P1489: high dose iron impairs malignant b-cell viability and improves immune antitumor functions in chronic lymphocytic leukemia

Background: Identification of novel strategies to improve Chronic Lymphocytic Leukemia (CLL) outcome is of primary importance as the disease remains incurable. Malignant B cells show high levels of reactive oxygen species (ROS), which favors adaptation 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 as an Achilles’ heel by exposing cells to iron excess to cause ROS generation, lipid peroxidation and ferroptosis. In addition, iron may activate the antitumor response of immune cells since T cells promote lipid peroxidation in target cells to succeed in their cell-killing activity, and iron up-taken by macrophages may promote their pro-inflammatory phenotype. Aims: We aim at investigating whether high-dose iron negatively affects malignant cell survival, increases the efficacy of current targeted therapies, and improves antitumor immune functions. Methods: We evaluated cell viability, ferroptosis markers and antioxidant gene expression levels in: 1) CLL cell lines (MEC-1, MEC-2 and PCL-12) cultured in the presence or the absence of 300 µM ferric ammonium citrate (FeAC; 2) peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) and B lymphocytes purified from patients treated in vitro with 300 μM FeAC alone or in combination with the BTK inhibitor ibrutinib (ibtr) or the BCL2 inhibitor venetoclax (vnx). Additionally, MEC-1 cells were treated with iron in presence of macrophages. In PBMC, we evaluated T cell response and proliferation rate induced by iron by measuring CD25 expression and CellTrace violet staining. In vivo, we evaluated disease development in MEC-1 xenografts generated in immunodeficient RAG2-/-γc-/- and in the CLL-prone Eμ-TCL1 mice treated with 20 mg/Kg ferric carboximaltose. Results:In vitro, iron treatment impaired cell proliferation in all CLL cell lines analyzed, inducing lipid peroxidation and cell death. Co-culturing MEC-1 cells with macrophages increased iron-induced cell death compared to the effect observed in MEC-1 cells mono-culture. In vivo, iron addition reduced MEC-1 cells in the bone marrow and spleen of xenografted mice and leukemic cells in the PB of Eμ-TCL1 mice compared to control mice. In patients primary samples, iron promoted leukemic cell death when cells were treated in vitro as either purified or bulk PBMC/BMMC (p<0.05). Moreover, the combination of iron with ibtr or vnx induced cell death at a higher extent than drugs or iron used as a single agent (p<0,05). Interestingly, patients samples with unmutated IGHV genes, which are more likely to experience aggressive disease, were more sensitive to iron treatment than IGHV-mutated patients. In addition, preliminary analysis showed that the expression levels of the ferroptosis-related gene LPCAT3 in the purified leukemic B lymphocytes directly correlate with the response to iron, suggesting a potential candidate marker of iron sensitivity. Finally, iron also promoted T cell proliferation and CD25 expression of CD8+ T cells, suggesting an increase in their cytotoxic potential. Summary/Conclusion: Our pre-clinical studies suggest the use of iron as a therapeutic agent in CLL, given its anti-leukemic toxicity either alone or in combination with the current CLL therapies. Further studies aimed at dissecting the molecular features to distinguish between highly iron sensitive versus poorly iron-sensitive patients will improve the personalization of this strategy in the clinical setting. Keywords: Iron, Chronic lymphocytic leukemia