bluuebunny/crossref_metadata_2025 huggingface dataset:
Fetal Maternal Hemorrhage As a Trigger for Fetal/Neonatal Alloimmune Thrombocytopenia: Evidence from a Murine Model
Authors: Zhi Huiying , Newman Debra K. , Newman Peter J.
Published: December 2023

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder caused by maternal alloantibodies targeting fetal platelet alloantigens. Among the 37 human platelet alloantigens (HPAs) identified, the HPA-1a/HPA-1b polymorphism, involving a Leu33Pro amino acid substitution in the integrin β3 subunit, is the most common cause of severe FNAIT, particularly in Caucasians. Leu33 serves as a key target for alloantibody binding, leading to fetal and neonatal thrombocytopenia and, in severe cases, spontaneous or trauma-induced intracranial hemorrhage. FNAIT shares a pathophysiologic basis with Hemolytic Disease of the Fetus and Newborn (HDFN), which stem from maternal immunization to alloantigens on fetal platelets and red blood cells (RBCs), respectively. While alloimmunization against RBC alloantigens typically occurs in response to fetal maternal hemorrhage (FMH), a clear link between FMH and maternal alloimmunization against HPA-1a on circulating fetal platelets has remained elusive. To assess whether FMH can induce platelet alloimmunization and lead to the development of FNAIT during pregnancy, we utilized recently developed transgenic mice expressing the HPA-1a allogeneic epitope on a murine GPIIIa backbone. To simulate varying degrees of FMH, we transfused different doses of HPA-1a platelets into wild-type (WT), non-pregnant female mice. We found that transfusing as few as 1x10 7 HPA-1a-positive platelets (equivalent to a FMH of approximately 30 ml in humans) resulted in production of high-titer HPA-1a-specific alloantibodies. Since >25% of the cases of FNAIT in humans are thought to occur in what are experienced as first pregnancies, we further investigated whether exposure to HPA-1a in pregnant female mice could still induce FNAIT. We found that when WT female mice pregnant with HPA-1a-positive fetuses were transfused with HPA-1a-positive platelets, they consistently developed HPA-1a-specific antibodies with similar titers as observed in non-pregnant females and delivered thrombocytopenic pups with FNAIT. Taken together, these findings demonstrate that exposure to HPA-1a-positive platelets in the maternal circulation during pregnancy is sufficient to trigger FNAIT, and that maternal tolerance, normally increased during pregnancy, does not play a significantly immunosuppressive role to prevent alloimmunization to the HPA-1a epitope. These findings support the notion that FMH may be a possible cause of alloimmunization in humans, even during a first pregnancy. Finally, the result of this study further validates the usefulness of this HPA-1a alloantigen-specific humanized transgenic mouse model to explore the etiology and pathophysiology of FNAIT. Further studies should significantly advance preventative and therapeutic strategies for this life-threatening immunologic disorder.
A Phase 1b Double-Blind, Placebo-Controlled Study of DISC-0974, an Anti-Hemojuvelin Antibody, in Patients with Non-Dialysis Dependent Chronic Kidney Disease and Anemia
Authors: Arora Samir , Monroy Jorge , Pergola Pablo , Silva Arnold , Buch Akshay , Novikov Natasha , Pelletier Olivia , Savage William
Published: December 2023

Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD) and anemia. Elevations in hepcidin contribute to the onset, maintenance, and severity of anemia. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin (HJV), a co-receptor in the bone morphogenetic protein-signaling pathway regulating hepcidin expression.

In a healthy volunteer study, DISC-0974 demonstrated dose-dependent reductions in serum hepcidin, increases in serum iron with doses up to 56 mg administered subcutaneously (SC), and increasing trends in reticulocyte count, reticulocyte hemoglobin, mean corpuscular hemoglobin, total hemoglobin (Hgb), and red blood cell count. In a rat model of anemia in CKD, DISC-0974 decreased hepcidin, increased serum iron, and normalized hemoglobin (Wu et al., ASH 2022).

DISC-0974-103 is a Phase 1b, double-blind, placebo-controlled, single-ascending dose study (NCT05745883) to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 in patients with NDD-CKD and anemia. Eligible participants include patients over 18 years of age with NDD-CKD, stages 2-5, hemoglobin (Hgb) <10.5 g/dL in women and <11.0 g/dL in men, serum ferritin ≥100 μg/L, and transferrin saturation (TSAT) ≤30%. Major exclusionary criteria include: concomitant treatment with therapeutic oral iron, intravenous iron, erythropoietin-stimulating agents, or blood transfusions; splenectomy; and nutritional, genetic, infectious, or autoimmune causes of anemia. Approximately 32 participants will be enrolled.

On Day 1, participants will receive a single dose of DISC-0974 SC with evaluations for safety, PK, and PD through 57 days of follow-up. Dose escalation is planned across 4 cohorts consisting of 8 participants each, randomized 3:1 active treatment to placebo, treated at single doses of 28, 40, 60, and 90 mg SC DISC-0974. Safety Review Committee assessments are planned to gate escalation decisions.

Primary endpoints include adverse events (AEs), clinical laboratory assessments, vital signs, physical examinations, and electrocardiograms. Secondary endpoints include changes in serum hepcidin-25, iron, TSAT, ferritin, Hgb, and other hematology biomarkers.

Enrollment is currently ongoing, and available study results will be presented at the meeting.
Racial Disparities in the Outcome of Tumor Lysis Sydrome (TLS) - a Five Year Population Based Analysis
Authors: Kamal Syeda Ashna Fatima , Prakash Vivek , Maiocco Giuseppe , Hussain Mohammad Junaid , Shah Ejaz , Agarwal Ishan , Goel Ruchika
Published: December 2023

DISCUSSION

Tumor lysis syndrome (TLS) is a potentially life-threatening complication that occurs in the setting of rapid tumor cell destruction. While several studies have explored the risk factors and management strategies for TLS [1], the impact of racial disparities on the incidence, severity, and outcomes of this syndrome remains understudied. We aim to investigate the impact of race on the outcomes of TLS.

Using weighted data from the Nationwide Inpatient Sample (NIS) database from 2016 to 2020, we assessed the outcomes (mortality, hospital utilization, total healthcare charges, complications) of individuals admitted with a primary diagnosis of TLS among races (non-Hispanic Whites, non-Hispanic Blacks, Hispanics, and Others including Native Americans, Asians, and other races/ethnicities). Baseline characteristics (table 1) were analyzed using T-test and Chi-Square, and a multivariate regression analysis was used to estimate outcomes between races, adjusted for patient and hospital confounders. Data analysis was performed using STATA® Version 17.0/SE Software, with statistical significance set at p < 0.05.

A total of 4340 were admitted with a primary diagnosis of TLS from 2016 to 2020. Among these, Blacks were the most prevalent (46%), followed by others (34%), Hispanics and whites (33% each). There was no significant difference in comorbid conditions between races. Moreover, no significant differences in the total healthcare expenditures or length of hospital stays were found among races. Pertaining to TLS outcomes, When compared to Whites, Blacks had a statistically significant increase in mortality (OR 2.35, 95%CI: 1.14-4.87). Considering manifestations of TLS, Others (Native Americans, Asians, and other races/ethnicities) had an increased likelihood of cardiac arrest (OR 9.14, 95%CI: 1.11-75.6). No statistically significant difference between races was found for gastrointestinal (GI) bleeding, acute kidney injury (AKI) or sepsis.

Our study highlights racial disparities in the outcomes of individuals hospitalized with TLS, including increased mortality in Blacks, and cardiac arrests in Others (Native Americans, Asians, and other races/ethnicities). Interestingly, there were no statistically significant differences in GI bleeding, or sepsis, which are commonly associated with increased mortality in TLS. Further investigations are warranted, particularly through prospective or retrospective studies, to elucidate the underlying factors contributing to these disparities.
Measuring Thrombin Generation in Platelet Rich Plasma with the ST Genesia
Authors: Ninivaggi Marisa , De Laat-Kremers Romy , De Laat Bas
Published: December 2023

Background: Thrombin generation (TG) is nowadays used more and more for diagnosing and monitoring of patients. The ST Genesia is a fully automated device for measuring TG in platelet poor plasma (PPP). As the samples are not being inverted prior to the measurement once loaded in the device, it was not known whether it was possible to measure TG in platelet rich plasma (PRP). Platelets can sediment in time possibly affecting TG results. The goal of this study was to evaluate the use of the ST Genesia for measuring TG in PRP in time and to investigate whether the assay is sensitive to platelet antagonists.

Methods: Blood was taken from 6 healthy donors and PRP was immediately prepared by centrifuging the blood once for 15 minutes at 220g. The PRP was divided into 14 tubes: 11 tubes for measuring TG in time; i.e. after 0, 15, 30, 45, 60, 75, 90, 105, 120, 180, 240 minutes after the samples were loaded on board; 1 tube for platelet count measurement, 1 tube for TG with the Calibrated Automated Thrombogram (CAT) assay and 1 tube for freezing the PRP. The PRP reagent of the CAT assay was used for triggering TG that contains a low tissue factor concentration. Tukey's multiple comparisons test was used for statistical analysis.

Results: The platelet count remained stable in time even without inverting the vial prior to the measurement up to 3 hours. After 4 hours, platelet count was significantly lower (p<0.01). TG in PRP remained stable in time up to 3 hours, after that lagtime and time-to-peak were extended by an average of 48% and 16%, respectively (p-values <0.0001 and 0.0121, respectively), while ETP and peak height remained stable (with low CV's of <6.0% and <7.3%, respectively). The ST Genesia proved to be sensitive to platelet antagonists aspirin (100µM) and Cangrelor (500nM) as peak height decreased with 22% and 24%, respectively. TG measured with the CAT and the ST Genesia was comparable. Measuring TG in PRP after being frozen at -80°C resulted TG kinetics similar to TG in PPP, with a shorter lagtime (-67%) and time-to-peak (-64%), with an increased peak height (+82%), and velocity index (+383%), and an unaffected ETP.

Conclusion: The ST Genesia can be used for the reproducible measurement of TG in PRP up to two hours after loading the samples in the device.
Venetoclax Combined with Daunorubicin and Cytarabine (2 + 6) in Acute Myeloid Leukemia: Updated Results of a Phase II Trial
Authors: Suo Xiaohui , Wang Dongmei , Zheng Fang , Zhang Suping , Zhang Congcong , Li Yinling , Shi Rui , Wu Yan , Yang Sisi , Zhao Liyun , Jiao Zongjiu , Liu Jie , Zhang Ling , Li Ling , Zhang Zhihua , Lu Xinxiao , Yuan Linyu , Gao Sifeng , Zhang Jilei , Zhao Xingli , Bai Guanchen , Mi Yingchang , Liu Kaiqi
Published: December 2023

INTRODUCTION

Venetoclax (Ven) combined with intensive chemotherapy had been proven effectively in the management of newly diagnosed acute myeloid leukemia (AML). Our previous study showed that Ven combined with DA (2 + 6) is a highly effective and safe induction therapy for adults with newly diagnosed AML. To further validate the efficacy and safety of this induction regimen, the study continued to enroll young patients with newly diagnosed AML. Now, we have updated the results of this phase 2, multicenter, single-arm trial.

METHODS

Details of the Study design, participants, inclusion and exclusion criteria, procedures, efficacy and safety evaluation were provided in our previous article “Venetoclax combined with daunorubicin and cytarabine (2 + 6) as induction treatment in adults with newly diagnosed acute myeloid leukemia: a phase 2, multicenter, single-arm trial” which published in the Journal of “Experimental Hematology & Oncology”.

RESULTS

Until June 30, 2023, seventy patients were enrolled and treated with Ven combined with DA (2 + 6) as induction therapy. The median age was 44 (range, 16-60) years, and 37 (52.9%) patients were male. According to the European Leukemia Network prognostic group (2022), 32 (45.7%), 9 (12.9%), and 29 (41.4%) patients were considered to belong to the favorable, intermediate, and adverse groups, respectively.

The ORR after one cycle of induction was 92.9% (65/70)with a composite complete response rate (CR + CRi) of 90.0% (CR 61/70, CRi 2/70). Moreover, 89.5% (51/57) of the patients reached CR with undetectable MRD. Grade 3 or worse adverse effects included neutropenia (100%), thrombocytopenia (100%), febrile neutropenia, and one mortality. The median neutrophil and platelet recovery times were 13 (5-36) and 13 (8-48) days, respectively. (Table 1)

Until Jan 30, 2023, with with a median follow-up of 9 (1-18) months, 6 patients proceed Allo-HSCT, the estimated 12-month OS, EFS, and DFS rates were 84.9%、81.0%、81.6%, respectively. (Figure 1)

Conclusion: Ven with DA (2 + 6) is a highly effective and safe induction therapy for adults with newly diagnosed AML.

Keywords Venetoclax, DA(2+6), induction treatment, acute myeloid leukemia
4D Intravital Imaging Studies Identify Platelets As the Predominant Cellular Procoagulant Surface in Hemostasis
Authors: Ballard Abigail , Bergmeier Wolfgang , Paul David , Lee Robert Hugh , Mackman Nigel , Flick Matthew J. , O'Shaughnessy Ellen , Adalsteinsson David , Kim Paul
Published: December 2023

Recent intravital imaging studies suggest that the vasculature is the predominant procoagulant surface during thrombotic plug formation. Similar studies investigating the interplay between endothelial cells (ECs), platelets, and coagulation/fibrinolytic factors during hemostatic plug formation are missing. We here describe a novel four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components with high spatiotemporal resolution. Fibrin accumulation following laser-induced endothelial ablation was observed at the endothelium-platelet plug interface. Fibrin levels were governed by the antagonistic balance between coagulation and fibrinolytic pathways. Phosphatidylserine (PS) was first detected in close physical proximity to the fibrin ring but then quickly spread across the endothelium. Impaired PS exposure in cyclophilinD -/- mice resulted in a significant reduction in fibrin accumulation. Adoptive transfer studies demonstrated a predominant role for platelets as a procoagulant surface during hemostatic plug formation, and fibrin accumulation was impaired by antiplatelet therapy (APT). Inhibition of fibrinolysis by tranexamic acid (TXA) led to increased fibrin accumulation in WT mice, but not in cyclophilinD -/- or WT mice treated with antiplatelet drugs. These studies indicate a predominant role of platelets as a procoagulant surface during hemostatic plug formation, and demonstrate that impaired procoagulant activity due to antiplatelet therapy is not reversed by TXA treatment.
Infection Outcomes and Hypogammaglobulinemia in Patients with Chronic Lymphocytic Leukemia Treated with Immunoglobulin Replacement Therapy
Authors: Soumerai Jacob , Gift Thais , Yousif Zaid , Desai Raj , Huynh Lynn , Belsky Gregory , Duh Mei Sheng , Sanchirico Marie
Published: December 2023

CONCLUSIONS

Hypogammaglobulinemia (HGG), a secondary immunodeficiency (SID), can manifest in patients with chronic lymphocytic leukemia (CLL) due to the disease and its treatments. Up to half of all deaths from CLL are attributed to infection; however, use of immunoglobulin replacement therapy (IgRT) for the prevention of infections in selected patients varies. We aimed to assess real-world IgRT utilization and infection outcomes in patients with CLL.

A retrospective, longitudinal study evaluating real-world IgRT use in adult patients with CLL diagnosed after 2010, with ≥ 3 visits/year and ≥ 12 months of clinical data, was conducted. De-identified clinical data came from the Massachusetts General Brigham Research Patient Data Registry. Immunoglobulin G (IgG) testing, infection outcomes, and antimicrobial use were compared before versus after IgRT initiation. Generalized estimating equation logistic regression models calculated odds ratios (ORs), 95% CIs, and P-values.

Of 3960 patients with CLL assessed, 2652 (67.0%) patients had IgG testing (29.0% tested at CLL diagnosis); 917 (34.6%) had HGG (IgG level < 500mg/dL). Median (interquartile range [IQR]) age was 68.0 (60.0, 76.0) years, 61.2% were men, 67.8% were treatment-naïve, and median (IQR) follow-up duration was 4.8 (2.4, 7.4) years. Among recipients of IgRT (n = 259), 56.8% received ≥ 2 administrations and the median (IQR) number of IgRT administrations was 2.0 (1.0, 4.0). Median (IQR) time from CLL diagnosis to IgRT initiation was 38.0 (11.4, 64.7) months. Significantly fewer patients had HGG 3 months after IgRT versus 3 months before (33.8% vs 72.8%; P < 0.0001). Significantly lower odds of infections and antimicrobial use were observed after IgRT initiation in 3- and 6-month time periods for: all infections, sinopulmonary and skin or soft tissue infections, infections requiring antimicrobials, all severe infections, severe sinopulmonary and skin or soft tissue infections, and severe infections requiring antimicrobials ( Table).

In this real-world study of patients with CLL, IgRT was associated with significant reductions in HGG, infection rates, and infections requiring antimicrobials. Guidelines are needed to optimize IgG testing, IgRT initiation, and SID management for patients with CLL.
Preclinical Activity of Novel TGF Beta Receptor I Kinase Inhibitors IOA-359 and IOA-360 for Treatment of Anemia in MDS/AML
Authors: Ramachandra Nandini , Vegivinti Charan Thej Reddy , Sahu Srabani , Rivera Bianca , Aminov Sarah , Fromowitz Ariel , Zhang Jinghang , Aodengtuya Fnu , Pradhan Kith , Zhao Rongbao , Schwartz Leya , Thiruthuvanathan Victor , Sanawar Rahul , Rabinovich Emma , Ajibade Opeyemi , Shastri Aditi , Lahn Michael , Verma Amit K.
Published: December 2023

Background:

Overactivation of the Transforming growth factor beta (TGF-β) superfamily has been associated with bone marrow failure in MDS. TGF-β binds to set of receptors that include the TGF-β receptor I kinase (also known as ALK5), that in turn phosphorylates and activates the downstream SMAD2/3 proteins. Activation of SMAD2/3 transcription factors has been shown to occur in MDS and is associated with anemia. Thus, we wanted to evaluate the preclinical efficacy of novel, clinic-ready, TGF-b receptor I kinase small molecule inhibitors IOA-359 and IOA-360 in MDS models.

Results:

Using the TREEspot™ SCAN Max Kinase Panel of 403 kinases, IOA-359 and IOA-360 were found to be highly selective for ALK5. Both, IOA-359 and IOA-360 have a blocking activity against ALK5 similar to clinical stage ALK5 inhibitors. To evaluate the efficacy of IOA-359 and IOA-360, we first used HEK 293- Blue-TGF-β cells that stably overexpresses a SMAD inducible secreted embryonic alkaline phosphatase (SEAP) reporter. Both IOA-359 and IOA-360 were able to significantly inhibit TGF-β driven reporter at nanomolar doses ( Fig1A). Next, we showed that both ALK5 inhibitors were able to block TGF-β-mediated SMAD2/3 downstream phosphorylation/activation in a dose dependent manner in MDS and leukemic cell lines. Functionally, the ALK5 inhibitors did not lead to any increase in viability or proliferation in leukemic cell lines.

We next tested the efficacy of the novel ALK5 inhibitors in primary patient samples from MDS and AML (N=8). Treatment with both ALK5 inhibitors led to increase in erythroid colony formation in majority of MDS samples in clonogenic assays. Colonies were picked up and examined for erythroid and myeloid differentiation by FACS analysis. We observed that ALK5 inhibition led to increase in Glycophorin A and CD71 expression suggesting enhanced erythroid differentiation ( Fig1B). Myeloid differentiation effects were modest in comparison. Furthermore, in selected samples, addition of Luspatercept led to greater maturation of erythrocytes. Treatment of healthy CD34 + hematopoietic stem and progenitors did not lead to significant changes in differentiation. Lastly, to evaluate downstream effects of ALK5 inhibition, we performed RNAseq analysis in MDS cells. Inhibition with ALK5 inhibitors led to numerous changes in genes affecting cellular differentiation, validating functional changes observed by us.

Conclusion:

Our current results support the preclinical in vitro efficacy of ALK5 inhibitors IOA-359 and IOA-360 alone and in combination with Luspartercept, highlighting their potential for further development and clinical testing in MDS/AML.
Functional Interrogation of Clinically Associated Venetoclax-Sensitive Genes Identifies an Essential Role of Transcription Factor ZNF740 in Therapeutic Response
Authors: Zhang Lixia , Lu Rui
Published: December 2023

Venetoclax, a targeted inhibitor of BCL-2, has shown therapeutic benefits in acute myeloid leukemia (AML). However, the emergence of drug resistance remains a significant clinical challenge, emphasizing the need for deeper understanding of its mechanisms. While comprehensive transcriptome analyses have identified many genes associated with venetoclax responsiveness, it remains crucial to distinguish between genes that play a direct role and those that are simply associated.

In this study, we focused on genes strongly linked to a positive venetoclax response in primary AML samples using the BeatAML2.0 dataset. We identified ~1,400 top genes whose expression inversely correlated with venetoclax AUC. We theorized that if any of these genes were essential for venetoclax sensitivity, their absence would lead to resistance. To explore their significance, we developed a focused CRISPR knockout library of these ~1,400 genes, with each gene represented by 7 guide RNAs, and an additional 250 guide RNAs as negative controls. We then performed CRISPR screens on AML cell lines, using either DMSO or venetoclax as treatments. Interestingly, the majority of the gene knockouts did not lead to resistance, suggesting that these genes likely have an associative rather than a direct impact on venetoclax sensitivity.

However, our screen did identify several genes that, when depleted, consistently resulted in resistance across different AML cell models. One of the top hits was the zinc finger transcription factor ZNF740, whose expression is strongly correlated with venetoclax AUC ( R = -0.52, p = 2.71e-26). For verification, we used CRISPR approach to knock out ZNF740 in Cas9-expressing OCI-AML2 and MOLM-13 cell lines. We then performed IC 50 and competitive proliferation assays under venetoclax treatment. We found that AML cells without ZNF740 exhibited a markedly higher IC 50 and demonstrated enhanced survival under venetoclax exposure. Conversely, enforced overexpression of ZNF740 rendered hypersensitivity to venetoclax. Intriguingly, our transcriptomic study showed that ZNF740 deletion upregulated monocyte differentiation signature genes, a change previously linked to venetoclax resistance.

Collectively, our findings indicate that ZNF740 serves as a newly identified, clinically relevant transcriptional modulator of venetoclax responsiveness. We are now investigating the molecular targets of ZNF740, particularly its role in guiding AML lineage decisions and its impact on venetoclax response. Gaining insight into ZNF740's function and mechanism could pave the way for innovative strategies to counteract venetoclax resistance observed in clinical settings.
Combination of Circulating Plasma Cells Enhances the Efficacy of R2-ISS Stage System for Risk Stratification of Newly Diagnosed Multiple Myeloma: A Single-Center Real-World Study
Authors: Chu Bin , Wang Yutong , Shi Lei , Sun Kai , Bao Li
Published: December 2023

Previous studies identified the prognostic significance of quantifying circulating plasma cells (CPCs) by multiparametric flow cytometry (MFC) in newly diagnosed multiple myeloma (NDMM) patients. Recently it was reported that the R2-ISS stage is a simple prognostic staging system allowing a better stratification of patients with intermediate-risk NDMM.

We evaluated if a similar quantification of CPCs could add prognostic value to the R2-ISS classification of 279 consecutive NDMM patients treated in our center from 2017 to 2022. Those patients with R2-ISS stage II and stage III disease were re-classified according to the CPCs numbers ≥0.05% (CPC high) or＜0.05% (CPC low) for the purposes of this study.

The median progression free survival (PFS) for patients were as follows: not reached (R2-ISS I), 43 months (R2-ISS II with CPC low),24 months(R2-ISS II with CPC high), 23 months (R2-ISS III with CPC low),12 months (R2-ISS III with CPC high) and 14 months (R2-ISS IV). The median overall survival (OS) for patients were as follows: not reached (R2-ISS I, R2-ISS II with CPC low, R2-ISS II with CPC high and R2-ISS III with CPC low) , 46 months (R2-ISS III with CPC high) and 32 months (R2-ISS IV).

Finally, ≥ 0.05% CPCs retained its adverse prognostic significance in a multivariable model for PFS and OS. Hence, quantifying CPCs by MFC can potentially enhance the R2-ISS classification of a subset of NDMM patients with stage II and III disease by identifying those patients with a worse than expected survival outcome.
Quantitative Model-Based Assessment of Multiple Sickle Cell Disease Therapeutic Approaches Alone and in Combination
Authors: Moody Amy , Maurer Tristan , Narula Jatin
Published: December 2023

In sickle cell disease (SCD) red blood cell sickling is driven by concentration dependent polymerization of mutation-carrying deoxygenated hemoglobin (deoxHbS). Multiple treatment strategies aim to reduce the concentration of deoxHbS to prevent sickling. Evidence for the utility of this approach is seen in patients endogenously expressing >30% HbF who are largely asymptomatic. In addition to inducing HbF, two other strategies, stabilizing oxygenated hemoglobin (oxyHbS) and lowering 2,3-BPG similarly aim to reduce deoxHbS. However, the degree of oxyHbS stabilization or 2,3-BPG lowering needed to achieve a similar reduction in deoxHbS as 30% HbF is not known. Furthermore, it may not be possible to reach the desired efficacy by a single therapeutic agent making it important to understand how these mechanisms would perform in combination. To address these questions, we extended the Monod-Wymann-Changeux (MWC) oxygen saturation model to include hemoglobin binding to 2,3-BPG and oxyHbS stabilizers and added an additional species to represent HbF. It was assumed that oxyHbS stabilizer occupancy provided equivalent polymerization protection as HbF, which may not be true for all molecules in this class. The model allows simulation of varying levels of oxyHbS stabilizer occupancy, 2,3-BPG lowering, or HbF intervention alone or in combination to calculate the deoxHbS concentration at oxygen tensions found in peripheral tissue and assess polymerization protection. By this approach, the model predicts 51% occupancy by an oxyHbS stabilizer or reduction of 2,3-BPG to 1.8 mM will give equivalent polymerization protection as 30% HbF. Simulations indicate combining any two mechanisms results in a further decrease in deoxHbS compared to either mechanism alone, suggesting combination therapy will have added benefit in the case of dose limitations. By including the main strategies aimed at reducing deoxHbS, this model represents a unified framework that is useful in supporting decisions across the R&D continuum and can be adapted as new clinical data becomes available to gain new and increasingly confident insights into treatment strategies for SCD patients.
Mechanisms of Red Blood Cell Splenic Filtration Revealed By Multiscale Modeling and in-Vitro Microfluidic Experiments
Authors: Peng Zhangli , Moreau Alexis , Yaya François , Lu Huije , Surendranath Anagha , Charrier Anne , Dehapiot Benoit , Helfer Emmanuelle , Viallat Annie
Published: December 2023

Interendothelial slits in the spleen fulfill the major physiological function of continuously filtering red blood cells (RBCs) from the bloodstream to remove abnormal and aged cells. To date, the process of passage of 8 µm RBCs through 0.3-µm wide slits remains enigmatic. Should the slits increase their caliber during RBC passage as sometimes proposed in the literature? Here, we elucidated the mechanisms that govern the passage dynamics or retention of RBCs in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with slits of defined physiological dimensions, including submicron width. We observed that healthy RBCs pass through 0.28-µm wide rigid slits at body temperature. To achieve this tour de force, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, they must be able to locally unfold their spectrin cytoskeleton inside the slits. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through slits scales with in-slit pressure drop and slit width to the -1 and -3 power, respectively. This transit dynamics is similar to that of a Newtonian fluid in a 2D Poiseuille flow, thus showing that it is controlled by the RBC cytoplasmic viscosity. Altogether, our results clearly show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.
The Role of CABIN1 in the Pathogenesis of Idiopathic Multicentric Castleman Disease
Authors: Laczko Dorottya , Fajgenbaum David C. , Kambayashi Taku
Published: December 2023

Introduction: Idiopathic Multicentric Castleman Disease (iMCD) is a rare disease characterized by flares of hypercytokinemia, leading to life threatening complications. Although several hypotheses have emerged including autoimmune, immune dysregulation, and/or infectious etiology, to date the exact pathogenesis of iMCD is unknown. IL-6 is thought to be the key cytokine that drives symptoms in a proportion of patients and current standard of care targets IL-6; however, it is only successful in one third of patients. Therefore, there is a dire need to identify additional driver cytokines/pathways to address treatment-refractory cases. Lately, the pathogenic role of CXCL13 in iMCD has emerged, as proteomic studies have shown that serum CXCL13 is elevated in iMCD and a biomarker of response to therapy. Given the heterogeneity of the disease, research into iMCD has been limited by the lack of an animal model. Through whole exome sequencing of an iMCD patient, we recently discovered a potential disease-causing mutation in a protein known as CABIN1. CABIN1 in known to play a role in T cell activation as a negative regulator of calcineurin and MEF2. Using Crispr/Cas9 technology, we generated a mouse model that harbors this gene mutation (CABIN1 V2185M).

Methods: Following LCMV infection or ovalbumin (OVA)-mRNA-LNP immunization serum cytokine levels were measured using Luminex assay or CXCL13 specific ELISA kit. Different immune cell populations and Nur77 levels were assessed by flow cytometry. MEF2D protein levels were determined by Western blot analysis.

Results: Although CABIN1 V2185M mice do not demonstrate spontaneous pathology at baseline, we found that upon LCMV CL13 infection, mice harboring the CABIN1 V2185M mutation displayed significantly higher morbidity and mortality compared to control mice. LCMV-infected CABIN1 V2185M mice displayed elevated serum levels of cytokines/chemokines including IL-6 and CXCL13, which are associated with human iMCD flares. Concurrently, we also measured a robust increase in the frequency of CD4 + T cells expressing CXCR5, which is the receptor for CXCL13. Similar to LCMV CL13, infection with LCMV Armstrong or immunization with OVA-mRNA-LNP also led to an increase in serum CXCL13 levels and CXCR5 +CD4 + T cells in the associated secondary lymphoid organ in CABIN1 V2185M compared to control mice. In terms of the underlying mechanism, we found that T cells of CABIN1 V2185M mice exhibit increased MEF2D protein and expression of a MEF2D-regulated gene, Nur77 following CD3/CD28 stimulation, suggesting that CABIN1 V2185M mutation acts through MEF2 dysregulation in T cells.

Conclusion: Our preliminary data shows that LCMV-infected CABIN1 mutant mice recapitulate the cytokine milieu of human iMCD. Therefore, CABIN1 could be the first ever described mutation that could drive iMCD disease. Furthermore, this mouse model could serve as a model platform to study the underlying pathogenic pathway(s) in iMCD.
Treatment Pattern and Survival of Human Immunodeficiency Virus Infection-Related Lymphoma in China 2008-2021
Authors: Liu Jun , Wang Chaoyu , Yang Tao , Wu Yan , Min Haiyan , Huang Yunhong , Wei Guo , Zhang Wei , Wang Min , Tang Xiaoqiong , Zhou Hui , Liu Yao
Published: December 2023

Background

Over the past few decades, little has been known about HIV-related lymphoma, and there has been a lack of treatment. However, with the development of cART therapy in the 1990s, the survival of HIV-related lymphoma patients has been significantly improved, but there is still a lack of large-scale data reports on HIV-related lymphoma patients in China.Therefore, we performed a multi-center study to analyze the clinical characteristics and outcomes of HIV-related lymphoma patients in China.

Methods

We retrospectively analyzed the data of 443 patients with human immunodeficiency virus infection-related lymphoma from eleven large academic centers in China from 2008 to 2021.

Results

In the entire cohort, the median age was 47 years (range, 18-90) at lymphoma diagnosis, and 364 patients were male (82.2%).More than half of the patients came from public health treatment centers. The most common type of pathology was DLBCL (61.6%)and BL (10.6%). Most patients had elevated lactate dehydrogenase (56.2%), bulky tumor(30.7%), B symptoms(39.3%), extranodal involvement(52.8%), and advanced Ann Arbor stage (60.3%) at diagnosis. High international prognostic index (IPI) score (3-5) at diagnosis was found in 58.7% of patients. The number of patients with CD4 cell count below 200/μl was 244, accounting for 55.1% of the total. The percentage of patients who gave up chemotherapy was as high as 15.6%.

The median follow-up of our cohort was 10.1 (0.1-160) months. The overall 1-year OS rates 70.2%. Cox univariate analysis showed that education level(HR=0.682, 95%CI 0.468-0.994; p=0.046),B symptoms(HR=0.730, 95%CI 0.562-0.948; p=0.018),PLR(ratio of the number of platelets to the number of lymphocytes)(HR=1.000, 95%CI 1.000-1.001; p=0.044),elevated LDH(HR=0.607, 95%CI 0.463-0.769; p=0.000),extranodal involvement(HR=1.498, 95%CI 1.141-1.968; p=0.004) were independent risk factor for adverse prognosis based on overall survival (OS).The overall 1-year PFS rates was 67.4%.Cox univariate analysis showed that age≥60(HR=1.819, 95%CI 1.197-2.763; p=0.005),anemia(Hb<120g/L)(HR=1.564, 95%CI 1.099-2.226; p=0.013),LDH(HR=3.646, 95%CI 2.115-6.286; p=0.000),CNS involvement(HR=2.209, 95%CI 1.384-3.524; p=0.001) were independent risk factor for adverse prognosis based on progression-free survival(PFS).

Conclusion

This is the largest retrospective study of HIV-associated lymphoma in China to date. Our results show that the proportion of men with HIV infection is much higher than that of women, which is positively correlated with the proportion of HIV infection. Patients with HIV-associated lymphoma have more risk factors at first diagnosis, including late stage, high risk layer, elevated LDH, extranodal involvement, and low CD4 cell count.
Predictive Modeling of Hematopoietic Stem Cell Mobilization and Collection Based on Peripheral Blood Marker
Authors: Ran Longrong , Peng Yu , Zhao Mingyu , Luo Xin , Chen Shuang , Yang Zailin , Liu Yao
Published: December 2023

Background: Autologous stem cell transplantation (ASCT) is the major transplantation therapy for patients with lymphoma and MM, and the success of auto-HSCT depends on the mobilization efficiency of peripheral blood HSC. In clinical practice, CD34 + cell count in peripheral blood is often monitored to predict the collection effect and determine the collection time. However, prediction of poor mobilizers is still difficult .

Methods: We retrospectively analyzed 108 patients with lymphoma and multiple myeloma(MM) at Chongqing University Cancer Hospital who underwent autologous hematopoietic stem cell (HSC) collection between January 2021 to August 2022. There are two groups: collection success group and collection failure group. Compared the differences of clinical characteristics and impact of peripheral blood related detection indexes before autologous HSC collection. Logistic regression analysis was used to establish a prediction model for the successful collection by multi-marker and further evaluated multi-marker prediction models.

Results : There was no significant difference in sex, disease type, disease stage, age, disease status before mobilization, mobilization drugs and chemotherapy courses between the successful collection group and the failed collection group ( P> 0.05). The proportion of patients with mobilization days less than 5 days was higher in the successful collection group ( P< 0.05). The platelet count (PLT), mononuclear cell count (MNC) and absolute CD34 + cell count in the successful collection group were significantly higher than those in the failure group. The sensitivity, specificity and area under the curve (AUC) of combining three indexes were 80.9%, 95.9% and 0.944, respectively. Applied the Logistic regression models to the absolute count of PLT, MNC and CD34 + cells to construct the multi-marker prediction algorithms. The 3-marker model of PLT+MNC+CD34 + cells had the lowest AIC, BIC and high AUC. A prediction model was established based on the absolute count of PLT, MNC and CD34 + cells.

Conclusion :Patients with less than 5 days of mobilization have a higher success rate of collection. PLT, MNC and CD34 + cell counts before collection can reflect HSC mobilization in patients with lymphoma and MM, and the combination of the three can improve the accuracy of prediction of collection effect.The predictive model based on the absolute number of PLT, MNC and CD34 + cells have good predictive value. Our study suggests that multi-markers are important diagnostic tools for predicting successful collection.
Screening for Intracellular Phosphorylation Cascades That Positively and Negatively Regulate the Self-Renewing Proliferation of Immature HPCs
Authors: Kitada Sayaka , Nishinaka-Arai Yoko , Nishikori Momoko , Niwa Akira , Saito Megumu K
Published: December 2023

For the purpose of studying hematological diseases involving complex lineages of cells, hematopoietic differentiation using patient-derived iPS cells has already proved to be powerful in the analysis of various diseases. However, in terms of sourcing for the expansion of its use to larger scale studies, the time-consuming and inefficient differentiation process still presents a significant obstacle. In this context, the development of a method that allows the proliferation of progenitor cells (HPCs) which maintains their ability to differentiate into multiple lineages is an important issue.

In vivo, immature multipotent hematopoietic stem cells (HSCs) reside in the bone marrow, where intracellular signals via phosphorylation cascades co-ordinately regulate the expression pattern of transcription factors (TFs), leading to fate decisions within the microenvironment between self-renewal and one-way differentiation into HPCs. In contrast, iPS cell-derived hematopoietic differentiation does not involve HSCs and the microenvironment differs from that of bona fide cells. Therefore, the signal combinations required to amplify iPS cell-derived HPCs without further differentiation should naturally differ from those observed in HSCs in vivo. Based on this assumption, we constructed a CRISPRa and CRISPRi screen for phosphorylation-related genes using iPS cell-derived differentiation to search for factors that enable long-term maintenance of multipotent HPCs.

First, Doxycycline-inducible dCas9-VPR (CRISPRa) or dCas9-KRAB-MecP2 (CRISPRi) expression cassettes were introduced into human iPS cells before, and then acted in combination with the phosphorylation-focused sgRNA libraries at the time when HPCs begin to be produced from mesodermal progenitors during their hematopoietic differentiation. Then, to identify the sgRNAs of interest with single-cell accuracy, colony formation assays were performed by isolating HPCs one by one by single-cell sorting. 2 weeks later, cells were collected only from wells that had formed CFU-Mix. Amplicon-sequences were performed using genomic DNA extracted from those cells and the sgRNAs that significantly contributed to the phenotype were identified by calculating the difference in enrichment with control sgRNAs. As a result, the CRISPRa screen identified a group of intracellular signals related with eight candidate genes known to be specifically activated in immature cells in the normal hematopoiesis. On the other hand, the results of CRISPRi showed that suppression of necroptosis-related signalling probably promotes long-term culture of HPCs in the stroma-free, artificial culture circumstances.

Our results showed the potential to obtain multipotent HPCs from iPS cells over a long period of time and in large quantities, simply by adjusting the intracellular signals without artificial adding or removing of individual genes.
Hypofractionated Radiotherapy in Aggressive B-Cell Lymphoma
Authors: Liu Ting-Bo , Li Xiaofan
Published: December 2023

Background and purpose: Radiotherapy (RT) is an effective and available local treatment for patients with refractory or relapsed (R/R) aggressive B-cell lymphomas. However, the value of comprehensive hypofractionated RT in this setting has not been confirmed.

Materials and methods : We retrospectively analyzed patients with R/R aggressive B-cell lymphoma who received comprehensive hypofractionated RT between January 2020 and August 2022 at a single institution. The objective response rate (ORR), overall survival (OS), progression-free survival (PFS) and acute side effects were analyzed.

Results : A total of 30 patients were included. The median dose for residual disease was 36 Gy, at a dose per fraction of 2.3-5 Gy. After RT, the ORR and complete response (CR) rates were 90% and 80%, respectively. With a median follow-up of 10 months (range, 2-27 months), 10 patients (33.3%) experienced disease progression and three died. The 1-year OS and PFS rates for all patients were 81.8% and 66.3%, respectively. The majority (8/10) of post-RT progressions involved out-field relapses. Patients with relapsed diseases, no response to systemic therapy, multiple lesions at the time of RT, and no response to RT were associated with out-field relapses. PFS was associated with response to RT (P=0.001) and numbers of residual sites (P<0.001). No serious non-hematological adverse effects (≥grade 3) associated with RT were reported.

Conclusion: These data suggest that comprehensive hypofractionated RT was effective and tolerable for patients with R/R aggressive B-cell lymphoma, especially for those that exhibited localized residual disease.
Acetyl Transferase EP300 Deficiency Leads to Chronic Replication Stress in Adult T-Cell Leukemia/Lymphoma
Authors: Niljikar Mrunmai , Juwarwala Aastha , Gagliardi Julia , Zhang Ranran , Ye B. Hilda , Madireddy Advaitha
Published: December 2023

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a disease model to study EP300 deficiency and an EP300-selective PROTAC degrader, as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, transmission of unrepaired inherited DNA lesions in the subsequent G1-phase and accumulation of cytosolic DNA in EP300-deficient cells. However, EP300 deficient cells show a significant defect in innate immune system activation. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress, persistent genomic instability and innate immune system evasion underlie aggressive chemo-resistant tumorigenesis in humans.