Ferroptosis escape in AML stem cells uncovered by single-cell profiling and reversed by STAT3/NRF2 inhibition Free

Chemo resistant Leukemic stem cells (LSCs) with leukemia re-initiating potential are a key contributor to relapse refractory disease in acute myeloid leukemia (AML), which confers an adverse outcome. These LSCs are primarily enriched in the CD34+CD38- compartment that also harbours healthy HSCs, posing a challenge in their specific elimination. Considering AML as a multi-clonal malignancy, single-cell multi-omics approach was undertaken to identify chemoresistance-associated genes/ pathways in AML LSCs for their potential targeted therapeutics.

For single cell whole transcriptome analysis, we enriched CD34+CD38- or CD34-CD117+CD38- leukemic stem cells isolated from bone marrow aspirates of adult patients with de novo AML (n=12) after informed consent. For differential gene expression analysis scRNAseq was also performed from CD34⁺ hematopoietic stem cells obtained from healthy donor controls. The DGE analysis of LSCs relative to HSCs identifiedsignificantly altered chemoresistance-associated genes of which, ferritin heavy chain 1 (FTH1) a key player in iron storage within the cells was substantially downregulated in LSCs indicating iron dependency. Altered Ferroptosis related gene (FRGs) expression including FTH1, TFRC, GPX4 and NFE2L2/NRF2 was validated by qPCR. Bioinformatic analysis revealed a common STAT3 binding motif in the promoter region of FRGs. Ferroptosis model was generated in AML cell lines including LSC-like AML cell line MA9 using Erastin or RSL3. These cells were treated with STAT3 inhibitor C188-9 or STAT3-NRF2 dual inhibitor (Brusatol) and the effect on cell proliferation was determined using CCK-8 assay, gene expression by qPCR, flow cytometry was used for the detection of lipid ROS levels by Bodipy dye and intracellular iron estimation were performed. Western blotting was carried out for pSTAT3 and GPX4 protein expression, and colony forming assay was performed with or without the indicated inhibitors.

We identified highly dysregulated FRGs, i.e., FTH1, GPX4, NRF2 and suppression of the ferroptosis pathway in LSCs relative to non-LSCs and HSCs. STAT3 emerged as a transcription factor with a common binding sequence present in the promoter region of FRGs. We demonstrated that STAT3 inhibitor C-188-9 reduced lipid ROS levels and altered FRG expression, but this approach was insufficient to promote ferroptosis. Treatment of AML cells with STAT3 inhibitor alone upregulated NRF2, a transcription factor involved in antioxidant response, thus opposing the ferroptosis-mediated cell death in AML cells. To overcome NRF2-mediated resistance, we further blocked NRF2-STAT3 axis in LSC-like MA9 cell line using Brusatol, which indeed promoted ferroptosis as indicated by increased lipid ROS, labile iron, reduced pSTAT3, GPX4 protein expression and significantly reduced colony forming units. Moreover, a combination of NRF2-STAT3 inhibitor potentiated the effect of ferroptosis inducers RSL3 and Erastin and enhanced the cytotoxicity in LSCs.

Our scRNAseq analysis of enriched AML LSCs revealed and dissected the mechanistic differences between LSCs and non-LSCs. Our data reveals a potential evasion strategy inherent to LSCs mediated by dependency on labile iron pool to produce suboptimal lipid ROS that supports their survival but prevent ferroptosis by upregulation of NRF2. Our findings suggest that promoting ferroptosis is indeed a promising strategy to combat chemoresistance in AML. Therefore, dual targeting of NRF2-STAT3 along with ferroptosis inducers hold potential as adjuvant therapy by promoting ferroptosis-mediated cell death in AML LSCs.