bluuebunny/crossref_metadata_2025 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 < 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.
Exposure of hiPSC-Derived Mature Megakaryocytes to Vincristine Boosts Platelet Biogenesis in Vitro
Authors: Harada Yasuo , Nakamura Emiri , Bingham Trevor , Zon Leonard I. , Daley George Q. , Frelinger Andrew , Eto Koji , Schlaeger Thorsten M.
Published: December 2023

Millions of healthy donor platelet (PLT) units are needed each year for therapeutic transfusions of patients with thrombocytopenia and bleeding disorders, but shortages are common. The pandemic worsened donor shortages in the US and Japan. To ensure the predictability of supply, and to mitigate clinical complications associated with conventional donor PLTs, we have developed human induced pluripotent stem cell (hiPSC)-derived megakaryocyte (MK) cell lines, imMKCLs, as a source of in vitro PLT production (iPSC-PLTs)(Nakamura et al., Cell Stem Cell, 2014; Sone et al., Stem Cell Rep, 2021), and manufactured platelets are now in clinical trials using a previously published protocol (Sugimoto et al., Blood, 2022; Sugimoto et al., Blood Adv, 2022). To optimize and improve the in vitro manufacturing protocol of iPSC-PLTs from imMKCLs, we conducted a comprehensive imaging-based compound screening which identified microtubule (MT) destabilizing agents, including the vinca alkaloid vincristine (VCR), that appear to boost proplatelet formation. Combined use of VCR at 10 μM and turbulent hydrodynamic culture conditions (Ito et al., Cell, 2018) allowed production of 23.0 PLTs per imMKCL, 2.5 fold higher than without VCR. This successful result was observed only when VCR was administered at day 3 of 6-days of the maturation phase, when imMKCLs have become polyploid, while exposure at earlier timepoints induced apoptosis. However, PLTs produced in this manner showed poor agonist-induced surface expression of P-selectin or the PAC1 epitope. Therefore, we next examined dose-dependent action of VCR on both yield of iPSC-PLTs and their function. At nanomolar concentrations (10 nM), VCR still exhibited significant PLT biogenesis-boosting activity, but without apparent impairment of PLT in vitro functionality. Interestingly, this low concentration of VCR still resulted in reduced marginal MT band staining in imMKCLs, suggesting a potential role of weakened membrane structure/status in enhanced iPSC-PLT generation. Currently, we are attempting to address if PLTs made from low-dose VCR-exposed imMKCLs have reduced marginal MT bands and whether they restore hemostasis when transfused into immunodeficient thrombocytopenic mice. Our study unveils the potential of VCR as a potent enhancer of PLT biogenesis in the imMKCL system, particularly when combined with optimized turbulent flow production systems. Our results provide a means to make production of iPSC-PLTs more cost-efficient and suggest a mechanism for the previously unexplained observation that vinca alkaloids raise PLT counts in vivo.
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.
Discovery of BT-114143, a Novel and Potent Small-Molecule Plasminogen Inhibitor for Hyperfibrinolysis
Authors: Tang Jun , Ji Sen , Hu Xiao , Wang Yan , Qi Jun , Wang Xiao , Wang Hao , Li Jianzong , Li Zhiqiang , Li Min , Zeng Shaomei , Yuan Xiong , Zhang Xiaodong
Published: December 2023

Blood coagulation and fibrinolysis are essential physiological functions, whose dysregulation is a life-threatening emergent situation requiring immediate medical intervention. In particular, hyperfibrinolysis-triggered bleeding occurs in certain medical conditions such as postpartum hemorrhage (PPH) or during perioperative procedures, natural disasters, traumatic accidents as well as battlefield situations, and is one of the major causes of mortalities worldwide during peacetime and war. Tranexamic acid (TXA) has been widely used as an antifibrinolytic drug since 1960s, however, the low potency of TXA has limited its clinical applications. Here, we have developed BT-114143, a novel and potent small-molecule plasminogen inhibitor. BT-114143 exhibited over four-fold higher affinity for recombinant human plasminogen than TXA in surface plasmon resonance (SPR) analysis. Similarly, affinity of BT-114143 was at least one magnitude higher than that of either epsilon-aminocaproic acid (EACA) or p-aminomethylbenzoic acid (PAMBA) (Table 1). In vivo efficacy studies were conducted in recombinant tissue plasminogen activator (rt-PA)-triggered rat tail bleeding model and rabbit liver bleeding model by hepatectomy. BT-114143 reduced bleeding time dose-dependently in both animal models, but also exhibited similar potency as compared to TXA. Similar potency between BT-114143 and TXA was further confirmed by rt-PA triggered fibrinolysis in either rat or rabbit whole blood in thromboelastography (TEG) assay, suggesting interspecies difference in response to bleeding. However, ex vivo studies on human plasma clot lysis assay and human whole blood TEG assay (Figure 1) demonstrated nearly ten-fold antifibrinolytic potency as compared to TXA, suggesting superior efficacy for treatment of hyperfibrinolysis-related bleedings. In addition, a panel of GLP toxicology studies has confirmed the preclinical safety of BT-114143. Moreover, electrophysiological analysis has demonstrated comparable inhibitory effect of BT-114143 and TXA on GABA A receptor and Glycine receptor. At present, BT-114143 is undergoing phase I clinical study in China (CTR20222910) and is expected to apply for FDA approval for clinical trial in 2024. Furthermore, BT-114143 is being developed for intramuscular (i.m.) injection, making it more suitable for emergency situations including PPH, traumatic injuries, natural disasters, and combat injuries.
SPI1-KLF1/LYL1 Axis Regulates Lineage Commitment during Human Endothelial-to-Hematopoietic Transition
Authors: Qu Kengyuan , Yen Kuangyu , Mo Shaokang , Huang Junfeng , Liu Shan , Shen Jun
Published: December 2023

PU.1 ( SPI1) is a crucial transcription factor in hematopoiesis, yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Given that the complexity of in vivo hematopoiesis can mask subtle cellular transitions, we sought to investigate the function of SPI1 in both complex in vivo environments and more controlled in vitro models of EHT to elucidate its precise role. By comparing the human in vivo and in vitro EHT transcriptomes, we observed similarities in the transcriptional profiles of SPI1, suggesting its regulatory role in EHT. Knocking down SPI1 in in vitro-generated hemogenic endothelial cells (HECs) led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis, we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells, respectively - as downstream targets ofSPI1. Concomitant overexpression of KLF1 or LYL1 with SPI1 knockdown in in vitro-generated HECs partially rescues the reduction in HPC formation induced by SPI1 knockdown. Specifically, KLF1 overexpression restores myeloid lineage potential, while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1- LYL1 axis in the regulatory network in in vivo EHT. Taken together, our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT, potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs).
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.
Characterization of Hemophagocytic Lymphohistio(HLH）in Relapsed Diffuse Large B-Cell Lymphoma Patients after CAR-T Therapy
Authors: Zhou Lili , Ye Shiguang , Dang Xiuyong , Li Shaoguang , Li Ping
Published: December 2023

Background:

Patients with chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL) have poor prognoses. The objective response rate of patients with standard salvage regimens is 26%, while CART therapy have complete remission (CR) rates of 40%- 54% against DLBCL. Half of DLBCL patients relapsed or progressed after chimeric CAR-T cell therapy, but only a few of them were accompanied by HLH. Its clinical characteristics are unclear. This study aimed to investigate the clinical characteristics of these patients.

Methods:

We retrospectively analyzed the clinical data of 8 patients with relapsed DLBCL lymphoma accompany by HLH after CAR-T therapy between May 2019 and July 2023(NCT02537977 and NCT04036019). We investigate the clinical characteristics of these patients, compare the survival between patients with and without CR after CAR-T therapy, compare the survival between patients with and without CRS after CAR-T therapy.

Results:

8 R/R DLBCL patients were enrolled. Median age was 59.5 years (range, 37-69), and 6(75%) patients had non-germinal center B-cell-like (non-GCB) DLBCL, one had GCB DLBCL, one had transformed DLBCL. 2 patients (25.0%) had HLH before CAR-T treatment previously. Among them, 3 patients (37.5%) achieved CR after CART therapy, while 3 patients (37.5%) achieved partial remission. ALL of the patients (100%) relapsed or progressed, and all of them died because of disease progression. With a median follow-up of 7 months, the median overall survival (OS) was 206.0 days (95%CI 106.2-305.8) and progress-free survival (PFS) was 125.0 days (95%CI 40.5-210.0). Particularly，the median time from HLH to death was only 45.0 days (95%CI: 22.8-67.2). 6 patients (75.0%) occurred grade 1-2 CRS, no patient occurred ≥grade 3 CRS, and 2 patients (25.0%) did not occurred any CRS. The median OS was 328.0 days and 142.0 days (95%CI 60.4-223.6) for patients without CRS and with CRS1-2, respectively. There is significantly different in OS for patients with and without CRS ( P=0.03). The median OS was 328.0 days (95%CI 217.6-438.4) and 142.0 days (95%CI 133.4-150.6) for patients with CR and without CR, respectively. The median PFS was 257.0 days (95%CI 103.4-410.6) and 77.0 days (95%CI 8.3-145.7) for patients with CR and without CR. There is significantly different in OS and PFS for patients with CR and other patients ( P=0.01 and P=0.01).

Conclusion:

Hemophagocytic lymphohistiocytosis (HLH) is a rare and life threatening disorder. The prognosis of patients with relapsed DLBCL lymphoma accompany by HLH after CAR-T therapy is very poor. Patients with CRS during CART therapy may more likely have HLH when DLBCL relapse. The patients without CRS have better survival time than those with CRS during CART therapy. We hypothesis that because both HLH and CRS is inflammatory syndrome resulting from T-cell activation.
Venetoclax Combined with Azacitidine and Homoharringtonine in Adults with Secondary Acute Myeloid Leukemia
Authors: Huang Fei , Chen Yu , Shi Zhongxun , Huang Huijun , Li Jianyong , Shen Wenyi
Published: December 2023

Secondary acute myeloid leukemia is an aggressive subset of acute myeloid leukemia (AML) that is, in general, is associated with a very poor prognosis and high mortality. Venetoclax combined with hypomethylating agents have showed relatively successful therapeutic effective in patients with relapse/refractory AML, while efficacy is limited in high-risk subset AML. We hypothesised that Venetoclax combined with Azacitidine and Homoharringtonine could have improved activity in patients with secondary acute myeloid leukemia, particularly in high-risk subgroups.

Methods

This single center, phase 2 trial was done at the first affiliated hospital of Nanjing medical university. The study enrolled adult patients with diagnosed of secondary AML (AML progressed after preexisting myeloid neoplasms or treatment AML arising from prior exposure of leukemogenic therapies). Patients were required to have an Easter Cooperative Oncology Group performance status of 3 or less, and adequate end-organ function. Patients who had received previous BLC2 inhibitor therapy were excluded. Patients received Azacytidine 75mg/m 2 and Homoharringtonine 3mg for 7 days with venetoclax 400mg daily. The primary endpoint was overall response rate. The secondary endpoints include safety, overall survival, and duration of response (according to recommendations of European LeukemiaNet 2017 guidelines). The trial was registered on ClinicalTrial.gov (NCT 05513131) and continues to accrue patients.

Results

Since now, we enrolled 11patients; 8(72.7%) patients had secondary AML, 3 (27.3%) patients had treatment related AML, while 2(18.2%) patients had TP53 mutation. The median age was 62 years (48-78 years). At the July 30, 2023 data cutoff, median follow-up time was 36.7 weeks (range 6.57-82.29 weeks). The overall response rate was 81.8% (9 of 11 patients). The most common treatment-emergent adverse events included bone marrow suppression with grades 3 or 4 neutropenia (n=11/11) and febrile neutropenia (n=10/11). The 30-day mortality for all patients was 9.1% (n=1). 2(18.2%) patients were eligible for allogeneic hematopoietic stem cell transplantation. The median overall survival was not achieved.

Conclusion

Venetoclax combined with Azacitidine and Homoharringtonine regimen had a tolerable safety profile and showed encouraging clinical activity characterised by a high response rate. further patients are needed to validate these findings.
Eosinophils to Basophils Rate, Mean Platelet Volume, and Neutrophils to Lymphocyte Rate for the Diagnosis of Systemic Amyloidosis
Authors: Quoc Trung Ly , Nguyen Viet Hoang , Takami Akiyoshi , Espinoza Luis
Published: December 2023

Methods: CBC, performed by an automated hematology analyzer, included hemoglobin

hematocrit, erythrocytes, MCV, MCH, MCHC, platelets, and white blood cell counts and

differentials (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), as well as red

blood cell distribution width (RDW) and mean platelet volume (MPV). Other indicators were

indirectly calculated (by dividing the numerator by the denominator) based on the values of CBC

and included neutrophils to lymphocyte rate (NLR), monocytes to lymphocyte rate (MLR)

platelets to leukocyte rate (PLR), leukocytes to eosinophil rate (LER), and leukocytes to basophil

rate (LBR). Among them, NLR, MLR, and PLR have been linked to various inflammatory

conditions, but their role in SA diagnosis is unknown. CBC values obtained at diagnosis from a

set of 128 Vietnamese patients with SA (mean age 43, 67% female) treated at Hanoi Medical

University Hospital were compared with those derived from 596 age-matched healthy controls.

Results: Among the values directly obtained from CBC, only neutrophil counts and MPV were

statistically significant between the two groups, with higher neutrophil counts (4,200 vs. 3,700, p

= 0.02) and lower MPV (7.41 vs. 8.35, p = 0.0002) observed in SA patients compared with

controls. The analysis of other indicators showed significantly higher NLR (2.3 vs. 1.8, p = 0.04)

and EBR (4.42 vs. 5.64; p = 0.03) in SA patients compared with controls. No differences in MLR

and PLR were observed between SA patients and controls. In multivariate analysis, a higher

NLR (p = 0.05), lower MPV (p = 0.001), and higher EBR (p = 0.02) remained associated with

the diagnosis of SA.

Conclusions: This study substantiates the relevance of a decreased MPV as well as an increased

NLR and EBR in patients with SA. CBC is a rapid and low-cost test, and NLR and EBR are

easily calculated; thus, these indexes could help clinicians in the diagnosis of SA.
Predicting Molecular Alterations from Blast Morphology of Acute Myeloid Leukemia Bone Marrow Aspirate Smears Using Machine Learning
Authors: Vadasz Brian , Gao Juehua , Lewis Joshua E , Jaye David L , Cooper Lee A.D.
Published: December 2023

Introduction

Acute Myeloid Leukemia (AML) has enormous molecular and cytogenetic heterogeneity. Evaluation of bone marrow morphology is essential in the diagnosis of AML, however, it requires significant training and there is wide inter-observer variability. Genomic testing has been progressively relied on for AML classification, ultimately helping to dictate prognosis and guide clinical management. However, genomic testing, if performed, can be subject to prolonged turnaround times and lacks clinical penetration in low-resource settings. To overcome these challenges, we aimed to evaluate the feasibility of predicting clinically relevant genomic alterations from bone marrow cell morphology using a machine learning approach.

Methods

We scanned 799 Wright-stained bone marrow aspirate smear slides, made for routine patient care, for subjects diagnosed with AML. These subjects were linked to clinical data, including pertinent cytogenetic/molecular alterations and subclassified as cases with mutated Nucleophosmin 1 ( NPM1), those with TP53 mutations and those with myelodysplastic-related changes (AML-MRC). Whole slide images were screened for containing spicules and blasts, and were analyzed to detect 180 million cells (blasts and non-blasts) excluding hemodilute and particle regions. We developed a multi-instance model to predict patient molecular subtype by repeatedly sampling 512 cells from each slide and using these to train a classifier that weights cells with morphology relevant to subtype. We also trained a model to predict NPM1 and TP53 mutations independently, as well as other frequent variants reported to impact blast morphology.

Results

Performance in predicting NPM1/TP53/AML-MRC subtype was moderate (0.80 AUROC). Performance of the single-gene model varied, with NPM1 having the highest performance (0.85 AUROC) followed by IDH (0.80 AUROC), FLT3 (0.79 AUROC) and TP53 (0.62 AUROC).

Conclusions

This study provides promising preliminary results for prediction of genetic alterations in AML in a large-scale single-institution dataset. Interestingly, this method did not rely on the classification of individual cells, avoiding the need for manually collecting annotations to train a blast cell classifier. Future work will evaluate the morphology of cells attended by the multi-instance model, and explore the value of enriching sampled cells with blasts using cell classification methods.
TAF1, the Largest Subunit of Tfiid, Is Dispensable for Adult Hematopoiesis
Authors: Liu Fan , Yue Jingyin , Tamiro Francesco , Sun Jun , Cingaram Pradeepkumar Reddy , Hossack Krystal Lisa , Caja Concepcion Martinza , Beckedorff Felipe , Shiekhattar Ramin , Nimer Stephen D.
Published: December 2023

Temporal and spatial control of gene expression is central to normal hematopoietic stem cell (HSC) biology. Many sequence-specific transcription factors have been identified as key regulators of HSC fate decision, while the function of general transcription factors in HSC behavior is poorly understood. We previously reported that TAF1, the largest subunit of the TFIID complex, plays a critical role in AML1-ETO driven acute myeloid leukemogenesis. To evaluate the function of TAF1 in normal fetal and adult hematopoiesis, we generated TAF1 conditional knockout (CKO) mice and identified an essential role of TAF1 in fetal erythropoiesis. Surprisingly, deletion of TAF1 in adult mice was not lethal to hematopoiesis; rather, we observed a marked expansion of hematopoietic stem and progenitor cell (HSPC) populations, with increased self-renewal and impaired differentiation capacity in these TAF1- null cells. Consistently, we found that TAF1-null HSPCs failed to up-regulate key differentiation-associated genes when induced to differentiate in vitro. Using a variety of genome-wide assays and biochemical approaches, we found that TAF1 loss not only disrupted TFIID complex formation and chromatin recruitment, but also reduced promoter accessibility, impaired RNA Polymerase II promoter recruitment and activity, as well as its promoter-proximal pausing mechanism. Thus, the effect of TAF1 loss on normal adult hematopoiesis primarily relates to the inability to upregulate genes involved in differentiation, while HSC self-renewal and the expression of self-renewal genes proceeds relatively normally.
Analysis of Safety and Efficacy of Avapritinib As Targeted Therapy for Pediatric Acute Myeloid Leukemia Patients with KIT Mutation after Transplantation
Authors: Wang Qingwei , Gao Li , Xiao Peifang , Lu Jun , Li Jie , Yao Yanhua , Hu Shaoyan
Published: December 2023

Objective: To investigate the safety and efficacy of Avapritinib in pediatric acute myeloid leukemia (AML) with KIT mutation after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Methods: The clinical data of 6 AML patients with KITmutation and RUNX1::RUNX1T1 fusion gene positive in Children's Hospital of Soochow University were retrospectively analyzed, including 2 cases of KIT:N822K, 3 cases of KIT: exon17 and 1 case of KIT: Asp816Tyr. After achieving complete remission in morphology, allo-HSCT was performed. The clinical outcomes of prophylactic therapy (2/6) and preemptive intervention (4/6) with Avapritinib after complete stem cell engraftment were observed, and the adverse events during treatment were recorded.

Results: Adverse events occurred in 4 of the 6 patients, among whom neutropenia was the most common (4/6), followed by facial edema (2/6) and thrombocytopenia (1/6). 2 patients who received prophylactic treatment achieved morphological complete remission, and minimal residual disease (MRD), fusion gene and KIT mutations were all negative. 4 patients received preemptive intervention because of RUNX1::RUNX1T1 fusion gene turned positive after allo-HSCT, and 1 patient's RUNX1::RUNX1T1 turned negative after single-agent Avapritinib treatment for 2 months. 2 patients did not achieve continuous RUNX1::RUNX1T1 negative after preemptive therapy with Decitabine (DAC) and donor lymph infusion (DLI), and then was treated with Avapritinib, one's RUNX1::RUNX1T1 fusion achieved continuously negative after 1 month treatment of Avapritinib, the other's achieved continuous negative after 7 months treatment of Avapritinib. One patient was treated with Avapritinib after DAC and DLI as preemtive intervention, and RUNX1::RUNX1T1 fusion did not turn negative, but the relative value of RUNX1::RUNX1T1 fusion was lower than before and lived stable for more than 10 months.

Conclusions: Avapritinib is safe and effective in the prophylactic and preemptive treatment of AML with KIT mutation after allo-HSCT in children, which provides a clinical drug for the prevention of relapse after transplantation.
Unrevealing Hematopoietic Reconstruction Discrepancies after Allo-UCBT and Allo-PBSCT at Single Cell Resolution
Authors: Li Qing , Wu Linjie , Liu Fang , Sun Huimin , Huo Yingying , Ma Shihui , Dong Fang , Zhu Xiaofan , Hao Sha , Cheng Tao
Published: December 2023

Umbilical cord blood (UCB) and G-CSF mobilizing peripheral blood stem cell (G-PBSC) are the main sources of hematopoietic stem cells for transplantation on clinic. Clinical data have shown that hematopoietic reconstruction in allogeneic cord blood transplantation (allo-UCBT) is significantly delayed compared with allogeneic peripheral blood stem cell transplantation (allo-PBSCT), suggesting that the cellular composition and reconstruction dynamics of UCB are significantly different from that of G-PBSC. Following on the heels of our recently study that found HSC/MPPs show differentiation bias to megakaryocytic-erythroid lineage and strong proliferation tendency, and defined a neutrophil progenitor population named S100A hi Neu2 which is inversely associated with the incidence of GVHD after allo-PBSCT (Yingying Huo et al, Science Immunology 2023), we next aimed to analyze the differences of hematopoietic reconstitution between allo-UCBT and allo-PBSCT and the causes of delayed hematopoietic reconstitution after allo-UCBT.

In this study, we collected peripheral blood (PB) and bone marrow (BM) samples from 8 patients with bone marrow failure syndrome (BMFs) who received allo-UCBT (n=4) or allo-PBSCT (n=4) at different time points (d14, d21, d30, d60, d90, d180, d365) after transplantation. At the same time, UCB, G-PBSC and BM of normal children were collected as controls. Lin -CD34 + cells from BM, G-PBSC and UCB were separated by FACS for STRT-seq, the total nucleated cells (TNCs) of all samples were isolated for 10X transcriptome sequencing. We used 276628 hematopoietic cells to map the single cell transcriptome profiles of hematopoietic reconstruction by different transplantation methods and annotated 44 cell types. Compared with the G-PBSC, the immunogenicity of HSC/MPPs in Lin -CD34 + cells derived from UCB decreased, the proportion increased, and the differentiation bias of HSC/MPPs to monocytic and erythroid lineage was stronger, but the differentiation ability to myeloid and megakaryocytic lineage was decreased, the proportion of EBM, MEP and CMP decreased. For TNCs, the proportion of T cells and NK cells increased, and NK_CD56dim_CD16 had a stronger immune function. Compared with allo-PBSCT, the differentiation of HSC/MPPs and CMP into neutrophils in the early stage after allo-UCBT was weakened. The proportion of MEP cells decreased, and the differentiation ability of HSC/MPPs to MEP, HSC/MPPs and MEP to Mk was weakened.

In summary, we found that the delayed reconstruction of neutrophils and platelets in patients with allo-UCBT are related to the nature of the graft, suggesting the importance of reasonable intervention of HSC derived from UCB to accelerate platelet implantation.