bluuebunny/crossref_metadata_2025_split huggingface dataset:
Embryonic Ectoderm Development (EED) Inhibitor APG5918 Improves Chronic Kidney Disease- (CKD)-Induced Hemoglobin Insufficiency in Preclinical Models of Anemia
Authors: Zhai Yifan , Yin Yan , Liang Eric , Wang Hengbang , Min Ping , Yang Dajun 
Published: December 2023

Introduction
Anemia can be caused by infections, malignancies, CKD, or chemotherapy. Increasing Hb or fetal hemoglobin (HbF) can prevent or reduce anemia-related pathophysiology. Polycomb inhibitory complex 2 (PRC2), consisting of EZH2, EED, and SUZ12, is a subunit of the major transcriptional corepressor complex of BCL11A, and targeting EED may be one novel approach for treatment of patients with anemia. We report the effects of APG-5918, a small-molecule selective EED inhibitor, on Hb and HbF induction in human CD34⁺ hematopoietic stem cells (HSCs) and the efficacy of APG-5918 in a CKD-induced anemia model in vivo.
Methods
The pharmacodynamic (PD) effects of APG-5918 were evaluated in human cord blood CD34⁺ HSCs by flow cytometry, RT-qPCR, and ELISA. In vivo, the antianemia effect of APG-5918 was evaluated in adenine-induced renal anemic Sprague-Dawley rats.
Results
APG-5918 inhibited H3K27me3 inCD34⁺ HSCs, the critical epigenetic mark catalyzed by PRC2. APG-5918 also increased γ-globin coding gene ( HBG) and β-globin coding gene ( HBB) mRNA levels in a dose-dependent manner. After 7 days of treatment with APG-5918 at 1 μM, HBG and HBB mRNA increased to 12.36-fold and 4.76-fold of the control group, respectively, which was pharmacologically superior to effects observed with hydroxyurea at 33 µM (~6.53-fold and 2.27-fold increases in HBG and HBB mRNA, respectively). Hb and HbF protein were also upregulated in a dose-dependent manner. A total of 1.69-fold and 1.9-fold increases in HbF and Hb protein expression were observed after 7 days of treatment with APG-5918 at 1 μM.HbF and Hb protein showed no significant difference after hydroxyurea treatment. In an adenine-induced renal anemic rat model, APG-5918 was administered orally once daily at 3 dose levels (5, 15, and 45 mg/kg). Both body weight and the blood parameters erythrocytes, Hb, hematocrit (HCT), and reticulocytes (RET) improved significantly in a dose-dependent manner compared to the model (adenine) group ( p &lt; 0.05, 0.01, or 0.001) (Figure 1). APG-5918 showed similar in vivo efficacy with erythropoietin (EPO): body weight and blood parameters in the group treated with APG-5918 45 mg/kg were comparable to those in the EPO group. In addition, blood parameters in the APG-5918 (15 mg/kg) plus EPO group recovered to the level of the control group, which was superior to the APG-5918 and EPO monotherapy groups.
Conclusions
APG-5918 demonstrated its effects as a single agent on inhibition of H3K27me3 inCD34⁺ HSCs, HbF and Hb induction, and normalization of hematologic parameters in a CKD-induced anemia model. APG-5918 also showed synergistic antianemia effects when combined with EPO. These findings support APG-5918 as a novel treatment option for CKD-induced anemia.
Multiple Myeloma in the Culturally and Linguistically Diverse Southwestern Sydney, Australia
Authors: Marivel A-M Joëlle , Roberts Tara L , Descallar Joseph , Aung Win Pa Pa , Cheng Jonathan , Becker Therese M , James Alexander , Ma Yafeng , Bryant Adam , Ling Silvia 
Published: December 2023

Multiple myeloma (MM) is an incurable haematological cancer with a median overall survival of 5 to 10 years. Multiple American studies have reported significant disparities in the incidence and clinical outcomes in MM patients that may be explained by race or ethnicity.
This study sought to characterise the racial/ethnic representation in myeloma patients who presented to the public hospitals in Southwestern Sydney between 2009 and 2020. This retrospective study describes the associations between race/ethnicity and age at diagnosis, cytogenetics, comorbidities, and survival outcomes.
Data for analysis was extracted from Mosaiq (electronic medical record system for oncology workflow management) and PathNet® (laboratory information system). Since neither race nor ethnicity are recorded in the NSW Healthcare system, race was defined based on the country of birth while ethnicity was defined as culturally and linguistically diverse (CALD), based on country of birth and language as well as use of an interpreter.
Seven regions of birth across 65 countries of birth and five CALD groups were identified in our cohort, with 26% of the cohort speaking a language other than English. Survival outcomes were similar regardless of race or CALD classification. Analysis of the cytogenetics and comorbidities are still in progress.
This is the first reported racial study in MM in NSW. The preliminary results presented here demonstrates that automatically classifying patients as Caucasian, solely based on country of birth can be misleading. In a multicultural region like Southwestern Sydney, it is important to consider patients' cultural and linguistic background.
A Single Center Experience for Clinical Evaluation of Paroxysmal Cold Hemoglobinuria and Donath-Landsteiner Testing
Authors: Villar-Prados Alejandro , Chien May , Yunce Muharrem , Abdelmonem Mohamed , Duda Molly 
Published: December 2023

Introduction: Paroxysmal cold hemoglobinuria (PCH) is a rare form of autoimmune hemolytic anemia (AIHA), mainly affecting children. The diagnosis and management is challenging due to similarities to other causes for AIHA and limited availability to Donath-Landsteiner (DL) testing.
Methods: In this single-center retrospective study, we aimed to characterize the clinical presentation and outcomes of PCH patients, defined as having positive Donath-Landsteiner antibodies, compared to a cohort of AIHA patients.
Results: DL-positive patients were observed to have higher lactose dehydrogenase levels and lower reticulocyte counts compared to DL-negative patients, although this was not statistically significant. We also observed that using steroids in DL-positive patients did not significantly impact their recovery.
Conclusions: Our findings support the limited published data on PCH patients and further prompt prompting larger multicenter studies to further characterize these patients so that they are more readily identified, especially in centers where DL antibody testing is not readily available.
Sars-CoV2 ORF7a Protein Stimulates Synthesis and Secretion of Endothelial Von Willebrand Factor
Authors: Zhang Quan , Huang Xiaohu , Liu Szumam , Yada Noritaka , Han Zhe , Zheng X. Long 
Published: December 2023

Introduction: Patients with SARS-CoV-2 show significantly elevated plasma levels of von Willebrand factor (VWF) and increased thrombosis. However, the molecular mechanism underlying increased plasma levels of VWF in COVID-19 patients is not fully understood.
Methods. Drosophila genetic screen, recombinant expression, endothelial culture, confocal microscopy, flow cytometry, and a microfluidic assay were employed for the study.
Results. Drosophila genetic screen for SARS-CoV-2 proteins allowed us to identify Orf7a to be the key factor that induces Hml (drosophila homolog of human VWF)-dependent hypercoagulability. Recombinant Orf7a tagged with mCherry (mCh) and FLAG was expressed transiently and stably in a murine endothelial cell line SVEC4-10 and a HEK293 cell line. The ORF7a protein is localized predominantly to the Golgi and trans-Golgi network (TGN) in HEK293 cells ( Fig. 1A) but shows its expression in perinuclear area and cell surface in SVEC4-10 cells ( Fig. 1B). Significantly higher numbers of VWF-strings were formed in the ORF7a transfected SVEC4-10 cell line under static state than in the non-transfected control cells. These cell surface or extracellular VWF strings were readily removed by a human recombinant ADAMTS13 ( Fig. 1C & 1D). Flow cytometry analysis revealed that significant higher levels of intracellular VWF signal in the cells following transient expression of Orf7a than in those non-expressed controls ( Fig. 2A & 2B). Microfluidic shear-based assay and confocal imaging analysis further demonstrated that dramatically increased rate and surface coverage of fluorescent labeled murine platelets over a histone-stimulated SVEC surface expressing Orf7 comparing with over the same cells without expression of Orf7, following perfusion of Adamts13 -/- murine whole blood (anticoagulated with PPACK) under arterial shear ( Fig. 2C & 2D). Using proteomics, we identified conserved host proteins ARF4, GBF1 and ERGIC-53 (a COPII pathway component) as direct interacting factors for Orf7a and demonstrated that these proteins have conserved roles in VWF secretion.
Conclusions:Our results demonstrate that ORF7a may upregulate VWF synthesis and secretion in endothelial cells, which enhances thrombus formation. Recombinant ADAMTS13 may rapidly remove endothelial surface ultra large VWF strings and exhibit therapeutic potential for COVID-19 associated thrombosis.
Preclinical Efficacy and Mechanism for a Novel BTK-Protacs Against Mantle Cell Lymphoma
Authors: Fei Yue , Wang Xiaohui , Yu Shangzhe , Yao Yixin , Zhao Guisen , Wang Xianhuo , Zhang Huilai 
Published: December 2023

Background: Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL) that exhibits diverse clinical courses and biological contexts. Given the significance of Bruton tyrosine kinase (BTK) as a therapeutic target in MCL, this study aimed to design a range of BTK-PROTACs. The objective was to develop efficient BTK-PROTACs capable of degrading BTK proteins, inhibiting the proliferation of MCL cells, and overcoming resistance to both covalent and non-covalent BTK inhibitors in both in vivo and in vitro settings.
Methods:A series of BTK-PROTACs were gradually screened by Western blot and MTS assays, and the best active drug was finally determined. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. RNA sequencing identified differentially expressed genes and important signaling pathways that influenced by BTK-PROTACs. BTKi-resistant cell lines were constructed to evaluate the ability of BTK-PROTACs to degrade BTK protein and suppress the proliferation of MCL cells.
R esults: BTK-PROTACs C23 demonstrated effective degradation of BTK protein and inhibition of tumor cell growth across various MCL cell lines. Additionally, C23 exhibited the ability to induce cell apoptosis, activate the ubiquitin pathway, and inhibit the B-cell receptor (BCR), NF-κB, and PI3K-AKT-mTOR signaling pathways. Importantly, C23 effectively degraded BTK in both Z138-BTK C481S and Z138-BTK L528W cells, suggesting its potential to overcome resistance to both covalent and non-covalent BTK inhibitors. In our in vivo experiments using MCL xenograft NSG mouse models, BTK-PROTACs C23 significantly impeded tumor proliferation in Z138-BTK WT, Z138-BTK C481S, and Z138-BTK L528W MCL models. Additionally, C23 induced the degradation of BTK in tumor tissues. Notably, HE staining revealed no significant adverse effects of BTK-PROTACs C23 on normal organs and tissues. Moreover, routine blood tests and liver function tests showed no impairment of hematopoietic or hepatic function caused by BTK-PROTACs C23.
C onclusion: In this study, we successfully designed and synthesized BTK-PROTACs C23, which demonstrated a remarkable ability to degrade BTK protein and effectively suppress the proliferation of MCL cells both in vitro and in vivo. Importantly, BTK-PROTACs C23 showed promising potential to overcome resistance to both covalent and non-covalent BTK inhibitors.
U2AF1 S34F Mutation Promote Megakaryopoiesis and Fibrogenesis in MDS
Authors: Zhang Wenjun , Liu Jinqin , Yang Lin , Yan Yiru , Qin Tiejun , Xu Zefeng , Huang Gang , Li Bing , Xiao Zhijian 
Published: December 2023

Aims: Exploring the clinical and pathological characteristics of MDS patients with U2AF1 mutation and investigating the impact of mutation on hematopoietic stem and progenitor cells.
Methods: Nine hundred and eighty-nine consecutive subjects with MDS who underwent targeted next generation sequencing (NGS) from August 2016 to September 2022 were included in this study. Clinical and pathological characteristics were collected and analyzed. U2AF1 S34F retroviral murine model was used to evaluate changes of hematopoiesis in the bone marrow and spleen.
Results: Among the 989 MDS patients, U2AF1 mutation occurs in 23% of them. Within the subgroup of patients diagnosed with MDS with fibrosis (MDS-f) according to the updated WHO 2022 criteria, the frequency of U2AF1 mutation rose to 42%. Patients with U2AF1 mutation exhibited lower levels of hemoglobin and higher degrees of fibrosis, along with a significant increase in megakaryocyte counts in bone marrow biopsy and smear ( Figure A).
Our U2AF1 S34F retroviral murine model also had MDS-like phenotype with pancytopenia in the peripheral blood and the significantly increased proportion of LSK, ST-HSCs and MPPs in bone marrow and spleen. A notable finding was the significant increase in the proportion of megakaryocyte progenitor cells (MkP). HE staining and CD41 immunohistochemistry staining demonstrated a pronounced elevation and clustering of megakaryocytes both in bone marrow and spleen ( Figure B).
Conclusion: Our study suggested that U2AF1 S34F mutation enhancing megakaryocytic differentiation in vivo. This finding may be associated with specific clinically and pathologically features in patients, including a significant increase of megakaryocytes and higher fibrosis grades, it could be a promising therapeutic target in fibrosis.
Bing Li and Zhijian Xiao are Co-corresponding authors.
Long-Term Eltrombopag in Children with Chronic Immune Thrombocytopenia: A Single-Center Extended Real-Life Observational Study in China
Authors: Wang Zhifa , Wang Nan , Ouyang Juntao , Ma Jingyao , Poon Man-Chiu , Chen Zhenping , Cheng Xiaoling , Wu Runhui 
Published: December 2023

Background: We have previously confirmed the efficacy and safety of eltrombopag (ELT) in children with chronic immune thrombocytopenia (cITP). However, there is still a lack of results on long-term ELT therapy. Here, we report the long-term follow-up results ( between September 2018 and June 2023 ) on our previous study.
Methods: This is a single-center real-life observational study that collected data from patients who previously enrolled in our children cITP study and had a follow-up period of more than 3 years by June 2023.
Results:
A total of 65 patients (28 males) were enrolled with a median age at ELT initiation of 6.34 (range 1.65, 14.13) years and a 47.07 (36.00, 57.00) months follow-up time with a 40.36 (10.53, 56.83) months of ELT therapy at the analysis time point.
In total, 29.23% (19/65) (of) patients discontinued ELT because of a stable response, and 18.46% (12/65) patients switched to another platelet-enhancing therapy due to loss of response to therapy after 19.13 (14.53, 26.37) months. Among the 19 patients who discontinued ELT because of a stable response, 24.62% (16/65) children achieved 12 m of off ELT treatment(SRoT), the last recorded platelet count ranged from 56-166×10 9/L (median value 107×10 9/L), and 4.62% (3/65) patients relapsed at the 5th, 6th, and 9th months after discontinuation, respectively. Of the 12 patients who lost response to ELT after 19.13 (14.53, 26.37) months of therapy, four switched to avatrombopag, three to hetrombopag, two to Chinse traditional medicine, one had splenectomy and two received additional prednisolone acetate. Thirty-four patients maintained a sustained response while continuing on ELT treatment but with tapering doses. No significant adverse events were reported. In the first 12 months of ELT, there was a higher rate of PLT ≥ 100×10 9/L in SRoT patients than in tapering patients (62.50% [10/16] vs. 5.88% [2/34]) and a higher rate of PLT 70-100×10 9/L (31.25% [5/16] vs. 27.78% [10/36]) ( P=0.01).
Conclusion:
We report more than 3 years long-term clinical data on cITP children taking ELT. We show that 24.62% of the children who had a stable response to ELT achieved 12 m of SRoT. The treatment response of children in the first 12 months after the initiation of treatment may be a predictive factor for successful discontinuation. These data highlight no new safety concerns regarding the long-term use of ELT in children with cITP.
Sphingomyelinase Regulates the Release of Protumoral Mitochondria Containing-Vesicles from AML Cells
Authors: Markham Matthew , Riley David , Maynard Rebecca S , Hampton Katherine , De Silva Ravindu , Hellmich Charlotte , Bowles Kristian , Rushworth Stuart 
Published: December 2023

Mitochondrial transfer from bone marrow stromal cells to acute myeloid leukaemia (AML) blasts is protumoral (Marlein et al., 2017; Moschoi et al., 2016) . We have previously shown that this was through a NADPH oxidase-2 (Nox2) dependent mechanism which increases AML fitness both in vitro and in vivo. Furthermore, we recently have shown that AML cells export mitochondria in vesicles which are subsequently phagocytosed by bone marrow macrophages (Moore et al., 2022) . Next, we have investigated how the release of mitochondria-containing vesicles is regulated and how this process contributes to the fitness of AML blasts.
Sphingolipids are a component of the cell membrane which impact the stability and curvature of the membrane. They have been shown to play a role in the biogenesis of vesicles and a number of the enzymes involved in sphingolipid metabolism are dysregulated in AML, therefore we investigated the role of sphingolipids in mitochondrial exocytosis via vesicle formation.
To understand the role of vesicles in regulating AML fitness, we used primary human AML (collected with approval on the East of England - Cambridgeshire and Hertfordshire Research Ethics Committee and the UK Health Research Authority, IRAS Project ID - 33753) and mouse syngeneic AML models (MN1 and HOXA9/Meis). We find that the myosin inhibitor blebbistatin inhibits vesicle release and results in an increased mitochondrial mass and increased apoptosis. In order to investigate the involvement of one of the major sphingolipids, ceramide, we targeted neutral sphingomyelinase (GW4869) which is responsible for ceramide synthesis. We treated the AML cells in vitro with GW4869 for 24 h which induced a large increase in vesicle release. Using the mitochondrial stain Mitotracker Green (MTG) we confirm that these vesicles contain mitochondria. The vesicles were isolated, DNA was extracted, and RT-qPCR performed which confirms increased mitochondrial DNA content. Others have previously shown that low levels of cellular reactive oxygen species (ROS) are associated with leukaemic stem cells (LSCs). DCF staining of AML treated with GW4869 shows low levels of cellular ROS. To assess how this process might impact the stemness of AML, we performed in vivo experiments. Pretreatment of MN1 cells with GW4869 accelerated engraftment in C57Bl/6 animals which corresponded with a decrease in cellular ROS.
In summary, we have demonstrated the mechanism of mitochondrial exocytosis is important for AML fitness and that by disrupting sphingolipid metabolism, dysregulation of this process leads to increased tumour burden.
Preclinical Assessment for Gene Addition Strategy in Familial Hemophagocytic Lymphohistiocytosis Related to Munc 13-4 Deficiency
Authors: Lagresle-Peyrou Chantal , Diana Jean-Sebastien , Magrin Elisa , Aussel Clotilde , Sorel Natael , Aslan Yetki , Roudaut Cécile , Schwartz Eden , Gabrion Aurélie , Tuchmann-Durand Caroline , Lefrere Francois , Galy Anne , Moshous Despina , Cavazzana Marina 
Published: December 2023

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHL) comprises a group of rare genetic disorders with defect cytotoxicity in T and NK cells predisposing to severe hyperinflammation with the clinical hallmark of the Hemophagocytic LymphoHistiocytosis (HLH) syndrome. The uncontrolled proliferation of activated T-CD8 lymphocytes and macrophages infiltrates lymphoid tissues, bone marrow, and multiple organs including the brain. Thus, if untreated full-scale FHL is usually fatal. FHL3 is due to mutations in the UNC13D gene coding for Munc 13-4 protein, one of the components of the perforin-dependent cytotoxicity apparatus. This condition accounts for 30% to 35 % of all genetic cases of HLH. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, remains difficult even when a compatible donor is available because of the patients' critical inflammatory background. In this context, gene therapy could be a promising therapeutic option, especially for those patients without HLA-compatible donor.
Method: The feasibility and efficacy of gene addition strategy for FHL3 have been previously demonstrated in mouse models. We then translated this approach to human cells using a third-generation self-inactivated lentivirus vector pseudo-typed with a conventional vesicular stomatitis virus-G (VSVG) expressing a codon-optimized UNC13D gene under the transcriptional control of EF1 alpha (LV-EF1a-UNC13D). HSPC or CD3+ T-cells were transduced with a GMP-like-grade LV-EF1a-UNC13D lentiviral vector. We studied the restoration of cytotoxic function.
Results: Our GMP transduction protocol led to efficient transcription of optimized mRNA codon and expression of Munc 13-4 protein as evidenced by RT qPCR and western blot, respectively. Interestingly, we show restored cytotoxicity activity after CD34+ differentiation toward CD3+ T-cells and NK cells. The vector copy number is compatible with a clinical approach. In vivo experiments are ongoing to validate the absence of genotoxicity in this setting.
Discussion conclusion: Our results support the feasibility and efficacy of UNCD13D gene transfer into hematopoietic stem and progenitor cells or T cells from Munc 13-4 deficient patients. Alternatively, gene therapy of HSPCs could be combined with transduced autologous T-cells infusion. The first attempts regarding this option are encouraging. UNC13D gene addition strategy could dramatically improve the care of FHL3 patients lacking an HLA-genoidentical donor.