Fig. 4: CLL cell sensitivity to ferroptosis is determined by the IGHV mutational status.

a On the basis of a combination of their mutational status for TP53 (mutated, MT, and wild-type, WT) and IGHV (unmutated, U-CLL, and mutated, M-CLL), the CLL cell lines were grouped accordingly into the following: TP53 MT and U-CLL, TP53 WT and U-CLL, TP53 MT and M-CLL, and TP53 WT and M-CLL. Ferroptosis was triggered by 100 nM ML162 or 100 nM RSL3, and specific cell death was assessed. Specific cell death was calculated relative to that of the control (=baseline) as follows: 100 × (% dead cells − % baseline)/(100 − % baseline). The baseline values were normalized to 0%. b Lipid peroxidation levels were measured in U-CLL (CII, HG3, PCL-12, and Wa-C3CD5+) and M-CLL (I83-E95, JVM-3, MEC-1, and PGA-1) cells under basal conditions in three independent experiments. c U-CLL and M-CLL cell lines were treated with 100 nM ML162 or 100 nM RSL3 in two independent experiments, and lipid peroxidation was assessed. d Baseline Fe²⁺ content (left panel, three independent experiments) and its relative change upon treatment with 100 nM ML162 and 100 nM RSL3 (right panel, two independent experiments) were assessed in U-CLL and M-CLL cell lines via FACS via Phen Green SK. Note that the Phen Green SK signal is quenched by Fe²⁺; thus, lower fluorescence indicates higher intracellular ferrous iron levels. e Primary patient U-CLL (n = 10) and M-CLL (n = 10) cells were treated with 500 nM ML162, and specific cell death was assessed. f Baseline lipid peroxidation is shown for primary patient U-CLL (n = 12) and M-CLL cells (n = 12). g Baseline Fe²⁺ content was assessed in primary patient U-CLL (n = 13) and M-CLL cells (n = 12). h Primary patient U-CLL (n = 10-12) and M-CLL cells (n = 10-12) were pretreated for 24 h with ibrutinib (Ibr, 10 µM) and subsequently treated with 500 nM ML162, and specific cell death and lipid peroxidation were assessed. i The expression of key pro- and anti-ferroptotic proteins was compared in primary patient U-CLL (n = 11–17) and M-CLL cells (n = 12–17) on the basis of the median fluorescence intensity (MdFI). The data are shown as the fold change between M-CLL/U-CLL cells, and significantly altered proteins are highlighted in red. Statistical significance was determined on the basis of groupwise comparisons of MdFI values. j TFRC/CD71 protein levels in primary patient U-CLL and M-CLL cells were analyzed via data retrieved from the proteome dataset by Meier-Abt et al. k The left panel shows a representative FACS-based gating strategy for B-cell subsets in HD-derived peripheral blood (n = 10): naïve B cells (NaB), transitional B cells (TrB), non-switched memory B cells (NSM), and switched memory B cells (SM). The TFRC/CD71 levels of the corresponding B-cell subsets measured by FACS are summarized in the right panel. l Kaplan‒Meier analysis of treatment-free survival in CLL patients stratified by TFRC/CD71 expression levels, which was performed on the basis of publicly available proteome data from Meier-Abt et al. Statistical analysis: Unpaired t-tests were applied for comparisons between independent groups (Fig. 4b–j), whereas one‒way ANOVA with multiple comparisons was used to assess differences across multiple treatment conditions (a, k). Kaplan–Meier survival analysis was applied for survival comparisons (l). Abbreviations: ‘n’ indicates the sample number; bars represent the mean; P-value: *P < 0.05; **P < 0.01; ***P < 0.001