New Therapeutic Options for the Treatment of Sickle Cell Disease.

Sickle cell disease (SCD; ORPHA232; OMIM # 603903) is a chronic and invalidating disorder distributed worldwide, with high morbidity and mortality. Given the disease complexity and the multiplicity of pathophysiological targets, development of new therapeutic options is critical, despite the positive effects of hydroxyurea (HU), for many years the only approved drug for SCD. New therapeutic strategies might be divided into (1) pathophysiology-related novel therapies and (2) innovations in curative therapeutic options such as hematopoietic stem cell transplantation and gene therapy. The pathophysiology related novel therapies are: a) Agents which reduce sickling or prevent sickle red cell dehydration; b) Agents targeting SCD vasculopathy and sickle cell-endothelial adhesive events; c) Anti-oxidant agents. This review highlights new therapeutic strategies in SCD and discusses future developments, research implications, and possible innovative clinical trials

Targeting lyn kinase in chorea-acanthocytosis: A translational treatment approach in a rare disease

Background: Chorea-acanthocytosis (ChAc) is a neurodegenerative disease caused by mutations in the VPS13A gene. It is characterized by several neurological symptoms and the appearance of acanthocytes. Elevated tyrosine kinase Lyn activity has been recently identified as one of the key pathophysiological mechanisms in this disease, and therefore represents a promising drug target. Methods: We evaluated an individual off-label treatment with the tyrosine kinase inhibitor dasatinib (100 mg/d, 25.8–50.4 weeks) of three ChAc patients. Alongside thorough safety monitoring, we assessed motor and non-motor scales (e.g., MDS-UPDRS, UHDRS, quality of life) as well as routine and experimental laboratory parameters (e.g., serum neurofilament, Lyn kinase activity, actin cytoskeleton in red blood cells). Results: Dasatinib appeared to be reasonably safe. The clinical parameters remained stable without significant improvement or deterioration. Regain of deep tendon reflexes was observed in one patient. Creatine kinase, serum neurofilament levels, and acanthocyte count did not reveal consistent effects. However, a reduction of initially elevated Lyn kinase activity and accumulated autophagy markers, as well as a partial restoration of the actin cytoskeleton, was found in red blood cells. Conclusions: We report on the first treatment approach with disease-modifying intention in ChAc. The experimental parameters indicate target engagement in red blood cells, while clinical effects on the central nervous system could not be proven within a rather short treatment time. Limited knowledge on the natural history of ChAc and the lack of appropriate biomarkers remain major barriers for “clinical trial readiness”. We suggest a panel of outcome parameters for future clinical trials in ChA

Duality of Nrf2 in iron-overload cardiomyopathy

Cardiomyopathy deeply affects quality of life and mortality of patients with b-thalassemia or with transfusion-dependent myelodysplastic syndromes. Recently, a link between Nrf2 activity and iron metabolism has been reported in liver ironoverload murine models. Here, we studied C57B6 mice as healthy control and nuclear erythroid factor-2 knockout (Nrf2-/-) male mice aged 4 and 12 months. Eleven-month-old wild-type and Nrf2-/- mice were fed with either standard diet or a diet containing 2.5% carbonyl-iron (iron overload [IO]) for 4 weeks. We show that Nrf2-/- mice develop an age-dependent cardiomyopathy, characterized by severe oxidation, degradation of SERCA2A and iron accumulation. This was associated with local hepcidin expression and increased serum non-transferrin-bound iron, which promotes maladaptive cardiac remodeling and interstitial fibrosis related to overactivation of the TGF-b pathway. When mice were exposed to IO diet, the absence of Nrf2 was paradoxically protective against further heart iron accumulation. Indeed, the combination of prolonged oxidation and the burst induced by IO diet resulted in activation of the unfolded protein response (UPR) system, which in turn promotes hepcidin expression independently from heart iron accumulation. In the heart of Hbbth3/+ mice, a model of b-thalassemia intermedia, despite the activation of Nrf2 pathway, we found severe protein oxidation, activation of UPR system and cardiac fibrosis independently from heart iron content. We describe the dual role of Nrf2 when aging is combined with IO and its novel interrelation with UPR system to ensure cell survival. We open a new perspective for early and intense treatment of cardiomyopathy in patients with b-thalassemia before the appearance of heart iron accumulation

Dietary ω-3 fatty acid supplementation improves murine sickle cell bone disease and reprograms adipogenesis

Sickle cell disease (SCD) is a genetic disorder of hemoglobin, leading to chronic hemolytic anemia and multiple organ damage. Among chronic organ complications, sickle cell bone disease (SBD) has a very high prevalence, resulting in long-term disability, chronic pain and fractures. Here, we evaluated the effects of ω-3 (fish oil-based, FD)-enriched diet vs. ω-6 (soybean oil-based, SD)-supplementation on murine SBD. We exposed SCD mice to recurrent hypoxia/reoxygenation (rec H/R), a consolidated model for SBD. In rec H/R SS mice, FD improves osteoblastogenesis/osteogenic activity by downregulating osteoclast activity via miR205 down-modulation and reduces both systemic and local inflammation. We also evaluated adipogenesis in both AA and SS mice fed with either SD or FD and exposed to rec H/R. FD reduced and reprogramed adipogenesis from white to brown adipocyte tissue (BAT) in bone compartments. This was supported by increased expression of uncoupling protein 1(UCP1), a BAT marker, and up-regulation of miR455, which promotes browning of white adipose tissue. Our findings provide new insights on the mechanism of action of ω-3 fatty acid supplementation on the pathogenesis of SBD and strengthen the rationale for ω-3 fatty acid dietary supplementation in SCD as a complementary therapeutic intervention

Tyrosine phosphorylation modulates peroxiredoxin-2 activity in normal and diseased red cells

: Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells

Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease

Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events

Fyn kinase is a novel modulator of erythropoietin signaling and stress erythropoiesis.

The signaling cascade induced by the interaction of erythropoietin (EPO) with its receptor (EPO-R) is a key event of erythropoiesis. We present here data indicating that Fyn, a Src-family-kinase, participates in the EPO signaling-pathway, since Fyn-/- mice exhibit reduced Tyr-phosphorylation of EPO-R and decreased STAT5-activity. The importance of Fyn in erythropoiesis is also supported by the blunted responsiveness of Fyn-/- mice to stress erythropoiesis. Fyn-/- mouse erythroblasts adapt to reactive oxygen species (ROS) by activating the redox-related-transcription-factor Nrf2. However, since Fyn is a physiologic repressor of Nrf2, absence of Fyn resulted in persistent-activation of Nrf2 and accumulation of nonfunctional proteins. ROS-induced over-activation of Jak2-Akt-mTOR-pathway and repression of autophagy with perturbation of lysosomal-clearance were also noted. Treatment with Rapamycin, a mTOR-inhibitor and autophagy activator, ameliorates Fyn-/- mouse baseline erythropoiesis and erythropoietic response to oxidative-stress. These findings identify a novel multimodal action of Fyn in the regulation of normal and stress erythropoiesis

The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a \u3b2-thalassemia mouse model

Anemia in \u3b2-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired \u3b1-globin chains impose substantial oxidative stress on \u3b2-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for \u3b2-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in \u3b2-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2\u3b1 axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing \u3b2-thalassemia\u2013related liver iron overload. In ex vivo studies on erythroid precursors from patients with \u3b2-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for \u3b2-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials

Peroxiredoxin-2 plays a pivotal role as multimodal cytoprotector in the early phase of pulmonary hypertension

Pulmonary-artery-hypertension (PAH) is a life-threatening and highly invalidating chronic disorder. Chronic oxidation contributes to lung damage and disease progression. Peroxiredoxin-2 (Prx2) is a typical 2-cysteine (Cys) peroxiredoxin but its role on lung homeostasis is yet to be fully defined. Here, we showed that Prx2 mice displayed chronic lung inflammatory disease associated with (i) abnormal pulmonary vascular dysfunction; and (ii) increased markers of extracellular-matrix remodeling. Hypoxia was used to induce PAH. We focused on the early phase PAH to dissect the role of Prx2 in generation of PAH. Hypoxic Prx2 mice showed (i) amplified inflammatory response combined with cytokine storm; (ii) vascular activation and dysfunction; (iii) increased PDGF-B lung levels, as marker of extracellular-matrix deposition and remodeling; and (iv) ER stress with activation of UPR system and autophagy. Rescue experiments with in vivo the administration of fusedrecombinant- PEP-Prx2 show a reduction in pulmonary inflammatory vasculopathy and in ER stress with down-regulation of autophagy. Thus, we propose Prx2 plays a pivotal role in the early stage of PAH as multimodal cytoprotector, targeting oxidation, inflammatory vasculopathy and ER stress with inhibition of autophagy. Collectively, our data indicate that Prx2 is able to interrupt the hypoxia induced vicious cycle involving oxidation-inflammationautophagy in the pathogenesis of PAH

Data demonstrating the role of peroxiredoxin 2 as important anti-oxidant system in lung homeostasis.

The data presented in this article are related to the research paper entitled "peroxiredoxin-2 plays a pivotal role as multimodal cytoprotector in the early phase of pulmonary hypertension" (Federti et al., 2017) [1]. Data show that the absence of peroxiredoxin-2 (Prx2) is associated with increased lung oxidation and pulmonary vascular endothelial dysfunction. Prx2 mice displayed activation of the redox-sensitive transcriptional factors, NF-kB and Nrf2, and increased expression of cytoprotective system such as heme-oxygenase-1 (HO- 1). We also noted increased expression of both markers of vascular activation and extracellular matrix remodeling. The administration of the recombinant fusion protein PEP Prx2 reduced the activation of NF-kB and Nrf2 and was paralleled by a decrease in HO-1 and in vascular endothelial abnormal activation. Prolonged hypoxia was used to trigger pulmonary artery hypertension (PAH). Prx2 precociously developed PAH compared to wildtype animals