Ironomycin induces mantle cell lymphoma cell death by targeting iron metabolism addiction

Abstract

Rationale: Mantle-cell lymphoma (MCL) remains an aggressive and incurable cancer. Accumulating evidence reveals that abnormal iron metabolism plays an important role in tumorigenesis and in cancer progression of many tumors. Based on these data, we searched to identify alterations of iron homeostasis in MCL that could be exploited to develop novel therapeutic strategies. Methods: Analysis of the iron metabolism gene expression profile of a cohort of patients with MCL enables the identification of patients with a poor outcome who might benefit from an iron homeostasis-targeted therapy. We analyzed the therapeutic interest of ironomycin, known to sequester iron in the lysosome and to induce ferroptosis. Results: In a panel of MCL cell lines, ironomycin inhibited MCL cell growth at nanomolar concentrations compared with conventional iron chelators. Ironomycin treatment resulted in ferroptosis induction and decreased cell proliferation rate, with a reduced percentage of cells in S-phase together with Ki67 and Cyclin D1 downregulation. Ironomycin treatment induced DNA damage response, accumulation of DNA double-strand breaks, and activated the Unfolded Protein Response (UPR). We validated the therapeutic interest of ironomycin in primary MCL cells of patients. Ironomycin demonstrated a significant higher toxicity in MCL cells compared to normal cells from the microenvironment. We tested the therapeutic interest of combining ironomycin with conventional treatments used in MCL. We identified a synergistic effect when ironomycin is combined with Ibrutinib, Bruton's tyrosine kinase (BTK) inhibitor, associated with a strong inhibition of B-Cell receptor (BCR) signaling. Conclusion: Altogether, these data underline that MCL patients my benefit from targeting iron homeostasis using ironomycin alone or in combination with conventional MCL treatments.

Keywords: B-cell receptor signaling; drug combination; iron metabolism; ironomycin; mantle cell lymphoma.

Figure 1

The iron score predicts the clinical outcome in MCL. (A) A list of 62 genes involved in the regulation of iron biology was established using previously published data ,. Gene expression microarray data from one cohort (Staudt cohort) of 71 newly-diagnosed MCL patients was used (accession number GSE10793). Data were analyzed with Microarray Suite version 5.0 (MAS 5.0), using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500. 4 iron-related genes were found to have a good prognostic value (in green) and 4 a bad prognostic value (in red). ABCG2 (ATP-binding cassette transporter G2), SCARA3 (Scavenger Receptor Class A Member 3), IREB2 (Iron Responsive Element Binding Protein 2) and SFXN4 (sideroflexin 4); (APEX1 (DNA-(apurinic or apyrimidinic site) lyase), TFRC (Transferrin Receptor Protein 1), SLC39A14 (Solute Carrier Family 39 Member 14), and HIF1A (Hypoxia inducible factor A 1). Scheme was created with BioRender. (B) Patients of the Staudt cohort GSE10793 (n = 71) were ranked according to increased iron score and a maximum difference in OS (overall survival) was obtained with iron score of -3.7798 (also named 'cut point') splitting patients into high-risk and low-risk groups. The iron score was significantly associated with high-risk in MCL patients. (C) Primary MCL cells from 9 patients were treated with ironomycin at the indicated concentrations for 4 days. Tumor cells were analyzed by flow cytometry and expressed in % of control. Results represent the median ± IQR. Statistical significance was tested using paired t-test: *** p value < 0.001, **** p value < 0.0001. (D,E) Peripheral blood mononucleated cells (PBMC) from healthy donors (n = 5) were treated with ironomycin or deferasirox for 4 days, counted in presence of trypan blue to visually distinguish dead cells (trypan blue positive) from living cells (trypan blue negative). (D) Viability was calculated as the percentage of living cells to total cells (living + dead) in each condition compared to control. (E) Populations of B-lymphocytes, T-lymphocytes and monocytes were quantified by flow cytometry and compared to control condition. The 3 populations are expressed as % of control. Asterisks indicate significant differences compared to control conditions after applying a Student's t-test for pairs. *: p-value < 0.05; **: p-value < 0.01; ***: p: value < 0.001; ns: not significant.

Figure 2

Ironomycin impairs the proliferation of MCL cells. (A) JEKO1, JVM2 and MAVER1 cell lines were treated as indicated for 48 h. Cells were counted at day 0 and at the end of the treatments, and the number of cells was normalized to day 0 to calculate the proliferation rate. Graphs show the average and standard deviation of 3-4 independent experiments. (B) Cells were treated or not with ironomycin (JEKO1: 10 and 50 nM; JVM2/MAVER1: 50 and 250 nM) and Deferasirox (80 µM) for 48 h and BrdU (10 μg/ml) was added during the last 1.5 h of treatment. Cells were fixed and processed to detect BrdU incorporation and total DNA. BrdU+ cells were assigned to S-phase. BrdU- cells were assigned to G0/G1 or G2/M phases based on their DNA content. Results are the mean of 3-4 independent experiments. (C,D) Cells were treated as indicated for 48 h, and the levels of Cyclin D1 and Cyclin D2 were analyze in cell lysates by western blot. Tubulin was used as a loading control. Figures show 1 representative experiment out of 3. (E) Total mRNA was extracted from cells treated as indicated for 48 h, subjected to retrotranscription and the levels of expression of CCND1, CCND2, RB1 and CDK4 genes were quantified by qPCR. Graphs show the average ± SD of 3 independent experiments. (F) Cells were treated or not with ironomycin (JEKO1: 50 nM; JVM2/MAVER1: 250 nM) for 48 h, collected and the indicated proteins were analyzed by western blot in whole cell lysates. In all the graphs in this figure, asterisks indicate significant differences compared to control conditions after applying a Student's t-test for pairs. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Figure 3

(A) Cells were treated as indicated for 48 h and Annexin V was detected by flow cytometry. Results are the mean ± SD of 3 independent experiments. (B) Cells were treated as in (A). The levels of the indicated proteins were analyzed by western blot. Figure shows 1 representative out of 3 independent experiments. (C) Cells were treated with ironomycin (JEKO1: 50 nM, JVM2/MAVER1: 250 nM) for 48 h, and the levels of the indicated proteins were analyzed by western blot. Tubulin was used as a loading control. Figure shows 1 representative out of 3 independent experiments. (D) BH3 profiling of JEKO1, JVM2 and MAVER1. Cells were treated with ironomycin (JEKO1: 50 nM, JVM2/MAVER1: 250 nM) or DMSO for 20 h. Then, BH3 mimetics (venetoclax: Bcl2i, AZD-5991: Mcl1i, A-1155463: Bcl-xLi) or vehicle DMSO (control) were added to the culture medium for 4 h. Annexin V+ cells were detected by flow cytometry. Graphs represent the difference (Δ) between the percentage of Annexin V+ cells in each condition and in the control (vehicle DMSO). Results are the mean ± SD of 3 independent experiments. (E) Cells were pre-treated with the ferroptosis inhibitor Ferrostatin-1 (10 μM, 30 min) before treatment with ironomycin (JEKO1: 50 nM; JVM2/MAVER1: 250 nM) or the ferroptosis inducer erastin (4 μM) for 48 h. Annexin V was detected by flow cytometry. Graphs show the mean ± SD of 3-4 independent experiments. In all the graphs in this figure, asterisks indicate significant differences compared to control conditions after applying a Student's t-test for pairs. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001; ns: not significant.

Figure 4

Ironomycin downregulates the expression of BCR-related genes and synergizes with BTK inhibitor ibrutinib. (A) JEKO1, JVM2 and MAVER1 cells were treated with ironomycin (JEKO1: 50 nM; JVM2/MAVER1: 250 nM) for 48 h. Total RNA was extracted and RNA-sequencing was performed. GSEA of down- and up-regulated pathways is shown. FDR: false discovery rate. (B) Cells were treated with ironomycin (JEKO1: 50 nM, JVM2/MAVER1: 250 nM) for 48 h, and the levels of the indicated proteins were analyzed by western blot. Tubulin was used as a loading control. Figure shows 1 representative out of 3 independent experiments. (C-E) Cells were seeded in flat-bottom 96-well plates, treated with increasing concentrations of ironomycin (1 - 4000 nM) and ibrutinib (0.125 - 32 μM), and incubated for 4 days. Cell growth was assessed by CellTiter Glo® assay. Drug synergy was calculated using R package “SynergyFinder”. Effect of drug combination on cell growth is shown in a pseudo-color scale from red (synergism) to green (antagonism). Matrixes show the mean of 3 independent experiments.

Figure 5

(A) JEKO1, JVM2 and MAVER1 cell lines were treated as indicated with ironomycin (JEKO1: 50 nM; JVM2/MAVER1: 250 nM) and ibrutinib (JEKO1: 0.5 μM; JVM2: 1.5 μM; MAVER1: 6.25 μM) for 48 h. Cells were counted at day 0 and at the end of the treatments, and the number of cells was normalized to day 0 to calculate the proliferation rate. Graphs show the mean ± SD of 3 independent experiments. (B) Cells were treated as in (A) and BrdU (10 μg/ml) was added during the last 1.5 h of treatment. Cells were fixed and processed to detect BrdU incorporation and total DNA. BrdU+ cells were assigned to S-phase. BrdU- cells were assigned to G0/G1 or G2/M phases based on their DNA content. Results are the mean ± SD of 3-4 independent experiments. (C) Cells were treated as in (A) and Annexin V was detected by flow cytometry. Graphs show the mean ± SD of 3-4 independent experiments. (A-C) Asterisks indicate a significant difference compared to control conditions after applying a Student's t-test for pairs. *: p-value < 0.05; **: p-value < 0.01; ***: p- value < 0.001; ****: p-value < 0.0001; ns: not significant. (D) Cells were treated as in (A). Total RNA was extracted, RNA-sequencing was performed and GSEA was applied to find upregulated and downregulated pathways in cells treated with ironomycin plus ibrutinib. FDR: false discovery rate.

Figure 6

Model of ironomycin cytotoxic effects alone and in combination with other drugs. (A) Ironomycin sequesters iron in lysosomes triggering different cellular responses: (1) the production of ROS through the Fenton reaction that cause peroxidation of lipids, which require GPX4 activity to be detoxified, and DNA damage that will cause cell cycle arrest; (2) impairment of mitochondrial metabolism and ATP production; (3) ER stress characterized by the activation of UPR, notably the IRE1α signaling pathway. High levels of lipid peroxidation, DNA damage, mitochondrial activity impairment and sustained ER stress lead to ferroptosis and apoptosis. Combination of ironomycin with BH3 mimetics have a synergistic toxic effect in MCL cells. (B) Ironomycin downregulates BCR-signaling and synergizes with ibrutinib. Combination of both drugs further increases a sustained UPR that leads to apoptosis. Figures were created with Biorender.com.