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3846 Differential Effects of Monoferric Transferrin Forms on Erythropoiesis Are Mediated By TFR2

Program: Oral and Poster Abstracts
Session: 112. Thalassemia and Globin Gene Regulation: Poster III
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Translational Research, Thalassemia, Hemoglobinopathies, Diseases
Monday, December 11, 2023, 6:00 PM-8:00 PM

Amaliris Guerra, PhD1*, Ariel Rivera, BA1*, Nolan Hamilton2*, Xiuli An, MD, PhD3, Nermi L. Parrow, PhD4, Yelena Ginzburg, MD5, Robert E. Fleming, MD6 and Stefano Rivella, phD7

1Children's Hospital of Philadelphia, Philadelphia, PA
2Children's Hospital of Philadelphia, Philadelphia
3Zhengzhou University, New York, NY
4Saint Louis University School of Medicine, St Louis, MO
5Division of Hematology & Medical Oncology, Tisch Cancer Institute/ Icahn School of Medicine at Mount Sinai, New York, NY
6Saint Louis University, Saint Louis, MO
7Department of Pediatrics, University of Pennsylvania, Philadelphia, PA

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), monoferric iron bound to the N-lobe or C-lobe (mono-TF) or to both lobes (holo-TF). All TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TFR2 which is mainly expressed by hepatocytes and erythroid precursors to modulate cellular signaling events regulating hepcidin expression and erythropoiesis. We previously generated N-lobe blocked (TfN-bl) or C-lobe blocked (TfC-bl) TF mutant mice, and observed marked differences between them in iron parameters, RBC count and EPO sensitivity (Parrow et al., 2019). In this current work, we investigate the effects of the mutated TFs on ineffective erythropoiesis in murine β-thalassemia intermedia. We moreover examine the consequences of erythroid knockout of TFR2 in the TF N-blocked and C-blocked mice.

Based on observations in the TF mutant mice, we hypothesized that Hbbth3/+ mice crossed with TfC-bl would demonstrate improved erythropoietic and iron parameters compared with Hbbth3/+TfN-bl. Hbbth3/+TfC-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbbth3/+TfC-bl relative to Hbbth3/+Tf+/+ controls. By contrast, similar improvements in RBC counts and hemoglobin were not observed in Hbbth3/+TfN-bl mice. Nonetheless, the Hbbth3/+TfN-bl mice displayed lower reticulocytes, increased platelets, decreased IE, decreased splenomegaly, and decreased serum ERFE compared to Hbbth3/+Tf+/+ controls and thus displayed a phenotype intermediate between Hbbth3/+TfC-bl and Hbbth3/+Tf+/+ controls. Serum EPO was elevated in Hbbth3/+TfN-bl compared to either Hbbth3/+TfC-bl or Hbbth3/+Tf+/+ mice. Moreover, the increased hepcidin observed in Hbbth3/+TfC-bl compared with Hbbth3/+Tf+/+ was not observed in Hbbth3/+TfN-bl mice.

To examine the contribution of erythroid TFR2 to the different phenotypes of the N-blocked and C-blocked TF mice, we generated a mouse line expressing TFR2-3xFLAG that is flanked by loxp sites (TFR2-3xFLAGfl/fl). These mice were used to generate mice with erythroid knockout of TFR2 in the TF mutant mice; i.e., EPORCre-tdtomTfN-blTFR2cko and EPORCre-tdtomTfC-blTFR2cko. N-blocked mice with conditional knockout of TFR2 demonstrated increases in RBC counts and Hb levels compared with the TF N-blocked mice (EPORCre-tdtomTfN-blTFR2cko vs TfN-bl/TfN-blTFR2-3xFLAGfl/fl), and had values similar to the C-blocked mice (TfC-bl/TfC-blTFR2-3xFLAGfl/fl or EPORCre-tdtomTfN-blTFR2cko). Likewise, conditional KO of TFR2 in the N-blocked mice resulted in lower EPO levels, which were similar to those in the C-blocked mice. These data support the hypothesis that differences observed between the mono-TF forms at the erythroid level are mediated via TFR2.

These data support a model by which lobe-specific iron occupancy of TF influences hematopoiesis via erythroid TFR2.

Disclosures: Ginzburg: Protagonist Therapeutics, Inc.: Consultancy, Research Funding. Rivella: GSK: Consultancy, Ended employment in the past 24 months; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Meira GTx: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Ended employment in the past 24 months; Incyte: Consultancy, Ended employment in the past 24 months; Celgene Corporation: Consultancy, Ended employment in the past 24 months; Rallybio, LLC: Consultancy, Ended employment in the past 24 months.

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