Ironomycin Induces Mantle Cell Lymphoma Cell Death By Targeting Iron Metabolism Addiction (2022)

Mantle-cell lymphoma (MCL) accounts for approximately 5 to 7% of all lymphomas, and in North America and Europe. MCL remains an aggressive and incurable cancer with existing therapies, presenting a significant unmet clinical need. Accumulating evidence reveals that abnormal iron metabolism plays an important role in tumorigenesis and in cancer progression of many tumors. In cancer cells, the demand for iron increases in response to sustained deregulation of cell proliferation and DNA synthesis. Based on these data, we searched to identify alterations of iron homeostasis in MCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of a cohort of patients with MCL (n=71) established the iron score (IS), a gene expression-based risk score enabling identification of patients with MCL with a poor outcome who might benefit from a suitable targeted therapy. We analyzed the therapeutic interest of ironomycin, a new promising iron depleting molecule. Ironomycin is known to sequester iron in the lysosome and to induce ferroptosis (Mai TT et al. Nature Chemistry 2017). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. In a panel of 6 MCL cell lines, ironomycin inhibited MCL cell growth at nanomolar concentrations compared with typical iron chelators. Then, we investigated if ironomycin induces cell death through apoptosis. Flow cytometry analysis revealed that ironomycin treatment resulted in AnnV+/7AAD+ and AnnV+/7AAD- death profiles in MCL. This effect was partially inhibited by the pan-caspase inhibitor Q-VD-Oph. Ironomycin also induced caspases 8 and 9 cleavage, and the activation of caspases 3/7. Furthermore, we found that ironomycin induces ferroptosis with lipid peroxidation and the production of ROS monitored by the fluorescence probe CM-H2DCFDA. Ironomycin induced cell death is inhibited by the ferroptosis inhibitor, ferrostatin-1. The treatment did not induce ferritinophagy, as there was no significant change on the expression of LC3B-II, ferritin and the transferrin receptor (CD71).Taken together, these data indicate that ironomycin kills MCL cells by apoptosis mediated by caspase activation and ferroptosis. Ironomycin significantly decreased cell proliferation rate, with a reduced percentage of cells in S-phase together with Ki67 and Cyclin D1 downregulation. Western blot analysis showed an increase of Chk1 and Chk2 phosphorylation, two central factors of DNA damage response, and an increase of the phosphorylation of γ-H2AX, indicative of DNA damage. With major importance, we validated the therapeutic interest of ironomycin in primary MCL cells of patients (n=5). Ironomycin (20 nM) demonstrated a significant higher toxicity in MCL cells compared to normal cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional chemotherapy used in MCL. Interestingly, we identified a synergistic effect when ironomycin is combined with Ibrutinib BTK inhibitor. Furthermore, Significant synergistic effects were also observed by combining ironomycin with BH3 mimetics or Doxorubicin. Altogether, these data underline that MCL patients my benefit from targeting iron homeostasis using ironomycin alone or in combination with conventional MCL treatments.