Multifunctionality of Prostatic Acid Phosphatase in Prostate Cancer Pathogenesis.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomic tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum, magnifying normally difficult to detect subsets of the protein of interest. For PAcP this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wild-type PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

Multifunctionality of prostatic acid phosphatase in prostate cancer pathogenesis.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

Voxelotor in adolescents and adults with sickle cell disease (HOPE): long-term follow-up results of an international, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: For decades, patients with sickle cell disease have had only a limited number of therapies available. In 2019, voxelotor (1500 mg), an oral once-daily sickle haemoglobin polymerisation inhibitor, was approved in the USA for the treatment of sickle cell disease in patients aged 12 years and older on the basis of HOPE trial data. To further describe the applicability of voxelotor as a treatment for this chronic illness, we report the long-term efficacy and safety of this drug at 72 weeks of treatment; the conclusion of the placebo-controlled HOPE trial. METHODS: HOPE is an international, randomised, double-blind, placebo-controlled, phase 3 trial done at 60 clinical sites in Canada, Egypt, France, Italy, Jamaica, Kenya, Lebanon, Netherlands, Oman, Turkey, the USA, and the UK. Patients (aged 12-65 years) with confirmed sickle cell disease, a haemoglobin concentration of 5·5-10·5 g/dL at enrolment, and who had between one and ten vaso-occlusive crisis events in the previous 12 months were enrolled. Patients receiving regularly scheduled transfusion therapy, who had received a transfusion in the previous 60 days, or who had been admitted to hospital for a vaso-occlusive crisis in the previous 14 days were excluded. Patients were randomly assigned (1:1:1) to receive either once-daily oral voxelotor 1500 mg, voxelotor 900 mg, or placebo for 72 weeks. Randomisation was done centrally by use of an interactive web response system, stratified by baseline hydroxyurea use (yes vs no), age group (adolescents [12 to <18 years] vs adults [18 to 65 years]), and geographic region (North America vs Europe vs other). The primary endpoint (already reported) was the proportion of patients who achieved a haemoglobin response at week 24. In this final analysis, we report prespecified long-term efficacy assessments by intention to treat, including changes in haemoglobin concentrations from baseline to week 72, changes in the concentration of haemolysis markers (absolute and percentage reticulocytes, indirect bilirubin concentrations, and lactate dehydrogenase concentrations) from baseline to week 72, the annualised incidence of vaso-occlusive crises, and patient functioning, as assessed with the Clinical Global Impression of Change (CGI-C) scale. Safety was assessed in patients who received at least one dose of treatment (modified intention-to-treat population). This trial is registered with ClinicalTrials.gov, NCT03036813. FINDINGS: Between Dec 5, 2016, and May 3, 2018, 449 patients were screened, of whom 274 were randomly assigned to the voxelotor 1500 mg group (n=90), the voxelotor 900 mg group (n=92), or the placebo group (n=92). At week 72, the adjusted mean change in haemoglobin concentration from baseline was 1·0 g/dL (95% CI 0·7 to -1·3) in the voxelotor 1500 mg group, 0·5 g/dL (0·3 to -0·8) in the voxelotor 900 mg group, and 0·0 g/dL (-0·3 to 0·3) in the placebo group, with a significant difference observed between the voxelotor 1500 mg group and the placebo group (p<0·0001), and between the voxelotor 900 mg group and the placebo group (p=0·014). Significant improvements in markers of haemolysis, as assessed by the difference in adjusted mean percentage change from baseline at week 72 versus placebo, were observed in the voxelotor 1500 mg group in indirect bilirubin concentrations (-26·6% [95% CI -40·2 to -12·9]) and percentage of reticulocytes (-18·6% [-33·9 to -3·3]). The proportion of patients in the voxelotor 1500 mg group who were rated as "moderately improved" or "very much improved" at week 72 with the CGI-C was significantly greater than in the placebo group (39 [74%] of 53 vs 24 [47%] of 51; p=0·0057). Serious adverse events unrelated to sickle cell disease were reported in 25 (28%) of 88 patients in the voxelotor 1500 mg group, 20 (22%) of 92 patients in the voxelotor 900 mg group, and 23 (25%) of 91 patients in the placebo group. Grade 3 or 4 adverse events were infrequent (ie, occurred in <10% of patients); anaemia occurred in five or more patients (two [2%] patients in the voxelotor 1500 mg group, seven [8%] patients in the voxelotor 900 mg group, and three [3%] patients in the placebo group). Of all 274 patients, six (2%) deaths occurred during the study (two deaths in each treatment group), all of which were judged as unrelated to treatment. INTERPRETATION: Voxelotor 1500 mg resulted in rapid and durable improvements in haemoglobin concentrations maintained over 72 weeks and has potential to address the substantial morbidity associated with haemolytic anaemia in sickle cell disease. FUNDING: Global Blood Therapeutics.

Accelerated approval of Oxbryta® (voxelotor): A case study on novel endpoint selection in sickle cell disease.

Sickle cell disease (SCD) is an inherited disease characterized by hemolysis, anemia, and vaso-occlusion leading to substantial morbidity and mortality. Development of prior pharmacologic therapies exclusively utilized vaso-occlusive crisis (VOC) as a clinical efficacy endpoint; however, this focus on VOC did not capture the full extent of disease symptomatology and complications and slowed the development of new therapies. Voxelotor, a hemoglobin S polymerization inhibitor, was recently approved in the United States for the treatment of SCD in adults and adolescents 12 years of age and older through an accelerated approval pathway. The rapid approval and availability of voxelotor was facilitated in a collaborative effort with the US Food and Drug Administration (FDA), using hemoglobin, a biologic surrogate endpoint, as reasonably likely to predict clinical benefit. Use of this new endpoint was supported by FDA-led multistakeholder discussions with physician and patient communities to identify unmet needs and potential clinical trial endpoints, as well as by a company-sponsored analysis of external patient-level data to demonstrate a correlation between hemoglobin change and stroke risk. A two-part phase 3 study was used to allow for rank ordering of key secondary endpoints based on a planned interim analysis. Continued open communication with the FDA was essential to gain agreement on hemoglobin as a novel endpoint and to address the unmet and urgent need of new therapies for SCD.

A Phase 3 Randomized Trial of Voxelotor in Sickle Cell Disease.

BACKGROUND: Deoxygenated sickle hemoglobin (HbS) polymerization drives the pathophysiology of sickle cell disease. Therefore, direct inhibition of HbS polymerization has potential to favorably modify disease outcomes. Voxelotor is an HbS polymerization inhibitor. METHODS: In a multicenter, phase 3, double-blind, randomized, placebo-controlled trial, we compared the efficacy and safety of two dose levels of voxelotor (1500 mg and 900 mg, administered orally once daily) with placebo in persons with sickle cell disease. The primary end point was the percentage of participants who had a hemoglobin response, which was defined as an increase of more than 1.0 g per deciliter from baseline at week 24 in the intention-to-treat analysis. RESULTS: A total of 274 participants were randomly assigned in a 1:1:1 ratio to receive a once-daily oral dose of 1500 mg of voxelotor, 900 mg of voxelotor, or placebo. Most participants had sickle cell anemia (homozygous hemoglobin S or hemoglobin Sß0-thalassemia), and approximately two thirds were receiving hydroxyurea at baseline. In the intention-to-treat analysis, a significantly higher percentage of participants had a hemoglobin response in the 1500-mg voxelotor group (51%; 95% confidence interval [CI], 41 to 61) than in the placebo group (7%; 95% CI, 1 to 12). Anemia worsened between baseline and week 24 in fewer participants in each voxelotor dose group than in those receiving placebo. At week 24, the 1500-mg voxelotor group had significantly greater reductions from baseline in the indirect bilirubin level and percentage of reticulocytes than the placebo group. The percentage of participants with an adverse event that occurred or worsened during the treatment period was similar across the trial groups. Adverse events of at least grade 3 occurred in 26% of the participants in the 1500-mg voxelotor group, 23% in the 900-mg voxelotor group, and 26% in the placebo group. Most adverse events were not related to the trial drug or placebo, as determined by the investigators. CONCLUSIONS: In this phase 3 randomized, placebo-controlled trial involving participants with sickle cell disease, voxelotor significantly increased hemoglobin levels and reduced markers of hemolysis. These findings are consistent with inhibition of HbS polymerization and indicate a disease-modifying potential. (Funded by Global Blood Therapeutics; HOPE ClinicalTrials.gov number, NCT03036813.).

A phase 1/2 ascending dose study and open-label extension study of voxelotor in patients with sickle cell disease.

New treatments directly targeting polymerization of sickle hemoglobin (HbS), the proximate event in the pathophysiology of sickle cell disease (SCD), are needed to address the severe morbidity and early mortality associated with the disease. Voxelotor (GBT440) is a first-in-class oral therapy specifically developed to treat SCD by modulating the affinity of hemoglobin (Hb) for oxygen, thus inhibiting HbS polymerization and downstream adverse effects of hemolytic anemia and vaso-occlusion. GBT440-001 was a phase 1/2 randomized, double-blind, placebo-controlled, single and multiple ascending dose study of voxelotor in adult healthy volunteers and patients with SCD, followed by a single-arm, open-label extension study. This report describes results of voxelotor (500-1000 mg per day) in patients with sickle cell anemia. The study evaluated the safety, tolerability, pharmacokinetic, and pharmacodynamic properties of voxelotor and established proof of concept by improving clinical measures of anemia, hemolysis, and sickling. Thirty-eight patients with SCD received 28 days of voxelotor 500, 700, or 1000 mg per day or placebo; 16 patients received 90 days of voxelotor 700 or 900 mg per day or placebo. Four patients from the 90-day cohort were subsequently enrolled in an extension study and treated with voxelotor 900 mg per day for 6 months. All patients who received multiple doses of voxelotor for ≥28 days experienced hematologic improvements including increased Hb and reduction in hemolysis and percentage of sickled red cells, supporting the potential of voxelotor to serve as a disease-modifying therapy for SCD. Voxelotor was well tolerated with no treatment-related serious adverse events and no evidence of tissue hypoxia. These trials were registered at www.clinicaltrials.gov as #NCT02285088 and #NCT03041909.

Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

BACKGROUND: Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus. RESULTS: Here, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures. CONCLUSIONS: These results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner. REVIEWERS: Reviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers' comments section.

Splenic metabolic activity predicts risk of future cardiovascular events: demonstration of a cardiosplenic axis in humans.

OBJECTIVES: This study sought to determine whether splenic activation after acute coronary syndrome (ACS) is linked to leukocyte proinflammatory remodeling and whether splenic activity independently predicts the risk of cardiovascular disease (CVD) events. BACKGROUND: Pre-clinical data suggest the existence of a cardiosplenic axis, wherein activation of hematopoietic tissues (notably in the spleen) results in liberation of proinflammatory leukocytes and accelerated atherosclerotic inflammation. However, it is presently unknown whether a cardiosplenic axis exists in humans and whether splenic activation relates to CVD risk. METHODS: (18)F-fluorodeoxyglucose ((18)FDG)-positron emission tomography (PET) imaging was performed in 508 individuals across 2 studies. In the first study, we performed FDG-PET imaging in 22 patients with recent ACS and 22 control subjects. FDG uptake was measured in spleen and arterial wall, whereas proinflammatory gene expression of circulating leukocytes was assessed by quantitative real-time polymerase chain reaction. In a second study, we examined the relationship between splenic tissue FDG uptake with subsequent CVD events during follow-up (median 4 years) in 464 patients who previously had undergone FDG-PET imaging. RESULTS: Splenic activity increased after ACS and was significantly associated with multiple indices of inflammation: 1) up-regulated gene expression of proinflammatory leukocytes; 2) increased C-reactive protein; and 3) increased arterial wall inflammation (FDG uptake). Moreover, in the second study, splenic activity (greater than or equal to the median) was associated with an increased risk of CVD events (hazard ratio [HR]: 3.3; 95% confidence interval [CI]: 1.5 to 7.3; p = 0.003), which remained significant after adjustment for CVD risk factors (HR: 2.26; 95% CI: 1.01 to 5.06; p = 0.04) and for arterial FDG uptake (HR: 2.68; 95% CI: 1.5 to 7.4; p = 0.02). CONCLUSIONS: Our findings demonstrate increased splenic metabolic activity after ACS and its association with proinflammatory remodeling of circulating leukocytes. Moreover, we observed that metabolic activity of the spleen independently predicted risk of subsequent CVD events. Collectively, these findings provide evidence of a cardiosplenic axis in humans similar to that shown in pre-clinical studies.

Anti-inflammatory strategies for plaque stabilization after acute coronary syndromes.

Despite dramatic advances in standard of care, the risk of recurrent myocardial infarction early after an acute coronary syndrome (ACS) remains high. This period of elevated risk after a cardiovascular event is associated with an acute inflammatory response. While post-ACS inflammation correlates with the risk for recurrent events and is likely to play a causal role in this period, the precise pathophysiologic mechanisms have been unclear. Recent studies have proposed that the cardiac event itself activates the sympathetic nervous system to directly mobilize hematopoietic stem cells to differentiate into inflammatory monocytes, acutely infiltrate plaque, and lead to recurrent plaque rupture. Here, we summarize the existing and emerging evidence implicating post-ACS activation of systemic inflammation in the progression of atherosclerosis, and identify possible targets for therapeutic intervention. We highlight experimental therapies and ongoing clinical studies that will validate these targets.

Spatial and temporal regulation of coronary vessel formation by calcineurin-NFAT signaling.

Formation of the coronary vasculature requires reciprocal signaling between endothelial, epicardially derived smooth muscle and underlying myocardial cells. Our studies show that calcineurin-NFAT signaling functions in endothelial cells within specific time windows to regulate coronary vessel development. Mouse embryos exposed to cyclosporin A (CsA), which inhibits calcineurin phosphatase activity, failed to develop normal coronary vasculature. To determine the cellular site at which calcineurin functions for coronary angiogenesis, we deleted calcineurin in endothelial, epicardial and myocardial cells. Disruption of calcineurin-NFAT signaling in endothelial cells resulted in the failure of coronary angiogenesis, recapitulating the coronary phenotype observed in CsA-treated embryos. By contrast, deletion of calcineurin in either epicardial or myocardial cells had no effect on coronary vasculature during early embryogenesis. To define the temporal requirement for NFAT signaling, we treated developing embryos with CsA at overlapping windows from E9.5 to E12.5 and examined coronary development at E12.5. These experiments demonstrated that calcineurin-NFAT signaling functions between E10.5 and E11.5 to regulate coronary angiogenesis. Consistent with these in vivo observations, endothelial cells exposed to CsA within specific time windows in tissue culture were unable to form tubular structures and their cellular responses to VEGF-A were blunted. Thus, our studies demonstrate specific temporal and spatial requirements of NFAT signaling for coronary vessel angiogenesis. These requirements are distinct from the roles of NFAT signaling in the angiogenesis of peripheral somatic vessels, providing an example of the environmental influence of different vascular beds on the in vivo endothelial responses to angiogenic stimuli.

Rates of behavior and aging specified by mitochondrial function during development.

To explore the role of mitochondrial activity in the aging process, we have lowered the activity of the electron transport chain and adenosine 5'-triphosphate (ATP) synthase with RNA interference (RNAi) in Caenorhabditis elegans. These perturbations reduced body size and behavioral rates and extended adult life-span. Restoring messenger RNA to near-normal levels during adulthood did not elevate ATP levels and did not correct any of these phenotypes. Conversely, inhibiting respiratory-chain components during adulthood only did not reset behavioral rates and did not affect life-span. Thus, the developing animal appears to contain a regulatory system that monitors mitochondrial activity early in life and, in response, establishes rates of respiration, behavior, and aging that persist during adulthood.