Hydroxyurea Research Articles

Efficacy and toxicity of hydroxyurea in mast cell activation syndrome patients refractory to standard medical therapy: retrospective case series

Determine efficacy and adverse events (AEs) of hydroxyurea (HU) in mast cell activation syndrome (MCAS) patients who were refractory to standard medical therapy. An electronic chart review was performed to find MCAS patients who received HU in a MCAS medical practice. Diagnosis of MCAS was established on the basis of mast cell (MC) activation symptoms in ≥ 5 systems plus ≥ 1 abnormal MC mediators and/or ≥ 20 MC/high power field on duodenal biopsies. Medicines not providing significant clinical improvement prior to HU were tabulated. The following symptoms were evaluated by patients on a 0–10 scale prior to and at the study conclusion: bone pain, abdominal pain, diarrhea, bloating, and nausea. Safety labs were obtained on a regular basis. Twenty out of three hundred ten (8.4%) MCAS patients received HU. Patients included 22 females, average age 42.4 years. Dysautonomia was present in 60%. An average of 10.6 (SD 1.7, range 8–13) medications were used prior to adding HU to various concomitant medications. Average dose of HU was 634 mg. In 20 patients who continued therapy for ≥ 2 months, there was statistically significant reduction of bone pain, abdominal pain, diarrhea, bloating, and nausea. Fourteen patients noted prolonged success with therapy. Six patients stopped HU within 6 weeks owing to AEs. Four patients treated ≥ 2 months had AEs and 2 led to HU cessation. All AEs were reversible. Refractory MCAS patients showed clear significant improvement in bone pain and gastrointestinal symptoms on HU. Systematic monitoring was effective in preventing the occurrence of severe HU-induced adverse events.

P-007: HYDROXYUREA FOR THE MANAGEMENT OF SICKLE CELL DISEASE IN LOW- AND MIDDLE-INCOME COUNTRIES: A SYSTEMATIC REVIEW (2000–2022)

Purpose: Hydroxyurea (HU) is a disease modifying oral therapy with demonstrated clinical efficacy and safety in patients with sickle cell disease (SCD) for over 30 years. In low- and middle-income countries (LMICs) where the disease burden is high, universal HU treatment is limited. Our aim for this systematic literature review was to summarise the available evidence conducted in low-resource settings on HU efficacy, safety and real-world effectiveness to demonstrate the need for wider access to HU for SCD patients in LMICs. The preliminary observations from clinical studies are presented here. Materials and methods: Articles published in Embase, PubMed, OVID and Web of Science between January 2000–2022 were reviewed; studies assessing the impact of HU in patients with SCD were considered. Results: Out of 3709 screened articles, 124 unique studies were included (Figure) with 20 clinical trials. Majority of the studies are from sub-Saharan Africa (SSA) and Latin America. The initial dose of HU ranged from 10–25 mg/kg/day and the most common initial dose was 15–20 mg/kg/day; the maximum tolerated dose (MTD) was 25–35 mg/kg/day. Across various age groups, as anticipated, a significant increase in haemoglobin, foetal haemoglobin and mean corpuscular volume levels with HU at both fixed (20 mg/kg/day) and escalated dose (≤35 mg/kg/day). HU at doses 15–30 mg/kg/day reduced the need for transfusions during the treatment period. HU treatment initiation reduced VOC-related pain, and reduction was better with escalated (30 mg/kg/day) versus fixed dosing (20–25 mg/kg/day). Similar reductions across various age groups were observed for atherosclerotic events. Initial low-dose HU therapy of 10 mg/kg/day and a 12-month MTD of 24.1±6.0 mg/kg/day reduced the primary stroke risk. Secondary strokes were prevented with HU (3 years; initial dose: 15–20 mg/kg/day; MTD: 30–35 mg/kg/day), and long-term treatment (3.5–9.3 years; 30–40 mg/kg/day) prevented recurrent strokes. HU treatment showed a reduction in the rates of hospitalisation, and this was?50% lower with escalated dose (29.0±3.5 mg/kg/day) when compared to fixed dose (19.3±1.7 mg/kg/day). Treatment with HU was also associated with significant reduction in malaria events and related hospitalisations. Significant reduction in the risk of mortality was observed after treatment with HU, assessed as events per 100 patients/person years, which can be attributed to fewer SCD–related clinical complications, infections, malaria and possibly transfusions. Various dosing of HU was well tolerated in children and adults, and the reported events of myelosuppression were mostly transient. Conclusion: Our preliminary observations suggest that clinical use of HU in low-resource settings is effective in improving haematological outcomes, reducing the risk of VOCs and primary and recurrent strokes, and reduction of malaria-related events. Need for transfusion, rates of hospitalisation and mortality were also reduced with HU treatment. These findings are in line with current consensus guidelines to initiate HU therapy to SCD patients starting as early as 9 months of age. We expect that the complete analysis of this systematic review will enable us to compile sufficient evidence for optimising the use of HU in LMICs.K. MARFO declares a conflict of interest: Stock shareholder: Novartis Other: Employee of Novartis S. KHAN declares a conflict of interest: Other: I am an employee of Novartis

P-025: ABNORMAL MITOCHONDRIA RETENTION IN MATURE RED BLOOD CELLS OCCURS NOT ONLY IN SS BUT ALSO SC PATIENTS AND IS ASSOCIATED WITH HEMOLYTIC MARKERS

Purpose: Sickle cell disease (SCD), the most frequent genetic disease worldwide, results from a unique point mutation leading to the synthesis of the abnormal hemoglobin S (HbS). Under hypoxic condition, HbS polymerization causes a mechanical distortion of red blood cells (RBCs) called sickling, which is accompanied by a decrease of their deformability and their life span. SCD is characterized by a wide variability of clinical expression and a complex pathophysiology including vaso-occlusive processes, intravascular hemolysis as well as pro-inflammation, pro-coagulant and vaso-constrictive states. Recently, the abnormal mitochondria retention in mature RBCs has been documented in small series of SS patients but the biological consequences of this abnormality have not been fully documented and not been documented in other sickle cell syndromes. In the present study, we analyzed mature RBC mitochondria retention in both SS and SC patients, the second most frequent sickle cell syndrome, taking into account the biological/clinical features associated with these abnormal RBCs. Materials and methods: Eighty-nine adult SCD patients were included: 35 SS patients treated with hydroxyurea (HU), 15 SS patients not treated with HU and 39 SC patients. All of them were at steady-state and regularly followed by the sickle center of Guadeloupe. Twenty-one Guadeloupean adult controls (AA) were also included. Mature RBCs containing mitochondria were identified by flow cytometry using MitoTracker® probe as well as thiazole orange and anti-CD71 antibody to discriminate total and stress reticulocytes respectively. Correlations between the percentage of mature mitochondria+-RBCs and the following parameters were then analyzed: RBC deformability (Ektacytometry), reticulocytes (both total and CD71+) counts and levels of hemoglobin, total bilirubin, aminotransferase aspartate and lactate dehydrogenase levels using routine procedures. The impact of splenic function was evaluated using Howell-Jolly bodies, assessed by classic May-Grünwald Giemsa smears, as a marker of splenic dysfunction. We also analyzed the relationships between mitochondria+-RBCs and HU treatment as well as the rates of hyper vaso-occlusive (painful vaso-occlusion, acute chest syndrome, osteonecrosis) and hyper hemolytic (micro/macro-albuminuria, leg ulcer) complications. Results: Abnormal mitochondria+ RBCs were detected not only in SS but also in SC patients, although at a less extend. In both sickle cell syndromes, positive correlations with mitochondria+ RBCs were detected with hemolysis markers (percentage of total and stress reticulocytes). In addition, a negative relationship with hemoglobin level was detected in SS patients. A negative correlation was also detected between these abnormal mitochondria+ RBCs and RBC deformability. In contrast, no association between the presence of this RBC sub-population and clinical manifestations, hydroxyurea treatment or Howell-Jolly bodies was detected. Conclusion: In the present report, we demonstrated that abnormal retention of mitochondria in mature RBCs occurred not only in SS but also in SC patients. We also presented evidence that this phenotype could be related to hemolysis rate. Further studies are warranted to reveal the molecular mechanisms involved. E. BERNIT declares a conflict of interest: Consultancy, Expert: for NOVARTIS PHARMA SAS

How Hydroxyurea Alters the Gut Microbiome: A Longitudinal Study Involving Angolan Children with Sickle Cell Anemia.

Sickle cell anemia (SCA) is an inherited hematological disorder and a serious global health problem, especially in Sub-Saharan Africa. Although hydroxyurea (HU) is the leading treatment for patients with SCA, its effects on the gut microbiome have not yet been explored. In this context, the aim of this study was to investigate this association by characterizing the gut microbiome of an Angolan SCA pediatric population before and after 6 months of HU treatment. A total of 66 stool samples were obtained and sequenced for the 16S rRNA gene (V3-V4 regions). Significant associations were observed in alpha and beta-diversity, with higher values of species richness for the children naïve for HU. We also noticed that children after HU had higher proportions of several beneficial bacteria, mostly short-chain fatty acids (SCFAs) producing species, such as Blautia luti, Roseburia inulinivorans, Eubacterium halli, Faecalibacterium, Ruminococcus, Lactobacillus rogosae, among others. In addition, before HU there was a higher abundance of Clostridium_g24, which includes C. bolteae and C. clostridioforme, both considered pathogenic. This study provides the first evidence of the HU effect on the gut microbiome and unravels several microorganisms that could be considered candidate biomarkers for disease severity and HU efficacy.

Unraveling the Mechanisms of Clinical Drugs-Induced Neural Tube Defects Based on Network Pharmacology and Molecular Docking Analysis.

Chemotherapeutic agents such as methotrexate (MTX), raltitrexed (RTX), 5-fluorouracil (5-FU), hydroxyurea (HU), and retinoic acid (RA), and valproic acid (VPA), an antiepileptic drug, all can cause malformations in the developing central nervous system (CNS), such as neural tube defects (NTDs). However, the common pathogenic mechanisms remain unclear. This study aimed to explore the mechanisms of NTDs caused by MTX, RTX, 5-FU, HU, RA, and VPA (MRFHRV), based on network pharmacology and molecular biology experiments. The MRFHRV targets were integrated with disease targets, to find the potential molecules related to MRFHRV-induced NTDs. Protein-protein interaction analysis and molecular docking were performed to analyze these common targets. Utilizing the kyoto encyclopedia of genes and genomes (KEGG) signaling pathways, we analyzed and searched the possible causative pathogenic mechanisms by crucial targets and the signaling pathway. Results showed that MRFHRV induced NTDs through several key targets (including TP53, MAPK1, HSP90AA1, ESR1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN) and multiple signaling pathways such as PI3K/Akt pathway, suggesting that abnormal proliferation and differentiation could be critical pathogenic contributors in NTDs induced by MRFHRV. These results were further validated by CCK8 assay in mouse embryonic stem cells and GFAP staining in embryonic brain tissue. This study indicated that chemotherapeutic and antiepileptic agents induced NTDs might through predicted targets TP53, MAPK1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN and multiple signaling pathways. More caution was required for the clinical administration for women with childbearing potential and pregnant.

Radiation stability and radiolysis mechanism of hydroxyurea in HNO3 solution: Alpha, beta, and gamma irradiations

Hydroxyurea (HU) is a novel salt-free reductant used potentially for the separation of U/Pu in the advanced PUREX process. In this work, the radiation stability of HU were systematically investigated in solution by examining the effects of the type of rays (α, β, and γ irradiations), the absorbed dose (10–50 kGy), and the HNO3 concentration (0–3 mol L−1). The influence degree on HU radiolysis rates followed the order of the absorbed dose > the ray type > the HNO3 concentration, but the latter two had moderate effects on HU radiolysis products where NH4+ and NO2− were found to be the most abundant ones, suggesting that the differences of α, β, and γ rays should be considered in the study of irradiation effects. The radiolysis mechanism was explored using density functional theory (DFT) calculations, and it proposed the dominant radiolysis paths of HU, indicating that the radiolysis of HU was mainly a free radical reaction among ·H, eaq–, H2O, intermediates, and the radiolytic free radical fragments of HU. The results reported here provide valuable insights into the mechanistic understanding of HU radiolysis under α, β, and γ irradiations and reliable data support for the application of HU in the reprocessing of spent fuel.

Prevalence and clinical outcomes of polycythemia vera and essential thrombocythemia with hydroxyurea resistance or intolerance

ABSTRACT Introduction To determine the prevalence, clinical outcomes, and factors associated with hydroxyurea (HU) resistance or intolerance among polycythemia vera (PV) and essential thrombocythemia (ET) patients. Methods This study was a retrospective cohort study including PV and ET patients diagnosed by WHO criteria and treated with HU between January 2000 and June 2020. Clinical features, laboratory data, and resistance or intolerance of HU were collected. The prevalence, clinical outcomes, and associated factors of HU resistance or intolerance were analyzed. Results There were 260 patients including 144 ET and 116 PV. The prevalence of HU resistance or intolerance was 11.9% (31 patients) which was more frequent in ET patients (14.6% vs. 8.6% in PV). Patients who had HU resistance or intolerance significantly increased the risk of bleeding events (HR 2.64; 95% CI 1.19–5.85, P = 0.017). The risk factors of HU resistance or intolerance were low baseline hemoglobin levels (HR 0.90; 95%CI 0.84–0.97, P = 0.01), age more than 60 years old (HR 3.98; 95% CI 2.08–7.62, P < 0.001) and splenomegaly (HR 2.08; 95% CI 1.03–4.21, P = 0.04). Conclusions The prevalence of HU resistance or intolerance in PV and ET patients was 11.9%. Patients with HU resistance or intolerance significantly increased the risk of bleeding complications.

The LAMMER Kinase MoKns1 Regulates Growth, Conidiation and Pathogenicity in Magnaporthe oryzae

Magnaporthe oryzae is an important pathogen that causes a devastating disease in rice. It has been reported that the dual-specificity LAMMER kinase is conserved from yeast to animal species and has a variety of functions. However, the functions of the LAMMER kinase have not been reported in M. oryzae. In this study, we identified the unique LAMMER kinase MoKns1 and analyzed its function in M. oryzae. We found that in a MoKNS1 deletion mutant, growth and conidiation were primarily decreased, and pathogenicity was almost completely lost. Furthermore, our results found that MoKns1 is involved in autophagy. The ΔMokns1 mutant was sensitive to rapamycin, and MoKns1 interacted with the autophagy-related protein MoAtg18. Compared with the wild-type strain 70−15, autophagy was significantly enhanced in the ΔMokns1 mutant. In addition, we also found that MoKns1 regulated DNA damage stress pathways, and the ΔMokns1 mutant was more sensitive to hydroxyurea (HU) and methyl methanesulfonate (MMS) compared to the wild-type strain 70−15. The expression of genes related to DNA damage stress pathways in the ΔMokns1 mutant was significantly different from that in the wild-type strain. Our results demonstrate that MoKns1 is an important pathogenic factor in M. oryzae involved in regulating autophagy and DNA damage response pathways, thus affecting virulence. This research on M. oryzae pathogenesis lays a foundation for the prevention and control of M. oryzae.

Health-Related Quality of Life and Adherence to Hydroxyurea and Other Disease-Modifying Therapies among Individuals with Sickle Cell Disease: A Systematic Review.

Background Sickle cell disease (SCD) is a hemoglobinopathy with increasing global prevalence resulting in pain episodes and multiorgan complications. Complications of SCD have been shown to adversely impact health-related quality of life (HRQOL) comprised of physical, social, and emotional domains; hence, HRQOL measures can serve as an effective evaluator of disease burden. Hydroxyurea (HU) and other disease-modifying therapies have demonstrated to significantly improve clinical outcomes in patients with SCD. Medication adherence is an essential mediator of the clinical benefits of these therapies; low adherence has been shown to increase disease burden and healthcare utilization. This systematic literature review intends to determine the association between adherence to disease-modifying therapies and HRQOL in patients with SCD. Methods We found a total of 12 articles involving 788 participants, which included both patients with SCD and caregivers/parents. Adherence was measured using self-report instruments, laboratory markers, such as fetal hemoglobin and mean corpuscular volume, and mHealth medication trackers. HRQOL was measured using self-report instruments. Results All studies demonstrated a correlation between higher HU adherence and better HRQOL scores. Higher HU adherence was associated with lower pain impact, less frequent pain episodes, less fatigue, and improved physical function and mobility, reflecting better physical HRQOL outcomes. Higher adherence was also associated with improved emotional response, decreased anxiety and depressive symptoms, and better social functioning and peer relationships. In addition, our findings indicated that having less frequent barriers to HU adherence was associated with better HRQOL scores. No studies evaluated HRQOL outcomes in relation to adherence to l-glutamine, voxelotor, or crizanlizumab. Conclusions Optimizing HU adherence has the potential to improve HRQOL in patients with SCD in addition to reducing healthcare utilization and improving treatment satisfaction. Addressing barriers to HU adherence can positively strengthen the relationship between adherence and HRQOL to potentially improve patient outcomes.

P1474: MOLECURAL MECHANISM OF CHEMOTHERAPEUTIC HYDROXYUREA IS MEDIATED BY NOS2

Background:: Hydroxyurea (HU) is a chemotherapeutic agent that reduces ribonucleotide reductase, stops DNA synthesis and repar, and therefore causes cell proliferation inhibition and apoptosis. Due to its cytostatic properties, HU is frequently used for treatment of myeloproliferative neoplasms, ovarian cancer, and sickle cell anemia. Nitric oxide (NO), produced by nitric oxide synthase (NOS) enzymes is a potent signaling molecule involved in blood flow regulation, neutrotransmission, and immunity. Although HU treatment increases NO levels, up to date it is not clear whether it originates from activation of NOS enzymes or HU degradation. Aims: The aim of this study was to determine the involvement of NOS2 enzyme in the cytostatic effect of HU. Methods: To examine the involvement of the NOS2 enzyme in the molecular mechanism of HU, we treated erythroleukemic HEL92.7.1 cells with pan-selective NOS inhibitor L-NAME (200µM, 1mM, and 5mM), NOS2 specific inhibitor 1400W (1, 10, and 100µM), or NOS2/NOS3 inhibitor DPI (1, 5, and 10µM), in combination with hydroxyurea (200µM), and monitored their effect on proliferation and cell cycle. Immunocytochemistry for the proliferation marker Ki67 was performed to assess proliferation, while cell distribution in cell cycle phases was determined by flow cytometry after propidium iodide staining. Colony forming assay have been performed with the bone marrow cells of Nos2 null mice after oral HU treatment to corroborate the data obtained by enzymatic inhibition. Results: In this study, we demonstrated that treatment of HEL92.7.1 cells with HU induces a dose-dependent increase in NOS2 protein levels and two products of the enzyme NOS - NO and citrulline. HU-induced citrulline levels can be reduced by treatment with the NOS inhibitor L-NAME, indicating that NO is produced de novo by the NOS enzyme rather than HU degradation. Inhibition of the NOS2 enzyme by L-NAME, 1400W, or DPI was sufficient to abolish HU-mediated inhibition of proliferation. While HU increased the number of cells in S-phase of the cycle at the expanse of the G0/G1 due to blocked DNA synthesis, combined treatment with HU and L-NAME or DPI inhibitor resulted in decreased G0/G1 phase and increased S and G2/M phases pointing to increased proliferation. These data indicate that the cytostatic properties of HU are mediated by the NOS2 enzyme. A colony formation assay showed that Nos2 deficient bone marrow cells isolated from mice treated orally with HU (200mg/kg) formed significantly more erythroid colonies (BFU-E and CFU-E) and granulocyte/macrophage progenitors (CFU-GM) compared to HU treated wild-type and untreated Nos2 null mice showing the involvement of Nos2 in the molecular mechanism of HU in vivo. Summary/Conclusion: Our results show that HU induces the enzymatic activity of the NOS2 protein which in turn is involved in the HU regulation of proliferation and cell cycle. Comprehensive knowledge of the molecular mechanism of HU might help to improve its beneficial properties and decrease adverse effects.

P1475: THE ROLE OF BCL11A AND HBS1L-MYB SINGLE NUCLEOTIDE POLYMORPHISMS IN EGYPTIAN SICKLE CELL DISEASE PATIENTS

Background: Sickle cell disease (SCD) is a debilitating blood disorder with a highly variable phenotype. Inter-individual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. The only currently FDA-approved HbF inducing drug for sickle cell disease (SCD) is hydroxyurea (HU). Genome wide association studies (GWAS) have identified three major quantitative trait loci (QTL) (intergenic region of HBS1L-MYB, BCL11A gene and XmnI site) associated with HbF. Several single nucleotide polymorphisms (SNPs) with persistence of HbF and amelioration of disease severity of β-thalassemia (β-thal) and SCD have been identified. Aims: The aim of the current study is to find the influence of these genetic modifiers, especially BCL11A (rs4671393) and HBS1L-MYB intergenic region (rs9399137) SNPs on the level of HbF, severity of SCD and response to hydroxyurea in Egyptian sickle cell disease patients. Methods: The present study included 100 patients with SCD; 56 patients were diagnosed as homozygous sickle cell anemia (SS) patients, while 44 patients were diagnosed as sickle beta thalassemia patients (Sβ). Genotyping for the genes was done by Real-Time PCR (RT-PCR) technique. Results: BCL11A (rs4671393) SNP showed higher hemoglobin and HbF levels when induced with HU, with significantly higher increment in HbF level post HU therapy as well as a milder clinical phenotype in our Sβ patients’ group. In our SS patients’ group it showed a lower incidence of pneumonia as well as a more effective response to HU treatment. HBS1L-MYB intergenic region (rs9399137) SNPs showed lower reticulocytic counts and serum ferritin levels in our Sβ patients’ group. Summary/Conclusion: In conclusion, our study shows that BCL11A (rs4671393) SNP showed higher hemoglobin and HbF levels when induced with HU, with significantly higher increment in HbF level post HU therapy as well as a milder clinical phenotype in our Egyptian study.

S196: ROPEGINTERFERON ALFA-2B ACHIEVES PATIENT-SPECIFIC TREATMENT GOALS IN POLYCYTHEMIA VERA: FINAL RESULTS FROM THE PROUD-PV/CONTINUATION-PV STUDIES

Background: Treatment of polycythemia vera (PV) aims to prevent thromboembolic complications, reduce the risk of progression to acute leukemia or myelofibrosis - of particular concern to patients - and ameliorate the symptom burden; specifically, to improve quality of life, therapy should address the most clinically important symptoms while reducing phlebotomies to avoid iron-deficiency symptoms. Long-term efficacy and safety of ropeginterferon alfa-2b have been demonstrated in PROUD-PV/CONTINUATION-PV; the final analysis applied a patient-focused approach. Aims: To analyze the patient-relevant benefit of ropeginterferon alfa-2b versus hydroxyurea (HU)/best available treatment (BAT) over 6 years. Methods: Patients diagnosed with PV according to WHO 2008 criteria who were cytoreduction-naïve or hydroxyurea pre-treated and gave written informed consent were randomized 1:1 to ropeginterferon alpha-2b or control treatment (HU) for one year in PROUD-PV. In CONTINUATION-PV, control arm patients could switch from HU to BAT. Patient-reported PV symptom burden was assessed based on adverse events documented in the patient diary and recorded at each visit; items defined in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-SAF TSS; fatigue, concentration problems, early satiety, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fevers) and medical synonyms were evaluated post-hoc. Efficacy assessments included Kaplan-Meier analysis of event-free survival, phlebotomy need and JAK2V617F allele burden. Analyses were conducted on the CONTINUATION-PV full analysis set over 6 years of treatment. Results: The full analysis set comprised 95 patients in the ropeginterferon alfa-2b arm and 74 in the control arm. Patient-reported symptoms defined in the MPN-SAF TSS were present in a small minority (9.5%) of patients per arm at baseline (up to Week 4 of treatment) in this early-stage PV population. Occurrence of the defined symptoms remained low over long-term treatment, reported in 15.7% of patients in the ropeginterferon alfa-2b arm and 20.7% in the control arm during the 6th year of treatment. No phlebotomies were required to maintain hematocrit <45% in the 6th year of treatment in 81.4% of patients receiving ropeginterferon alfa-2b compared with 60.0% of patients in the control arm (p=0.005). Depletion of the JAK2V617F alle burden, which may lower the risk of progression to myelofibrosis, was observed in ropeginterferon alfa-2b treated patients; JAK2V617F allele burden <1% at 6 years was achieved in 19/92 (20.7%) patients in the ropeginterferon alfa-2b arm with baseline allele burden >10%. One patient met this threshold in the control arm (1/70 [1.4%]; p=0.0001). Event-free survival (risk events: disease progression, death and thromboembolic events) over ≥6 years of treatment was significantly higher among ropeginterferon alfa-2b treated patients than the control group (risk events reported in 5/95 vs. 12/74 patients, respectively; p=0.04 [Log-Rank]; Fig 1). Image:Summary/Conclusion: Long-term ropeginterferon alfa-2b therapy fulfils treatment goals important to patients with PV: a good quality of life as indicated by a low symptom burden and phlebotomy requirement, the potential to influence myelofibrosis risk, and better event-free survival versus BAT.

P1503: REAL WORLD DATA ON EFFICACY OF PHARMACEUTICAL-GRADE L-GLUTAMINE IN PREVENTING SICKLE CELL DISEASE-RELATED ACUTE COMPLICATIONS AND HEMOLYSIS IN PEDIATRIC AND ADULT PATIENTS.

Background: Oxidative stress is one of the key contributors to the pathophysiology of sickle cell disease (SCD) and related complications including acute pain (vaso-occlusive crisis or VOC), tissue ischaemia, stroke, and acute chest syndrome (ACS). L-glutamine (L-gln), a precursor of nicotinamide adenine dinucleotide (NAD), has been shown to play an important role in the regulation of oxidative stress. In the pivotal Phase 3 clinical trial, L-gln demonstrated significant reduction in VOC, hospitalizations, and ACS events compared to placebo in patients with SCD, with or without hydroxyurea (HU) use, over a 48-week period. In September 2021, a re-analysis of the Phase 3 trial data revealed that L-gln decreased the number of VOCs by 45%. Aims: To confirm the efficacy on clinical observations and hemolysis markers of oral therapy with pharmaceutical-grade L-gln in pediatric and adult patients with SCD at follow-up time points of 24, 48 and 72 weeks. Methods: In the observational study conducted from October 2019 through April 2021, 19 patients (4 patients from Qatar and 15 patients from French Guiana) were treated with L-gln (0.3mg/kg) twice daily. Laboratory parameters (hemoglobin (Hb) levels, hematocrit, WBC counts, reticulocyte counts, and LDH levels) were measured at baseline and follow-up time points. Clinical parameters (number of VOCs, hospitalizations, days hospitalized, ACS events, and blood transfusions) were documented for the year prior to treatment initiation as baseline values. These parameters were also collected at 24, 48, and 72 weeks from treatment initiation. Adverse events (AEs) were also collected during the treatment period. The data values at 24, 48, and 72 weeks have been annualized. Statistical analysis was performed using MedCalc 20.015. Results: Compared to baseline, patients had significantly fewer number of VOCs at 24, 48, and 72 weeks following L-gln therapy (median change from 3.0 to 0;p<0.00001). Compared to baseline, there were fewer hospitalizations (median change from 3.0 to 0;p<0.00001) and patients spent fewer days in hospital (median change from 15.0 to 0;p<0.00001). Furthermore, at 24, 48, and 72 weeks, the number of blood transfusions was considerably lower than at baseline (median, from 3.0 to 0.0;p<0.00001). Following treatment with L-gln, the mean Hb level increased significantly from baseline to 72 weeks (8.2 to 8.8 g/dL;p<0.001) with peak mean increase from baseline of 11.2% at 48 weeks. A similar increasing trend was observed for hematocrit proportions from baseline to 72 weeks (24% to 27%;p<0.001) with highest mean improvement from baseline of 15.5% at 48 weeks. Conversely, mean reticulocyte counts and LDH levels were significantly reduced at follow-up time points compared to baseline (p=0.003 and p<0.001, respectively). Image:Summary/Conclusion: This study demonstrated for the first time that L-gln therapy in SCD patients from Qatar and French Guiana resulted in significant improvements in clinical outcomes (number of VOCs, number and duration of hospitalizations, and number of blood transfusions) accompanied by a noteworthy increase in Hb levels and a reduction in markers of hemolysis (reticulocyte counts and LDH levels). Moreover, while 11 ACS events were reported the year before starting L-gln, only 2 ACS events occrred after 12 months of initiation of L-gln. Treatment with L-gln resulted in clinically significant outcomes combined with significant improvement of the hemolysis parameters from baseline through 72 weeks suggesting sustained long-term efficacy. Only a few number of AEs occurred and were mild. No compliance issue was reported.

P1467: HEALTHCARE PROFESSIONALS (HCP) OPINIONS ON TREATMENTS FOR SICKLE CELL DISEASE (SCD) AND THE HCP–PATIENT RELATIONSHIP: RESULTS FROM THE INTERNATIONAL SICKLE CELL WORLD ASSESSMENT SURVEY (SWAY)

Background: SCD is an inherited blood disorder that has a significant impact on morbidity and mortality. Care for patients (pts) with SCD may be influenced by HCP specialty and care setting (Smeltzer et al. BMJ Open 2021). Aims: To assess potential differences in treatment and management of SCD by HCPs participating in SWAY, based on HCP specialty and care setting. Methods: SWAY was a cross-sectional survey developed by international SCD experts, pt advocates and Novartis to assess pt and HCP experiences of SCD. Pts with SCD ≥6 years old (N=2145) and HCPs (N=365) completed the survey between Apr and Oct 2019. Eligible HCPs had qualified in their primary specialty by 2014 and were managing ≥10 SCD pts at the time of survey (≥5 pts per HCP in Canada; ≥2 pts per HCP in the Netherlands). Data reflect only the experiences of the surveyed HCPs (recruited by Adelphi Real World Network). Results: HCPs were grouped by specialty (hematology, n=150; hematology–oncology, n=101; pediatric, n=31; general practitioner [GP], n=50 and other [internal medicine specialist, emergency department physician, physician assistant, nurse/nurse practitioner, other], n=33) and care setting (SCD center, n=46; university/teaching hospital, n=105; regional center/community hospital, n=57; private hospital/office, n=109 and other [health center and other], n=48). Most hematologists (82%), hematologist–oncologists (79%) and pediatricians (87%) reported acute (vaso-occlusive crisis [VOC]) pain as one of the most common symptoms discussed with their pts vs 54% of GPs and 42% of other HCPs (chronic pain was reported by 78% of GPs and 70% of other HCPs). Accordingly, 97% of hematologists, 88% of hematologist–oncologists and 90% of pediatricians reported ever initiating hydroxyurea (HU) – a treatment for VOCs – vs 14% of GPs and 30% of other HCPs. More hematologists, hematologist–oncologists and pediatricians ranked reducing VOCs as a top treatment goal (other than a cure for SCD) than reducing chronic pain levels, while the opposite was true for GPs or other HCPs (Table). More HCPs at SCD centers and university/teaching hospitals initiated standard and exchange blood transfusions to help manage their pts SCD than other care settings (Table), yet fewer HCPs at SCD centers than at other care settings reported iron overload as a complication their pts’ experienced (SCD center, 26%; university/teaching hospital, 40%; regional center/community hospital, 47%; private hospital/office, 31% and other, 42%). HCPs at SCD centers were also more likely to provide information to pts in the form of leaflets (SCD center, 70%; university/teaching hospital, 60%; regional center/community hospital, 49%; private hospital/office, 59% and other, 60%) or direct them to pt support groups (SCD center, 63%; university/teaching hospital, 47%; regional center/community hospital, 30%; private hospital/office, 33% and other, 33%). Image:Summary/Conclusion: Hematologists, hematologist–oncologists and pediatricians in SWAY were more likely to discuss VOCs (the hallmark of SCD) during consultation with their pts than GPs or other HCPs. This may be a reason why fewer GPs and other HCPs reported initiating HU, the mainstay of SCD therapy, compared with the other groups analyzed. Moreover, HCPs at SCD centers vs other care settings appeared to be more likely to initiate certain SCD treatments. These results support a multidisciplinary and collaborative approach to SCD care in order to optimize pt outcomes, where each HCP speciality and care setting fulfills distinct but vital aspects of pts’ needs.

PB2045: POLYCYTHEMIA VERA IN YOUNG SUBJECTS: ABOUT A COHORT OF 11 CASES

Background: Polycythemia Vera (PV) is a typical Philadelphia-negative (Ph_) myeloproliferative disease. It is secondary in 95% of cases to the JAK 2 mutation on exon 14. The median age at diagnosis is 65 years. PV is unusual in young patients, so that little information is available on long-term clinical evolution in this particular group. Aims: The aim of this study is to describe the clinical presentation, biologic features, treatment approaches, and long-term outcome especially the risk of thrombosis. Methods: Of all the 64 patients followed in our department for PV, we report 11 cases aged less than 55 years. The inclusion criteria were a diagnosis satisfying the PVSG criteria and, from 2016 on, the WHO criteria. Secondary causes of erythrocytosis were carefully ruled out in all patients. At diagnosis, cytogenetic and molecular analysis were not done in our patients. Search for JAK2 mutation and serum EPO dosage were performed in all cases. Results: PV in young patients represents 17% of all PV followed. Median age of our population is 44 years, range 32-52, with 27% of patients under 40 years old. slightly more often in men with (sex ratio 1.75). Arterial hypertension is the most common comorbidity and it is a cardiovascular risk factor in the majority of patients. A thromboembolic event revealing PV is found in 2 patients. The main clinical signs were significant burning sensation in fingers and palms due to the increased blood viscosity, splenomegaly which observed in 63% of patients, and the occurrence of severe itching on contact with warm bath. Thromboembolic events occurred in 3 patients during the evolution. All patients had the diagnosis of absolute erythrocytosis with moderate hyperleukocytosis and thrombocytosis. Bone marrow biopsy showed hypercellular bone marrow with erythroid and megakaryocytic lineage hyperplasia. The JAK2 V617F mutation was positive with an average allelic load high at 63.1% [22.9 - 93.6]. The dosage of serum EPO was low with an average of 1 mU/ML. During the follow-up period, no patient had progression into acute leukemia or myelofibrosis with myeloid metaplasia, thus no death. The median survival is 49 months [3-99] and the survival without thromboembolic event is 42 months [3-99]. The majority of patients were treated by phlebotomy with 1 or 2 units per month, before starting hydroxy urea. The use of the latter in association with Aspirin (n=10 pts) reduced the use of bloodletting. The thrombotic events required treatment with antithrombotic drug. No patient was treated with Ruxolitinib (treatment not available). Summary/Conclusion: PV remains rare in patients under 55 years of age (17% in our cohort). This has an impact on overall survival and thromboembolic event-free survival with a good prognosis in the young population.

WASHC1 interacts with MCM2-7 complex to promote cell survival under replication stress.

WASHC1 is a member of the Wiskott-Aldrich syndrome protein (WASP) family and is involved in endosomal protein sorting and trafficking through the generation of filamentous actin (F-actin) via activation of the Arp2/3 complex. There is increasing evidence that WASHC1 is present in the nucleus and nuclear WASHC1 plays important roles in regulating gene transcription, DNA repair as well as maintaining nuclear organization. However, the multi-faceted functions of nuclear WASHC1 still need to be clarified. We show here that WASHC1 interacts with several components of the minichromosome maintenance (MCM) 2-7 complex by using co-immunoprecipitation and in situ proximity ligation assay. WASHC1-depleted cells display normal DNA replication and S-phase progression. However, loss of WASHC1 sensitizes HeLa cells to DNA replication inhibitor hydroxyurea (HU) and increases chromosome instability of HeLa and 3T3 cells under condition of HU-induced replication stress. Re-expression of nuclear WASHC1 in WASHC1KO 3T3 cells rescues the deficiency of WASHC1KO cells in the chromosomal stability after HU treatment. Moreover, chromatin immunoprecipitation assay indicates that WASHC1 associates with DNA replication origins, and knockdown of WASHC1 inhibits MCM protein loading at origins. Since efficient loading of excess MCM2-7 complexes is required for cells to survive replicative stress, these results demonstrate that WASHC1 promotes cell survival and maintain chromosomal stability under replication stress through recruitment of excess MCM complex to origins.

Prophylactic Hydroxyurea Treatment Is Associated with Improved Cerebral Hemodynamics as a Surrogate Marker of Stroke Risk in Sickle Cell Disease: A Retrospective Comparative Analysis.

Sickle cell disease (SCD) increases the incidence of childhood stroke eighty-fold. Stroke risk can be estimated by measurement of the blood velocity through the middle cerebral artery (MCA) using transcranial doppler ultrasound (TCD). A high MCA blood velocity indicates increased stroke risk due to cerebral vasculopathy, and first-line treatment to prevent primary or recurrent strokes in high-risk children with SCD has classically been chronic blood transfusions. Research has more recently shown that many of these patients may safely transition from transfusions to oral hydroxyurea (HU) treatment while maintaining a decreased risk of stroke. However, the effect on stroke risk of truly prophylactic HU treatment beginning in infancy, prior to the onset of cerebral vasculopathy, is less well understood. Our retrospective study aimed to document the long-term effects of HU treatment compared with no HU treatment in children with SCD, using TCD measurements as our primary outcome and a surrogate marker of stroke risk. Our results showed that when accounting for age-related variability and duration of treatment, prophylactic HU treatment was independently associated with lower TCD MCA velocities compared with no HU treatment, providing further evidence supporting its early initiation for patients with SCD.

Hydroxyurea protects against diabetic cardiomyopathy by inhibiting inflammation and apoptosis

Hydroxyurea (HU), a small molecule with various biological properties, was used in myeloproliferative, tumorous, and non-hematological diseases. However, whether HU plays a role in diabetic cardiomyopathy (DCM) remains unclear. Our study aimed to investigated whether HU could ameliorate DCM or not. Induction of type 1 diabetes mellitus (T1DM) in C57BL/6 J mice was achieved by intraperitoneal injection of streptozotocin (STZ). Mice in control and diabetic groups were treated with HU (20 mg/kg) in drinking water for 16 weeks. Our data showed that diabetic mice had significantly increased FBG level and decreased body weight, along with abnormal diastolic function and myocardial fibrosis. Inflammatory factors including TNF-α, IL-1β, IL-6, ICAM, VCAM, and apoptosis-related proteins including caspase-3 and BAX were significantly up-regulated in heart tissues. HU treatment remarkably improved these changes. Similarly, application of HU (5 µM) significantly improves the survival rate of high glucose (HG)-treated H9C2 cells. Thus, HU rescued the cardiomyocytes via inhibition of apoptosis and inflammation in DCM.

Effects of Hydroxyurea on Skeletal Muscle Energetics and Function in a Mildly Anemic Mouse Model.

Hydroxyurea (HU) is a ribonucleotide reductase inhibitor most commonly used as a therapeutic agent in sickle cell disease (SCD) with the aim of reducing the risk of vaso-occlusion and improving oxygen transport to tissues. Previous studies suggest that HU may be even beneficial in mild anemia. However, the corresponding effects on skeletal muscle energetics and function have never been reported in such a mild anemia model. Seventeen mildly anemic HbAA Townes mice were subjected to a standardized rest-stimulation (transcutaneous stimulation)-protocol while muscle energetics using 31Phosphorus magnetic resonance spectroscopy and muscle force production were assessed and recorded. Eight mice were supplemented with hydroxyurea (HU) for 6 weeks while 9 were not (CON). HU mice displayed a higher specific total force production compared to the CON, with 501.35 ± 54.12 N/mm3 and 437.43 ± 57.10 N/mm3 respectively (+14.6%, p < 0.05). Neither the total rate of energy consumption nor the oxidative metabolic rate were significantly different between groups. The present results illustrated a positive effect of a HU chronic supplementation on skeletal muscle function in mice with mild anemia.

Abstract 803: Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers

Abstract Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 are associated with an increased lifetime risk of breast and ovarian cancers. While the BRCA proteins play a well-established role in double-stranded DNA break repair, recent studies have revealed an emerging role of BRCA1/2 in replication stress response. While replication forks are extensively degraded by nucleases in BRCA-deficient cancer cells, activation of specialized fork recovery mechanisms enables resumption of DNA synthesis and promotes cell survival. My project aims to determine this fork recovery mechanism in BRCA1-deficient cells and to identify potential recovery factors that can be targeted to improve chemotherapeutic response in BRCA1-mutated breast and ovarian cancers. To monitor perturbations in replication fork dynamics on a genome-wide scale, we utilize a DNA fiber assay technique measuring rates of fork recovery and replication fork degradation. In parallel, electron microscopy analysis allows direct visualization of replication fork intermediates. Cell survival assays are employed to test how loss of fork recovery factors impacts cell proliferation and chemotherapeutic response in BRCA1-deficient cells. Our results reveal that RAD18 and UBC13, which catalyze ubiquitination of Proliferating Cellular Nuclear Antigen (PCNA), promote fork recovery in BRCA1-deficient, but not BRCA2-deficient, cancer cells. Previous work has also shown that PCNA polyubiquitination by UBC13 is important for reversed fork formation in BRCA-proficient cells. However, our findings show that extensive degradation of reversed fork substrates still occurs in BRCA1-deficient cells lacking RAD18 or UBC13, indicating that PCNA polyubiquitination is not essential for fork reversal in this genetic background. In addition, loss of RAD18 in BRCA1-deficient cells significantly slows cell proliferation, and UBC13 inhibition further sensitizes cells lacking BRCA1 to the replication stress inducer Hydroxyurea (HU). Based on our findings, we hypothesize that RAD18, UBC13, and PCNA ubiquitination may represent novel targets to improve chemoresponse in BRCA1-deficient cancers that rely on fork recovery mechanisms for survival. Citation Format: Emily Cybulla, Jessica Jackson, Stephanie Tirman, Annabel Quinet, Delphine Lemacon, Alessandro Vindigni. Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 803.