ALBERT

Description: Abstract LBA-3 Sickle cell disease (SCD) is a debilitating hemolytic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was first identified a century ago, we still lack effective mechanism-based safe therapies to treat this disease. Thus, identification of specific molecules triggering sickling, the central pathogenic process of the disease, is extremely important to advance our understanding of the molecular basis for the pathogenesis of SCD and to develop novel therapeutics. Using non-biased metabolomic screening, we found that sphingosine-1-phosphate (S1P) is significantly elevated in the blood of SCD mice. Further analysis revealed that the activity of sphingosine kinase 1 (Sphk1, the enzyme that produces S1P) is significantly elevated in erythrocytes of SCD mice. Chronic treatment of SCD mice with a SphK1 inhibitor significantly attenuated sickling, hemolysis, inflammation and multiple tissue damage by reducing erythrocyte and plasma S1P levels. Erythrocyte S1P levels were further elevated following hypoxia/reoxygenation-induced acute sickle crisis (ASC) in SCD mice and blocking its elevation by a Sphk1 specific inhibitor significantly reduced hallmark features associated with ASC. As with SCD mice, we found that erythrocyte Sphk1 activity and erythrocyte and plasma S1P levels were significantly elevated in humans with SCD compared to normal individuals. Inhibition of SphK1 in cultured primary human erythrocytes isolated from SCD patients inhibited hypoxia-induced elevation of erythrocyte S1P levels and reduced sickling. Thus, we have revealed for the first time that SphK1-mediated S1P elevation in SCD erythrocytes is a key contributor to sickling in SCD and that Sphk1 inhibition can attenuate both acute and chronic sickling events and disease progression. S1P is an important signaling molecule regulating diverse biological processes. Although S1P is predominantly produced and stored in RBCs, nothing was known about the physiological role of S1P in normal RBCs or the pathophysiological role of S1P in SCD until we conducted a metabolomic screen. In an effort to determine the molecular mechanism underlying S1P-induced sickling, we unexpectedly found that S1P directly binds with Hb and results in a reduced Hb-O2 affinity. This finding led us to further discover that 2,3-diphosphoglycerate, another erythrocyte specific allosteric modulator, is required for S1P-mediated allosteric modulation and that these two endogenous heterotropic modulators work cooperatively to induce a substantial reduction in Hb-O2 affinity. Supporting the biochemical and functional findings, molecular modeling predicts that S1P binds near the water filled central cavity of HbA at a site that is different from the Hb-2,3-DPG binding site. Thus, our discovery adds a significant new chapter to erythrocyte physiology by revealing S1P is a novel allosteric modulator of Hb-O2 affinity and also providing a mechanism underlying S1P-mediated sickling by promoting the formation of deoxyHbS. Thus, the work reported here could be the foundation leading to future human trials and a possible therapy for SCD, a life-threatening hemolytic disorder for which the current treatment is extremely limited. The significance of our findings extends well beyond SCD. Our findings reveal a previously unrecognized important role for S1P in erythrocyte physiology and indicate a new concept for the regulation of O2 release from Hb under normal and sickle cell disease conditions. For SCD, elevated S1P is detrimental because reduced Hb-O2 affinity leads to more deoxygenation of HbS, increased sickling and subsequent multiple life-threatening complications. However, for normal erythrocytes, elevated S1P is likely beneficial by decreasing Hb-O2 affinity allowing for more O2 release to hypoxic tissues. Thus, for humans with normal Hb, if elevated S1P can induce O2 release to hypoxic tissues it may be a novel therapeutic target for a range of disorders, from chronic heart failure to diabetic retinopathy, traumatic blood loss, pulmonary disease and even cancer. In this way our findings reveal important novel opportunities to treat and prevent not only SCD but also multiple cardiovascular and pulmonary diseases associated with hypoxia. Thus, the impact of our novel finding is significant and enormous. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1006 Objective: Priapism is abnormal prolonged penile erection occurring without sexual interest. The condition is prevalent among men with sickle cell disease (SCD). Priapism is a urological emergency which needs early intervention to avoid the risks of penile fibrosis and eventual erectile dysfunction. Due to poorly understood pathogenesis of priapism no effective approaches to manage the disorder. Recent studies have revealed excess adenosine (Ado) in priapism via Ado A2B receptor (ADORA2B), suggesting novel therapeutic possibilities. Here, we aim to conduct preclinical studies to assess the efficacy and safety of Ado-based therapy in priapism. Materials and Methods: ADA-deficient mice (ADA−/−) and SCD Berkeley mice are two independent priapism animal models. We treated both mice with polyethylene glycol-modified ADA (PEG-ADA) to lower penile adenosine level or PSB1115, a selective ADORA2BR antagonist. The erectile function are measured by the changes of intracavernosal pressure (ICP) induced by cavernous nerve stimulation (CNS). Penile fibrosis is evaluated by histological studies and RT-PCR analysis of fibrotic marker gene. In vitro human microvascular endothelial cells (HMECs) were used to determine mechanism underlying ADORA2B-induced priapism. Results: Both ADA−/− and SCD mice with elevated Ado levels in penile tissue displayed priapic feature defined by prolonged and heightened erectile in response to CNS. Chronic reduction of accumulation of penile Ado levels by PEG-ADA enzyme therapy or PSB1115 corrected the priapic feature in both priapic animal models and further prevented progression of penile fibrosis. Significantly, both HIF-1α and eNOS mRNA level were elevated but reversed by PSB1115 treatment in penile tissues of ADA−/− mice and SCD mice. Finally, we provide in vitro direct evidence that PSB1115 treatment or siRNA knockdown HIF-1α in HMECs significantly reduced Ado-induced eNOS mRNA, implicating that ADORA2B-mediated HIF-1α induction contributes to elevated eNOS mRNA and underlies Ado-mediated priapism. Conclusions: PEG-ADA and PSB1115 are effective and safe to treat priapism and exacerbation of the disease by decreasing penile Ado levels or interfering its signaling. This study provides direct preclinical evidence for the novel and general utility of PEG-ADA enzyme therapy or ADORA2B antagonists for priapism and sets up a solid foundation for future clinical trials to assess the usefulness of Adobased therapeutics to treat priapism. Disclosures: No relevant conflicts of interest to declare.

Description: Sphingosine 1-phosphate (S1P) is a bioactive signaling lipid highly enriched in mature erythrocytes. Previous study has revealed that levels of S1P are significantly elevated in patients and mice with Sickle Cell Disease (SCD), a devastating and highly prevalent genetic hemolytic disorder that causes life-threatening hemolysis, tissue damage, and organ dysfunction with very limited treatment. Moreover, the activity of S1P generating enzyme-Sphingosine Kinase 1 (SphK1) is increased in human and mouse SCD erythrocytes, and inhibition of SphK1 decreased erythrocyte sickling. However, the structural and functional basis for the pathogenic nature of S1P in SCD remains obscure. Here, we report that increased erythrocyte S1P promotes pathogenic metabolic reprogramming coupled to increased channeling of glucose to glycolysis rather than through the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increasing deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, hemolysis and disease progression. S1P functioning intracellularly binds to deoxyHbS, facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the PPP. Extending from SCD, we unexpectedly found that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure of HbA complexed with S1P alone or in combination with 2,3-BPG at 1.9 Å resolution revealed the overall architecture and unique features of S1P-2,3-BPG-deoxyHbA complex. In the presence of 2,3-BPG, S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to a1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with b2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying pathogenic consequences of elevated S1P in SCD and its potential role in normal erythrocyte physiology. Disclosures Kato: Mast Therapeutics: Consultancy; Bayer: Research Funding.

Description: Proteasome machinery is a conserved cellular component to maintain normal protein homeostasis. Hypoxia is well known to control hypoxia inducible factor levels by proteasomal machinery in nucleated cells. However, the specific targets and regulation of proteasomal machinery in non-nucleated mature erythrocytes under hypoxia remain poorly understood. To determine if hypoxia regulates erythrocyte proteasomal machinery, we conducted Western blot to detect total ubiquitinated and K48 specific ubiquitinated proteins on the erythrocyte membrane in both human and mice with or without sickle cell disease (SCD), a hemolytic genetic disease with a high mortality, morbidity and frequently facing hypoxia. We found that ubiquitinated, especially K48 specific ubiquitinated proteins were significantly accumulated in both SCD Berkeley mice and humans compared to WT mice and normal controls, indicating that proteasomal machinery is impaired in SCD. Next, to determine specific ubiquitinated proteins accumulated on the membrane of human sickle erythrocytes (sRBC), we conducted immunoprecipitation of sRBC membrane proteins with total ubiquitin antibody followed by an robust and nonbiased proteomic profiling. We found significant accumulation of several categories of ubiquitinated proteins on human mature sRBC membrane, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), reactive oxygen species (ROS) related enzyme (catalase), components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Mechanistically, we revealed that the impaired proteasomal machinery found in mature sRBC was due to the blockage of trafficking of p97 bound ubiquitinated-proteins from membrane to cytosolic proteasome. As such, inhibition of p97 by CB-5083 or proteasome by MG132 led to further induction of hypoxia-induced ubiquitinated membrane proteins and sickling in cultured human sRBC. Given the fact that sphingosine-1-phosphate (S1P) contributes to sickling by binding with deoxygenated sickle Hb (deoxy-HbS), triggering deoxy-HbS anchoring membrane and releasing glycolytic enzymes, we immediately hypothesized that S1P may be involved in proteasomal machinery by regulating trafficking of p97-bound ubiquitinated proteins from membrane to cytosol in sRBC. To test this intriguing possibility, we generated SCD/Sphk1-/- mouse. Intriguingly, we found that the genetic deletion of SphK1 attenuated impaired proteasomal machinery in sRBC with less accumulation of p97 and ubiquitinated proteins on sRBC membrane, indicating that elevated S1P is detrimental in sRBC by inducing accumulation of p97 and ubiquitinated proteins on the membrane. Moreover, to determine if S1P-regulated p97 trafficking from membrane to the cytosol is unique to sRBC, we exposed wild type and SphK1-/- mice to 8% hypoxia up to 72 hours. In contrast to sRBC, we found that genetic deletion of SphK1 abolished p97 trafficking from membrane to cytosol in normal erythrocytes under hypoxia. Finally, we conducted in vitro proof-of-principle genetic studies to determine if S1P directly involves in translocation of membrane anchored p97 to cytosol using inverted ghost membrane (IGM). We demonstrated that S1P treatment induces deoxy-HbA translocation from cytosol to membrane and in turn restoring p97 release from membrane to the cytosol in IGM isolated from SphK1-/- mice only under hypoxia but not normoxia. Thus, we have provided both human and mouse genetic evidence supporting a working model: in normal individuals under hypoxia, S1P is a key factor regulating the efficient proteasomal machinery by binding deoxy-HbA, promoting deoxy-HbA anchoring membrane and in turn triggering release of p97-ubiqutinated proteins to cytosol for its degradation. With mutation in HbS, S1P promotes deoxy-HbS anchoring the membrane and forms polymers, thus blocks membrane bound p97 release and impairs proteasomal machinery in sRBC. Overall, our findings identified that S1P is a missing key component of proteasome machinery by its differential mechanism regulating p97 trafficking from membrane to cytosol in normal erythrocyte physiology under hypoxia and the pathophysiology of SCD that open up new and differential therapies for the SCD and normal individuals facing hypoxia. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Adenosine signaling via ADORA2B induces SphK1 activity in sickle and normal erythrocytes via PKA-mediated ERK1/2 activation. Lowering adenosine by PEG-ADA or interfering ADORA2B activation by specific antagonist decreases SphK1 activity in normal and sickle RBCs.

Description: Abstract 3232 Sickle cell disease (SCD) is a severe genetic disorder with a high morbidity and mortality. Understanding the molecular basis responsible for sickling, a central pathogenesis of SCD, is critical for developing new therapeutic strategies. Using a nonbiased high throughput metabolomic screen, coupled with genetic and pharmacological approaches, recent studies demonstrated that excessive adenosine signaling through the A2B adenosine receptor triggers sickling by induction of 2,3-bisphosphoglycerate (2,3-BPG), an erythroid specific metabolite that induces O2 release from hemoglobin. Adenosine is a signaling nucleoside that elicits many physiological effects by engaging membrane receptors. Notably, equlibrative nucleoside transporters (ENTs) on erythrocytes have been long speculated to regulate extracellular adenosine concentrations under hypoxic conditions. Thus, we hypothesize that ENT is likely a key molecule responsible for elevated circulating adenosine levels and contributing to pathophysiology of SCD. To test this hypothesis, we first conducted western blot analysis to compare expression profiles of ENTs on normal and sickle erythrocytes. We found that ENT1 is the major ENT expressed on both mouse and human erythrocytes. Unexpectedly, ENT1 levels were significantly reduced in sickle erythrocytes compared to normal erythrocytes in both humans and mice, suggesting that ENT1 may contribute to increased adenosine levels seen in SCD. Next, we performed pharmacological studies to determine the exact role of ENT in normal and sickle erythrocytes. We found that treatment with dipyridamole or an ENT1 specific inhibitor (NBMPR) enhanced adenosine-induced elevation of 2,3-BPG in cultured mouse RBCs. Using Hemox Analyzer, we found that co-treatment of adenosine with either dipyridamole or NBMPR resulted in a further right shift of oxygen equilibrium curve (OEC) and further increase in P50 compared to the cells treated with adenosine alone. Similar to our pharmacological studies, we found that genetic deletion of ENT1 further enhanced adenosine-induced 2,3-BPG production in cultured erythrocytes, additional right shift of OEC and increased P50. Extending mouse studies to human, we demonstrate that co-treatment of adenosine with either dipyridamole or NBMPR further enhanced the adenosine alone-mediated 2,3-BPG induction in cultured erythrocytes isolated from normal individuals and SCD patients. Finally, we found that dipyridamole treatment significantly enhanced hypoxia-mediated 2,3-BPG production, right shift of OEC and substantial sickling in cultured erythrocytes isolated from SCD patients. Overall, our studies demonstrate that 1) ENT1 is a major transporter expressed by RBCs and that inhibition or deletion of ENT1 results in enhanced adenosine-mediated 2,3-BPG induction and deoxygenation in normal RBCs; 2) Decreased ENT1 expression in sickle erythrocytes is responsible for elevated circulating adenosine and thereby contributes to sickling by promoting 2,3-BPG production and triggering deoxygenation. Therefore, our findings reveal a previously unrecognized role of ENT1 in erythrocyte physiology, add a new insight to the pathophysiology of SCD and open up new therapies for the disease. Disclosures: No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) is a prevalent hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although it is well accepted that deoxygenation and polymerization of sickle hemoglobin (HbS) are initial triggers for sickling, it has been known for more than three decades that abnormal membrane lipid organization and composition are found in sickle erythrocyte. Early studies showed some lipids are altered in sickle erythrocytes, however, no studies have identified overall membrane lipid alteration and functional role of those altered specific lipids in SCD. Using unbiased metabolomic profiling, we found that lysophospholipids (LPLs), particularly lysophosphocholines (LysoPCs), were significantly elevated inside erythrocytes of mice with SCD due to imbalanced Lands' cycle. Lands' cycle containing two concerted enzymes: phospholipases A2 (PLA2s) and lysophospholipid acyltransferases (LPLATs) was initially discovered in 1958. However, its function and cellular regulation in membrane homeostasis in SCD remain unrecognized prior to our metabolomics screening. Here, we demonstrated that enhancing imbalanced Lands' cycle promotes a process of sickling and disease progression in mice by inducing LysoPC content inside erythrocytes. Significantly, correcting impaired Lands' cycle reduced LysoPC levels within erythrocytes and attenuated sickling and disease progression in mice. Mechanistically, we revealed that hypoxia-mediated MEK/ERK activation underlies imbalanced Lands' cycle by preferentially inducing activity of PLA2 but not LPCAT1 in mouse sickle erythrocytes. Additionally, the detrimental role of impaired Lands' cycle-induced LysoPC production in sickling via MEK/ERK-dependent activation of PLA2 in SCD patients mirrors our mouse finding. Overall, our studies have identified a pathological role of imbalanced Lands' cycle in SCD, revealed molecular basis regulating Lands' cycle and immediately provided novel therapeutic possibilities for the disease. Disclosures No relevant conflicts of interest to declare.