Marrow CD34 Research Articles

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.

Linear programming based computational technique for leukemia classification using gene expression profile.

Cancer is a serious public health concern worldwide and is the leading cause of death. Blood cancer is one of the most dangerous types of cancer. Leukemia is a type of cancer that affects the blood cell and bone marrow. Acute leukemia is a chronic condition that is fatal if left untreated. A timely, reliable, and accurate diagnosis of leukemia at an early stage is critical to treating and preserving patients' lives. There are four types of leukemia, namely acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic in extracting, and chronic myelogenous leukemia. Recognizing these cancerous development cells is often done via manual analysis of microscopic images. This requires an extraordinarily skilled pathologist. Leukemia symptoms might include lethargy, a lack of energy, a pale complexion, recurrent infections, and easy bleeding or bruising. One of the challenges in this area is identifying subtypes of leukemia for specialized treatment. This Study is carried out to increase the precision of diagnosis to assist in the development of personalized plans for treatment, and improve general leukemia-related healthcare practises. In this research, we used leukemia gene expression data from Curated Microarray Database (CuMiDa). Microarrays are ideal for studying cancer, however, categorizing the expression pattern of microarray information can be challenging. This proposed study uses feature selection methods and machine learning techniques to predict and classify subtypes of leukemia in gene expression data CuMiDa (GSE9476). This research work utilized linear programming (LP) as a machine-learning technique for classification. Linear programming model classifies and predicts the subtypes of leukemia Bone_Marrow_CD34, Bone Marrow, AML, PB, and PBSC CD34. Before using the LP model, we selected 25 features from the given dataset of 22283 features. These 25 significant features were the most distinguishing for classification. The classification accuracy of this work is 98.44%.

Insight into the mechanism of CD34+ cell mobilisation impairment in multiple myeloma patients treated with anti-CD38 therapy.

Induction therapy followed by CD34+ cell mobilisation and autologous transplantation represents standard of care for multiple myeloma (MM). However, the anti-CD38 monoclonal antibodies daratumumab and isatuximab have been associated with mobilisation impairment, yet the mechanism remains unclear. In this study, we investigated the effect of three different regimens (dara-VCd, isa-KRd and VTd) on CD34+ cells using flow cytometry and transcriptomics. Decreased CD34+ cell peak concentration and yields, longer collection and delayed engraftment were reproduced after dara-VCd/isa-KRd versus VTd induction in 34 patients in total. Using flow cytometry, we detected major changes in the proportion of apheresis product and bone marrow CD34+ subsets in patients treated with regimens containing anti-CD38 therapy; however, without any decrease in CD38high B-lymphoid progenitors in both materials. RNA-seq of mobilised CD34+ cells from 21 patients showed that adhesion genes are overexpressed in CD34+ cells after dara-VCd/isa-KRd and JCAD, NRP2, MDK, ITGA3 and CLEC3B were identified as potential target genes. Finally, direct in vitro effect of isatuximab in upregulating JCAD and CLEC3B was confirmed by quantitative PCR. These findings suggest that upregulated adhesion-related interactions, rather than killing of CD34+ cells by effector mechanisms, could be leading causes of decreased mobilisation efficacy in MM patients treated with anti-CD38 therapy.

Deferoxamine promotes recovery of bone marrow hematopoietic function in mice exposed to a sublethal dose of X-ray irradiation

To evaluate the effect of deferoxamine (DFO) on bone marrow hematopoietic function in C57 mice exposed to a sublethal dose of X-ray irradiation. C57 mice exposed to a sublethal dose (5.4 Gy, 1.0 Gy/min) of total body X-ray irradiation (TBI) were treated with subcutaneous injection of 100 mg/kg DFO, with normal saline as the control, on a daily basis for 10 and 20 consecutive days. Body weight changes of the mice were monitored every 3 days. Five mice were selected from each group at 10 and 20 days for examination of blood cell counts, bone marrow nucleated cell counts, percentage of bone marrow CD34+ cells, bone marrow pathology, and expressions of cleaved PARP-1, cleaved caspase-3, VEGF, GPX4, and SLC7A11 in the nucleated cells. The body weight of the mice decreased significantly on day 3 in TBI and DFO groups (P<0.05), and to the lowest on day 6 in TBI group (P<0.01). Blood cell counts and bone marrow nucleated cell counts of the mice were significantly decreased at 10 and 20 days following TBI (P<0.01). On day 10 following TBI, the mice showed significantly decreased nucleated cells and the presence of adipocytes in the bone marrow, where increased expressions of cleaved PARP-1 and cleaved caspase-3 and lowered expressions of GPX4 and SLC7A11 were detected in the nucleated cells (P<0.05). In the mice exposed to TBI, treatment with DFO significantly increased CD34+ cell percentage (P<0.001), decreased the expressions of cleaved PARP-1 and cleaved caspase-3, and increased the expressions of GPX4, SLC7A11 and VEGF in the bone marrow nucleated cells (P<0.05). DFO treatment significantly increased blood cell counts and bone marrow nucleated cells in mice at 20 days following TBI (P<0.05). DFO improves bone marrow hematopoiesis in mice with sublethal-dose TBI by inhibiting apoptosis and ferroptosis of bone marrow nucleated cells and promoting VEGF expression and CD34+ cell proliferation.

Host CD34+ cells are replacing donor endothelium of transplanted heart

Endothelium dysfunction is a central problem for early rejection due to the host alloimmune response and the late status of arteriosclerosis in heart transplantation. However, reliable pieces of evidence are still limited concerning the source of the regenerated endothelium within the transplanted heart. We analyzed single-cell RNA sequencing data and constructed an inducible lineage tracing mouse, combined heart transplantation with bone marrow transplantation and a parabiosis model, cellular components, and endothelial cell populations in cardiac graft lesions. Our single-cell RNA sequencing analysis of a transplanted heart allowed for the establishment of an endothelial cell atlas with a heterogeneous population, including arterial, venous, capillary, and lymphatic endothelial cells. Along with genetic cell lineage tracing, we demonstrated that the donor cells were mostly replaced by recipient cells in the cardiac allograft, up to 83.29% 2weeks after transplantation. Furthermore, recipient nonbone marrow CD34+ endothelial progenitors contributed significantly to extracellular matrix organization and immune regulation, with higher apoptotic ability in the transplanted hearts. Mechanistically, peripheral blood-derived human endothelial progenitor cells differentiate into endocardial cells via Vascular endothelial growth factor receptor-mediated pathways. Host circulating CD34+ endothelial progenitors could repair the damaged donor endothelium presumably through CCL3-CCR5 chemotaxis. Partial depletion of host CD34+ cells resulted in delayed endothelial regeneration. We created an annotated fate map of endothelial cells in cardiac allografts, indicating how recipient CD34+ cells could replace the donor endothelium via chemokine CCL3-CCR5 interactions. The mechanisms we discovered could have a potential therapeutic effect on the long-term outcomes of heart transplantation.

Transcriptomic comparison of bone marrow CD34 + cells and peripheral blood neutrophils from ET patients with JAK2 or CALR mutations

BackgroundEssential thrombocythemia (ET) is one of the most common types of Ph-negative myeloproliferative neoplasms, an infrequent group of blood cancers that arise from a CD34 + hematopoietic stem cell (HSC) in the bone marrow (BM) primarily due to driver mutations in JAK2, CALR or MPL. These aberrations result in an overproduction of mature myeloid cells in peripheral blood (PB). To date, no targeted therapies have been approved for ET patients, so the study of the molecular mechanisms behind the disease and the identification of new therapeutic targets may be of interest. For this reason, in this study, we have compared the transcriptomic profile of undifferentiated CD34 + cells and mature myeloid cells from ET patients (CALR and JAK2-mutated) and healthy donors deposited in publicly available databases. The study of the similarities and differences between these samples might help to better understand the molecular mechanisms behind the disease according to the degree of maturation of the malignant clone and the type of mutation and ultimately help identify new therapeutic targets for these patients.ResultsThe results show that most of the altered hallmarks in neutrophils were also found in CD34 + cells. However, only a few genes showed a similar aberrant expression pattern in both types of cells. We have identified a signature of six genes common to patients with CALR and JAK2 mutations (BPI, CRISP3, LTF, MMP8, and PTGS1 upregulated, and PBXIP1 downregulated), a different signature of seven genes for patients with CALR mutations (BMP6, CEACAM8, ITK, LCN2, and PRG2 upregulated, and MAN1A1 and MME downregulated) and a signature of 13 genes for patients with JAK2 mutations (ARG1, CAST, CD177, CLEC5A, DAPP1, EPS15, IL18RAP, OLFM4, OLR1, RIOK3, SELP, and THBS1 upregulated, and IGHM downregulated).ConclusionsOur results highlight transcriptomic similarities and differences in ET patients according to the degree of maturation of the malignant clone and the type of mutation. The genes and processes altered in both CD34 + cells and mature neutrophils may reveal altered sustained processes that could be studied as future therapeutic targets for ET patients.

Upregulation of S100A6 and its relation with CD34+ cells apoptosis in high-risk myelodysplastic syndromes patients

ObjectivesMyelodysplastic syndromes (MDS) are a group of myeloid malignancies characterized by peripheral blood cytopenia and hematopoietic dysplasia that often progress to acute myeloid leukemia (AML). Increased apoptosis of normal hematopoietic cells and decreased apoptosis of malignant clonal hematopoietic cells in patients with MDS is some of the mechanisms leading to ineffective hematopoietic cells in the bone marrow. S100 calcium-binding protein A6 (S100A6) is upregulated in many malignancies. The overexpression of S100A6 in these malignancies has been associated with proliferation, migration, and invasion phenotypes in cancer cells, and we aimed to investigate the expression of S100A6 in CD34+ cells and the relationship between S100A6 expression and apoptosis of CD34+ cells in high-risk patients with MDS. MethodsWe measured S100A6 mRNA expression in bone marrow (BM) CD34+ cells from high-risk patients with MDS using RT-PCR. Next, we examined S100A6 expression in CD34+ cells using flow cytometry. We also analyzed the correlation between CD34+ cell apoptosis and S100A6 expression in high-risk patients with MDS. ResultsOur data showed increased S100A6 mRNA expression in CD34+ cells in patients with MDS (1.05 ± 0.69 vs. 0.17 ± 0.12; P＜0.01). The expression of S100A6 in BM CD34+ cells also increased (58.40 ± 13.18 vs. 45.83 ± 15.01). The expression of S100A6 in CD34+ cells and apoptosis of CD34+ cells were negatively correlated in patients (r = −0.75; P < 0.01). ConclusionsCollectively, S100A6 may be a potential marker of CD34+ cells in high-risk patients with MDS and may participate in the pathological behaviors of CD34+ cells, such as evasion of apoptosis. Thus, S100A6 may be a potential target for eliminating minimal residual disease.

Integrated Genomic and Transcriptomic Analysis Improves Disease Classification and Risk Stratification of MDS with Ring Sideroblasts.

Ring sideroblasts (RS) define the low-risk myelodysplastic neoplasm (MDS) subgroup with RS but may also reflect erythroid dysplasia in higher risk myeloid neoplasm. The benign behavior of MDS with RS (MDSRS+) is limited to SF3B1-mutated cases without additional high-risk genetic events, but one third of MDSRS+ carry no SF3B1 mutation, suggesting that different molecular mechanisms may underlie RS formation. We integrated genomic and transcriptomic analyses to evaluate whether transcriptome profiles may improve current risk stratification. We studied a prospective cohort of MDSRS+ patients irrespective of World Health Organization (WHO) class with regard to somatic mutations, copy-number alterations, and bone marrow CD34+ cell transcriptomes to assess whether transcriptome profiles add to prognostication and provide input on disease classification. SF3B1, SRSF2, or TP53 multihit mutations were found in 89% of MDSRS+ cases, and each mutation category was associated with distinct clinical outcome, gene expression, and alternative splicing profiles. Unsupervised clustering analysis identified three clusters with distinct hemopoietic stem and progenitor (HSPC) composition, which only partially overlapped with mutation groups. IPSS-M and the transcriptome-defined proportion of megakaryocyte/erythroid progenitors (MEP) independently predicted survival in multivariable analysis. These results provide essential input on the molecular basis of SF3B1-unmutated MDSRS+ and propose HSPC quantification as a prognostic marker in myeloid neoplasms with RS.

5612617 EFFICACY AND SAFETY OF A SINGLE DOSE OF EXAGAMGLOGENE AUTOTEMCEL FOR TRANSFUSION-DEPENDENT-THALASSEMIA AND SEVERE SICKLE CELL DISEASE

Background: Elevated fetal hemoglobin(HbF) is associated with improved outcomes in patients(pts) with transfusion-dependent β-thalassemia(TDT) and sickle cell disease(SCD). Exagamglogene autotemcel(exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells(HSPCs). Early data from the pivotal trials CLIMB THAL-111(NCT03655678) and CLIMB SCD-121(NCT03745287) showed that a single dose of exa-cel increased HbF and total Hb sufficiently to eliminate red blood cell(RBC) transfusions(TDT) and vaso-occlusive crises(VOCs; SCD). Aims: To report efficacy and safety data from the first 75 pts dosed with exa-cel in the ongoing CLIMB THAL-111 and CLIMB SCD-121 trials. Methods: Following pharmacokinetic-adjusted busulfan myeloablation and exa-cel infusion, pts(12-35y) are monitored for engraftment, total Hb, HbF, BCL11A edited alleles, transfusions, VOCs(SCD only), and adverse events(AEs). Data presented as mean(min-max) unless noted. Results: 44 pts with TDT(age 21.3 [12-35] y) and 31 pts with SCD(age 22.5 [12-34} y) had been infused with exa-cel at data cut(follow-up 12.3 [1.2-37.2] mo and 9.6 [2.0-32.3] mo, respectively). 26/44 pts with TDT(59.1%) had β0/β0 or a β0/β0-like genotype(β0/IVS-I-110, IVS-I-110/IVS-I-110). In the 2-yr period before screening, pts with TDT received 36.0(15.0-71.0) units RBC/yr and pts with SCD had 3.9(2.0-9.5) severe VOCs/yr. After exa-cel infusion, all pts engrafted neutrophils and platelets. Median time to neutrophil and platelet engraftment was 29 and 43 days in pts with TDT and 27 and 32 days in those with SCD, respectively. Overall, 42 of 44 pts with TDT stopped RBC transfusions(duration 0.8 to 36.2 mo); 2 pts had not yet stopped transfusions but had 75% and 89% reductions in transfusion volume. By Month 3, increases in HbF and mean total Hb levels(>9 g/dL) were achieved, with mean total Hb levels increasing to >11 g/dL thereafter and were maintained. All pts with SCD(n=31) no longer had severe VOCs after exa-cel infusion(duration 2.0 to 32.3 mo). The mean proportion of HbF was >20% by Month 3, increasing to ~40% at Month 4 and was stable thereafter, with mean total Hb levels >11 g/dL after Month 3. Pts with TDT and SCD with ≥1 yr follow-up had stable proportions of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells. Two pts with TDT had serious AEs(SAEs) considered related to exa-cel. The first pt was previously reported. The second pt had delayed neutrophil engraftment and thrombocytopenia, which were considered related to both exa-cel and busulfan. All SAEs resolved. No pts with SCD had SAEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to elimination of transfusions in almost all patients with TDT and elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles remained stable after more than 1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with busulfan myeloablation and autologous transplant. These results indicate exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for TDT and severe SCD. Reference 1. Frangoul et al, N Engl J Med 2021; 384:252-260.

Bone marrow CD34+ molecular chimerism as an early predictor of relapse after allogeneic stem cell transplantation in patients with acute myeloid leukemia.

Minimal residual disease (MRD) monitoring is an important tool to optimally address post-transplant management of acute myeloid leukemia (AML) patients. We retrospectively analyzed the impact of bone marrow CD34+ molecular chimerism and WT1 on the outcome of a consecutive series of 168 AML patients submitted to allogeneic stem cell transplantation. The cumulative incidence of relapse (CIR) was significantly lower in patients with donor chimerism on CD34+ cells ≥ 97.5% and WT1 < 213 copies/ABL x 10^4 both at 1st month (p=0.008 and p<0.001) and at 3rd month (p<0.001 for both). By combining chimerism and WT1 at 3rd month, 13 patients with chimerism < 97.5% or WT1 > 213 showed intermediate prognosis. 12 of these patients fell in this category because of molecular chimerism < 97.5% at a time-point in which WT1 was < 213. Our results confirm that lineage-specific molecular chimerism and WT1 after allo-SCT (1st and 3rd month) are useful MRD markers. When considered together at 3rd month, CD34+ molecular chimerism could represent an earlier predictor of relapse compared to WT1. Further studies are necessary to confirm this preliminary observation.

The density of bone marrow mononuclear cells and CD34+ cells in patients with three neurologic conditions

BackgroundThis study aimed to identify the density of mononuclear cells (MNCs) and CD34+ cells in the bone marrow of patients with three neurologic conditions.MethodsThe study included 88 patients with three neurologic conditions: 40 with cerebral palsy (CP) due to oxygen deprivation (OD), 23 with CP related to neonatal icterus (NI), and 25 with neurological sequelae after traumatic brain injury. Bone marrow aspiration was conducted from the patients’ bilateral anterior iliac crest under general anesthesia in an operating theater. MNCs were isolated by Ficoll gradient centrifugation and then infused intrathecally.ResultsThere was a significant difference in the average MNC per ml and percentage of CD34+ cells by the type of disease, age group, and infusion time (p value < 0.05). The multivariable regression model showed the percentage of CD34+ association with the outcome (gross motor function 88 items- GMFM-88) in patients with CP.ConclusionsThe density of MNCs was 5.22 million cells per mL and 5.03% CD34+ cells in patients with three neurologic conditions. The highest density of MNCs in each ml of bone marrow was found in patients with CP due to OD, whereas the percentage of CD34+ cells was the highest among patients with CP related to NI.

TET1-mediated microRNA-188-5p promoter hydroxymethylation regulates PTEN/PI3K/AKT signaling pathway in acute myeloid leukemia cells

ObjectiveAcute myeloid leukemia (AML) remains a common hematopoietic malignancy, and drug resistance greatly blunts the efficacy of chemotherapy in AML treatment. Adriamycin (ADM, also called doxorubicin), is one of the most widely used chemotherapeutics for treating cancers. Herein, we studied the molecular mechanisms underlying microRNA-188-5p (miR-188-5p)-mediated ADM resistance in AML. MethodsDifferentially expressed miRNAs were screened in normal and malignant hematopoietic cells by bioinformatics tools. MiR-188-5p expression in primary bone marrow CD34+ cells and AML cells was evaluated. AML/ADM cells were established using THP-1 and Kasumi-1 cells. The effect of miR-188-5p on the drug resistance in AML/ADM cells was examined by delivery of miR-188-5p-inhibitor. The binding relationship between TET1 and miR-188-5p was analyzed by ChIP, and the downstream target of miR-188-5p was predicted by bioinformatics analysis and validated by dual-luciferase assay. Finally, rescue experiments were carried out in vitro and in vivo. ResultsmiR-188-5p was highly expressed in AML cells, and miR-188-5p-inhibitor sensitized the AML/ADM cells to ADM. Inhibition of TET1 reduced miR-188-5p promoter hydroxymethylation and downregulated miR-188-5p. miR-188-5p bound to the 3′UTR of PTEN to inhibit PTEN expression, and the PI3K/AKT signaling was activated upon inhibition of PTEN. Suppression of PTEN conferred resistance again to AML/ADM cells in the presence of miR-188-5p inhibitor. ConclusionTET1 elevates miR-188-5p expression by promoting miR-188-5p promoter hydroxymethylation, and miR-188-5p inhibits PTEN expression to induce PI3K/AKT signaling pathway activation, leading to ADM resistance in AML.

Uncovering Transcriptome Characteristics in Bone Marrow Hematopoiesis in Immune Thrombocytopenia at Single-Cell Resolution

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we hypothesized that bone marrow (BM) CD34+ hematopoietic stem and progenitor cells (HSPCs) participated in the defective megakaryogenesis in ITP patients, and we performed single-cell RNA-seq (scRNA-seq) of BM CD34+ HSPCs to reveal the overall transcriptome alterations in HSPCs in ITP. Dimensional reduction using uniform manifold approximation and projection (UMAP) showed the effective integration of the datasets from different samples. We manually annotated the cell clusters into 17 different cell types with distinct gene expression patterns. By performing differential expression analysis, enrichment analysis, trajectory analysis, transcriptional regulatory network analysis and cell-cell interaction analysis, we found that gene expression, transcriptional regulatory networks and cell-cell interactions varied in HSPCs of ITP, particularly in a pre-B cell population and natural killer/T cell progenitors, highlighting the selective immune aberration associated with defective megakaryopoiesis in ITP. Differentially expressed gene (DEG) analysis indicated that the top upregulated genes in a megakaryocytic progenitors (MkP) population of ITP were associated with erythropoiesis, suggesting a bias toward erythrocytes in megakaryopoiesis in ITP. Moreover, Flow cytometry confirmed that the number of CD9+ cells from CD34+ HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+ CD34+ HSPCs from healthy controls tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients. The percentage of megakaryocytes differentiated from CD9+CD34+ HSPCs was higher than that of the CD9- counterparts from healthy controls, whereas in ITP patients, the percentage decreased to only 1/4th of that in the healthy controls and was comparable to that from the CD9- HSPCs, indicating that differentiation of CD9+ HSPCs toward the megakaryopoietic lineage was impaired in ITP. Further analysis revealed the universality of their heterogeneity and provided an analytical basis for the study of megakaryocyte-related diseases, including ITP. Enrichment analysis confirmed the definition of MkP in our ITP integrated dataset. To further analyze the transcriptional difference between ITP and HC, we pooled MkP from all samples. The expression of several molecular features of MkP was visualized, and seven transcriptionally heterogeneous subpopulations of MkP were identified. We then assessed the expression of marker genes in each subpopulation and characterized the gene sets enriched in different clusters, which showed pronounced heterogeneity within MkP, consistent with the results of previous analyses of MkP/Mk from multi-hematopoietic tissues. We evaluated ITP-associated DEGs in each MkP subcluster, and we found that most of these DEGs were subtype-specific, indicative of the subtype-specific effects of the disorder. In summary, this study dissects the significant heterogeneity and disease characteristics of BM CD34+ HSPCs from ITP patients at single-cell resolution, revealing new insights regarding the pathophysiology of ITP.

Correlation of Clonal Hematopoiesis in Vexas Syndrome with Immunophenotype Alterations in CD34+ HSPCs (Hematopoietic Stem Progenitors Cells): Multiparametric Flow Cytometry Heterogeneity in CD34+CD38- Stem Cells Fraction and Upregulation of CD90 Thy-1 By Unsupervised Strategy Using PCA, Tsnee and Flowsom

Introduction: VEXAS (vacuoles,E1enzyme,X-linked, autoinflammatory,somatic) syndrome is caused by somatic UBA1mutations affecting myeloid compartment of hematopoietic stem and progenitor cells (HSPC).VEXAS syndrome is a unique systemic autoinflammatory disease associated with cytopenias with marrow dysplasia(1).There is currently no published data concerning the immunophenotypic characteristics pattern of CD34+ HSC in UBA1 mutated patients w/wo MDS.The identification of an easily-accessible profile in CD34+CD38- most immature HSC for UBA1 mutations might help to identify UBA1 patients beside molecular testing and potential target of therapeutic interest. Methods: Here we report flow cytometry of bone marrow CD34+ HSPCs in a cohort of 11 UBA1 mutated patients from the Lyon University Hospital. Our routine 2-tubes panel used in the follow-up of AML MRD, were applied with minimal mandatory 8 markers per tube, according to ELN recommendations (to identify the pattern of LAIP/DfN in bulk and LSC in CD34+CD38- fraction). A «backbone» of CD34/CD38/CD45/CD117 , and CD7,CD56, CD13, CD33, CD19 for the 1st tube and CD90,CLL1+TIM3+CD97, CD123, CD45RA for the 2nd one. Acquired data in BD Canto and Lyric cytometers were analysed using DIVA/FlowJo and KALUZA softwares for classical strategy completed with Tsnee and FlowSom software for unsupervised analyse. Control group was healthy donor or regenerating marrow and MDS patients UBA1 negative. A total of minimal 500000 to one million of cells was acquired in each tube. Results: The clinical and biological characteristics of the 11 UBA1 mutated patients are summarized in Fig1A. Parameters as MFI in cell lineage and LSC markers, percentage of targeted populations (lympho-myeloid primed progenitor (LMPP), Multipotential Progenitor (MPP), Common myeloid progenitor (CMP), granulocyte/monocyte progenitor (GMP) and megakaryocyte/erythroid progenitor (MEP), were integrated in principal component analysis (PCA) (Fig1B). PCA analysed compared our VEXAS cohort with normal marrow (n=10) and MDS patients without UBA1 mutation (n=23), and clusterized properly UBA1 mutated patients from normal marrowand MDS patients. The most contributive variables for appropriate clusterizing were LSC/progenitors markers CD90 Thy1/ CD45RA and CLL1/TIM3 /CD97, CD123 and GMP, MEP, MPP, LMPP-like populations. Interestingly, all normal marrow clusterized together, highly separated from UBA1 mutated cases. However, MDS UBA1 negative patients were heterogeneously distributed, suggesting a strong variability in CD34+ fraction. No lineage marker (i.e. CD13) or aberrant markers (i.e. CD7) was involved in PCA clustering, suggesting that alteration in LSC:HSCP fractions are mostly relevant for distinction between clonal haematopoiesis in UBA1 mutated patients compared to MDS UBA1 negative and normal marrow. A correlation matrix showed a close correlation between LSC/progenitors markers and CD34+CD38- frequency, mostly based on CD90/Thy-1 overexpressed in clonal hematopoiesis observed in UBA1 mutated patients (Fig 1C). We merged data from 2 UBA1 mutated, 2 MDS UBA1 negative and 4 healthy donors to perform unsupervised analysis Tsnee and FlowSom (Fig1D). Interestingly, both UBA1 mutated patients showed different pattern in CD34+ compared to MDS and normal healthy donors suggesting most primitive alteration in CD90 Thy-1 expression in CD34+CD38-cells. Flowsom map allowed to identify nodes of stem cell with abnormal phenotype clearly different in UBA1 mutated patients, backgating of this node showed the overexpression of CD90 in CD34+CD38- cells in UBA1 pos. Conclusions: This is the first large immunophenotypic characterisation by flow of clonal haematopoiesis in UBA1 mutated patients. Our results showed that VEXAS syndrome is characterized by overexpression of CD90 Thy-1 primitive Stem cell markers in CD34+ HSPCs and in most immature CD34+CD38- fraction. Altogether, we can hypothesize that such phenotype might represent specific disease hallmark involved in physiopathology of niche deregulation, which should be confirmed in a larger cohort of patients in a future multicentric clinical trials to improve biological diagnosis and comprehension of kinetics of clonal haematopoiesis in VEXAS patients. 1) Beck DB, Ferrada MA, Sikora KA, et al. Somatic mutations in UBA1 and severe adult-onset autoinflammatory disease. N Engl J Med. 2020;383:2628-2638. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment with Oral Azacitidine in Elderly Patients with Higher Risk Myelodysplastic Syndromes after Response to Subcutaneous Azacitidine

Background Hypomethylating agents (HMAs) are the standard of care for (elderly) IPSS-R higher risk MDS (HR-MDS) patients, but HMA treatment for those patients who are not eligible for hematopoietic stem cell transplant has to be continued for very long time to maintain efficacy. This prolonged continuous treatment renders patients dependent on hospital and on caregivers, and as a result, decreases QoL and too often provokes premature interruption of therapy. Oral azacitidine is approved for maintenance therapy of acute myeloid leukemia patients in complete remission after intensive chemotherapy, and it has shown activity in lower risk MDS. Oral decitabine has been recently approved by FDA for treatment of MDS. There are no data regarding maintenance of response achieved by sc azacitidine in HR MDS by switching to the oral formulation. Aim The primary objective of the study is to explore the feasibility of replacing subcutaneous (sc) azacitidine by the oral formulation, in patients with HR-MDS and responding to sc azacitidine as well as the maintenance or improvement of the achieved hematological response (time to relapse/progression).Methods A monocentric, pilot phase 2 study (ClinicalTrials.gov: NCT04806906) was planned to enroll 10 subjects with confirmed diagnosis of MDS or dysplastic non proliferative CMML, IPSS-R higher risks, aged ≥ 65 years, in CR/CRi, PR or SD with HI stable for additional 2 cycles after response at 4-6 cycles of azacitidine therapy, hence a total of at minimum 6 to 8 cycles of sc azacitidine. At screening, morphological MDS diagnosis, cytogenetic analysis, IWG response assessment, bone marrow and peripheral blood sampling for flow cytometric analysis, NGS evaluation of somatic mutations, DNA methylation analysis and HRQoL are performed. During treatment, subjects are assessed for safety, tolerability and efficacy of oral azacitidine. Assessments during treatment include monitoring for AEs, monitoring for loss of response and SPMs, physical examination, vital signs and weight measurement, ECOG performance status, hematology and serum chemistry, serum ferritin level, concomitant medications, therapies and procedures, review of bone marrow aspirate or biopsy. Patients receive 300 mg oral azacitidine for the first 14 days of each 28-day treatment cycle until no longer receiving the benefit or until progression. Dose of oral azacitidine can be de-escalated based on toxicity. Besides feasibility of treatment with oral azacitidine, we evaluate safety/tolerability, time to treatment discontinuation, and patient-reported outcomes, utilizing the EQ-5D, and measures of healthcare resource utilization. DNA methylation pattern of bone marrow CD34 positive cells at diagnosis, after response to sc azacitidine, and after 2 and 4 cycles of oral azacitidine is also determined by ERRBS for all patients. Results As per August 1, 2022, with a follow up ranging from 1-10 months, 5 HR MDS patients have been enrolled in the study. Median age is 81 yrs (79-86). Male/female ratio: 4/1. At start of therapy with sc azacitidine IPSS-R risk was high 3/5, very high 2/5. Median number of cycles of sc azacitidine is 25 (7-56), median number of cycles of oral azacitidine is 4 (1-9). The dose of 300mg/day was maintained for all patients. Pattern of DNA methylation determined by ERRBS is available for all treated patients and confirms the activity of oral azacitidine. Two patients evolved to AML after 9 and 4 cycles of oral azacitidine, with progression free survival (PFS) from onset of therapy of 17 and 12 months respectively. None of those 2 patients had adverse events during therapy. Treatment was interrupted after 2 cycles in 1 patient following a GI event grade 3 and following patient decision; 2 patients are still ongoing without report of hematological or non-hematological toxicity. At present 1/5 patients died with AML. Conclusions. The patients included in this study were HR MDS, particularly frail, not eligible to investigational studies because of poor compliance and very old median age. The administration of oral azacitidine in patients who responded to sc azacitidine is feasible, the treatment is very well tolerated with no AEs different from those observed in sc azacitidine treatment. Two of five treated patients experienced progressive disease after >12 months of HMA therapy from diagnosis. Oral azacitidine may allow prolonged and continuous treatment in frail HR MDS, optimizing outcome.

Efficacy and Safety of a Single Dose of Exagamglogene Autotemcel for Severe Sickle Cell Disease

Background: Elevated fetal hemoglobin (HbF) is associated with improved outcomes in patients with sickle cell disease (SCD). Exagamglogene autotemcel (exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). Recent data from the pivotal CLIMB SCD-121 (NCT03745287) trial showed that a single dose of exa-cel increased HbF and total hemoglobin (Hb) sufficiently to eliminate vaso-occlusive crises (VOCs) in patients with severe SCD. Here, we report efficacy and safety data from the first 31 patients dosed with exa-cel in the ongoing CLIMB SCD-121 trial. Methods: Patients aged 12 to 35 years with severe SCD and a history of ≥2 VOCs per year in the previous 2 years before screening were eligible. Following pharmacokinetic-adjusted, busulfan myeloablation and infusion of exa-cel, patients are monitored for engraftment, total Hb, HbF, BCL11A-edited alleles, transfusions, VOCs, and adverse events (AEs). The primary endpoint is the proportion of patients who have not experienced a severe VOC for at least 12 months after the infusion of exa-cel, starting 60 days after their last RBC transfusion. Updated efficacy and safety results on all dosed patients will be included in the presentation. Data are reported as mean (min-max), unless noted. Results: At the most recent data cut in February 2022, 31 patients with SCD (age 22.5 [12-34] years) had been infused with exa-cel (follow-up 9.6 [2.0-32.3] months), of whom 6 (19.4%) were between the ages of 12 and <18 years and 29 (93.5%) had the βs/βs genotype. In the 2-year period before screening, patients experienced 3.9 (2.0-9.5) severe VOCs per year. After exa-cel infusion, all patients engrafted neutrophils and platelets with a median time of 27 and 32 days, respectively. All patients were VOC-free at the time of the data cut (duration of follow-up 2.0-32.3 months after exa-cel infusion; Figure). Median time from exa-cel infusion to last RBC transfusion was 19 (11-52) days. The mean proportion of HbF was >20% by Month 3, with mean total Hb levels >11 g/dL on and after Month 3. All 11 patients who have at least 12 months of follow-up after exa-cel infusion have maintained HbF levels >20% while experiencing no VOCs. At Month 6, the mean proportion of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells was 86.6% and 76.0%, respectively. These proportions remained stable in all patients who had ≥1 year of follow-up (Figure). There were no patients who had serious AEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to the elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles have remained stable after ≥1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with that of busulfan myeloablation and autologous transplant. Exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for SCD. Figure. Clinical data for patients with SCD (N=31) infused with exa-cel. Patients were VOC-free after exa-cel infusion. Time (months) since exa-cel infusion is indicated by the dark blue bar. Inset depicts the percentage of BCL11A-edited alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells of patients with SCD infused with exa-cel over time. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD59-Regulated Ras Compartmentalization Orchestrates Antitumor T-cell Immunity.

T cell-mediated immunotherapy represents a promising strategy for cancer treatment; however, it has achieved satisfactory clinical responses in only a limited population. Thus, a broader view of the T-cell immune response is required. The Ras/MAPK pathway operates in many important signaling cascades and regulates multiple cellular activities, including T-cell development, proliferation, and function. Herein, we found that the typical membrane-bound complement regulatory protein CD59 is located intracellularly in T cells and that the intracellular form is increased in the T cells of patients with cancer. When intracellular CD59 is abundant, it facilitates Ras transport to the inner plasma membrane via direct interaction; in contrast, when CD59 is insufficient or deficient, Ras is arrested in the Golgi, thus enhancing Ras/MAPK signaling and T-cell activation, proliferation, and function. mCd59ab deficiency almost completely abolished tumor growth and metastasis in tumor-bearing mice, in which CD4+ and CD8+ T cells were significantly increased compared with their proportions in wild-type littermates, and their proportions were inversely correlated with tumor growth. Using bone marrow transplantation and CD4+ and CD8+ T-cell depletion assays, we further demonstrated the critical roles of these cells in the potent antitumor activity induced by mCd59ab deficiency. Reducing CD59 expression also enhanced MAPK signaling and T-cell activation in human T cells. Therefore, the subcellular compartmentalization of Ras regulated by intracellular CD59 provides spatial selectivity for T-cell activation and a potential T cell-mediated immunotherapeutic strategy.

Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in immune cell progenitors. Differentially expressed gene (DEG) analysis indicated that there was an impaired megakaryopoiesis of ITP. Flow cytometry confirmed that the number of CD9+ and HES1+ cells from Lin−CD34+CD45RA− HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+Lin−CD34+CD45RA− HSPCs tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients and more erythrocytes were produced. The percentage of megakaryocytes differentiated from CD9+Lin−CD34+CD45RA− HSPCs was 3-fold higher than that of the CD9− counterparts from healthy controls (HCs), whereas, in ITP patients, the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9− HSPCs. Additionally, when co-cultured with pre-B cells from ITP patients, the differentiation of CD9+Lin−CD34+CD45RA− HSPCs toward the megakaryopoietic lineage was impaired. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. This study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.

Subclones of bone marrow CD34+ cells in acute myeloid leukemia at diagnosis confer responses of patients to induction chemotherapy.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with a prognosis that varies with genetic heterogeneity of hematopoietic stem/progenitor cells (HSPCs). Induction chemotherapy with cytarabine and anthracycline has been the standard care for newly diagnosed AML, but about 30% of patients have no response to this regimen. The resistance mechanisms require deeper understanding. In our study, using single-cell RNA sequencing, we analyzed the heterogeneity of bone marrow CD34+ cells from newly diagnosed patients with AML who were then divided into sensitive and resistant groups according to their responses to induction chemotherapy with cytarabine and anthracycline. We verified our findings by TCGA database, GEO datasets, and multiparameter flow cytometry. We established a landscape for AML CD34+ cells and identified HSPC types based on the lineage signature genes. Interestingly, we found a cell population with CRIP1high LGALS1high S100Ashigh showing features of granulocyte-monocyte progenitors was associated with poor prognosis of AML. And two cell populations marked by CD34+ CD52+ or CD34+ CD74+ DAP12+ were related to good response to induction therapy, showing characteristics of hematopoietic stem cells. Our study indicates the subclones of CD34+ cells confers for outcomes of AML and provides biomarkers to predict the response of patients with AML to induction chemotherapy.

Dioscin Regulating Bone Marrow Apoptosis in Aplastic Anemia.

BackgroundAplastic anemia (AA), a disease of bone marrow failure, is caused by CD8+T mediated apoptosis of hematopoietic cells. However, traditional immunosuppressive therapy (IST) has severe liver and kidney toxicity and even cannot achieve the expected therapeutic effect in some patients.PurposeOur study is aimed to investigate the effect and mechanism of dioscin (DNS) for treating AA.MethodsBriefly, we established and evaluated the AA mouse model, DNS and positive control drugs were used for intervention treatment. After 14 days of intervention, femoral bone marrow pathology, bone marrow mononuclear cells (BMMCs) apoptosis rate, bone marrow CD34+ cell surface Fas (CD95) expression and Fas signaling pathway key proteins were detected.ResultsAfter the establishment of the AA mouse model, the number of peripheral blood cells including granulocytes, erythrocytes, hemoglobin, platelets and reticulocytes in the AA group model was significantly decreased compared with the group control (P < 0.01). The degree of bone marrow hyperplasia in the sternum and femur is extremely low. After different drug interventions, compared with the group model, the number of peripheral blood cells in the AA mice rebounded significantly in group DNS (P < 0.01). Not only that the apoptosis rate of BM-MCs decreased (P < 0.01), meanwhile, the CD95 molecule expressed on the CD34+ bone marrow cells had a significant decline (P < 0.01), and the expression level of the key proteins of Fas signaling pathway was also significantly decreased (P < 0.01).ConclusionDNS recovered the peripheral pancytopenia and bone marrow failure in AA mice. DNS reduced the key protein of Fas signaling pathway level to inhibit apoptosis of bone marrow cells to treat AA.