Hematopoietic Precursors Research Articles

A case of sudden death due to pulmonary embolism of bone marrow origin, masked as acute drug toxicity: A rare case in a bewildering clinical scenario

Sudden death is unexpected death within 24 hours from the onset of symptoms with or without known preexisting conditions. Respiratory pathologies are the second most common cause of such deaths. 39 years old male patient with a history of mental illness for the last 10 years and on regular treatment with antipsychotic medications, suffered sudden deterioration of higher functions and was admitted to the Medical College, and Hospital, Kolkata with a provisional diagnosis of drug toxicity. He succumbed to his condition within 24 hours of admission and the dead body was referred to Kolkata Police Morgue for a medicolegal autopsy.Shows multi-system involvement in the form of Pulmonary Oedema, consolidation, broncho-pneumonic changes and subpleural petechiae, Cardiomegaly due to biventricular hypertrophy, Cerebral Oedema, Enlarged kidneys with loss of cortico-medullary differentiation and streaky cortico-medullary haemorrhages. Stomach findings were unremarkable.1) Toxicological Examination of blood, bile, vitreous and routine viscera was negative for any poison or pharmacological agents.2)Histopathological examination with routine H&E staining showed Pulmonary oedema with lymphocytic infiltration and the presence of fat and hematopoietic precursor cells in pulmonary vessels, Interstitial Oedema and focal glomerulosclerosis and Periportal inflammation in the liver.

Paratesticular Extramedullary Hematopoiesis in Children.

Extramedullary hematopoiesis (EMH) is an uncommon occurrence, usually associated with hematologic disorders, but it rarely presents as an isolated finding. To determine the frequency, immunomorphologic features, and clinicopathologic background of EMH in orchiectomies from pediatric patients. All orchiectomy specimens removed from children from 2008 to 2020 in our institution were retrospectively reviewed. Biopsies and neoplasias were excluded. The EMH diagnosis was rendered when hematopoietic cell precursors were present. Immunohistochemical stainings were performed to characterize the hematopoietic components. Seventy-nine orchiectomies from 77 children (mean age, 5 years; range, 0-17 years) were included in our study. Forty-three patients (55.8%) underwent surgery for testicular atrophy, 30 (39.0%) for torsion, and 4 (5.2%) for intersex conditions. EMH was identified in 6 of 79 orchiectomies (7.6%), all performed for testicular torsion. All patients but one were newborns, and the remaining patient was 15 years old. No patient had evidence of a hematologic disorder. All EMH foci were in a background of reactive changes with a variable extension, either in the epididymis (4 cases) or in the deferens duct (2 cases). Immunostaining confirmed an association of myeloid (myeloperoxidase+) and erythroid precursors (E-cadherin+) in all 6 cases. One case also presented rare megakaryocytes, and one showed benign TdT+ B-cell precursors. To our knowledge, this is the first study that demonstrates EMH as a common finding in orchiectomy samples, especially from newborns. Despite the lack of pathologic potential, it is important to recognize EMH in order to avoid misdiagnosis.

Isolation and Analysis of B-cell Progenitors from Bone Marrow by Flow Cytometry.

B cells play a critical role in host defense, producing antibodies in response to microbial infection. An inability to produce an effective antibody response leaves affected individuals prone to serious infection; therefore, proper B-cell development is essential to human health. B-cell development begins in the bone marrow and progresses through various stages until maturation occurs in the spleen. This process involves several sequential, complex events, starting with pre- and pro-B cells, which rearrange the heavy and light chain genes responsible for producing clonally diverse immunoglobulin (Ig) molecules. These cells then differentiate into immature B cells, followed by mature B cells. The bone marrow is a complex ecological niche of supporting stromal cells, extracellular matrix components, macrophages, and hematopoietic precursor cells influencing B-cell development, maturation, and differentiation. Once fully mature, B cells circulate in peripheral lymphoid organs and can respond to antigenic stimuli. As specific cell surface markers are expressed during each stage of B-cell development, researchers use flow cytometry as a powerful tool to evaluate developmental progression. In this protocol, we provide a step-by-step method for bone marrow isolation, cell staining, and data analysis. This tool will help researchers gain a deeper understanding of the progression of B-cell development and provide a pertinent flow gating strategy.

The Generation and Functional Characterization of Human Microglia-Like Cells Derived from iPS and Embryonic Stem Cells.

The following protocol describes the generation of microglia cells from human-induced pluripotent stem cells (hiPSCs) using commercially available kits by StemCell Technologies. This protocol consists of three major steps: (1) Differentiation of hematopoietic precursor cells, (2) Microglia differentiation, and (3) Microglia maturation. Assays are described to characterize hematopoietic precursor cells and mature microglia.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Regulation of eIF4E guides a unique translational program to control erythroid maturation.

Translation control is essential in balancing hematopoietic precursors and differentiation; however, the mechanisms underlying this program are poorly understood. We found that the activity of the major cap-binding protein eIF4E is unexpectedly regulated in a dynamic manner throughout erythropoiesis that is uncoupled from global protein synthesis rates. Moreover, eIF4E activity directs erythroid maturation, and increased eIF4E expression maintains cells in an early erythroid state associated with a translation program driving the expression of PTPN6 and Igf2bp1. A cytosine-enriched motif in the 5' untranslated region is important for eIF4E-mediated translation specificity. Therefore, selective translation of key target genes necessary for the maintenance of early erythroid states by eIF4E highlights a unique mechanism used by hematopoietic precursors to rapidly elicit erythropoietic maturation upon need.

An ELF4 hypomorphic variant results in NK cell deficiency.

NK cell deficiencies (NKD) are a type of primary immune deficiency in which the major immunologic abnormality affects NK cell number, maturity, or function. Since NK cells contribute to immune defense against virally infected cells, patients with NKD experience higher susceptibility to chronic, recurrent, and fatal viral infections. An individual with recurrent viral infections and mild hypogammaglobulinemia was identified to have an X-linked damaging variant in the transcription factor gene ELF4. The variant does not decrease expression but disrupts ELF4 protein interactions and DNA binding, reducing transcriptional activation of target genes and selectively impairing ELF4 function. Corroborating previous murine models of ELF4 deficiency (Elf4-/-) and using a knockdown human NK cell line, we determined that ELF4 is necessary for normal NK cell development, terminal maturation, and function. Through characterization of the NK cells of the proband, expression of the proband's variant in Elf4-/- mouse hematopoietic precursor cells, and a human in vitro NK cell maturation model, we established this ELF4 variant as a potentially novel cause of NKD.

221P The role of TNF-alpha in thymus dysfunction during acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by an increased proliferation of hematopoietic progenitors or precursors (blasts) of the different myeloid lineages. Studies performed in AML-affected patients revealed a T-cell immunodeficiency, characterized by a decreased number of peripheral T lymphocytes' TRECs and a restricted repertoire.

Cellular Plasticity As Mechanisms to Escape from NOTCH1-Inhibition in T-ALL

Cellular plasticity has recently emerged as an underlying mechanism for treatment refraction in cancer. Hence, characterizing cell states and the transcriptional changes required for state transitions is critical for the development of efficient targeted therapies. Acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and adolescents that is associated with high rates of treatment failure and early relapse. T-ALL patients frequently harbor NOTCH1 activating mutations as the driving oncogene in this disease. Using T-ALL patient-derived xenograft (PDX) models carrying activating NOTCH1 mutations, we aim to u First, to determine how cell state diversity drives treatment refraction in T-ALL, we performed full-length single-cell transcriptome analysis on 3188 immune cells collected from 2 sensitive and 3 refractory PDX models. PDX models were treated with either with NOTCH inhibitor DBZ (Dipenzazepine; 10 μM/kg every other day intravenously) or vehicle. Profiled cells were collected at both a short (5 days) and a late time point (x-150 days) post-treatment to assess the effects of DBZ. Analysis of early hematopoietic and thymic precursor signatures demonstrate expression of immature hematopoietic signatures and acquisition of alternative lineage identities coexisting within the same cell, predominantly in refractory models. Analyses of pre-treatment samples indicated that disruption of the normal developmental hierarchy was associated with blockade at an early stage of T-cell commitment and can predict response to NOTCH inhibition in vivo. Upon treatment, cells underwent dramatic transcriptional reprogramming resulting in expansion of 2 distinct populations that greatly differ in both differentiation stage and developmental trajectory. One population expresses lymphoid differentiation programs, whereas the other population is enriched in genes associated with RNA processing and more immature hematopoietic signatures. Cell fate trajectories assessment using Monocle 2, revealed that consistent with their immature hematopoietic signature, refractory cells map at the start point of the differentiation trajectory, while sensitive cells clustered at the endpoint. We next sought to characterize the differentiation hierarchies that define the immature states. Developmental potential as assessed by CytoTRACE was higher in refractory cells when compared to their sensitive counterpart. To identify determinants of cell fate in a functionally relevant manner, we inferred gene regulatory network configurations using SCENIC and defined state specific regulon activity. Among the most prominently expressed regulons present in mature states, we identified transcriptional regulators involved in T-cell differentiation such as IKAROS, GATA3, and SOX5, while in immature states we observed high transcriptional regulon activity in transcription factors associated with renewal of hematopoietic progenitors such as ATF4, BCLAF1, and MYC in addition to the chromatin remodelers SMARCA4 and EZH2. To assess whether transcriptional rewiring in refractory cells results from greater chromatin accessibility, we performed single-cell ATAC sequencing (10x Genomics) on splenic cells from 1 sensitive and 1 refractory T-ALL PDX model treated with DBZ or vehicle. After integration with corresponding single-cell RNA-sequencing data, annotated ATAC profiles demonstrated greater chromatin accessibility, predominantly in active promotor and enhancer regions, in the refractory model compared to the sensitive model. Peak to gene linkage analysis revealed widespread alterations in cis-regulatory regions near genes associated with immature hematopoietic precursor signatures such as CD33. Furthermore, greater chromatin accessibility in refractory cells was associated with an increased number of enhancer loops per expressed gene as determined by H3K27ac HiChIP analysis. In conclusion, we demonstrated that presence of highly plastic cellular states defined by aberrant differentiation trajectories, distinct transcriptional circuitries, and remodeled chromatin architecture results in treatment escape in T-ALL.

Identification and Separation of Distinct Subpopulations of Hematopoietic Stem Cells and Multipotent Progenitors By Deep Learning

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are crucial for maintaining lifelong hematopoiesis. Several decades of successful clinical HSC transplantation have demonstrated the therapeutic importance of HSCs and MPPs. For basic research, HSCs and MPPs are routinely analyzed and separated by fluorescence-activated cell sorting (FACS) based on cell surface marker staining, a procedure that requires incubation with various antibodies and expensive laser-equipped flow cytometers. Both antibody staining and laser impair cell viability and stem cell activity. To develop a new staining-free, laser-free method for predicting and separating subpopulations of HSCs and MPPs, we used a deep learning approach that can extract minutiae from large-scale datasets of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) to distinguish subpopulations of hematopoietic precursor solely based on their morphology. Remarkably, our deep learning model can achieve predictable identification of subsets of HSCs with at least 85% accuracy; this demonstrates for the first time that HSC subpopulations can be differentiated solely based on their morphology exhibited in light microscopic images through deep learning. We anticipate that our robust and accurate deep-learning-based platform for hematopoietic precursors will provide a basis for the future development of a next-generation cell sorting system.

Interim Safety and Efficacy Results from a Phase 1/2 Study of Zinc Finger Nuclease-Modified Autologous Hematopoietic Stem Cells for Sickle Cell Disease (PRECIZN-1)

Introduction Sickle cell disease (SCD) is caused by pathologic variants in both alleles of the β-globin gene. Elevated fetal hemoglobin (HbF) levels inhibit sickle cell formation and ameliorate symptoms and improve survival in patients with SCD (Hebert, Am J Hematol, 2020;95:1235-45). BIVV003 is a novel therapeutic product comprising autologous CD34+ hematopoietic stem precursor cells (HSPC) modified ex vivo by zinc finger nucleases (ZFN) targeting specifically the BCL11A gene erythroid-specific enhancer (ESE) to increase endogenous HbF production in erythrocytes (RBC). Methods PRECIZN-1 (NCT03653247) is an ongoing first-in-human, open label, single arm, multi-center study evaluating safety and tolerability of BIVV003 (n=8; aged 18-40 years) with severe SCD. Eligible subjects underwent mobilization and apheresis with plerixafor 240 ug/kg/day for up to 3 days to collect autologous CD34+HSPCs for manufacturing of BIVV003. HSPCs were transfected ex vivo with ZFN mRNAs targeting the ESE region of the BCL11A locus to manufacture BIVV003. After myeloablative conditioning with busulfan 3.2 mg/kg/day intravenously (IV) for 4 days with dose adjustment based on pharmacokinetics, subjects received BIVV003 as a single minimum dose of 3× 106 CD34+ HSPC/kg IV. Subjects were monitored for stem cell engraftment and hematopoietic recovery, adverse events (AEs), clinical and laboratory hemolysis markers, total hemoglobin (Hb) and HbF, RBC containing HbF (F cells), and SCD-related events for up to 104 weeks post-BIVV003 infusion. Results As of the cutoff date of 03 May 2022, 4 subjects have received BIVV003 infusions up to 125 weeks of follow-up. A fifth subject was dosed with BIVV003 on 04 May 2022 and has engrafted. Among the 5 subjects, the male:female ratio is 3:2, the age range is 18-35 years, and 4 have genotype HbSS and 1 HbS-β0. No subject has required HSPC rescue; all subjects had hematopoietic reconstitution without requiring RBC or platelet transfusions for more than one month post-BIVV003 infusion. The average time to neutrophil and platelet engraftment was 23 days and 33 days, respectively. In bone marrow aspirates 26 weeks post-infusion, the frequency of cells containing indels at the BCL11A ESE target locus was in the range of 17.1-34.1%. No clonal dominance has been observed in bone marrow or peripheral blood cells. By Week 26, all 4 subjects reached a protective level of ≥ 10 pg HbF/F cell that inhibits HbS polymerization (Steinberg, Blood, 2014;123:481-485), and this level was sustained in 3 out of 4 subjects through the most recent assessment at Week 104, 78, and 52, respectively, along with F cell levels of 90% or higher. The change in total Hb from baseline (at screening) to the latest visit was -0.3-3.1 g/dL, and the HbF fraction increased to 12.2-41.2% (Figure 1). Three subjects have had no recurrence of severe vaso-occlusive crises (VOCs) post-infusion, compared to experiencing 6 to 22 severe VOCs in the 2 years pre-study. The one subject who did not sustain a HbF/F level ≥ 10 pg experienced 2 severe VOCs at 9 and 16 months post-infusion. The adverse events (AEs) reported were consistent with plerixafor mobilization and busulfan myeloablative conditioning. No AEs related to BIVV003 were reported. Conclusions The interim safety and efficacy results reported here confirm the potential therapeutic value of ZFN-mediated modification of the BCL11A ESE region and BIVV003 infusion to address current unmet needs of patients with SCD. BIVV003 has been well tolerated in the 5 subjects infused to date, with no AEs related to BIVV003. Three of the 4 subjects infused and with over 52 weeks of follow-up have had stable engraftment of ZFN-modified HSPCs resulting in sustained elevated HbF levels and absence of severe VOCs post-BIVV003 administration. A fifth subject received BIVV003 manufactured using an improved process that has shown in internal experiments to increase the number of ZFN-modified long-term progenitors in the drug product. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Functional in Vitro 3D Model of Human Hematopoietic Niches

Niches in the bone marrow (BM) regulate hematopoietic stem, progenitor and precursor cell fate and behaviour through cell-cell interactions and soluble factor secretion. Niche biology and niche-hematopoietic crosstalk are very complex processes not completely elucidated yet, due essentially to human material accessibility difficulties and limits of existing in vitro culture models. Here, we develop a 3D fully human bone marrow model recapitulating all hematopoietic and non-hematopoietic niche elements to aid further investigation of normal and pathological haematopoiesis and to move away from expensive, time-consuming and imperfect animal models. At first, we have developed a protocol to in vitro production and culture of all human main cellular non-hematopoietic components of bone marrow microenvironment. This comprehensive stroma is generated from human primary cells from femoral head, without any external cell lines contribution, and it reflects accurately the characteristics of the donor. In a 2-step culture process, mesenchymal stromal cells and endothelial cells are first selected and amplified simultaneously in an amplification medium, then they are cultured in the amplification medium as control or in a specific differentiation medium patented by the authors in conventional 2D culture plates or in 3D cultures: in spheroids, by self-organization in ultra-low-adherence plates or on a biomaterial (β-TriCalcium Phosphate), in the U-CUP perfused bioreactor. The resulting cellular stroma composition is analysed by quantifying the gene expression of markers of the osteoblastic (RUNX2, OSX, BSP), adipocytic (LPL, PPARγ, ADIPOQ) and endothelial (CD31) lineages . Moreover, specific markers of hematopoietic niche as ANG1, SCF, CXCL12, LEPR, VCAM, OPN are investigated by RT-qPCR. The spatial organization and the proportion of the different cellular stroma components are analysed by immunofluorescence (Osteoblastic cells: OSX marker, adipocytic cells: Bodipy probe, endothelial cells: CD31). The resulting tissues exhibited all compositional and structural features of human physiological BM: all stroma cells are presents and they express the main niche soluble factors. So, we explore if medullary stroma differentiated using our protocol is able to support the maintenance of HSPCs, to do this we performed coculture experiments in 2D with CD34+ cells sorted from umbilical cord blood. After 14 and 21 days of coculture, hematopoietic cells are recovered, counted and analysed by flow cytometry. Results show that our differentiated stroma is capable of maintaining, expanding and functionally regulated healthy human cord blood-derived HSPCs. An immature compartment (CD34+CD38-) is still present at day 21. Importantly, our primary in vitro niche model supports HSPCs with no cytokine addition. The same results are obtained with healthy human peripheral blood-derived HSPCs cocultured in 3D with the stroma organised in spheroids. Moreover, we demonstrated that our BM model is capable not only to maintain HSPC compartment but also to drive hematopoietic differentiation. In 2D coculture we detected mature blood cells and in 3D cultures, peripheral blood-derived HSPCs cocultured with spheroids are able to engage in erythroid lineage and recapitulate the early phases of erythropoiesis. These results demonstrate the fully complexity and huge versatility of our functional in vitro 3D model of human hematopoietic niches. Our exhaustive fully human hematopoietic niche model prepared from the primary cells of single donor, enables the design of advanced, tuneable in vitro BM proxies for the study of human physiological and pathological haematopoiesis and for screening molecules for predictive purposes in personalized medicine.

Mucosal-Associated Invariant T (MAIT) Cells Are Functionally Impaired in Pediatric Patients Following HCT and Their Recovery Is Associated with the Onset of GvHD

Delayed quantitative and qualitative immune recovery, in particular of the T-cell lineage, is associated with an increased risk of infections, disease relapse and adverse clinical outcomes in patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). This delay is particularly relevant for recipients of T cell-depleted grafts who do not benefit from the adoptive transfer of mature donor T cells and experienced profound and prolonged immunodeficiency which lasts until the T-cell pool is newly generated from donor hematopoietic precursor cells. A better understanding of the reconstitution dynamics of all the components of the immune system after allo-HCT is crucial to optimize the control of infections, leukemia relapse and graft-versus-host disease (GvHD). We performed a longitudinal observational study to explore the kinetics of T-cell recovery in patient peripheral blood by combining multiparameter flow-cytometry and functional ex vivo assays. Ninety-three children with hematological malignancies who received transplantation after myeloablative conditioning regimen were included in the study: 59 patients received T-cell receptor (TCR) alpha-beta/CD19-depleted HLA-haploidentical HCT (alpha/beta-haploHCT), while 34 unmanipulated matched unrelated donor (MUD) HCT (32 MUD recipients received BM and 2 received PBSC). Among the different T-cell populations assessed, we found that mucosal-associated invariant T (MAIT) cells displayed profound alterations in frequency and function across all time points analyzed. MAIT cells are a class of unconventional αβ T cells that recognize microbial proteins presented by non-polymorphic MHC class I related-molecule (MR1) and possess innate-like capacity. While total TCRαβ T cells gradually started to recover approximately from 3 months reaching normal levels between one to two years post transplantation (Figure 1), MAIT cells defined as CD3+ TCRαβ+ MR1-5-OP-RU-tetramer+ showed long-term impaired reconstitution, and their numbers did not reach normal levels up to two-years post-HCT (Figure 1). Compared to MUD HCT, MAIT cell recovery after alpha/beta-haploHCT was significantly delayed, in agreement with the fact that TCRab reconstitution in this clinical setting is primarily driven by de novo T cell formation. Previous studies demonstrated that MAIT cells are characterized by the binding to MR1-Ag tetramers and simultaneous expression of the C-type lectin-like receptor CD161, a marker associated with memory phenotype and trafficking of immune cells. Interestingly, across all time points of the study, we observed that most MAIT cells following were CD161-. We also found increased expression of the activation marker CD38 and the senescence marker CD57, and downregulation of CD28 in MAIT cells following allo-HCT, suggesting a dysfunctional or hyperactivated status. In agreement with these data, ex vivo stimulation of PBMCs at day +360 post allo-HCT with Escherichia Coli or anti-CD3/anti-CD28 showed that MAIT cells had suboptimal upregulation of the activation markers CD25 and CD69, and reduced production of pro-inflammatory cytokines IFNg, TNFa and IL2, when compared to healthy control samples. Previous studies suggested that a low MAIT cell number was associated with an increased risk of acute GvHD, In the MUD cohort of our study, we did not observe significant correlations between the kinetics of MAIT cell recovery and the occurrence of GvHD. On the other hand, we found a significantly higher frequency of CD161+ MAIT cells at day +30 in alpha/beta-haploHCT patients who developed acute GvHD (grade 1 or greater). Trends toward higher frequency of CD161+ MAIT cells in patients developing acute GvHD were also observed at day +60 and +90, although not statistically significant. Our study shows that MAIT cell recovery is delayed in children for up to two years following allo-HCT. MAIT cells exhibited altered functional status as demonstrated by the expression of activation/senescence makers as well as the suboptimal response to TCR stimulation. Interestingly, we found that a large proportion of MAIT cells are CD161- after transplantation, which can be the result of recruitment of CD161+ cells to peripheral tissues or down regulation of CD161 after cell activation. Finally, our data suggested that the evaluation of CD161 expression on MAIT cells after alpha/beta-haploHCT may identify patients at risk of acute GvHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Characteristics of AML1-ETO Fusion Gene Induced Pre-Leukemia Immune Microenvironment

Objective: The immune-privileged microenvironment plays a critical role in immune escape of acute myeloid leukemia (AML), however the characteristics of the immune micro-environment in pre-leukemia states have not yet been clarified. In this study, the Aml1Eto/+; Mx1-Cre mouse model was employed to explore the remodeling of the immune microenvironment by leukemic precursor cells, and to elucidate the characteristics of the immune microenvironment in the pre-leukemic state. Methods: Four weeks after PIPC induction (5mg/kg), bone marrow-derived immune cells such as T cells, B cells and macrophages(Mφ) that do not express the AML1-ETO (AE) fusion gene were sorted from Aml1Eto/+; Mx1-Cre mice (experimental group) and Aml1Eto/+;w/o Mx1-Cre mice (control group), respectively. The immune cell subsets were analyzed using CIBERSORT after bulk RNA-sequence, and TCR and BCR information was reconstructed by TRUST4. Results: After PIPC induction, the proportion of the AE fusion gene expressed hematopoietic precursors cells expanded significantly in the experiment group. The absolute counts of T cells and dendritic cells that did not express the fusion gene showed a considerable increment (p<0.0001; p=0.0067), and the monocyte-macrophage cells decreased (p<0.0001; p=0.0009). In addition, the expressions of Gzma and other cytotoxicity-related genes were significantly up-regulated in the T cells of experiment group. As for reconstructed TCR, the clonal diversity of TCR expanded. Although the proportion and absolute count of T cell increased when AE fusion protein induced, there didn't appear some hyperexpanded clones. The clonotype relative abundance and gene usage preference of TCR & BCR are similar between experiment and control group. There were 11 pre-leukemia-specific TCR amino acid sequences appeared, though no dominant clone was formed, suggesting that T cells produced relevant but not strong enough immune responses to pre-leukemia precursor cells. Further analysis found that the T cells in experiment group were composed of more Treg cells and expressed higher exhaustion-related genes such as PD-1, indicated that T cells began to accumulate negative immune regulation pathways in the bone marrow microenvironment of pre-leukemic state. For the antigen presentation, the expression of MHC-I molecules on AE fusion gene expressed LT-HSC and GMP in the experimental group was up-regulated, while the expressions of MHC-I and MHC-II molecules in dendritic cells were not significantly different from those in the control group. Meanwhile, CIBERSORT and flow cytometry analysis showed more classically activated M1-Mφ in the experiment group, in which the expressions of INF-γ, IL-1, and other genes were up-regulated. In terms of B cells, the production of immunoglobulins related genes and antigen processing and presenting genes were up-regulated. The BCR clonal diversity expanded, and 73 pre-leukemia-specific BCR amino acid sequences appeared, but no dominant clones were formed. Among the heavy chain sequences, the clonal diversity of IgD and IgM in the experimental group increased, the relative abundance of IgM increased, and the relative abundance of IgA decreased. Conclusion: We performed a comprehensive cell-type based dissection of the AML1-ETO fusion gene induced pre-leukemia immune microenvironment. The results demonstrates that in pre-leukemia stage, activated immune microenvironment is involved in the fight with AML1-ETO expressed cells. And there is also an accumulation of negative immune regulation mechanisms which may potentially contribute to weaken "the power" of immune surveillance. Although further validation is required, restoring dysfunctional immune cells or enhance the AML1-ETO specific immune response might be a potential early therapeutic approach for patients.

Lactate Utilization Provides a Metabolic Escape to Resist the Antileukemic Activity of BET Inhibition in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly heterogeneous malignant disease of the bone marrow resulting from arrest of hematopoietic precursors and uncontrolled proliferation of myeloblasts. Inhibition of the BET-family co-activator BRD4 (BETi) shows encouraging pre-clinical activity but limited clinical activity as a single agent. We previously demonstrated that MYC suppression by BETi induces metabolic and mitochondrial changes in AML blasts leading to a collapse in mitochondrial respiration. AML blasts that are resistant to BETi undergo comparable mitochondrial respiration despite impaired glycolysis and glutaminolysis. The metabolic pathway allowing BETi-resistant AML blasts to maintain metabolic integrity is unclear. Therefore, to identify how AML blast cells metabolically bypass BET inhibition, we used two different cell lines: BETi-resistant MOLM-13 and BETi-susceptible MV-4-11 cells. AML cell lines were treated with BETi for 48 hr and global untargeted metabolic mass spectrometry revealed that glycolysis, glutaminolysis, and fatty acid metabolism were not leveraged to maintain tricarboxylic acid (TCA) cycle activity (Fig. 1A). Rather, an increased NADH/NAD+ ratio and intracellular lactate accumulation suggested that lactate was being utilized as an alternative carbon source to sustain mitochondrial respiration. In support, MOLM-13 cells treated with BETi accumulated higher levels of mitochondrial lactate dehydrogenase, which preferentially converts lactate into pyruvate to fuel the TCA cycle. The heightened capacity of MOLM-13 cells to utilize lactate better maintained cellular viability but failed to support cellular replication, suggesting that lactate utilization promotes cellular senescence during BET inhibition. Therefore, we employed three chemical inhibitors of lactate utilization to couple with BETi: AZD3965 to prevent extracellular lactate uptake, UK5099 to block lactate import into the mitochondria, and oxamate to broadly impair lactate dehydrogenase activity. All three chemical inhibitors of lactate utilization were sufficient to render MOLM-13 cells susceptible to BET inhibition, as mitochondrial respiration, ATP production, and cellular viability were decreased to levels comparable to MV-4-11 cells. A similar correlation was observed in patient samples where AML blasts with increased resistance to BETi were predisposed to utilize lactate as an alternative carbon source and rendered susceptible to BET inhibition with lactate utilization inhibitors. Furthermore, these results were translated in vivo as low-dose combination of BETi and oxamate significantly reduced burdens of BETi-resistant cells in murine models (Fig. 1B). These results demonstrate that some AML blasts metabolically adapt to overcome BET inhibition by consuming lactate but that combinations of BETi and lactate utilization inhibitors can stymie this resistance. Recognizing the metabolic switching to utilize lactate in AML therapy resistance, and studying lactate utilization inhibitors in clinical settings, especially in BETi-based therapies should be considered. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of Red Blood Cell Metabolism in Patients with Low-Risk Myelodysplastic Syndrome (LR-MDS): A Proof-of-Concept Study

Low risk myelodysplastic syndromes (MDS) show high clinical heterogeneity ranging from mild anemia with nearly normal performance status to life-threatening cytopenias, frequent hospitalization, and disability. Anemia occurs in 80%-85% of patients and therapeutic strategies are still very limited mainly consisting of of erythropoiesis stimulating agents and red blood cell (RBC) transfusion support. Starting from the working hypothesis that the defective maturation of hematopoietic precursors may dampen the "functionality" of MDS-RBCs, we aimed at evaluating RBC metabolic pathway in anemic patients with LR-MDS. We focused on the glycolytic pathway, which is the prominent metabolic cascade in RBCs and is currently targeted with allosteric stimulators of pyruvate kinase (PK) enzyme in clinical trials of PK deficiency, thalassemia, and sickle cell diseases, which are also marked by ineffective erythropoiesis like MDS. Sixty-six consecutive anemic patients (i.e. Hb<11 g/dL) diagnosed with MDS according to WHO 2016 classification that met IPSS-R criteria for very low, low, or intermediate-risk MDS were enrolled in the study. All patients were sampled for peripheral blood at least 4 weeks after the last RBC unit to reduce possible blood donor contamination. The determination of activities of the glycolytic RBC enzymes pyruvate kinase (PK) and hexokinase (HK) (the terminal and initial steps of the glycolytic pathway), and of ATP levels was performed by standard spectrophotometric method according to Beutler et al, 1984. 2,3 DPG determination was tested by AssayGenie Kit in a subgroup of patients. Ektacytometric analysis by LoRRca MaxSis (Laser-Assisted Optical Rotational Cell Analyzer, Mechatronics, NL) was also performed in a subgroup of patients. As shown in table 1, patients were mainly elderly males, and mostly belonged to IPSS low (78%) and IPSS-R very low/low groups (85%). Median levels of PK activity were 13.7 IU/gHb, with a wide range (7.5-35.9) and were reduced in 23 cases (35%; normal reference range 11.9-16.7 IU/gHb); median values of HK activity were 2.3 (0.8-8.1), increased in 60 patients (91%; normal reference range 0.78-1.32 IU/gHb). This resulted in an abnormally reduced PK/HK ratio (median 5.45, range 2-15) in 63 subjects (95%; (normal reference range calculated on 167 normal subjects: 11.55 - 17.65). To asses if impaired glycolysis may result in altered RBC deformability or in lack of energy, ATP levels, 2,3 DPG, and ektacytometric curve were evaluated. Median ATP levels were 3.4 mcM/gHb (1.3-6.5), reduced in 33/56 tested patients (59%; normal reference range 3.6-4.9 mcM/gHb), of whom 32 had simultaneous reduction of PK/HK activity ratio (figure 1), and 11 decreased PK activity. Concerning 2,3 DPG levels preliminarily analysed in a subgroup of patients, they were close to the upper limit of the normal range (median, 389 nM/gHb, 318-639). Osmoscan, performed in 10 patients did not show altered RBC deformability. Preliminary data show decreased glycolytic activity in RBCs from patients with LR-MDS highlighted by reduced PK activity in more than 1/3 of cases, and increased HK activity in almost all subjects. This results in reduced RBC ATP content in up to 2/3 of patients (possibly due to reduced production or to increased consumption) indicating an overall lack of energy in MDS-RBCs. The latter may be an interesting target for allosteric PK stimulators and warrants further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Attenuation of Graft-Versus-Host-Disease Via Genetic or Pharmacologic Inhibition of the RNA Splicing Factor RBM39

Recent studies have identified pharmacologic modulation of RNA splicing via degradation of the splicing factor RBM39 to mediate anticancer effects and enhance responses to immune checkpoint blockade through generation of novel RNA-splicing derived immunogenic peptides in tumors. However rigorous evaluation of the role of RBM39 in immune cells has not been performed. Here we identify that RBM39 modulates exon recognition in T cells to regulate their function. Although essential for the survival of hematopoietic precursors and tumors, RBM39 is dispensable for peripheral T cell survival. Functional evaluation of the T cell alloantigen response in allogeneic hematopoietic stem transplantation (allo-HSCT) revealed that Rbm39 loss attenuated graft-versus-host-disease (GVHD) and improved overall survival while partially sparing graft-versus-tumor (GVT) effects. To rigorously evaluate the role of RBM39 in immune cells, we generated an Rbm39 conditional knockout mouse by floxing Rbm39 exon 7, which encodes its first RNA recognition motif. Pan-hematopoietic deletion of Rbm39 resulted in rapid complete failure of hematopoiesis and impaired hematopoietic stem cell (HSC) self-renewal. We next analyzed the phenotype of animals with T cell specific deletion of RBM39 and compared this with the effects of pharmacologic RBM39 degradation on T cells. Strikingly, in contrast to the profound effects of genetic RBM39 loss in HSCs and the inability of cancer cells to tolerate RBM39 deletion, RBM39 deletion under CD4-cre (deletion early in thymopoiesis) or distal Lck-cre (late in thymopoiesis) yielded modestly fewer viable peripheral T cells, which skewed towards a CD44+ effector/memory phenotype. This observation was also recapitulated with extended treatment of mice with the RBM39 degrader E7820 (in phase II trials currently; clinicaltrials.gov NCT05024994). Deep RNA-seq analysis of splenic CD3+ T cells from distal Lck-cre Rbm39fl/fl mice revealed widespread decreased efficiency of RNA splicing in Rbm39 null T cells compared to controls, indicative of altered RNA splicing in T cells with Rbm39 deletion. Rbm39 KO T cells experienced global increases in exon skipping at genomic regions where upstream introns had reduced GC content. These data identify that RBM39 is required in a cell and tissue-specific manner for cell survival and further highlights the selectivity of therapeutic approaches targeting RBM39. We next assessed the functional consequences of pharmacologic RBM39 degradation or genetic RBM39 deletion in T cells. Consistent with observations in genetic mouse models, T cells could tolerate extensive pharmacologic RBM39 degradation without loss of proliferative capacity in response to PMA/Ionomycin or anti-CD3+CD28 stimulation. Similarly, genetic Rbm39 null T cells had an intact proliferative response to PMA/Ionomycin. Furthermore, when we evaluated the effects of RBM39 loss in the MHC-mismatched C57BL/6 => Balb/c allo-HSCT system, Rbm39 deletion in donor T cells under either CD4- or distal Lck-cre profoundly reduced GVHD severity and improved overall survival in recipients (Fig. A). These genetic findings were recapitulated via pharmacologic degradation of RBM39 in the same HSCT system (Fig. B). Similar experiments performed in the setting of Balb/c hosts engrafted with A20 lymphoma cells revealed partial preservation of GVT activity using Rbm39 KO T cells. Single cell RNA sequencing analyses of CD4-cre Rbm39fl/fl primary T cells revealed upregulation of the integrin beta1 subunit in both CD4 and CD8 T cells. This suggests alteration of T cell trafficking away from GVHD target organs as one mechanism by which RBM39 loss may alter T cell alloreactivity in allo-HSCT. Our results identify cell-type specific roles for RBM39 in hematopoiesis and immune cells and suggest a therapeutic approach for GVHD via direct modulation of RNA splicing. Of note, as the RBM39 degrader E7820 is currently in phase II clinical trials for relapsed myeloid neoplasms, our previous studies and this data identify a treatment for myeloid neoplasms in the post-transplant setting, where RBM39 degraders mediate both direct and immune-mediated anti-tumor effects while also attenuating GVHD via modulation of RNA splicing in T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intracranial Extramedullary Hematopoiesis: a Rare Entity

Abstract Introduction/Objective Extramedullary hematopoiesis (EMH) is characterized by ectopic proliferation and differentiation of hematopoietic precursor cells outside the bone marrow. In postnatal life, EMH occurs due to hematopoietic disorders, infection or advanced tumors and is usually seen in the spleen, liver, and lymph nodes. Intracranial EMH is an extremely rare entity that has been reported to be associated with hematological disorders, meningioma, subdural hematoma and pilocytic astrocytoma. Methods/Case Report To highlight the significance of identifying EMH as one of the differential diagnoses of brain lesions, we present a 67-year-old female with a history of headaches and generalized malaise. Her past medical history was significant for hypertension and mild anemia. As part of the headache workup, magnetic resonance imaging (MRI) of the brain showed a 3.7cm cystic mass in the right inferomedial parietal lobe with medial mural enhancement, associated with edema and mass effect. A similar 3mm left parietal lobe enhancing focus was also noted. These findings were suggestive of a hemangioblastoma. Resection of the larger mass showed a benign vascular lesion with endothelial hyperplasia without the appearance of cavernous hemangioma or vascular malformation. Foci of erythroid and myeloid precursors, and megakaryocytes were seen. Immunohistochemical stains were negative for GFAP, CK AE1/AE3 and inhibin ruling out glial neoplasms, metastatic carcinoma and hemangioblastoma. Hematopoietic cells were positive for CD45, while immature red cells were positive for E-cadherin and endothelial cells were positive for CD31, consistent with EMH. Further workup was negative for any underlying hematological disorders, resulting in the diagnosis of idiopathic intracranial EMH. The patient received intensity- modulated radiation therapy (IMRT) for her remaining left parietal lesion. After 3 months of regular follow-up, her headache and malaise significantly improved. Results (if a Case Study enter NA) NA. Conclusion To our knowledge, this is the first reported case of idiopathic intracranial EMH.

Receptor activator of nuclear factor-κB ligand-mediated osteoclastogenesis signaling pathway and related therapeutic natural compounds.

Osteoclast is a hematopoietic precursor cell derived from the mononuclear macrophage cell line, which is the only cell with bone resorption function. Its abnormal activation can cause serious osteolysis related diseases such as rheumatoid arthritis, Paget's disease and osteoporosis. In recent years, the adverse effects caused by anabolic anti-osteolytic drugs have increased the interest of researchers in the potential therapeutic and preventive effects of natural plant derivatives and natural compounds against osteolytic diseases caused by osteoclasts. Natural plant derivatives and natural compounds have become major research hotspots for the treatment of osteolysis-related diseases due to their good safety profile and ability to improve bone. This paper provides an overview of recent advances in the molecular mechanisms of RANKL and downstream signaling pathways in osteoclast differentiation, and briefly outlines potential natural compounds with antiosteoclast activity and molecular mechanisms.

Immunostimulatory effects of a subcritical water extract of Ganoderma.

Ganoderma, a medicinal mushroom with various physiological activities, has been extensively investigated regarding its effectiveness. The aim of the present study was to examine the effects of a subcritical water extract of Ganoderma (SWEG) on the immune system. The use of subcritical water with a higher temperature and pressure than hot water allows efficient elution of components from natural products. As an evaluation of the effectiveness of SWEG, a cell proliferation and a cell differentiation test were carried out using A-6 cells, a model of hematopoietic stem cells. Furthermore, an oral administration test in mice was conducted to examine the effects of SWEG on the number and function of immune cells. As a result, SWEG was revealed to promote both self-renewal and differentiation into immune cells such as T cells and natural killer (NK) cells in experiments with A-6 cells. These results were not obtained in experiments using hot water extract of Ganoderma lucidum and Ganoderma sinense. The oral administration test in mice demonstrated that SWEG increased hematopoietic precursor cells, immature B cells, and NK cells in the bone marrow, and T cells in the thymus. In addition, SWEG enhanced the immune functions in the spleen by promoting granzyme B expression and NK cell activity. SWEG was demonstrated to be a food material that acts on HSCs and regulates immunity in vivo.