Edward Jachimowicz

Targeting EPO and EPO receptor pathways in anemia and dysregulated erythropoiesis

Introduction: Recombinant human erythropoietin (rhEPO) is a first-line therapeutic for the anemia of chronic kidney disease, cancer chemotherapy, AIDS (Zidovudine therapy), and lower-risk myelodysplastic syndrome. However, rhEPO frequently elevates hypertension, is costly, and may affect cancer progression. Potentially high merit therefore exists for defining new targets for anti-anemia agents within erythropoietin (EPO) and EPO receptor (EPOR) regulatory circuits. Areas covered: EPO production by renal interstitial fibroblasts is subject to modulation by several regulators of hypoxia-inducible factor 2a (HIF2a) including Iron Response Protein-1, prolyl hydroxylases, and HIF2a acetylases, each of which holds potential as anti-anemia drug targets. The cell surface receptor for EPO (EPOR) preassembles as a homodimer, together with Janus Kinase 2 (JAK2), and therefore it remains attractive to develop novel agents that trigger EPOR complex activation (activating antibodies, mimetics, small-molecule agonists). Additionally, certain downstream transducers of EPOR/JAK2 signaling may be druggable, including Erythroferrone (a hepcidin regulator), a cytoprotective Spi2a serpin, and select EPOR-associated protein tyrosine phosphatases. Expert opinion: While rhEPO (and biosimilars) are presently important mainstay erythropoiesis-stimulating agents (ESAs), impetus exists for studies of novel ESAs that fortify HIF2a’s effects, act as EPOR agonists, and/or bolster select downstream EPOR pathways to erythroid cell formation. Such agents could lessen rhEPO dosing, side effects, and/or costs.

Brief Report: Serpin Spi2A as a Novel Modulator of Hematopoietic Progenitor Cell Formation

Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HPs) and their Janus kinase‐coupled receptors (HP‐Rs). For HP/HP‐R studies, one central challenge in determining specific effects involves the delineation of nonredundant signal transduction factors and their lineage restricted actions. Via loss‐of‐function studies, we define roles for an HP‐regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B‐cell, and hematopoietic stem cell (HSC) formation. In granulomyelocytic progenitors, granulocyte macrophage colony stimulating factor (GMCSF) strongly induced Serpina3g expression with Stat5 dependency. Spi2A‐knockout (KO) led to 20‐fold decreased CFU‐GM formation, limited GMCSF‐dependent granulocyte formation, and compromised neutrophil survival upon tumor necrosis factor alpha (TNF‐α) exposure. In B‐cell progenitors, Serpina3g was an interleukin‐7 (IL7) target. Spi2A‐KO elevated CFU‐preB greater than sixfold and altered B‐cell formation in competitive bone marrow transplant (BMT), and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A‐KO compromised LT‐HSC proliferation (as well as lineageneg Sca1pos Kitpos (LSK) cell lysosomal integrity), and skewed LSK recovery post 5‐FU. Spi2A therefore functions to modulate HP‐regulated immune cell and HSC formation post‐5‐FU challenge. Stem Cells 2014;32:2550–2556

Novel roles for podocalyxin in regulating stress myelopoiesis, Rap1a, and neutrophil migration

Podocalyxin (Podxl) is a CD34 orthologue and cell surface sialomucin reported to have roles in renal podocyte diaphragm slit development; vascular cell integrity; and the progression of blood, breast, and prostate cancers. Roles for Podxl during nonmalignant hematopoiesis, however, are largely undefined. We have developed a Vav-Cre Podxl knockout (KO) mouse model, and report on novel roles for Podxl in governing stress myelopoiesis. At steady state, Podxl expression among hematopoietic progenitor cells was low level but was induced by granulocyte colony-stimulating factor (G-CSF) in myeloid progenitors and by thrombopoietin in human stem cells. In keeping with low-level Podxl expression at steady state, Vav-Cre deletion of Podxl did not markedly alter peripheral blood cell levels. A G-CSF challenge in Podxl-KO mice, in contrast, hyperelevated peripheral blood neutrophil and monocyte levels. Podxl-KO also substantially heightened neutrophil levels after 5-fluorouracil myeloablation. These loss-of-function phenotypes were selective, and Podxl-KO did not alter lymphocyte, basophil, or eosinophil levels. Within bone marrow (and after G-CSF challenge), Podxl deletion moderately decreased colony forming units-granulocytes, eyrthrocytes, monocyte/macrophages, megakaryocytes and CD16/32posCD11bpos progenitors but did not affect Gr-1pos cell populations. Notably, Podxl-KO did significantly heighten peripheral blood neutrophil migration capacities. To interrogate Podxls action mechanisms, a co-immunoprecipitation plus liquid chromatography-mass spectrometry approach was applied using hematopoietic progenitors from G-CSF-challenged mice. Rap1a, a Ras-related small GTPase, was a predominant co-retrieved Podxl partner. In bone marrow human progenitor cells, Podxl-KO led to heightened G-CSF activation of Rap1aGTP, and Rap1aGTP inhibition attenuated Podxl-KO neutrophil migration. Studies have revealed novel roles for Podxl as an important modulator of neutrophil and monocyte formation and of Rap1a activation during stress hematopoiesis.

ErbB2 promotes endothelial phenotype of human left ventricular epicardial highly proliferative cells (eHiPC)

The adult human heart contains a subpopulation of highly proliferative cells. The role of ErbB receptors in these cells has not been studied. From human left ventricular (LV) epicardial biopsies, we isolated highly proliferative cells (eHiPC) to characterize the cell surface expression and function of ErbB receptors in the regulation of cell proliferation and phenotype. We found that human LV eHiPC express all four ErbB receptor subtypes. However, the expression of ErbB receptors varied widely among eHiPC isolated from different subjects. eHiPC with higher cell surface expression of ErbB2 reproduced the phenotype of endothelial cells and were characterized by endothelial cell-like functional properties. We also found that EGF/ErbB1 induces VEGFR2 expression, while ligands for both ErbB1 and ErbB3/4 induce expression of Tie2. The number of CD31posCD45neg endothelial cells is higher in LV biopsies from subjects with high ErbB2 (ErbB2high) eHiPC compared to low ErbB2 (ErbB2low) eHiPC. These findings have important implications for potential strategies to increase the efficacy of cell-based revascularization of the injured heart, through promotion of an endothelial phenotype in cardiac highly proliferative cells.

SERPIN SPI2A AS A NOVEL MODULATOR OF HEMATOPOIETIC PROGENITOR CELL FORMATION

Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HPs) and their Janus kinase‐coupled receptors (HP‐Rs). For HP/HP‐R studies, one central challenge in determining specific effects involves the delineation of nonredundant signal transduction factors and their lineage restricted actions. Via loss‐of‐function studies, we define roles for an HP‐regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B‐cell, and hematopoietic stem cell (HSC) formation. In granulomyelocytic progenitors, granulocyte macrophage colony stimulating factor (GMCSF) strongly induced Serpina3g expression with Stat5 dependency. Spi2A‐knockout (KO) led to 20‐fold decreased CFU‐GM formation, limited GMCSF‐dependent granulocyte formation, and compromised neutrophil survival upon tumor necrosis factor alpha (TNF‐α) exposure. In B‐cell progenitors, Serpina3g was an interleukin‐7 (IL7) target. Spi2A‐KO elevated CFU‐preB greater than sixfold and altered B‐cell formation in competitive bone marrow transplant (BMT), and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A‐KO compromised LT‐HSC proliferation (as well as lineageneg Sca1pos Kitpos (LSK) cell lysosomal integrity), and skewed LSK recovery post 5‐FU. Spi2A therefore functions to modulate HP‐regulated immune cell and HSC formation post‐5‐FU challenge. Stem Cells 2014;32:2550–2556

Brief Report: Serpin Spi2A as a Novel Modulator of Hematopoietic Progenitor Cell Formation: Spi2A Regulation of HPC Expansion, and Survival

Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HPs) and their Janus kinase‐coupled receptors (HP‐Rs). For HP/HP‐R studies, one central challenge in determining specific effects involves the delineation of nonredundant signal transduction factors and their lineage restricted actions. Via loss‐of‐function studies, we define roles for an HP‐regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B‐cell, and hematopoietic stem cell (HSC) formation. In granulomyelocytic progenitors, granulocyte macrophage colony stimulating factor (GMCSF) strongly induced Serpina3g expression with Stat5 dependency. Spi2A‐knockout (KO) led to 20‐fold decreased CFU‐GM formation, limited GMCSF‐dependent granulocyte formation, and compromised neutrophil survival upon tumor necrosis factor alpha (TNF‐α) exposure. In B‐cell progenitors, Serpina3g was an interleukin‐7 (IL7) target. Spi2A‐KO elevated CFU‐preB greater than sixfold and altered B‐cell formation in competitive bone marrow transplant (BMT), and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A‐KO compromised LT‐HSC proliferation (as well as lineageneg Sca1pos Kitpos (LSK) cell lysosomal integrity), and skewed LSK recovery post 5‐FU. Spi2A therefore functions to modulate HP‐regulated immune cell and HSC formation post‐5‐FU challenge. Stem Cells 2014;32:2550–2556