The Clinical Relevance of the EPH/Ephrin Signaling Pathway in Pediatric Solid and Hematologic Malignancies.

Pediatric neoplasms represent a complex group of malignancies that pose unique challenges in terms of diagnosis, treatment, and understanding of the underlying molecular pathogenetic mechanisms. Erythropoietin-producing hepatocellular receptors (EPHs), the largest family of receptor tyrosine kinases and their membrane-tethered ligands, ephrins, orchestrate short-distance cell-cell signaling and are intricately involved in cell-pattern morphogenesis and various developmental processes. Unraveling the role of the EPH/ephrin signaling pathway in the pathophysiology of pediatric neoplasms and its clinical implications can contribute to deciphering the intricate landscape of these malignancies. The bidirectional nature of the EPH/ephrin axis is underscored by emerging evidence revealing its capacity to drive tumorigenesis, fostering cell-cell communication within the tumor microenvironment. In the context of carcinogenesis, the EPH/ephrin signaling pathway prompts a reevaluation of treatment strategies, particularly in pediatric oncology, where the modest progress in survival rates and enduring treatment toxicity necessitate novel approaches. Molecularly targeted agents have emerged as promising alternatives, prompting a shift in focus. Through a nuanced understanding of the pathway's intricacies, we aim to lay the groundwork for personalized diagnostic and therapeutic strategies, ultimately contributing to improved outcomes for young patients grappling with neoplastic challenges.

Targeting Interleukin-13 Receptor α2 and EphA2 in Aggressive Breast Cancer Subtypes with Special References to Chimeric Antigen Receptor T-Cell Therapy.

Breast cancer (BCA) remains the leading cause of cancer-related mortality among women worldwide. This review delves into the therapeutic challenges of BCA, emphasizing the roles of interleukin-13 receptor α2 (IL-13Rα2) and erythropoietin-producing hepatocellular receptor A2 (EphA2) in tumor progression and resistance. Highlighting their overexpression in BCA, particularly in aggressive subtypes, such as Her-2-enriched and triple-negative breast cancer (TNBC), we discuss the potential of these receptors as targets for chimeric antigen receptor T-cell (CAR-T) therapies. We examine the structural and functional roles of IL-13Rα2 and EphA2, their pathological significance in BCA, and the promising therapeutic avenues their targeting presents. With an in-depth analysis of current immunotherapeutic strategies, including the limitations of existing treatments and the potential of dual antigen-targeting CAR T-cell therapies, this review aims to summarize potential future novel, more effective therapeutic interventions for BCA. Through a thorough examination of preclinical and clinical studies, it underlines the urgent need for targeted therapies in combating the high mortality rates associated with Her-2-enriched and TNBC subtypes and discusses the potential role of IL-13Rα2 and EphA2 as promising candidates for the development of CAR T-cell therapies.

Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry.

BACKGROUND: Calcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN. METHODS: Intracellular Ca2+ levels were measured over a time frame of 0-1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0-108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109-594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595-1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann-Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test. RESULTS: Following EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells. CONCLUSIONS: This report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling.

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimerizes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface. Artificial intelligence-guided modeling and simulations revealed that the pseudokinase mutations cause differences in the pathogenic full-length JAK2 dimers, particularly in the FERM-SH2 domains. A detailed molecular understanding of mutation-driven JAK2 hyperactivation may enable novel therapeutic approaches to selectively target pathogenic JAK2 signaling.

Erythropoietin-producing hepatocellular receptor B6 is highly expressed in non-functioning pituitary neuroendocrine tumors and its expression correlates with tumor size.

BACKGROUND: Erythropoietin-producing hepatocellular (EPH) receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The Erythropoietin-producing hepatocellular receptor tyrosine kinase member EphB6 is a pseudokinase which has not attracted an equivalent amount of interest as its enzymatically-active counterparts. The aim of this study was to assess the expression of EphB6 in pituitary tumors. METHODS AND RESULTS: Human normal pituitaries and pituitary tumors were examined for EphB6 mRNA expression using real-time PCR and for EphB6 protein by immunohistochemistry and Western blotting. EphB6 was highly expressed in non-functioning pituitary neuroendocrine tumors (NF-PitNETs) versus the normal pituitary and GH-secreting PitNETs. EphB6 mRNA expression was correlated with tumor size. CONCLUSIONS: Our results suggest EphB6 aberrant expression in NF-PitNETs. Future studies are warranted to determine the role and significance of EphB6 in NF-PitNETs tumorigenesis.

Development of a 18F-Labeled Bicyclic Peptide Targeting EphA2 for Molecular Imaging of PSMA-Negative Prostate Cancer.

Although PSMA PET/CT imaging has great potential for noninvasively detecting prostate cancer (PCa), limitations exist for patients with low PSMA expression, caused by androgen deprivation treatment or neuroendocrine differentiation. Analysis of The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data found that erythropoietin-producing hepatocellular receptor A2 (EphA2), a receptor overexpressed in most PCa could be a potential target for PSMA-negative PCa. A fluorescent ligand ETF and a radiolabeled ligand [18F]AlF-ETN derived from a EphA2-targeting bicyclic peptide were synthesized and investigated. ETF could selectively stain and visualize the EphA2-positive but PSMA-negative PC3 cells, in complementary to the PSMA-targeting probe. PET/CT imaging and biodistribution experiments demonstrated that [18F]AlF-ETN specifically accumulated in PC3 tumors with a high contrast (tumor-to-muscle ratio: 21.29 ± 6.55). In conclusion, we have demonstrated the potential for using EphA2 to detect PSMA-negative PCa and developed a radiolabeled ligand [18F]AlF-ETN to specifically image EphA2 expressing PCa with high contrast.

Erythropoietin receptor is a risk factor for prognosis: A potential biomarker in lung adenocarcinoma.

Lung cancer has the highest mortality rate of all cancers, and LUAD's survival rate is particularly poor. Erythropoietin receptor (EPOR) can be detected in lung adenocarcinoma (LUAD), however, the expression levels and prognostic value of EPOR in LUAD are still unclear. In our study, clinicopathological data of 92 LUAD patients between January 2008 and June 2016, multiple bioinformatics databases and immunohistochemistry were used to explore the EPOR expression, the mutant genes affecting EPOR expression, and the correlation of EPOR expression with oxidative stress - related genes, prognosis, immune microenvironment. All statistical analyses were performed in the R version 4.1.1. The study found that EPOR expression might be down-regulated at the mRNA levels and significantly up-regulated at the protein levels in LUAD, which indicates that the mRNA and protein levels of EPOR are inconsistent. The muTarget showed that the expression of EPOR was significantly different between the mutant group and the wild group of 15 genes, including DDX60L and C1orf168. Importantly, we found that EPOR was associated with VEGF and HIF family members, and had significant positive correlation with oxidative stress - related genes such as CCS, EPX and TXNRD2. This suggests that EPOR may be involved in the regulation of oxidative stress. The Kaplan-Meier 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 overall survival (29.5 vs 46 months) and had a good predictive ability for 4-year and 5-year survival probability. EPOR is expected to be a potential new prognostic marker for LUAD.

A critical role for erythropoietin on vagus nerve Schwann cells in intestinal motility.

BACKGROUND: Dysmotility and postoperative ileus (POI) are frequent major clinical problems post-abdominal surgery. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine that promotes recovery of the intestine in various injury models. While EPO receptors (EPOR) are present in vagal Schwann cells, the role of EPOR in POI recovery is unknown because of the lack of EPOR antagonists or Schwann-cell specific EPOR knockout animals. This study was designed to explore the effect of EPO via EPOR in vagal nerve Schwann cells in a mouse model of POI. RESULTS: The structural features of EPOR and its activation by EPO-mediated dimerization were understood using structural analysis. Later, using the Cre-loxP system, we developed a myelin protein zero (Mpz) promoter-driven knockout mouse model of Schwann cell EPOR (MpzCre-EPORflox/flox / Mpz-EPOR-KO) confirmed using PCR and qRT-PCR techniques. We then measured the intestinal transit time (ITT) at baseline and after induction of POI with and without EPO treatment. Although we have previously shown that EPO accelerates functional recovery in POI in wild type mice, EPO treatment did not improve functional recovery of ITT in POI of Mpz-EPOR-KO mice. CONCLUSIONS: To the best of our knowledge, this is the first pre-clinical study to demonstrate a novel mouse model of EPOR specific knock out on Schwan cells with an effect in the gut. We also showed novel beneficial effects of EPO through vagus nerve Schwann cell-EPOR in intestinal dysmotility. Our findings suggest that EPO-EPOR signaling in the vagus nerve after POI is important for the functional recovery of ITT.

Differentially Expressed Genes Induced by Erythropoietin Receptor Overexpression in Rat Mammary Adenocarcinoma RAMA 37-28 Cells.

The erythropoietin receptor (EPOR) is a transmembrane type I receptor with an essential role in the proliferation and differentiation of erythroid progenitors. Besides its function during erythropoiesis, EPOR is expressed and has protective effect in various non-hematopoietic tissues, including tumors. Currently, the advantageous aspect of EPOR related to different cellular events is still under scientific investigation. Besides its well-known effect on cell proliferation, apoptosis and differentiation, our integrative functional study revealed its possible associations with metabolic processes, transport of small molecules, signal transduction and tumorigenesis. Comparative transcriptome analysis (RNA-seq) identified 233 differentially expressed genes (DEGs) in EPOR overexpressed RAMA 37-28 cells compared to parental RAMA 37 cells, whereas 145 genes were downregulated and 88 upregulated. Of these, for example, GPC4, RAP2C, STK26, ZFP955A, KIT, GAS6, PTPRF and CXCR4 were downregulated and CDH13, NR0B1, OCM2, GPM6B, TM7SF3, PARVB, VEGFD and STAT5A were upregulated. Surprisingly, two ephrin receptors, EPHA4 and EPHB3, and EFNB1 ligand were found to be upregulated as well. Our study is the first demonstrating robust differentially expressed genes evoked by simple EPOR overexpression without the addition of erythropoietin ligand in a manner which remains to be elucidated.

The indispensable role of the RNA helicase DDX5 in tumorigenesis induced by the myeloproliferative neoplasm-associated JAK2V617F mutant.

A point mutation (V617F) in the Janus kinase 2 (JAK2) gene results in the production of disorderly activated tyrosine kinase, which causes myeloproliferative neoplasms (MPN). We herein demonstrated that the RNA helicase DDX5 was highly expressed at the mRNA and protein levels through the activation of signal transducer and activator of transcription 5 (STAT5) in Ba/F3 cells expressing a JAK2V617F mutant and erythropoietin receptor (V617F/EpoR cells) and MPN patient-derived HEL cells. A treatment with the JAK1/2 inhibitor, ruxolitinib and STAT5 inhibitor, pimozide significantly inhibited DDX5 mRNA expression and enhanced the degradation of DDX5 in these cells, suggesting that the JAK2V617F mutant positively regulates DDX5 mRNA expression and DDX5 protein stability by activating STAT5. The knockdown of DDX5 specifically inhibited the activation of mechanistic target of rapamycin (mTOR) in V617F/EpoR cells and HEL cells and significantly suppressed the proliferation of these cells. Furthermore, the knockdown of DDX5 markedly suppressed tumorigenesis, splenomegaly, and liver hypertrophy caused by an inoculation of V617F/EpoR cells in nude mice. Collectively, these results revealed that JAK2V617F exhibits transforming activity by inducing the expression of DDX5 in a STAT5-dependent manner, indicating the potential of the JAK2V617F/STAT5/DDX5 axis as a therapeutic target in the treatment of MPN.

Targeting the innate repair receptor axis via erythropoietin or pyroglutamate helix B surface peptide attenuates hemolytic-uremic syndrome in mice.

Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli and is characterized by microangiopathic hemolytic anemia and acute kidney injury. Hitherto, therapy has been limited to organ-supportive strategies. Erythropoietin (EPO) stimulates erythropoiesis and is approved for the treatment of certain forms of anemia, but not for HUS-associated hemolytic anemia. EPO and its non-hematopoietic analog pyroglutamate helix B surface peptide (pHBSP) have been shown to mediate tissue protection via an innate repair receptor (IRR) that is pharmacologically distinct from the erythropoiesis-mediating receptor (EPO-R). Here, we investigated the changes in endogenous EPO levels in patients with HUS and in piglets and mice subjected to preclinical HUS models. We found that endogenous EPO was elevated in plasma of humans, piglets, and mice with HUS, regardless of species and degree of anemia, suggesting that EPO signaling plays a role in HUS pathology. Therefore, we aimed to examine the therapeutic potential of EPO and pHBSP in mice with Stx-induced HUS. Administration of EPO or pHBSP improved 7-day survival and attenuated renal oxidative stress but did not significantly reduce renal dysfunction and injury in the employed model. pHBSP, but not EPO, attenuated renal nitrosative stress and reduced tubular dedifferentiation. In conclusion, targeting the EPO-R/IRR axis reduced mortality and renal oxidative stress in murine HUS without occurrence of thromboembolic complications or other adverse side effects. We therefore suggest that repurposing EPO for the treatment of patients with hemolytic anemia in HUS should be systematically investigated in future clinical trials.

Effect of Erythropoietin on Mononuclear Cells of the Bone Marrow and Spleen.

We studied the effect of preconditioning of human bone marrow mononuclear cells with erythropoietin on the immunophenotype of immunocompetent cells and paracrine activity of mouse splenocytes. The expression of erythropoietin receptors on immunocompetent human bone marrow cells was shown to change after a short-term (60 min) exposure to erythropoietin. The number of T helpers carrying erythropoietin receptors decreased and the number of T suppressors, B lymphocytes, and monocytes carrying erythropoietin receptors increased. The presence of 30% conditioned medium from human bone marrow mononuclear cells or 33.4 U/ml of erythropoietin reduced apoptosis/necrosis, increased intracellular activity of NADPH-dependent oxidoreductases of splenocytes, and did not affect oxidative phosphorylation (did not enhance lactate production and glucose uptake by cells).

Epo-IGF1R cross talk expands stress-specific progenitors in regenerative erythropoiesis and myeloproliferative neoplasm.

We found that in regenerative erythropoiesis, the erythroid progenitor landscape is reshaped, and a previously undescribed progenitor population with colony-forming unit-erythroid (CFU-E) activity (stress CFU-E [sCFU-E]) is expanded markedly to restore the erythron. sCFU-E cells are targets of erythropoietin (Epo), and sCFU-E expansion requires signaling from the Epo receptor (EpoR) cytoplasmic tyrosines. Molecularly, Epo promotes sCFU-E expansion via JAK2- and STAT5-dependent expression of IRS2, thus engaging the progrowth signaling from the IGF1 receptor (IGF1R). Inhibition of IGF1R and IRS2 signaling impairs sCFU-E cell growth, whereas exogenous IRS2 expression rescues cell growth in sCFU-E expressing truncated EpoR-lacking cytoplasmic tyrosines. This sCFU-E pathway is the major pathway involved in erythrocytosis driven by the oncogenic JAK2 mutant JAK2(V617F) in myeloproliferative neoplasm. Inability to expand sCFU-E cells by truncated EpoR protects against JAK2(V617F)-driven erythrocytosis. In samples from patients with myeloproliferative neoplasm, the number of sCFU-E-like cells increases, and inhibition of IGR1R and IRS2 signaling blocks Epo-hypersensitive erythroid cell colony formation. In summary, we identified a new stress-specific erythroid progenitor cell population that links regenerative erythropoiesis to pathogenic erythrocytosis.

Pulpal expression of erythropoietin and erythropoietin receptor after direct pulp capping in rat.

This study aimed to evaluate the effect of direct pulp capping on the expression of erythropoietin (Epo) and Epo-receptor (Epor) genes in relation to the expression of inflammatory and osteogenic genes in rat pulp. Dental pulps of the first maxillary molars of Wistar Albino rats were exposed and capped with either calcium hydroxide or mineral trioxide aggregate, or were left untreated. After 4 wk, animals were euthanized, and maxillae were prepared for histological and real-time polymerase chain reaction analysis. Histological scores of pulp inflammation and mineralization, and relative expressions of Epo, Epor, inflammatory cytokines, and pulp osteogenic genes were evaluated. The capped pulps showed higher expressions of Epo, while the untreated pulps had the highest expression of Epor. Both calcium hydroxide and mineral trioxide aggregate downregulated the expression of tumor necrosis factor alpha compared to untreated controls, and upregulated transforming growth factor beta compared to healthy controls. Alkaline phosphatase expression was significantly higher in experimental groups. Relative expression of Epo negatively correlated with pulp inflammation, and positively correlated with pulp mineralization. Pulp exposure promoted expression of Epor and pro-inflammatory cytokines, while pulp capping promoted expression of Epo, alkaline phosphatase, and downregulated Epor and pro-inflammatory cytokines.

Iron Deficiency in Heart Failure and Effect of Dapagliflozin: Findings From DAPA-HF.

BACKGROUND: Iron deficiency is common in heart failure and associated with worse outcomes. We examined the prevalence and consequences of iron deficiency in the DAPA-HF trial (Dapagliflozin and Prevention of Adverse-Outcomes in Heart Failure) and the effect of dapagliflozin on markers of iron metabolism. We also analyzed the effect of dapagliflozin on outcomes, according to iron status at baseline. METHODS: Iron deficiency was defined as a ferritin level <100 ng/mL or a transferrin saturation <20% and a ferritin level 100 to 299 ng/mL. Additional biomarkers of iron metabolism, including soluble transferrin receptor, erythropoietin, and hepcidin were measured at baseline and 12 months after randomization. The primary outcome was a composite of worsening heart failure (hospitalization or urgent visit requiring intravenous therapy) or cardiovascular death. RESULTS: Of the 4744 patients randomized in DAPA-HF, 3009 had ferritin and transferrin saturation measurements available at baseline, and 1314 of these participants (43.7%) were iron deficient. The rate of the primary outcome was higher in patients with iron deficiency (16.6 per 100 person-years) compared with those without (10.4 per 100 person-years; P<0.0001). The effect of dapagliflozin on the primary outcome was consistent in iron-deficient compared with iron-replete patients (hazard ratio, 0.74 [95% CI, 0.58-0.92] versus 0.81 [95% CI, 0.63-1.03]; P-interaction=0.59). Similar findings were observed for cardiovascular death, heart failure hospitalization, and all-cause mortality. Transferrin saturation, ferritin, and hepcidin were reduced and total iron-binding capacity and soluble transferrin receptor increased with dapagliflozin compared with placebo. CONCLUSIONS: Iron deficiency was common in DAPA-HF and associated with worse outcomes. Dapagliflozin appeared to increase iron use but improved outcomes, irrespective of iron status at baseline. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03036124.

Erythropoietin in Optic Neuropathies: Current Future Strategies for Optic Nerve Protection and Repair.

Erythropoietin (EPO) is known as a hormone for erythropoiesis in response to anemia and hypoxia. However, the effect of EPO is not only limited to hematopoietic tissue. Several studies have highlighted the neuroprotective function of EPO in extra-hematopoietic tissues, especially the retina. EPO could interact with its heterodimer receptor (EPOR/ßcR) to exert its anti-apoptosis, anti-inflammation and anti-oxidation effects in preventing retinal ganglion cells death through different intracellular signaling pathways. In this review, we summarized the available pre-clinical studies of EPO in treating glaucomatous optic neuropathy, optic neuritis, non-arteritic anterior ischemic optic neuropathy and traumatic optic neuropathy. In addition, we explore the future strategies of EPO for optic nerve protection and repair, including advances in EPO derivates, and EPO deliveries. These strategies will lead to a new chapter in the treatment of optic neuropathy.

Developmental changes in iron metabolism and erythropoiesis in mice with human gain-of-function erythropoietin receptor.

Iron availability for erythropoiesis is controlled by the iron-regulatory hormone hepcidin. Increased erythropoiesis negatively regulates hepcidin synthesis by erythroferrone (ERFE), a hormone produced by erythroid precursors in response to erythropoietin (EPO). The mechanisms coordinating erythropoietic activity with iron homeostasis in erythrocytosis with low EPO are not well defined as exemplified by dominantly inherited (heterozygous) gain-of-function mutation of human EPO receptor (mtHEPOR) with low EPO characterized by postnatal erythrocytosis. We previously created a mouse model of this mtHEPOR that develops fetal erythrocytosis with a transient perinatal amelioration of erythrocytosis and its reappearance at 3-6 weeks of age. Prenatally and perinatally, mtHEPOR heterozygous and homozygous mice (differing in erythrocytosis severity) had increased Erfe transcripts, reduced hepcidin, and iron deficiency. Epo was transiently normal in the prenatal life; then decreased at postnatal day 7, and remained reduced in adulthood. Postnatally, hepcidin increased in mtHEPOR heterozygotes and homozygotes, accompanied by low Erfe induction and iron accumulation. With aging, the old, especially mtHEPOR homozygotes had a decline of erythropoiesis, myeloid expansion, and local bone marrow inflammatory stress. In addition, mtHEPOR erythrocytes had a reduced lifespan. This, together with reduced iron demand for erythropoiesis, due to its age-related attenuation, likely contributes to increased iron deposition in the aged mtHEPOR mice. In conclusion, the erythroid drive-mediated inhibition of hepcidin production in mtHEPOR mice in the prenatal/perinatal period is postnatally abrogated by increasing iron stores promoting hepcidin synthesis. The differences observed in studied characteristics between mtHEPOR heterozygotes and homozygotes suggest dose-dependent alterations of downstream EPOR stimulation.

Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia.

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia characterized by prominent erythroid proliferation whose molecular basis is poorly understood. To elucidate the underlying mechanism of erythroid proliferation, we analyzed 121 AEL using whole-genome, whole-exome, and/or targeted-capture sequencing, together with transcriptome analysis of 21 AEL samples. Combining publicly available sequencing data, we found a high frequency of gains and amplifications involving EPOR/JAK2 in TP53-mutated cases, particularly those having >80% erythroblasts designated as pure erythroid leukemia (10/13). These cases were frequently accompanied by gains and amplifications of ERG/ETS2 and associated with a very poor prognosis, even compared with other TP53-mutated AEL. In addition to activation of the STAT5 pathway, a common feature across all AEL cases, these AEL cases exhibited enhanced cell proliferation and heme metabolism and often showed high sensitivity to ruxolitinib in vitro and in xenograft models, highlighting a potential role of JAK2 inhibition in therapeutics of AEL. SIGNIFICANCE: This study reveals the major role of gains, amplifications, and mutations of EPOR and JAK2 in the pathogenesis of pure erythroleukemia. Their frequent response to ruxolitinib in patient-derived xenograft and cell culture models highlights a possible therapeutic role of JAK2 inhibition for erythroleukemia with EPOR/JAK2-involving lesions. This article is highlighted in the In This Issue feature, p. 369.

A Full Target-Mediated Drug Disposition (TMDD) Model to Explain the Changes in Recombinant Human Erythropoietin (rhEpo) Pharmacokinetics in Patients with Different Bone Marrow Integrity Following Hematopoietic Transplantation.

Our aim was to build a mechanistic full target-mediated drug disposition (TMDD) model for rhEpo to better understand rhEpo disposition, Epo receptor (EpoR) synthesis, and degradation in hematopoietic transplant patients with four distinct bone marrow conditions. All PK data were analyzed simultaneously using the nonlinear mixed effect modeling approach with NONMEM. The final model was a two-compartmental full TMDD model, which adequately characterizes rhEpo PK in patients and provides insight into the dynamics of free EpoR, rhEpo-EpoR, and total EpoR. The model predicted association rate constant (kon), dissociation rate constant (koff), and internalization rate constant (kint) were 0.0276 pM-1h-1, 0.647 h-1, and 0.255h-1, respectively, which were supported by experimental data. Also, the EpoR degradation rate constant (kdeg) was estimated to be 0.461 h-1. EpoR production rate was estimated to be 37.5 pM/h for adults at pre-ablation baseline and 5.91 pM/h, and 4.19 pM/h in the early post-transplant post-engraftment, and late post-transplant full engraftment. Our model provides extensive information on the dynamics of free EpoR, total EpoR and rhEpo-EpoR, and proven to be more robust and can provide more physiologically relevant binding parameters than previous models.