• Cells expressing JAK2 E846D or R1063H exhibit pathologic STAT5 activation in the specific context of EPOR.• Cooperation of germ line JAK2 mutations E846D and R1063H defines a JAK2-signaling threshold for induction of erythrocytosis.The role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/ STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.

Mutations of the truncated cytoplasmic domain of human erythropoietin receptor (EPOR) result in gain-of-function of erythropoietin (EPO) signaling and a dominantly inherited polycythemia, primary familial and congenital polycythemia (PFCP). We interrogated the unexplained transient absence of perinatal polycythemia observed in PFCP patients using an animal model of PFCP to examine its erythropoiesis during embryonic, perinatal, and early postnatal periods. In this model, we replaced the murine EpoR gene (mEpoR) with the wild-type human EPOR (wtHEPOR) or mutant human EPOR gene (mtHEPOR) and previously reported that the gain-of-function mtHEPOR mice become polycythemic at 3~6 weeks of age, but not at birth, similar to the phenotype of PFCP patients. In contrast wtHEPOR mice had sustained anemia. We report that the mtHEPOR fetuses are polycythemic, but their polycythemia is abrogated in the perinatal period and reappears again at 3 weeks after birth. mtHEPOR fetuses have a delayed switch from primitive to definitive erythropoiesis, augmented erythropoietin signaling, and prolonged Stat5 phosphorylation while the wtHEPOR fetuses are anemic. Our study demonstrates the in vivo effect of excessive EPO/EPOR signaling on developmental erythropoiesis switch and describes that fetal polycythemia in this PFCP model is followed by transient correction of polycythemia in perinatal life associated with low Epo levels and increased expression of erythrocytes’ phosphatidylserine. We suggest that neocytolysis contributes to the observed perinatal correction of polycythemia in mtHEPOR newborns as embryos leaving the hypoxic uterus are exposed to normoxia at birth.

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.

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Obstructive sleep apnea (OSA), characterized by intermittent hypoxia, causes cardiovascular, metabolic, neurocognitive and cancer complications. Hypoxia expands the red cell mass by stimulating erythropoietin (EPO) production; yet in our analysis of 527 OSA patients, <1% had OSA-related polycythemia (Gangaraju et al Blood 2016 128:2444). Typical hypoxia-induced red blood cells (RBCs) mass normalized upon return to normoxia by neocytolysis i.e. preferential destruction of young hypoxia-born RBCs. In an animal model, we demonstrated that neocytolysis is caused by excessive generation of reactive oxygen species (ROS) from increase of mitochondrial retention and accumulation of ROS. ROS increase resulted from decreased antioxidant enzyme catalase mediated by hypoxia-induced miR-21. We hypothesized that polycythemia in OSA was prevented by neocytolysis. It is also well-known that OSA induces systemic inflammation markers including C-reactive protein, IL-6, TNF-α, IL-8, and NF-κb. Inflammation participates in the control of the number of RBCs by inducing hepcidin, the principal regulator of iron metabolism. Increased hepcidin suppresses erythropoiesis by inhibiting iron release from macrophages. Based on this evidence, we also hypothesized that the absence of polycythemia in OSA might be caused by an independent contribution of inflammation-mediated suppression of erythropoiesis. We studied OSA patients before and after treatment with continuous positive airway pressure (CPAP). Increased erythropoiesis was evidenced by increased EPO and reticulocytosis. EPO levels correlated with time spent below sPO2 89 %, indicating that severe OSA patients had more augmented erythropoiesis. However, hematocrit levels were normal. Hemolysis was detected in some but not all OSA patients by end tidal carbon monoxide (a product from heme catabolism). After CPAP treatment, these changes diminished but hematocrits did not change. Conditions favoring neocytolysis were confirmed by increased ROS from expanded reticulocytes' mitochondria which correlated with time spent below sPO2 89 %. Downregulated catalase resulting from increased miR-21 was also detected. Also these changes normalized with CPAP. These results indicate that hemolysis of hypoxia-born RBCs prevents OSA patients from becoming polycythemic. Increased ROS was not only found in reticulocytes but also in leukocytes; these also normalized with CPAP. Expression of inflammatory markers (NFKB1, TNF, and IL6) in granulocytes was higher in OSA compared to controls and normalized by CPAP; these levels correlated with apnea-hypopnea Index (AHI). OSA patients had higher hepcidin levels, correlating with inflammatory marker levels and inversely correlated with EPO. Iron and transferrin saturation levels were lower in OSA compared to controls, inversely correlating with high hepcidin levels. These data indicated that besides neocytolysis, coexistent suppression of erythropoiesis by inflammation contributed to the lack of polycythemia in OSA. In OSA, inflammation mediated increase of ROS in leukocytes is a known causative factor of cardiovascular disease. We now report increase of both ROS and inflammatory markers in leukocytes. We conclude that the absence of polycythemia in OSA is the result of hemolysis via neocytolysis and inflammation-mediated suppression of erythropoiesis. Increased ROS in blood cells and systemic inflammation from OSA-constitute mechanisms likely contributing to the pathophysiology of OSA. Disclosures Ganz: Vifor: Consultancy; Ablynx: Consultancy; Keryx Pharma: Consultancy, Research Funding; Silarus Pharma: Consultancy, Equity Ownership; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA; Akebia: Consultancy, Research Funding; Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy.