Regulation of Iron Homeostasis and Efficacy of Rusfertide Analog Peptide in a Mouse Model for Polycythemia Vera

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 <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.

Potent antitumor activity of a glutamyltransferase-derived peptide via an activation of oncosis pathway

Hepatocellular carcinoma (HCC) still presents poor prognosis with high mortality rate, despite of the improvement in the management. The challenge for precision treatment was due to the fact that little targeted therapeutics are available for HCC. Recent studies show that metabolic and circulating peptides serve as endogenous switches for correcting aberrant cellular plasticity. Here we explored the antitumor activity of low molecular components in human umbilical serum and identified a high abundance peptide VI-13 by peptidome analysis, which was recognized as the part of glutamyltransferase signal peptide. We modified VI-13 by inserting four arginines and obtained an analog peptide VI-17 to improve its solubility. Our analyses showed that the peptide VI-17 induced rapid context-dependent cell death, and exhibited a higher sensitivity on hepatoma cells, which is attenuated by polyethylene glycol but not necrotic inhibitors such as z-VAD-fmk or necrostatin-1. Morphologically, VI-17 induced cell swelling, blebbing and membrane rupture with release of cellular ATP and LDH into extracellular media, which is hallmark of oncotic process. Mechanistically, VI-17 induced cell membrane pore formation, degradation of α-tubulin via influx of calcium ion. These results indicated that the novel peptide VI-17 induced oncosis in HCC cells, which could serve as a promising lead for development of therapeutic intervention of HCC.

ACTH(6–9)PGP Peptide Protects SH-SY5Y Cells from H2O2, tert-Butyl Hydroperoxide, and Cyanide Cytotoxicity via Stimulation of Proliferation and Induction of Prosurvival-Related Genes

Stabilized melanocortin analog peptide ACTH(6–9)PGP (HFRWPGP) possesses a wide range of neuroprotective activities. However, its mechanism of action remains poorly understood. In this paper, we present a study of the proproliferative and cytoprotective activity of the adrenocorticotropic hormone fragment 6–9 (HFRW) linked with the peptide prolyine–glycyl–proline on the SH-SY5Y cells in the model of oxidative stress-related toxicity. The peptide dose-dependently protected cells from H2O2, tert-butyl hydroperoxide, and KCN and demonstrated proproliferative activity. The mechanism of its action was the modulation of proliferation-related NF-κB genes and stimulation of prosurvival NRF2-gene-related pathway, as well as a decrease in apoptosis.

ACTH(6-9)PGP Peptide Protects SH-SY5Y Cells from the H2O2, tert-Butyl Hydroperoxide, and Cyanide Cytotoxicity via Prolif-eration Stimulation and Antioxidant-related Genes Induction

Stabilized melanocortin analog peptide ACTH(6-9)PGP (FRWGPGP) possess a wide range of neuroprotective activities. However, its mechanism of action remains poorly understood. In this paper, we studied the pro-proliferative and cytoprotective activity of the adrenocorticotropic hormone fragment 6-9 (FRWG) linked with the peptide Prolyl-Glycyl-Proline on the SH-SY5Y cells in the model of oxidative stress-related toxicity. The peptide dose-dependently protected cells from H2O2, tert-butyl hydroperoxide, and KCN. The mechanism of its action was the modu-lation of proliferation-related (NF-kB and Nrf-2) and antioxidant-related (HO-1, Nqo1, Gclc) genes and apoptosis decrease.