Human Bone Marrow Cells Research Articles

Identification of CD141+vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells

BackgroundCritical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI.MethodsEx vivo cultured VPCs and BM-MSCs from bone marrow were characterized and their effects on neovascularization and long-term tissue regeneration were tested in a mouse CLI model.ResultsVPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity in vitro but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141+VPCs are present in BM aspirates as a rare population and can be expanded ex vivo with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141+VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 and 12 weeks, likely indicating their lineage commitment to endothelial cells and vascular smooth muscle, respectively, in vivo.ConclusionDual-cell therapy using BM-derived CD141+ VPCs and BM-MSCs holds potential for further development in clinical trials to treat peripheral artery disease and diabetic ulcers.

In vitro models of the crosstalk between multiple myeloma and stromal cells recapitulate the mild NF-κB activation observed in vivo

Multiple myeloma (MM) is linked to chronic NF-κB activity in myeloma cells, but this activity is generally considered a cell-autonomous property of the cancer cells. The precise extent of NF-κB activation and the contributions of the physical microenvironment and of cell-to-cell communications remain largely unknown. By quantitative immunofluorescence, we found that NF-κB is mildly and heterogeneously activated in a fraction of MM cells in human BMs, while only a minority of MM cells shows a strong activation. To gain quantitative insights on NF-κB activation in living MM cells, we combined advanced live imaging of endogenous p65 Venus-knocked-in in MM.1S and HS-5 cell lines to model MM and mesenchymal stromal cells (MSCs), cell co-cultures, microfluidics and custom microbioreactors to mimic the 3D-interactions within the bone marrow (BM) microenvironment. We found that i) reciprocal MM-MSC paracrine crosstalk and cell-to-scaffold interactions shape the inflammatory response in the BM; ii) the pro-inflammatory cytokine IL-1β, abundant in MM patients’ plasma, activates MSCs, whose paracrine signals are responsible for strong NF-κB activation in a minority of MM cells; iii) IL-1β, but not TNF-α, activates NF-κB in vivo in BM-engrafted MM cells, while its receptor inhibitor Anakinra reduces the global NF-κB activation. We propose that NF-κB activation in the BM of MM patients is mild, restricted to a minority of cells and modulated by the interplay of restraining physical microenvironmental cues and activating IL-1β-dependent stroma-to-MM crosstalk.

Erythropoietin-dependent Acquisition of CD71 hi CD105 hi Phenotype within CD235a - Early Erythroid Progenitors.

The development of committed erythroid progenitors and their continued maturation into mature erythrocytes requires the cytokine erythropoietin (Epo). Here, we describe the immunophenotypic identification of a unique Epo-dependent colony-forming unit-erythroid (CFU-E) cell subtype that forms during early erythropoiesis (EE). This previously undescribed CFU-E subtype, termed late-CFU-E (lateC), lacks surface expression of the characteristic erythroid marker CD235a (glycophorin A) but has high levels of CD71 and CD105. LateCs could be prospectively detected in human bone marrow (BM) cells and, upon isolation and reculture, exhibited the potential to form CFU-E colonies in medium containing only Epo (no other cytokines) and continued differentiation along the erythroid trajectory. Analysis of ex vivo cultures of BM CD34 + cells showed that acquisition of the CD7 hi CD105 hi phenotype in lateCs is gradual and occurs through the formation of four EE cell subtypes. Of these, two are CD34 + burst-forming unit-erythroid (BFU-E) cells, distinguishable as CD7 lo CD105 lo early BFU-E and CD7 hi CD105 lo late BFU-E, and two are CD34 - CFU-Es, also distinguishable as CD71 lo CD105 lo early CFU-E and CD7 hi CD105 lo mid-CFU-E. The transition of these EE populations is accompanied by a rise in CD36 expression, such that all lateCs are CD36 + . Single cell RNA-sequencing analysis confirmed Epo-dependent formation of a CFU-E cluster that exhibits high coexpression of CD71, CD105, and CD36 transcripts. Gene set enrichment analysis revealed the involvement of genes specific to fatty acid and cholesterol metabolism in lateC formation. Overall, in addition to identifying a key Epo-dependent EE cell stage, this study provides a framework for investigation into mechanisms underlying other erythropoiesis-stimulating agents.

Genetic analysis from multiple cohorts implies causality between 2200 druggable genes, telomere length, and leukemia

BackgroundClinical therapeutic targets for leukemia remain to be identified and the causality between leukemia and telomere length is unclear. MethodsThis work employed cis expression quantitative trait locus (eQTL) for 2,200 druggable genes from the eQTLGen Consortium and genome-wide association studies (GWAS) summary data for telomere length in seven blood cell types from the UK Biobank, Netherlands Cohort as exposures. GWAS data for lymphoid leukemia (LL) and myeloid leukemia (ML) from FinnGen and Lee Lab were used as outcomes for discovery and replication cohorts, respectively. Robust Mendelian randomization (MR) findings were generated from seven MR models and a series of sensitivity analyses. Summary-data-based MR (SMR) analysis and transcriptome-wide association studies (TWAS) were further implemented to verify the association between identified druggable genes and leukemia. Single-cell type expression analysis was employed to identify the specific expression of leukemia casual genes on human bone marrow and peripheral blood immune cells. Multivariable MR analysis, linkage disequilibrium score regression (LDSC), and Bayesian colocalization analysis were performed to further validate the relationship between telomere length and leukemia. Mediation analysis was used to assess the effects of identified druggable genes affecting leukemia via telomere length. Phenome-wide MR (Phe-MR) analysis for assessing the effect of leukemia causal genes and telomere length on 1,403 disease phenotypes. ResultsCombining the results of the meta-analysis for MR estimates from two cohorts, SMR and TWAS analysis, we identified five LL causal genes (TYMP, DSTYK, PPIF, GDF15, FAM20A) and three ML causal genes (LY75, ADA, ABCA2) as promising drug targets for leukemia. Univariable MR analysis showed genetically predicted higher leukocyte telomere length increased the risk of LL (odds ratio [OR] = 2.33, 95 % confidence interval [95 % CI] 1.70–3.18; P = 1.33E-07), and there was no heterogeneity and horizontal pleiotropy. Evidence from the meta-analysis of two cohorts strengthened this finding (OR = 1.88, 95 % CI 1.06–3.05; P = 0.01). Multivariable MR analysis showed the causality between leukocyte telomere length and LL without interference from the other six blood cell telomere length (OR = 2.72, 95 % CI 1.88–3.93; P = 1.23E-07). Evidence from LDSC supported the positive genetic correlation between leukocyte telomere length and LL (rg = 0.309, P = 0.0001). Colocalization analysis revealed that the causality from leukocyte telomere length on LL was driven by the genetic variant rs770526 in the TERT region. The mediation analysis via two-step MR showed that the causal effect from TYMP on LL was partly mediated by leukocyte telomere length, with a mediated proportion of 12 %. ConclusionOur study identified several druggable genes associated with leukemia risk and provided new insights into the etiology and drug development of leukemia. We also found that genetically predicted higher leukocyte telomere length increased LL risk and its potential mechanism of action.

BCG vaccination alters the epigenetic landscape of progenitor cells in human bone marrow to influence innate immune responses

Although the Bacille-Calmette-Guérin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90days after BCG vaccination or placebo. We showed that BCG alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occurred primarily within uncommitted stem cells. By contrast, changes in chromatin accessibility were most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF) and early growth response (EGR) transcription factors and were highly correlated (r>0.8) with the interleukin (IL)-1β secretion capacity of paired peripheral blood mononuclear cells (PBMCs). Our findings shed light on BCG vaccination's profound and lasting effects on HSPCs and its influence on innate immune responses and trained immunity.

Atranorin is a novel potential candidate drug for treating myelodysplastic syndrome

This work is devoted to the study of biocompatibility, cyto- and genotoxicity, mechanism of action and prospects for the use of atranorin, which is an AKT kinase inhibitor, for the treatment of myelodysplastic syndrome. Atranorin was isolated by preparative flash chromatography; identification was carried out by UV, IR, and NMR spectroscopy, mass spectrometry, and elemental analysis. Biocompatibility studies included studies of haemocompatibility, genotoxicity, antioxidant activity, cytotoxicity against ECV340 and HEK293 cell lines. Computer modelling of the interaction of atranorin with AKT kinase was carried out using docking followed by molecular dynamics of the resulting complexes; the ADMET properties of atranorin were also calculated. Flow cytometry included analysis of the expression level of PD-L1 and TIM-3 in the presence of atranorin on THP-1, Mono-Mac-1 and KG-1 cell lines, as well as human bone marrow cells.

Analysis of the TID-I and TID-L Splice Variants' Expression Profile under In Vitro Differentiation of Human Mesenchymal Bone Marrow Cells into Osteoblasts.

Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the TID splice variants (TID-L, TID-I) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the TID gene on the expression of ALPL and SPP1 was also investigated. The TID proteins and the expression of TID splice variants were detected. After differentiation, the expression of TID-L and TID-I increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of SPP1. Three days after transfection, the expression of SPP1 increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of TID-L and TID-I changes under differentiation of B-MSCs into osteoblasts and may influence the expression of SPP1. However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.

Instructive interaction between myelodysplastic hematopoiesis and the bone marrow microenvironment at the single-cell level

AbstractMyelodysplastic neoplasms (MDS) are hypothesized to remodel their bone marrow (BM) microenvironment to reinforce conditions for their propagation. In this study, we investigated interactions between MDS cells and the BM niche at single-cell level. In a patient-derived xenograft (PDX) model, we analyzed 13 000 cells from different murine niche cell populations after long-term (>24 weeks) exposure to MDS vs healthy human grafts. Subsequently, we analyzed over 24 000 primary human BM cells enriched for the nonhematopoietic compartment by using whole bone fragments from n = 8 patients with MDS and n = 7 healthy, age-matched donors. In PDX who received MDS transplantation, mesenchymal cell (MSC) subpopulations were forced to overexpress hematopoietic factors such as Cxcl12 and Il7 upon contact with hematopoietic MDS cells as compared with healthy grafts. Single-cell analyses of primary in situ BM cells from patients with MDS showed highly heterogeneous MSC subpopulations on a patient-individual level. We identified inflammatory gene expression profiles as well as overexpression of C-X-C Motif Chemokine Ligand 12, KIT ligand, and Interleukin 7 in MDS MSCs and endothelial cells. In conclusion, we demonstrate reprogramming of the BM microenvironment by MDS cells, pointing to altered MSC subpopulations with increased growth factor expression profiles in a subgroup of patients with MDS.

Abstract 3333: Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index

Abstract Aim We aimed to design an MDM2-p53 antagonist with a differentiated tolerability profile that could be used to treat patients with wild-type TP53 malignancies. As part of an alliance between Newcastle University, Astex Pharmaceuticals, and Cancer Research Horizons, we discovered ASTX295, a potent inhibitor of the MDM2-p53 interaction that is currently under clinical investigation in patients with solid tumors (NCT03975387). We selected ASTX295 as a compound with a predicted short plasma half-life, which we hypothesised would help to mitigate the dose-limiting neutropenia and thrombocytopenia observed with earlier MDM2-p53 antagonists in clinical studies. To examine this hypothesis in vitro, we determined time- and concentration-dependent responses to ASTX295 treatment in healthy volunteer-derived human bone marrow cells, megakaryocytes, and in a panel of human tumor cell lines. Methods Samples containing bone marrow cells from healthy patients undergoing hip surgery were obtained under the ethical approval of the Newcastle Biobank (REC 12/NE/0395). Following Lymphoprep™ separation, cells were treated ex vivo with ASTX295 and seeded for Granulocyte-macrophage (GM) colony-forming assays in methylcellulose. Megakaryocytes were obtained from in vitro differentiation of CD34+ stem/progenitor cells. Human tumor cell lines (including MDM2-amplified SJSA-1) were treated with ASTX295 in vitro and seeded at low density for colony-forming assays. Exposures of 6, 12, or 24h were examined, and the data plotted to calculate LC50 values. Results The clonogenic survival of tumor cells was time-dependent, with LC50 values (mean ± SEM) in SJSA1 cells being 238 ± 46nM and 75 ± 7nM respectively (n = 3-4), following a 12h or 24h exposure to ASTX295. Time-dependent effects were also evident in five human bone marrow samples but with LC50 values of 1.9, >3, >10, >10, and >10uM being achieved at 12h, and 860 ± 268nM at 24h. Megakaryocytes showed similar time-dependent sensitivities in which daily treatment of 2 or 6h over three days did not induce apoptosis while significant cell death was observed when the treatment time was extended to 16-24h daily. In contrast, short, daily pulse treatment of 2-6h in cell lines (MV4-11, MOLM-13, SJSA-1) over three days was sufficient to induce cell death. Conclusions ASTX295 is a potent antagonist of the MDM2-p53 interaction. Collectively, our in vitro data suggest that a shorter exposure to ASTX295 (up to 12h), may help to spare healthy bone marrow cells whilst killing tumor cells. Hence, intermittent exposure to an MDM2-p53 antagonist could favourably modulate its therapeutic index. The predicted short plasma half-life of ASTX295 should provide flexibility in controlling the duration of exposure in vivo, potentially enabling a more bone-marrow sparing approach to MDM2-p53 antagonism to be utilised. Citation Format: Elaine Willmore, Maria Ahn, Suzanne Kyle, Yan Zhao, Huw Thomas, Kenneth S. Rankin, Luke Bevan, Lynsey Fazal, Keisha Hearn, Nicola Wilsher, Justyna Kucia-Tran, Nicola Ferrari, Nicola Wallis, Neil Thompson, John Lyons, Duncan Miller, Celine Cano, Martin E. Noble, Ian R. Hardcastle, Steven Howard, Gianni Chessari, John Lunec, David R. Newell, Steve R. Wedge. Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3333.

Abstract 5701: AU2-94: A CDK4-specific inhibitor with the marked anti-tumor efficacy

Abstract Cyclin-dependent kinases (CDKs) are a vital family of proteins in the cell development. Several CDK4/6 inhibitor drugs significantly extended the lives of subtypes breast cancer patients. However, they have some shortcomings. Many patients do not respond to them well mainly due to off-target toxicity requiring dose reductions and treatment interruptions, and thus building resistance, which limit their effectiveness. Here, we describe AU2-94, a highly selective CDK4 inhibitor, for the treatment of CDK4-dependent tumours including breast cancer (ER+ and TNBC), colorectal cancer and glioblastoma (GBM). AU2-94 exhibits high selectivity for CDK4 (Ki = 2 nM) over CDK6 (Ki = 279 nM). Consistent with the CDK4-targeted mechanism, AU2-94 arrested the G1 cells and inhibited cancer cell proliferation. Interestingly, AU2-94 reduced cellular CDK2, Cyclin E2, Cyclin A2, Cyclin B1, and induced apoptosis via reduction of Bcl-2, FOXM1 etc. In contrast to cancer cells, AU2-94 has little impact on the cell cycle, senescence, or apoptosis of human bone marrow cells, whereas the known CDK4/6 inhibitors increased the G1 cells. AU2-94 showed little or no effect on lymphocyte and neutrophil counts whilst the clinical CDK4/6 inhibitors reduced them. This lower effect of AU2-94 on the bone marrow, neutrophils and lymphocytes might be due to its lesser effect on CDK6 compared to the clinical CDK4/6 inhibitors. AU2-94 was highly efficacious against multiple in vivo tumour models. In the ER+ breast cancer T47D xenografts, AU2-94 caused tumour regression. In the model of MDA-MB-453 AR+ luminal breast cancer (CDK4, KRASmut, PI3KCAmut), AU2-94 demonstrated remarkable efficacy, resulting in the 6 out of 7 (86%) mice tumour-free. In the colorectal cancer HCT116 (CDK4/6, KRASmut) xenografts, AU2-94 inhibited tumour growth markedly in a dose-dependent manner from 25 to 200 mg/kg without any overt toxicity. The pharmacokinetic and pharmacodynamic study revealed up to 500-fold higher concentration of AU2-94 in the tumours than in the plasma, suggesting its tumour targeting specificity. Several robust biomarkers/PD markers were identified. The Rb-CDK4-Cyclin D pathway is altered in the majority of GBM due to homozygous deletion of CDKN2A/B. AU2-94 has an excellent therapeutic potential as it crossed the blood-brain-barrier effectively. In the both U87 subcutaneous and orthotopic models, AU2-94 reduced the tumour burden and increased the survival of GBM bearing mice significantly compared to vehicle. Moreover, AU2-94 synergised the anti-cancer efficacy of PI3K/Akt/mTOR and HER2 inhibitors in combination. In summary, AU2-94 is a CDK4 inhibitor with high selectivity against a panel of 360 human kinases. This selectivity can be a reason for the better safety of AU2-94 compared to the clinical CDK4/6 inhibitors. The high oral bioavailability, robust antitumor efficacy and excellent safety profiles make AU2-94 a highly attractive candidate for clinical development. Citation Format: Laychiluh Bantie, Jimma Likisa, Muhammed Rahaman, Theodosia Teo, Ava Safaroghliazar, Sunita KC Basnet, Ramin Hassankhani, Benjamin Noll, Robert Milne, Shudong Wang. AU2-94: A CDK4-specific inhibitor with the marked anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5701.

An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Lactiplantibacillus plantarum K8 lysates regulate hypoxia-induced gene expression

Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1α accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1α target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. Intriguingly, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1α destabilization under normoxic conditions, suggesting that K8 may regulate HIF1α stability in a non-canonical pathway. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1α-mediated hypoxic gene activation.

Activated mesenchymal stem/stromal cells promote myeloid cell differentiation via CCL2/CCR2 signaling

Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation invivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the invivo effects of MSC transplantation.

Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

Effects of HMGB1/TLR4 on secretion IL-10 and VEGF in human jaw bone-marrow mesenchymal stem cells.

We aimed to investigate the regulatory effects of HMGB1/TLR4 signaling pathway on the expression of IL-10 and VEGF in human bone marrow mesenchymal stem cells. Human JBMSCs were isolated and cultured. Then, HMGB1 was added into the JBMSCs culture medium, and the protein and mRNA expression levels of IL-10 and VEGF were assessed. Moreover, cells were pretreated with a specific TLR4 inhibitor (TAK-242), and the expression changes of IL-10 and VEGF were compared. Compared with the control group, exposure to HMGB1 in human JBMSCs up-regulated TLR4, IL-10, and VEGF secretion at both protein and mRNA levels (P<0. 05). In addition, the increased expression of IL-10 and VEGF could be restrained in TAK-242 group compared with the HMGB1 group (P<0.05). The results indicated that HMGB1 activate TLR4 signaling pathway in Human JBMSCs, which plays a regulatory role in cytokines expression.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4–CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis

AbstractSystemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor–independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Instructive Interaction between Myelodysplastic Hematopoiesis and the Bone Marrow Microenvironment at Single Cell Level

Myelodysplastic Neoplasms (MDS) are hypothesized to re-model their bone marrow (BM) microenvironment to re-inforce conditions for their propagation. In this study, we investigated interactions between MDS cells and the BM niche at single cell level in a MDS patient-derived xenograft model (PDX) as well as primary human MDS and healthy (HY) bone marrow (BM samples in order to identify cues of disturbed crosstalk between MDS hematopoiesis and the BM microenvironment. In the PDX model, we analyzed &amp;gt;13,000 cells of n=15 different murine niche cell populations after long-term (&amp;gt;24 weeks) exposure to n=3 MDS patient samples versus n=2 age-matched HY human grafts. Subsequently, we analyzed over 24,000 primary human BM cells enriched for the non-hematopoietic compartment by using whole bone fragments from n=8 MDS patients and n=7 HY age-matched donors. All cells were analyzed with combined 10x single cell RNA sequencing and human “Cellular Indexing of the Transcriptome and Epitopes by Sequencing” (CITE Seq). Results were validated by flow cytometry and gene expression analyses. Mesenchymal cell (MSC) subpopulations from PDX mice exposed to MDS versus HY hematopoiesis could be subdivided into distinct “Cxcl12+ abundant reticular” (CAR) cells with osteogenic and adipogenic differentiation trajectories. Various CAR cell populations displayed an increased frequency in MDS transplanted mice as compared to PDX transplanted with HY grafts ( Panel A). Upon contact with hematopoietic MDS cells, CAR cell populations overexpressed numerous genes involved in extracellular matrix reorganization and hematopoietic supporting factors, such as Cxcl12, Il7, Lox and Loxl1 as compared to mice bearing HY grafts. In situ, BM cells from MDS patients showed highly heterogeneous MSC subpopulations with similar differentiation trajectories to the ones observed in our PDX models on a patient-individual level. We detected two non-hematopoietic cell populations, designated as endothelial cells (ECs) and MSCs characterized by expression of CXCL12, IGFBP5 or COL1A2 and quantification with epitope sequencing (CITE Seq) with mutually exclusive detection of CD31 and CD271 expression (n=581 cells in total, Panel B). In n=3 MDS patients, we identified an MDS-specific MSC subpopulation with high IGFPB4 and CXCL12 expression as well as overexpression of KITLG and IL7. This suggests that this altered specialized secretory mesenchymal state is present in a subset of MDS patients and may support the malignant hematopoietic clones by overexpression of key hematopoietic factors. Interactome analyses using spatial receptor:ligand bioinformatic tools such as CellphoneDB, RNAMagnet and omnipath further confirmed that stromal cells alter their cellular profile and secretory behavior in response to MDS hematopoiesis. In conclusion, we show that murine BM stroma is re-programmed by exposure to human MDS xenografts to upregulate extracellular matrix (ECM) re-modeling and HSC supporting factors. In native human MDS BM in vivo, rare primary mesenchymal cells are re-programmed to increase hematopoietic support in a patient-individual manner pointing to altered MSC subpopulations with increased growth factor expression profiles in a subgroup of MDS patients.

Glycosylation as regulator of human B-cell leukaemias in bone marrow

Leukemic B-cells are lodged in the bone marrow [BM], a complex organ composed of many cell types and extracellular matrix. Determining how the reciprocal interactions between these components are regulated is critical to our understanding of the factors that allow leukemia cells to survive, multiply and withstand treatment. All cells in the bone marrow are surrounded by a glycocalyx, a glycan-rich layer of high complexity, which regulates such cell-cell and cell-matrix interactions. However, the structure and function of the glycan components of the biomolecules that constitute this layer have not been explored in much detail. Gaps are difficult to fill due to technical limitations as well as the fact that the composition of the BM in health, disease and aging is not static. This also applies to B-lineage malignancies that develop or persist in BM such as B-cell precursor acute lymphoblastic leukemia and Multiple Myeloma, and the effects of their treatment. In contrast, the proteomes and transcriptomes of different human bone marrow cells have been studied more extensively. A combination of technologies now increasingly allows correlations to be made between the expression of glycosyltransferases and glycan structures in cell lines, which could be extrapolated to RNAseq data from primary cells. Glycopeptide analysis will also be invaluable in providing details of specific glycan occupancy on glycoproteins, even if only as a snapshot in time. Functional studies on CD19, CD138/SDC1 and BCMA/TNFRSF17 have already demonstrated the importance of their glycosylation. Additional studies using such approaches are likely to find many more other instances in which malignant B-cell homeostasis is regulated by glycosylation, and lead to the identification of new targets to treat B-cell malignancies.

RTID-05. SURGICAL WINDOW OF OPPORTUNITY TRIAL REVEALS MECHANISMS OF RESPONSE AND RESISTANCE TO NAVTEMADLIN (KRT-232) IN PATIENTS WITH RECURRENT GLIOBLASTOMA

Abstract Over half of glioblastoma multiforme (GBM) patients retain wild-type p53, resulting in potential susceptibility to MDM2 inhibitors. These molecules disrupt the interaction between p53 and its negative regulator MDM2, resulting in downstream pathway signaling and cell fate decisions such as growth inhibition and death from cellular stress. We have analyzed clinical samples from 21 patients with recurrent GBM treated with the MDM2 inhibitor KRT-232, all of which relapsed despite the biological promise of this small molecule. By analyzing on-treatment biopsy samples, we detected upregulation of p53 transcriptional targets, suggesting blood-brain barrier penetrance and drug pharmacodynamics. However, in patient-derived cell lines, treatment with KRT-232 at the clinically achieved concentration was only sufficient to stall but not reduce tumor growth. Cell death via apoptosis was achieved in these cell line models when MDM2 inhibition was combined with the chemotherapeutic agent temozolomide. These drugs have non-overlapping mechanisms of action and exhibit synergy in our cell line models. Normal human bone marrow cells underwent a reversible growth inhibition phenotype when treated with KRT-232 and TMZ, supporting the idea that a therapeutic window exists with this combination. We additionally observed that tumor progression occurred in the absence of p53 inactivating mutations, suggesting that other mechanisms of resistance to KRT-232 may modulate drug response and resistance in GBM. Indeed, drug treatment induced an adaptive response in human tumors, characterized by the upregulation of genes involved in glial cell differentiation processes. Ongoing work is focused on understanding how tumor heterogeneity and cell state modulate response and resistance of GBMs to MDM2 inhibition and other therapeutic strategies that reactivate p53 signaling. Our study highlights the utility of tissue sampling during clinical trials and represents the first clinical effort to detect adaptive changes in human tumor tissue associated with p53 reactivation.