and TECR KD MA9 cells revealed significant attenuation of ERK-1/2/ NF-κB signaling in both models. These results indicated that Azin1 stabilizes TECR to regulate AML by promoting fatty acid chain elongation.
In summary, our study defines a non-polyamine axis which Azin1 stabilizes TECR to reprogram fatty acid metabolism, fueling AML progression via TECR-ERK1/2/NF-κB axis. Pharmacological or genetic inhibition of Azin1 offers a novel precision intervention strategy for AML by targeting its metabolic vulnerabilities.
