Erythropoietin Receptor is a Risk Factor for Prognosis: A Potential Biomarker in Lung Adenocarcinoma

Background: Lung cancer has the highest mortality rate of all cancers, and LUAD's survival rate is particularly poor. Erythropoietin receptor (EPOR) is a member of the cytokine class I receptor family and can be detected in cancers such as lung adenocarcinoma (LUAD), however, the expression levels and prognostic value of EPOR in LUAD are still unclear.Methods: Multiple bioinformatics databases such as TIMER, Kaplan-Meier Plotter and TCGA databases, immunohistochemical method, and clinicopathological data of 92 LUADpatients between January 2008 and June 2016 were used to explore the EPOR expression, gene mutations affecting EPOR expression, EPOR-interacting or coexpressed genes, potential biological functions and the correlation of EPOR expression with prognosis, immune microenvironment and so on.All statistical analyses were performed in the R version 4.1.1.Results: In this study, the EPOR mRNA expression in LUAD tissues was possibly downregulated compared with that in normal lung tissues, but the EPOR protein expression in LUAD tissues was higher than that in paired normal lung tissues. Mutations in five genes, DDX60L, LGR6, POTEB3, RIF1 and SOX5, resulted in downregulation of EPOR expression, mutations in 10 genes includingC1orf168, DBX2 and EIF5B, resulted in upregulation of EPOR expression. Erichment analyses showed that EPOR is involved in neural tissue ligand-receptor interactions, MAPK and PI3K/Akt signaling pathways and cancer pathways. The KM Plotter and PrognoScan databases consistently concluded that EPOR was associated with prognosis in LUAD patients. Our clinicopathological data showed that high EPOR expression was associated with poorer OS (29.5 vs 46 months) and had a good predictive ability for 5-year survival probability. Conclusions: EPOR expression might be downregulated at the mRNA levels and significantly upregulated at the protein levels in LUAD, which showed that the mRNA and protein levels of EPOR are inconsistent.The high expression of EPOR was associated with poor prognosis and is expected to be a potential new prognostic marker for LUAD.

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.

Haemangioma: A Study of the Biology

<p>Infantile haemangioma (IH), considered a primary tumour of the microvasculature, is the most common tumour of infancy affecting about 10% of Caucasian infants. IH predominantly affects white, female and premature infants. IH typically undergoes an initial rapid proliferation during infancy (proliferative phase) characterised by aggressive angiogenesis, followed by spontaneous involution over the next 1-5 years (involuting phase) and continued improvement up to 10 years (involuted phase), often with a fibro-fatty residuum. IH consists of cells of various lineages, with the presence of mesenchymal stem cells, endothelial progenitor cells, endothelial cells, myeloid haematopoietic cells, and pericytes. This thesis demonstrates the expression of primitive (stem/progenitor cell) markers on the endothelium of IH. The expression of the transcription factors brachyury, Tal-1 and GATA-2, along with the demonstration of erythropoiesis in IH explants in vitro supports the hypothesis that IH consists of a primitive endothelium similar to an embryonic haemogenic endothelium. The expression of the erythropoietin receptor and haemoglobin zeta chain by the endothelium of IH further strengthens the notion that IH is a haemogenic endothelium. Consistent with the primitive embryonic origin, the expression of the placental markers human chorionic gonadotrophin (hCG) and human placenta lactogen (hPL), but not cytokeratin 7 (CK7) or human leucocyte antigen- G (HLA-G) by the endothelium in IH, supports a placental chorionic villous mesenchymal core cell, and not a trophoblast, origin for IH. IH thus has an extraembryonically derived primitive mesodermal origin. This primitive mesoderm is able to account for the haemogenic endothelium phenotype of the endothelium of proliferating IH microvessels with its capacity for both erythropoietic and mesenchymal differentiation. Additionally, data are presented to show that IH expresses key components of the renin-angiotensin system (RAS), angiotensin converting enzyme (ACE), angiotensin II (ATII), angiotensin receptor 2 (ATR2). Cultured IH-derived stem cells can be induced to proliferate and form blast colonies in response to ATII treatment. The crucial regulatory role of RAS in the proliferation and differentiation of the stem/progenitor cell population within IH accounts for the natural progression of IH. A model is proposed to provide a rational explanation for the serendipiditous discovery of the dramatic effect that the β-blocker, Propranolol has in accelerating involution of IH. The hypothesis that Propranolol exerts its action on IH through modulation of the RAS by blocking renin activity and preventing the conversion of angiotensinogen to angiotensin I, thereby reducing ATII levels, has led to a clinical trial using Captopril, an ACE inhibitor in the treatment of problematic proliferating IH. The observed accelerated involution of IH by Captopril which blocks the conversion of angiotensin I to ATII confirms a key regulatory role for RAS in the biology of IH This discovery underpins the development of potentially safer and novel treatment modalities for this enigmatic condition.</p>

Repurposing of High-Dose Erythropoietin as a Potential Drug Attenuates Sepsis in Preconditioning Renal Injury

Due to (i) the uremia-enhanced sepsis severity, (ii) the high prevalence of sepsis with pre-existing renal injury and (iii) the non-erythropoiesis immunomodulation of erythropoietin (EPO), EPO was tested in sepsis with pre-existing renal injury models with the retrospective exploration in patients. Then, EPO was subcutaneously administered in mice with (i) cecal ligation and puncture (CLP) after renal injury including 5/6 nephrectomy (5/6Nx-CLP) and bilateral nephrectomy (BiNx-CLP) or sham surgery (sham-CLP) and (ii) lipopolysaccharide (LPS) injection, along with testing in macrophages. In patients, the data of EPO administration and the disease characteristics in patients with sepsis-induced acute kidney injury (sepsis-AKI) were evaluated. As such, increased endogenous EPO was demonstrated in all sepsis models, including BiNx-CLP despite the reduced liver erythropoietin receptor (EPOR), using Western blot analysis and gene expression, in liver (partly through hepatocyte apoptosis). A high-dose EPO, but not a low-dose, attenuated sepsis in mouse models as determined by mortality and serum inflammatory cytokines. Furthermore, EPO attenuated inflammatory responses in LPS-activated macrophages as determined by supernatant cytokines and the expression of several inflammatory genes (iNOS, IL-1β, STAT3 and NFκB). In parallel, patients with sepsis-AKI who were treated with the high-dose EPO showed favorable outcomes, particularly the 29-day mortality rate. In conclusion, high-dose EPO attenuated sepsis with preconditioning renal injury in mice possibly through the macrophage anti-inflammatory effect, which might be beneficial in some patients.

Haemangioma: A Study of the Biology

<p>Infantile haemangioma (IH), considered a primary tumour of the microvasculature, is the most common tumour of infancy affecting about 10% of Caucasian infants. IH predominantly affects white, female and premature infants. IH typically undergoes an initial rapid proliferation during infancy (proliferative phase) characterised by aggressive angiogenesis, followed by spontaneous involution over the next 1-5 years (involuting phase) and continued improvement up to 10 years (involuted phase), often with a fibro-fatty residuum. IH consists of cells of various lineages, with the presence of mesenchymal stem cells, endothelial progenitor cells, endothelial cells, myeloid haematopoietic cells, and pericytes. This thesis demonstrates the expression of primitive (stem/progenitor cell) markers on the endothelium of IH. The expression of the transcription factors brachyury, Tal-1 and GATA-2, along with the demonstration of erythropoiesis in IH explants in vitro supports the hypothesis that IH consists of a primitive endothelium similar to an embryonic haemogenic endothelium. The expression of the erythropoietin receptor and haemoglobin zeta chain by the endothelium of IH further strengthens the notion that IH is a haemogenic endothelium. Consistent with the primitive embryonic origin, the expression of the placental markers human chorionic gonadotrophin (hCG) and human placenta lactogen (hPL), but not cytokeratin 7 (CK7) or human leucocyte antigen- G (HLA-G) by the endothelium in IH, supports a placental chorionic villous mesenchymal core cell, and not a trophoblast, origin for IH. IH thus has an extraembryonically derived primitive mesodermal origin. This primitive mesoderm is able to account for the haemogenic endothelium phenotype of the endothelium of proliferating IH microvessels with its capacity for both erythropoietic and mesenchymal differentiation. Additionally, data are presented to show that IH expresses key components of the renin-angiotensin system (RAS), angiotensin converting enzyme (ACE), angiotensin II (ATII), angiotensin receptor 2 (ATR2). Cultured IH-derived stem cells can be induced to proliferate and form blast colonies in response to ATII treatment. The crucial regulatory role of RAS in the proliferation and differentiation of the stem/progenitor cell population within IH accounts for the natural progression of IH. A model is proposed to provide a rational explanation for the serendipiditous discovery of the dramatic effect that the β-blocker, Propranolol has in accelerating involution of IH. The hypothesis that Propranolol exerts its action on IH through modulation of the RAS by blocking renin activity and preventing the conversion of angiotensinogen to angiotensin I, thereby reducing ATII levels, has led to a clinical trial using Captopril, an ACE inhibitor in the treatment of problematic proliferating IH. The observed accelerated involution of IH by Captopril which blocks the conversion of angiotensin I to ATII confirms a key regulatory role for RAS in the biology of IH This discovery underpins the development of potentially safer and novel treatment modalities for this enigmatic condition.</p>

Aging-Related Changes in Erythropoietic Activity and Iron Metabolism in a Mouse Model of Congenital Erythrocytosis with Human Gain-of-Function Erythropoietin Receptor

We previously created and characterized a mouse model of congenital erythrocytosis with low erythropoietin (EPO) levels from a gain-of-function mutation of the human erythropoietin receptor gene (mtHEPOR) (Divoky et al. PNAS. 2001; 98:986; Divoky et al. JMM Berl. 2016; 94:597). These mice develop fetal erythrocytosis, followed by transient amelioration of erythrocytosis in perinatal life, and reappearance at 3-6 weeks of age. Similarly, erythrocytosis is observed in heterozygous mtHEPOR patients postnatally but not at birth. We previously reported dynamic changes of the erythron with iron homeostasis during ontogenesis in these mice (Kralova et al. Blood 2017; 130: 170). We observed that while perinatal mtHEPOR mice exhibit relative iron deficiency, aged mice had iron overload. Here, we evaluated developmentally-determined factors associated with hyperactivation of EPOR signaling which could cause a transition from iron deficiency (neonates) to hyperferremia and increased iron deposition (aged mice). To assess the consequences of different levels of EPOR-JAK2-STAT5 signaling, we studied hetero- and homozygous mtHEPOR mice that differ in their severity of erythrocytosis. We found that prenatally and perinatally, mtHEPOR hetero- and homozygous mice have increased erythroferrone (Erfe) transcripts and reduced hepcidin, consistent with the known inverse correlation between Erfe and hepcidin and in accordance with increased numbers of immature erythroid progenitors in the fetal hepatic circulation. At birth, previously normal Epo expression decreased and remained low in adulthood. Iron deficiency, observed in mtHEPOR hetero- and homozygotes at postnatal day 7, was likely related to increased iron consumption by augmented erythropoiesis at this stage. Postnatally, hepcidin levels increased in mutant mice, accompanied by low Erfe induction and iron accumulation in the liver and spleen as reflected by the upregulation of hepatic Bmp6 expression in mature adult (aged ~6.5 months) and old (~16 months) mtHEPOR homozygotes. We hypothesized that this could be a consequence of diminished iron consumption due to a progressive decline of erythropoiesis in mtHEPOR mice, possibly mediated by premature aging of erythroid progenitors with cell-autonomously increased proliferative history and/or increased inflammation. Indeed, young mutant erythrocytes had decreased erythrocyte survival and expression of a senescent marker CD47, an inhibitor of erythrocytes' phagocytosis. Additionally, a progressive decline in the percentage of Ter119-positive bone marrow cells and immature erythroblasts was observed in mtHEPOR hetero- and homozygotes with aging. Clonogenic assays of old mice revealed suppression of early (BFU-E) and late (CFU-E) erythroid progenitors and myeloid bias of hematopoiesis, paralleled by the up-regulation of PU.1 expression, elevation of platelet counts, and an increase in megakaryocytes chiefly in the bone marrow of mtHEPOR homozygotes. Serum levels of inflammatory cytokines did not indicate systemic inflammation; however, induced transcripts of IL-6, Inf-γ, Tgf-β, and Tnf-α, mainly in mtHEPOR homozygotes showed local bone marrow inflammatory stress. These data indicate progressive attenuation of erythroid drive in mtHEPOR homozygotes, and less so in mtHEPOR heterozygotes, paralleled by a decline in hematocrit levels with aging. In response to attenuated erythropoietic activity, iron consumption was reduced in mtHEPOR mice, leading to iron accumulation in the liver and spleen accompanied by markedly increased hepcidin synthesis. Our data suggest that even in the absence of systemic inflammation, albeit with possible paracrine inflammatory signals, known to affect bone marrow remodeling and hematopoietic aging, life-lasting prolonged activation of EPOR-JAK2-STAT5 signaling promoted exhaustion of erythroid progenitors and resulted in an age-related decline of accelerated erythropoiesis in this mouse model of congenital erythrocytosis with human gain-of-function EPOR. Grant support: Czech grant agencies projects GA17-05988S, NV19-07-00412 and LTAUSA17142, Palacky University project IGA_LF_2021_004. Disclosures No relevant conflicts of interest to declare.

Rker-050 Rescued Ruxolitinib (Rux)-Associated Reductions in Red Blood Cell Volume

Myelofibrosis (MF) is characterized by the dysfunctional Janus kinase/signal transducers and activators of transcription signaling (JAK/STAT) pathways leading to progressive proliferation of granulocytic and megakaryocytic cells in the bone marrow at the expense of other hematopoietic lineages. Clinical signs of MF include cytopenias, splenomegaly and transformation to acute leukemia. Jakafi® (ruxolitinib, or rux), a JAK2 inhibitor, is a therapeutic for MF and functions to impair the activated mutations that cause the expansion of megakaryocytic precursors. However, JAK2 also transduces signals of the erythropoietin receptor, thrombopoietin receptor, and the granulocyte colony-stimulating factor receptor. Therefore, individuals being treated with rux are susceptible to treatment-associated effects on normal hematopoiesis resulting in thrombocytopenia, neutropenia and anemia. The TGF-β superfamily plays a vital role in the regulation of hematopoiesis; specifically, SMAD 2/3 activation results in cell quiescence and inhibits precursors from progressing through later stages of hematopoiesis. KER-050, a modified ActRIIA extracellular domain fused to the Fc of human IgG1, is designed to inhibit ligands including activin A, activin B, GDF8 and GDF11, that activate SMAD 2/3. In a preclinical study, administration of KER-050 in mice led to upregulation of erythropoiesis by mobilizing early- and late-stage erythroid precursors and facilitating their terminal maturation into red blood cells (RBCs). In a Phase 1 clinical study, administration of KER-050 to healthy volunteers led to sustained increases in RBCs and hemoglobin (HGB) along with increases in platelets. Given the observed effect of KER-050 on increasing RBCs, we evaluated whether treatment with a research form of KER-050 (RKER-050) could reverse rux-associated reductions in RBCs. Additionally, preclinical studies have shown that KER-050 potentially functioned as a muscle anabolic by increasing lean mass in rodents. We first established anemia in C57Bl/6 mice by dosing with rux before administering RKER-050. Anemia was confirmed on study day 37; mice receiving 120 mg/kg rux via oral gavage (PO) BID had significantly lower RBC (-7.4%, p=0.0001), HGB (-4.0%, p=0.002) and hematocrit (HCT; -5.7%, p=0.0006) levels compared to the control group. Treatment with RKER-050 was initiated on study day 41 and mice received 7.5 mg/kg RKER-050 or vehicle intraperitoneally (IP) twice weekly for approximately 14 days. Mice receiving rux alone continued their decline in RBCs and, on day 55, continued to have significant reductions in RBC (-6.7%, p&lt;0.0001), HGB (-6.0, p&lt;0.00001) and HCT (-5.6%, p=0.0002) levels compared to the control group. These findings are consistent with the progressive effect of JAK2 inhibition on suppressing erythrocyte development and production. In contrast, treatment with RKER-050 abrogated the observed rux-associated reductions in RBCs, HGB, and HCT in the rux-RKER-050 cohort with significant observed increases (+15.8%, +12.2%, +11.2%, respectively, all p&lt;0.0001) when compared to the rux-vehicle group. The rux-RKER-050 cohort also had significantly increased body mass, measured between study day 41 and study day 55 versus the rux-vehicle group (+9.9%, p= 0.006, and +0.69%, respectively). These data demonstrate that rux treatment reduced RBCs, HGB, and HCT in mice, and that coadministration of RKER-050 reversed rux-associated reductions in RBC parameters. Therefore, treatment with KER-050 has the potential to mitigate the dose limiting effects of rux and enhance duration of therapy in MF patients. RKER-050 also increased body weight in mice receiving rux through its anabolic effect on muscle, a potential benefit in elderly MF patients. These data support the potential benefit of KER-050 as a monotherapy and in combination with rux in patients with MF and anemia. KER-050 will be assessed in a Phase 2 clinical trial (KER050-MF-301), which we expect to commence in 2021. Disclosures Nathan: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Feigenson: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lamora: Keros Therapeutics: Current Employment. Tseng: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Mercury Chloride Impacts on the Development of Erythrocytes and Megakaryocytes in Mice

Inorganic mercury (Hg2+) is a highly toxic heavy metal. The aim of this study was to investigate the impact of Hg2+ on the development of erythrocytes and megakaryocytes. B10.S mice (H-2s) and DBA/2 mice (H-2d) were administrated with 10 μM HgCl2 or 50 μM HgCl2 via drinking water for four weeks, and erythro-megakaryopoiesis was evaluated thereafter. The administration of 50 μM HgCl2 increased the number of erythrocytes and platelets in B10.S mice, which was not due to a reduced clearance for mature erythrocytes. The administration of 50 μM HgCl2, but not 10 μM HgCl2, increased the number of progenitors for erythrocytes and megakaryocytes in the bone marrow (BM) of B10.S mice, including erythroid-megakaryocyte progenitors (EMPs), burst-forming unit-erythroid progenitors (BFU-Es), colony-forming unit-erythroid progenitors (CFU-Es), and megakaryocyte progenitors (MkPs). Moreover, 50 μM HgCl2 caused EMPs to be more proliferative and possess an increased potential for differentiation into committed progenies in B10.S mice. Mechanistically, 50 μM HgCl2 increased the expression of the erythropoietin receptor (EPOR) in EMPs, thus enhancing the Jak2/STAT5 signaling pathway to promote erythro-megakaryopoiesis in B10.S mice. Conversely, 50 μM HgCl2 did not impact erythro-megakaryopoiesis in DBA/2 mice. This study may extend our current understanding for hematopoietic toxicology of Hg.