Regulation of Iron Homeostasis and Efficacy of Rusfertide Analog Peptide in a Mouse Model for Polycythemia Vera
Authors: Taranath Roopa , Zhao Li , Vengalam Jayanthi , Lee Lawrence , Tang Tenny , Dion Celino , Su Ahu , Tovera James , Bhandari Ashok , Cheng Xiaoli , Mattheakis Larry , Liu David Y 
Published: November 2021

Polycythemia Vera (PV) is a rare blood disease where mutations in JAK2 kinase confer constitutive JAK2 activity leading to abnormally elevated erythropoiesis that is independent of erythropoietin. PV patients present with iron deficiency at diagnosis due to increased iron utilization for erythropoiesis (Ginzburg YZ, Leukemia 2018) which worsens after repeated therapeutic phlebotomy (TP) performed to maintain hematocrit below 45%. The resulting suppression of hepcidin, the body's main negative regulator of iron metabolism, fuels expanded erythropoiesis resulting in a continued need for TP and thereby exacerbating patients' iron deficiency.

Rusfertide targets iron exporter membrane protein ferroportin to trigger its degradation, preventing iron export from cells responsible for dietary iron absorption and cells that store and recycle iron. The resulting pharmacodynamic effect of lowered serum iron has disease-modifying effects in PV (Ginzburg YZ, Leukemia 2018). Rusfertide essentially eliminated the need for therapeutic phlebotomy in all PV patients (Kremyanskaya M, Blood 2020 136 Suppl 1: 33). Rusfertide also reversed iron deficiency, as indicated by increased serum ferritin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) in these patients.

We present results from studies in a mouse PV model with JAK2-V617F mutation as in human PV (Mullaly A, Cancer Cell 2010; JAX stock #031658). We show that rusfertide analog Peptide A is efficacious in lowering hematocrit (HCT) while modulating other hematological parameters. Further, we show redistribution of iron away from erythropoiesis and renormalization of iron homeostasis as evidenced by ferrokinetic parameters.

PV mice were treated over 6-weeks (thrice per week) with Vehicle or Peptide A at 2.5 or 7.5 mg/kg. At the end of 6 weeks, hematology parameters HCT, hemoglobin, RBC counts, were elevated in the PV-Vehicle group as compared to wild type (WT-Vehicle) mice (Table 1). Hematology parameters in PV-2.5 mg/kg group were lowered to WT-Vehicle values. In PV-7.5 mg/kg group, these parameters were lower than WT-Vehicle values, indicating that excessive iron restriction (EIR) leads to the expected anemic conditions. MCH and mean corpuscular hemoglobin concentration (MCHC) in PV-Vehicle group and PV-2.5 mg/kg treated were comparable to WT-Vehicle, indicating a lack of EIR. For the PV-7.5 mg/kg treated group, MCH and MCHC were significantly lower than WT-Vehicle, suggesting EIR at a high dose impacts hemoglobin concentration of RBC.

To investigate the impact of iron restriction with Peptide A on erythroblast precursor cells in bone marrow, we conducted flow-cytometry analysis by gating on CD71 and TER-119 expression, and measuring intracellular iron using Ferro Far Red (FFR) dye. The CD71 + early precursor cell population did not change with Peptide A treatment however, the CD71 -/TER-119 + late precursor cell population was significantly lowered (~4-fold and 7.5-fold, in 2.5 and 7.5 mg/kg Peptide A treated PV groups respectively). Iron levels of CD71 + cells were dose-dependently and statistically significantly reduced in the Peptide A treated groups as compared to PV-Vehicle group. Iron levels of CD71 - cells were marginally lowered only in the PV-7.5 mg/kg group.

We investigated the nature of iron redistribution induced by Peptide A, by using flow assay to assess iron concentration in splenic macrophages (F4/80 +/CD11b +). Iron was ~2-fold higher in the PV-7.5 mg/kg group as compared to PV-vehicle, and marginally higher in PV-2.5 mg/kg group. Total tissue iron concentration in the spleen was elevated in a dose-related manner in Peptide A treated groups compared to PV-Vehicle group, and in commensuration serum ferritin was increased. Serum iron was ~2-fold lower in PV-Vehicle group as compared to WT-Vehicle indicating iron depletion due to increased iron utilization for erythropoiesis. Serum iron measured after clearance of Peptide A from circulation (48 hr post-dose), was marginally increased for both Peptide A treated groups compared to PV-Vehicle.

These data demonstrate that treatment with rusfertide and analogs, restricts iron from erythropoiesis by sequestering it in macrophage storage compartments. These effects along with normalization of iron homeostasis contribute to usefulness of rusfertide dose titration treatment in maintaining HCT &lt;45% and improving symptoms related to iron deficiency in human PV.

Figure 1 Figure 1.

Disclosures

Taranath: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhao: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Vengalam: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tang: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dion: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Su: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tovera: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhandari: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cheng: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mattheakis: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company.