Inhibition of NFS1 Induces Ferroptosis and Activates Immune Response in Acute Myeloid Leukemia Cells through STAT1/ISG Pathway
Authors: Liu Ying , Qian Bingxin , Cai Xiaoya , Huang Guiqing , Li Dengju 
Published: December 2024

Iron, as a fundamental trace element, is involved in a variety of physiological processes. Its homeostatic imbalance is closely associated with the formation of reactive oxygen species(ROS), and is a key factor in the development of various diseases, including leukemia. Leukemia cells require more iron than normal cells to sustain rapid growth and proliferation, making them more sensitive to ferroptosis, an iron-dependent cell death mechanism. Therefore, targeting iron metabolism-related proteins to selectively kill leukemia cells through regulation of iron homeostasis is expected to be a novel strategy for leukemia treatment. The NFS1 cysteine desulfurase is a rate-limiting enzyme involved in the biosynthesis of iron-sulfur clusters and participates in regulating iron homeostasis, energy metabolism, and lipid biosynthesis among other cellular processes.

Through the detection of samples from 44 patients in a single center, the results indicated that, compared with 10 healthy volunteers, the mRNA expression level of NFS1 was elevated in acute myeloid leukemia(AML) patients. The analysis based on public database determined that heightened expression of NFS1 correlated significantly with poorer overall survival rates for these individuals. Given our uncertainty about how exactly NFS1 influences AML cells' behavior, we utilized lentiviruses for stable knockdown cells within AML cell lines SKM-1 and THP-1. This intervention led to reduced proliferation but increased mortality among these cells, along with enhanced susceptibility towards various chemotherapeutic agents such as azacitidine and venetoclax.

Further examination involving regulatory cell death biomarkers alongside inhibitors rescue experiments revealed that suppressing NFS1 triggered both ferroptosis as well as apoptosis within these cells. Subsequent assessments focusing on Fe2+ and ROS metabolic levels confirmed that knockdown of NFS1 induced ferroptosis by eliciting an iron starvation response and subsequent oxidative stress in AML cells.

GO enrichment analysis revealed that the differentially expressed genes that were significantly up-regulated after knockdown of NFS1 found by transcriptome sequencing were mainly enriched in response to virus, cytokine pathway and type I interferon signaling, specifically including multiple genes of IFIs and OASs. IFI, also called Interferon-stimulated gene(ISG), is any gene induced during an interferon (IFN) response. IFN binds to the cognate cell-surface receptor activated JAK/STAT pathway to activate ISGs. By PCR verification on significantly increased gene expression of ISGs and STAT1 after knockdown of NFS1. Then, targeted inhibition of STAT1 was performed to indicate that knockdown of NFS1 induced apoptosis in AML cells by activating the STAT1/ISG pathway. Meanwhile, the ELISA tests confirmed that knockdown of NFS1 in AML cells regulated the release of pro-inflammatory cytokines and affected the polarization of macrophages.

Taken together, this study provides insights into the mechanisms of NFS1 in AML development and suggests the promise of inhibiting NFS1 in AML therapeutic strategies.