Investigations into the Mechanisms and Clinical Implications of Modulation of Hepcidin Levels By Luspatercept in TD MDS and TD b-Thalassemia

Abstract

Introduction: Multiple blood transfusions lead to significant iron overload in transfusion dependent (TD) thalassemia (TDT) and TD-MDS. This excess iron is deposited in various organs leading to end organ damage, morbidity, and mortality. Iron overload patients are treated with iron chelation therapy (ICT) owing to lack of effective physiological iron removal mechanisms. Furthermore, chronic transfusions lead to paradoxical changes in hepcidin, the master regulator of iron homeostasis. While hepcidin is upregulated under conditions of increased intracellular and extracellular iron concentrations through BMP6 and IL6 dependent signaling; it is downregulated during expanded erythropoiesis through Erythroferrone (ERFE) to increase iron availability for developing erythron. Increased levels of hepcidin sequesters iron within organs leading to inefficiency of iron chelation therapy in TD-MDS and TDT patients. Luspatercept is a modified activin receptor fusion protein (modified ActRIIB-Fc) that sequesters certain members of TGF-b superfamily ligands, thus enhancing late-stage erythroid-maturation. Luspatercept is approved for TDT and TD-MDS patient populations-based on significant reduction in transfusion burden in two independent phase 3 trails (BELIEVE and MEDALIST, respectively). Here, we evaluated modulation of hepcidin levels with luspatercept treatment and its clinical implications in anemias due to elevated levels of hepcidin including TD-MDS and TDT patients. Methods: Luspatercept was administered sub-cutaneously every three weeks. Hepcidin and ERFE levels were measured in serum or plasma samples from BELIEVE (NCT02604433) and MEDALIST (NCT02631070) trial patients using specific ELISA and or chemiluminescence assay kits at week 25 following treatment. The regulation of hepcidin by luspatercept, BMP6 and IL6 in HepG2 cell-line model was determined by ELISA (in conditioned media) and q-PCR at increasing doses and time points. Results: Serum hepcidin levels at baseline across both arms in MEDALIST and BELIEVE were 80.65 ng/ml and 43.1 ng/ml respectively. These were significantly higher than normal healthy individuals (range of 5-10 ng/ml; P < 0.001), reported previously (Ilkovska etal JIMB 2016). Patients treated with luspatercept in both TDT and TD-MDS showed significant reduction in serum levels of hepcidin by 49% and 53% respectively (P<0.001) at week 25 compared to baseline. Hepcidin levels in the placebo group in TD-MDS patients demonstrated an increase of 119% (P<0.001) and a decrease of 12% in TDT patients (P=0.004) at the same time point. In addition, ERFE protein levels in serum, were elevated in response to Luspatercept treatment in both TD-MDS (30%) and TDT (51%) (P<0.001) in comparison to the placebo arms of decrease of 28% in TD-MDS and an increase of 2% in TDT. In HepG2 cells, treatment with recombinant BMP6 and IL6 increased hepcidin protein expression in a dose dependent manner. Luspatercept treatment inhibited this increase in hepcidin expression mediated by BMP6 and IL6. Time course analyses (2, 6, 24 and 72 hours), revealed that luspatercept inhibited hepcidin transcription and secretion levels starting at 24 hours. Conclusions: Luspatercept treatment reduced transfusion burden and iron loading rate due to improved erythropoiesis and release of ERFE by maturing erythroblasts consequently leading to reductions in elevated hepcidin levels in both TD-MDS and TDT patients. Our data provides a mechanistic rationale for downregulation of hepcidin levels with luspatercept treatment. Interestingly, cell-based assay data suggest that luspatercept directly modulates hepcidin transcription suggesting that in diseased conditions with abnormally higher levels of hepcidin and TGFβ family ligands, luspatercept might also modulate hepcidin transcription by sequestering TGFβ family ligands implicated in hepcidin synthesis. We postulate that decreased hepcidin levels results in release of excess iron into peripheral blood, making it available for effective chelation. These observations implicate sequencing of luspatercept, and ICT for effective management of iron overload in these TD patients. Furthermore, the data provide a mechanistic rationale for evaluating luspatercept in hepcidin dependent anemia of inflammation and iron refractory iron deficiency anemia (IRIDA) disease indications.