Fig. 5: The specificity of the combination therapy is shaped by the decisive presence of intracellular iron.

a Viability of U937 cells treated haemin (H) in combination with ART and ATO + ART for 48 h (n = 4; ATO = 2 μM; ART = 5 μM; Haemin (H) = 5 μM and 20 μM). b Viability of primary AML cells treated with haemin (H), ART, ATO, and combinations for 48 h (n = 27; ATO = 2 μM; ART = 5 μM; Haemin (H) = 5 μM). (Independent biological replicates on the freshly isolated bone marrow specimens). c Immunoblot of FTH in U937 cells post-exposure to ATO, ART and ATO + ART for 24 h (n = 3; ATO = 2 μM; ART = 5 μM). d Heatmap of TMT-labelled proteome (FTH, FTL, TFRC, FECH, TOMM20) of primary AML, APL, and ALL cells compared to healthy control mononuclear cells. (5 samples per group, and the differentially expressed proteins and fold change were calculated compared to healthy control mononuclear cells). e Kaplan-Meier curves of overall survival in TCGA AML patients in the FTH-high red (red) and FTH-low (blue) groups. f Structure highlighting the endoperoxide moiety of artesunate (ART), active metabolite dihydroartemisinin (DHA), and the inactive metabolite deoxy dihydroartemisinin (Deoxy DHA). Histograms illustrating FL2 shift on flowcytometry in the U937 cells treated with these three agents for 24 h and the fluorescent intermediates that accumulate as a result with ART and DHA but not with Deoxy-DHA (n = 3; 5 μM = ART, DHA, and Deoxy derivative). g Protoporphyrin –IX (PPIX) is a fluorescent intermediate of the heme biosynthesis pathway. The precursor of heme biosynthesis, a-ALA, enhanced ART activity, as seen in heme-based supplementation. The ART utilization of iron results in less/lack of iron for the production of heme, which, in turn, leads to the accumulation of fluorescent intermediate PPIX that is observed with ART and its active intermediate DHA, not with the deoxy form ART/DHA. Hemin/Heme containing degrading enzyme HMOX-1 and ferritin is increased by ATO, thereby increasing the availability of iron for ART activity (Created with Adobe Illustrator). Data are presented as mean ± SEM. n.s., P > 0.05; *P < 0.05; ***P < 0.001; ***P < 0.0001, with a two-tailed unpaired t-test or one-way analysis of variance.