PF369 EXPLOITING IRON TOXICITY TO INCREASE VULNERABILITY OF MALIGNANT B CELLS
Authors: J. Bordini, M. Brambilla, F. Cerruti, P. Cascio, P. Ranghetti, L. Scarfò, C. Camaschella, P. Ghia, A. Campanella
Journal: HemaSphere (2019)

Background: Redox homeostasis plays a crucial role in B cells maturation, activation and survival. Moreover, H2O2 levels and proteasome activity are ultimately involved in antibody production. This equilibrium could be affected by iron balance since iron excess works as pro-oxidant. Cancer cells rearrange iron trafficking proteins to promote iron uptake, which favors proliferation. This adaptation might be exploited as an Achilles’ heel by exposing cells to iron excess, which may promote ROS generation and ferroptosis, a form of iron-dependent cell death induced by excessive formation of lipid hydroperoxides. Aims: We aim at exploiting this strategy to negatively affect survival of malignant B-cells and increase efficacy of therapeutic agents. Methods: Multiple myeloma (MM)(MM.1S and U266) and chronic lymphocytic leukemia (CLL) (MEC-1, MEC-2 and PCL12) cell lines and primary cells were exposed to iron excess (300 μM ferric ammonium citrate, FeAC) for 48 hours either alone or in combination with the proteasome inhibitor bortezomib (BTZ, 10 nM) or the BCL2 inhibitor venetoclax (VNTX, 1,25–2,5 nM) in the case of MM or CLL cells, respectively. We evaluated cell proliferation, cell viability, lipid peroxidation by measuring malondialdehyde (MDA) levels and antioxidant gene expression. We measured the activity of purified 26S proteasome exposed to FeCl2 and the effect of iron on protein degradation in MM cells, evaluating poly-Ub proteins. In selected in vivo experiments, we tested, in the MM murine model Vk∗MYC, the combination of iron dextran (100 mg/Kg) with the VMP regimen (BTZ, melphalan, and prednisone). Results: All MM and CLL cell lines analyzed were sensitive to iron excess as indicated by an impaired cell proliferation, increased cell death and an accumulation of MDA, a well-recognized event associated to ferroptosis. Besides inducing oxidative stress, iron also affected the proteasome. Iron induced a dose-dependent reduction of chymotrypsin-like activity of the 26S proteasome. In accordance, exposure to 300 μM FeAC promoted poly-Ub proteins accumulation in MM cells. Importantly, combination of iron with BTZ promoted higher poly-Ub, MDA levels and cell death than separate treatments. In vivo, VMP-Iron treated Vk∗MYC mice showed more evident reduction of monoclonal component (p < 0,01) as compared to VMP-Saline mice. In CLL cell lines, which were the most iron sensitive cells among the cell lines analyzed, iron sensitivity was associated to antioxidant capacity. All CLL cell lines had lower basal expression of NFE2L2, HMOX1, and GPX4 genes compared to typical iron resistant cell lines, such as prostate cancer PC-3 cells. Iron sensitivity was more heterogeneous in CLL primary cells, with 3/5 cases showing iron sensitivity as documented by increased AnV-PI positive cells after iron exposure. In 2/5 cases, irrespective of iron sensitivity, the combination of iron and VNTX showed addictive efficacy in promoting cell death then separate treatments. We are currently investigating whether differences in antioxidant gene expression levels may correlate with the iron response and the effect of iron-VNTX combination in primary cells. In addition, we are currently testing the effect of different iron doses in a CLL xenograft immunodeficient mouse model, using MEC-1 cells. Summary/Conclusion: Iron excess induces oxidative damage and can affect cellular pathways important for malignant cell survival ultimately leading to cell death. Combination of high-dose iron to standard therapies is worth exploring as it may increase efficacy of the current treatment by adding limited toxicity.