Metformin-Induced Ferroptosis Is a Therapeutic Vulnerability in IDH2-Mutant AML Linked to Metabolic Rewiring Towards Fatty Acid Oxidation
Authors: Sternadt Dominique , Pereira-Martins Diego , Silveira Douglas RA , Weinhaeuser Isabel , Yang Ming , Chatzikyriakou Prodromos , Casares Alaez Pilar , Ammatuna Emanuele , Oudejans Lieve , Huls Gerwin A. , Frezza Christian , Quek Lynn , Schuringa Jan Jacob 
Published: December 2023

Metabolic rewiring is an essential feature of leukemic cells to sustain tumorigenesis and is largely influenced by mutational status. We previously demonstrated this phenomenon in FLT3-ITD acute myeloid leukemia (AML) upon combined inhibition of complex II activity and lactate import (Erdem et al., 2022). Mutations in isocitrate dehydrogenase ( IDH1/2mut) occur in about 20% of AML cases, and despite the emergence of targeted therapies, patients often relapse. The molecular and epigenetic outcomes of 2-R-hydroxyglutarate accumulation and competitive inhibition of histone demethylases have been extensively studied in IDH1/2mut AMLs, but the metabolic consequences are still largely unexplored. Here, we applied multi-omics studies on cell line models and primary AML samples combined with functional studies to unravel the metabolic rewiring of IDH1/2mut AMLs.
Transcriptional characterization of isogenic TF1 wt and IDH2R140Q cell lines (10978 genes) revealed increased expression of CD36, a major fatty acid (FA) transporter, and gene set enrichment analysis (GSEA) associated IDH2R140Q cells with terms related to FA processes and activity of mitochondrial complex I. Quantitative metabolome analysis of TF1 wt and IDH2R140Q (180 metabolites) revealed lower levels of acylcarnitines of variable carbon lengths, suggesting increased FA oxidation, and upregulation of glycerophospholipid and glycerolipid metabolism in IDH2R140Q cells. Next, we validated our findings by performing a metabolomic profiling (172 metabolites) on primary AML samples (n=26, including 4 IDH1mut and 2 IDH2mut), for which label-free quantitative proteome data (11272 proteins) was also generated. Proteome analysis confirmed increased CD36 expression and enrichment for FA and mitochondrial processes in IDH1/2mut patients. In line, metabolome data also revealed increased oxidation of branched-chain FAs in this sample group. Altogether, these findings suggested a profound disturbance in lipid metabolism and supported the notion that IDH1/2 mutant cells rely on mitochondrial respiration, whereby FAs seem to be the preferred carbon source.
Next, we performed an in vitro drug screen targeting the main metabolic pathways, which revealed increased sensitivity of IDH2R140Q cells to the FDA-approved complex I inhibitor metformin. Extracellular flux analysis indicated no significant difference in the oxygen consumption rate (OCR) between TF1 wt and IDH2R140Q upon metformin treatment, while the viability of mutant cells was significantly diminished. Contrarily, the basal extracellular acidification rate (ECAR) was increased in TF1 wt cells upon complex I inhibition, suggesting that these cells rewire their metabolism towards glycolysis more efficiently than mutant cells. Furthermore, we knocked down CD36 expression to investigate whether disrupting lipid metabolism in IDH2R140Q cells would constitute a metabolic vulnerability. Notably, CD36 knockdown resulted in increased resistance to metformin in IDH2R140Q cells. These data suggested that enhanced lipid uptake mediated by CD36 in IDH2R140Q cells not only boosts OCR but may also disrupt lipid homeostasis, causing cells to become more susceptible to lipid peroxidation. Since the role of metformin-induced ferroptosis in AML is still unclear, we performed an RNA-seq analysis on TF1 wt and IDH2R140Q cells treated with metformin (5 mM) for 72 hours (10326 genes). Single-sample GSEA associated the transcriptome of metformin-treated cells with ferroptosis signatures in both cell lines but to a significantly higher extent in IDH2R140Q. We functionally validated this finding by using the BODIPY C11 probe, a lipid peroxidation sensor. After 24h of metformin treatment, both TF1 wt and IDH2R140Q displayed increased lipid ROS. Lipid peroxidation levels were further enhanced by combining metformin with palmitate, a saturated FA, supporting the notion that elevated lipid availability and uptake may increase cell death via ferroptosis.
Altogether, we identified a new metabolic vulnerability in IDH1/2mut AMLs associated with a profound disturbance in lipid metabolism. Moreover, we show that treatment with metformin not only inhibits complex I but also induces cell death via ferroptosis in IDH2mut cells providing an alternative treatment option in combination with available IDH2mut targeted therapies for this subgroup of patients.