P597: HIGH DOSE IRON IMPAIRS MALIGNANT B-CELL VIABILITY IN CHRONIC LYMPHOCYTIC LEUKEMIA

Abstract

Background: Identification of new strategies to improve Chronic Lymphocytic Leukemia (CLL) outcome is fundamental since the disease remains incurable. Malignant B-cells show high levels of reactive oxygen species (ROS), which favors adaptation and survival but also enhances sensitivity to pro-oxidant therapeutics. Redox homeostasis is affected by iron balance since iron excess works as pro-oxidant, and cancer cells rearrange iron trafficking proteins to promote iron uptake. This adaptation might be exploited by exposing malignant cells to iron excess to cause ROS generation, lipid peroxidation, and eventually ferroptosis. We previously demonstrated the feasibility of this strategy in multiple myeloma and prostate cancer pre-clinical models. Aims: We aim at exploiting high dose iron to negatively affect malignant cell survival and increase therapeutic efficacy of current target therapies (BTK and BCL2 inhibitors). We aim at dissecting the molecular mechanisms underlying iron toxicity and at testing whether iron can improve immune dysfunctions. Methods: CLL cell lines (MEC-1, MEC-2 and PCL-12), patients peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) and B lymphocytes purified from patients PB were treated with 300 µM ferric ammonium citrate (FeAC) alone or in combination with the BTK inhibitor ibrutinib (IBR, 5-10 µM) or the BCL2 inhibitor venetoclax (VNTX, 1.25-2.5 nM). We evaluated cell viability by annexin PI staining, lipid peroxidation and ferroptosis by measuring malondialdehyde (MDA) levels and antioxidant gene expression levels by qRT-PCR. We additionally evaluated T cell activation by measuring CD25 expression. MEC-1 xenografts generated in immunodeficient RAG2-/-yc-/- mice were treated with 20 mg/Kg ferric carboximaltose. Results:In vitro, iron treatment impaired cell proliferation in all CLL cell lines analyzed, inducing accumulation of MDA and cell death (p<0.01). In vivo, iron supplementation reduced the amount of MEC-1 cells in bone marrow and spleen of xenograft mice as compared to control saline-treated mice. In patients primary samples, iron induced cell death of leukemic lymphocytes (CD19+CD5+) either as purified cells or within bulk PBMC (p<0.01). Moreover, combination of iron with IBR or VNTX induced cell death at a higher extent compared to each single drug or to iron alone (p<0.01). Leukemic cells in BMMC samples were also iron sensitive and the effect became more evident upon combination with IBR or VNTX (p<0.05). Neither iron nor iron-drug combinations induced cell death in non-malignant cells. Iron also increased CD25 expression in CD8+ T cells and we are currently exploring whether this might indicate that iron can also improve cytotoxic mediated immune response against leukemic cells. As single patient analysis revealed heterogeneity of response, we investigated whether we might predict iron sensitivity by analyzing the antioxidant gene expression profile. All CLL cell lines analyzed showed a lower basal expression of NFE2L2, HMOX1, and GPX4 antioxidant genes than typical iron-resistant cell lines, such as prostate cancer PC-3 cells, suggesting that differences in antioxidant gene expression levels may mediate iron response and that this association is worth to be explored in CLL primary samples to explain heterogeneous iron response. Summary/Conclusion: Our pre-clinical studies suggests that exploiting iron toxicity might be a valuable strategy to improve current target therapies in CLL. Additional studies aimed at distinguishing iron sensitive patients from poor responders will improve the translational potential of this therapeutic approach.