Fig. 1: CLL cells are sensitive to ferroptosis induction.

a The CLL cell lines CII, HG3, I83-E95, JVM-3, Mec-1, PCL-12, PGA-1, and Wa-C3CD5+ were treated in two independent experiments with the GPX4 inhibitor ML162 (100 nM) or RSL3 (100 nM) for 4 h, and compound-triggered specific cell death was determined. Specific cell death was calculated relative to the untreated control as follows: 100 × (% dead cells − % baseline)/(100 − % baseline). The baseline values were normalized to 0%. b The same set of CLL cell lines was subjected to two independent experiments with the GPX4 inhibitors ML162 or RSL3 in the presence/absence of zVAD (apoptosis inhibitor, 10 µM), necrostatin-1/Nec-1 (necroptosis inhibitor, 100 µM), and deferoxamine (DFO, 100 µM) or ferrostatin-1/Fer-1 (ferroptosis inhibitors, 25 µM). c In the same samples, lipid peroxidation was assessed by flow cytometry (FACS) using BODIPY™ 581/591 C11, as shown in the representative analysis in the left panel. The data are summarized in the right panel. d Specific cell death of B cells from healthy donors (HD, n = 10) and CLL cells from patients (CLL, n = 20) was assessed via FACS following 24 h of ML162 treatment (500 nM), as shown in the representative analysis in the left panel. The data are summarized in the right panel. e Lipid peroxidation of B cells from healthy donors (HD, n = 10) and CLL cells from patients (CLL, n = 20) was assessed via FACS following 24 h of ML162 treatment (500 nM), as shown in the representative analysis in the left panel. The data are summarized in the right panel. f Expression of key pro- and anti-ferroptotic proteins was analyzed in primary CLL cells (CLL, n = 31) and HD B cells (HD, n = 12) on the basis of the median fluorescence intensity (MdFI) directly ex vivo. The data are shown as the fold change between CLL/HD patients. Statistical significance was determined on the basis of groupwise comparisons of MdFI values. Significantly differentially expressed proteins are highlighted in red. g Intracellular ferrous iron (Fe²⁺) (HD, n = 10 and CLL, n = 24, left panel) and glutathione (HD, n = 8 and CLL, n = 34, right panel) baseline levels were measured via FACS via Phen Green SK and Thioltracker™, respectively. Note that the Phen Green SK signal is quenched by Fe²⁺; thus, lower fluorescence indicates higher intracellular ferrous iron levels. h Specific cell death was assessed in B cells and CLL cells from nontransgenic littermates (TCL1-, n = 5) and transgenic Eµ-TCL1 mice (TCL1+, n = 5) following ex vivo treatment for 24 h with 500 nM ML162. Statistical analysis: paired t-tests were applied for comparisons involving dependent (matched) samples (a), whereas unpaired t-tests were used for comparisons between independent groups (d–h). One-way ANOVA with multiple comparisons was used to assess differences across multiple treatment conditions (b, c). ‘n’ indicates the sample number, bars represent the mean; P-value: *P < 0.05; **P < 0.01; ***P < 0.001