PB1748: DIFFERENTIAL IRON HOMEOSTASIS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN VITRO MODELS

Abstract

Background: Iron is an essential element which plays several physiological activities in different human organs. Current studies suggest that dysregulation of iron proteins and subsequent accumulation of iron are associated with the response of leukemia to treatments. In particular, it has been reported that low levels of ferroportin-1 (FPN1), lead to an increased sensitivity to chemotherapy in acute myeloid leukemia. Aims: We aimed at analyzing the gene expression profile of iron-related genes in childhood B acute lymphoblastic leukemia (ALL) and characterize the iron metabolism in vitro models. Methods: Iron related genes (Ceruloplasmin (CP), Ferroportin-1 (SLC40A1), Transferrin Receptor 1 (TFR1) and Ferritin Light Chain (FTL)) were analyzed by qRT-PCR in BM samples of 87 childhood ALL and 8 healthy donors. Cases were selected to be representative of t(12;21), t(4;11), t(9;22) or t(1;19) subgroups, as well as MRD-based standard-risk (SR), medium-risk (MR) and high-risk (HR) response. Furthermore, we evaluated the cellular viability in REH, RS4;11, 697 and TOM-1 cell lines after exposure to iron as ferric ammonium citrate (FAC 100 to 400 µM), deferoxamine (DFO 25 µM) and FAC+DFO (100 µM + 25 µM), using MTT assay at 24-48 and 72h. TFR1 and FTL mRNA levels were assessed at 8h, 16h, 24h, 48h. Labile Iron Pool (LIP) was measured by flow-cytometry using Calcein-AM technique after the exposure to FAC (100-200-400 µM) at 48h. Results:TFR1, SLC40A1 and CP were up-regulated in HR group vs. controls. SLC40A1 and CP were down-regulated in all genetic subgroups, except for CP in t(9;22). TFR1 was slightly expressed in all subgroups. In the t(1;19) subgroup all the iron genes were downregulated, suggesting that this subgroup may not be responsive to iron, as also confirmed by our in vitro data. Iron supplementation by FAC [100 µM] resulted in a boost of viability for TOM-1, REH and RS4;11. TOM-1 and REH viability decreased with FAC 200-400 µM, while RS4;11 proliferation was stimulated by all concentrations. Together with low transcriptional levels of iron genes, 697 viability did not change with different FAC treatments. DFO reduced cell viability in a time-dependent manner. At 48h the viability started to drop for all cell lines (TOM-1 80% - RS4;11 66% - REH 71% - 697 51%) and the co-administration of iron restored the viability to levels similar or even higher than the untreated (TOM-1 100%-RS4;11 80% -REH 92% - 697 81%). FTL expression was significantly up-regulated in all cell lines, while the TFR1 was down-regulated at 8, 16 and 24h vs. untreated. Still, some differences were detected within the cellular models: RS4;11 showed highest level of FTL expression, while in 697 the transcript was slightly upregulated. In 697 cell line, the LIP increased with higher dosages of FAC, suggesting that this cell line was not able to handle high amount of free iron. TOM-1, RS4;11 and REH presented LIP levels higher than the untreated condition, still no significant differences were noticed between the concentrations. Summary/Conclusion:SLC40A1 and CP high levels of HR group potentially suggest that their refractory behavior may be correlated with ability to keep regulate iron intracellular concentration. TOM-1, REH and RS4;11 behave as resistant cell lines, whilst the 697 appear as not sensitive to iron metabolism. In conclusion, leukemia cell lines can be considered as trustable in vitro models for further studies in ALL to investigate whether iron metabolism can be determinant for survival and resistance, and consequently a target for treatment.