Bone Marrow Hematopoietic Progenitor Research Articles

Biomarkers for Radiation Biodosimetry and Correlation with Hematopoietic Injury in a Humanized Mouse Model.

After a large-scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation and require subsequent medical management. Acute exposure to moderate doses (2-6 Gy) of radiation can lead to the hematopoietic acute radiation syndrome, in which the bone marrow (BM) is severely compromised, and severe hemorrhage and infection are common. Previously, we have developed a panel of intracellular protein markers (FDXR, ACTN1, DDB2, BAX, p53 and TSPYL2), designed to reconstruct absorbed radiation dose from human peripheral blood (PB) leukocyte samples in humanized mice up to 3 days after exposure. The objective of this work was to continue to use the humanized mouse model to evaluate biomarker dose-/time- kinetics in human PB leukocytes invivo, at an earlier (day 2) and later (day 7) time point, after exposure to total-body irradiation (TBI) doses of 0 to 2 Gy of X rays. In addition, to assess hematological sensitivity and radiation-induced injury, PB leukocyte cell counts, human BM hematopoietic stem cell (HSC) and progenitor cell [multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte myeloid progenitor (GMP), megakaryocyte/erythrocyte progenitor (MEP) and multi-lymphoid progenitor (MLP)] levels were measured, and their correlation was also examined as the BM damages are difficult to assess by routine tests. Peripheral blood B-cells were significantly lower after TBI doses of 0.5 Gy on day 2 and 2 Gy on days 2 and 7; T-cells were significantly reduced only on day 2 after 2 Gy TBI. Bone marrow HSCs and MPP cells showed a dose-dependent depletion after irradiation with 0.5 Gy and 2 Gy on day 2, and after 1 Gy and 2 Gy on day 7. Circulating B cells correlated with HSCs, MPP and MLP cells on day 2, whereas T cells correlated with MPP, and myeloid cells correlated with MLP cells. On day 7, B cells correlated with MPP, CMP, GMP and MEP, while myeloid cells correlated with CMP, GMP and MEP. The intracellular leukocyte biomarkers were able to discriminate unirradiated and irradiated samples at different time points calculated by receiver operating characteristic (ROC) curve. Using machine learning algorithm methods, combining ACTN1, p53, TSPYL2 and PB-T cell and PB-B cell counts served as a strong predictor (area under the ROC >0.8) to distinguish unirradiated and irradiated samples independent of the days after TBI. The results further validated our biomarker-based triage assay and additionally evaluated the radiation sensitivity of the hematopoietic system after TBI exposures.

Decoding human bone marrow hematopoietic stem and progenitor cells from fetal to birth

Decoding human bone marrow hematopoietic stem and progenitor cells from fetal to birth

Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3weeks to 18months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=.01) and hemoglobin levels (p<.05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p=.001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Radioprotective effectiveness of a novel delta-tocotrienol prodrug on mouse hematopoietic system against 60Co gamma-ray irradiation through inducing granulocyte-colony stimulating factor production

Considering the increasing risk of nuclear attacks worldwide, the development of develop potent and safe radioprotective agents for nuclear emergencies is urgently needed. γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have demonstrated a potent radioprotective effect by inducing the production of granulocyte-colony stimulating factor (G-CSF) in vivo. However, their application is limited because of their low bioavailability. The utilization of ester prodrugs can be an effective strategy for modifying the pharmacokinetic properties of drug molecules. In this study, we initially confirmed that DT3 exhibited the most significant potential for inducing G-CSF effects among eight natural vitamin E homologs. Consequently, we designed and synthesized a series of DT3 ester and ether derivatives, leading to improved radioprotective effects. The metabolic study conducted in vitro and in vivo has identified DT3 succinate 5b as a prodrug of DT3 with an approximately seven-fold higher bioavailability compared to DT3 alone. And DT3 ether derivative 8a were relatively stable and approximately 4 times more bioavailable than DT3 prototype. Furthermore, 5b exhibited superior ability to mitigate radiation-induced pancytopenia, enhance the recovery of bone marrow hematopoietic stem and progenitor cells, and promote splenic extramedullary hematopoiesis in sublethal irradiated mice. Similarly, 8a shown potential radiation protection, but its radiation protection is less than DT3. Based on these findings, we identified 5b as a DT3 prodrug, and providing an attractive candidate for further drug development.

Interplay between Inflammatory Microenvironment and RUNX1-Mediated Transcriptomic Changes Drives Defective Hematopoiesis in Familial Platelet Disorder

Background: Familial platelet disorder (FPD) is caused by germline loss-of-function mutations in RUNX1 an essential transcription factor regulating hematopoiesis. The absence of functional RUNX1 leads to mild-to-moderate thrombocytopenia, platelet dysfunction, and increased bleeding in FPD patients. In ~40% of cases, the patients progress to overt leukemia at median age of 33 years through unknown mechanisms. FPD patients also present with skin allergy and eczema which are associated with systematic elevation of cytokines. Given the well-established connection between inflammation and leukemia initiation and progression, we sought to investigate whether inflammatory stress play a role in regulating abnormal hematopoiesis and eventually cause clonal evolution in FPD. Methods: We acquired bone marrow cells from FPD patients who had not developed leukemia yet. We then compared FPD bone marrow hematopoietic stem and progenitors (HSPCs) with healthy progenitors using in vitro differentiation and colony formation assays. For comprehensive mechanistic evaluation, we performed 10x single-cell RNA sequencing (scRNA-seq) on FPD (n=10) and healthy (n=4) bone marrow cells, analyzing &amp;gt;100,000 cells using Seruat package. We evaluated cytokine levels in bone marrow fluid and peripheral blood using 65-plex Luminex assay. The impact of the inflammatory cytokines and intervention strategies were tested using in vitro differentiation and colony formation assays. The results were validated in vivo using mice with germline Runx1 mutation. Results: We identified that FPD progenitors are impaired in megakaryocytic (&amp;gt;2.0-fold, p&amp;lt;0.05) and erythroid differentiation with increased myeloid differentiation (&amp;gt;1.7-fold, p&amp;lt;0.05) compared to healthy control. In addition, FPD HSPCs show increased myeloid colony formation (&amp;gt;1.6-fold, p&amp;lt;0.05) and serial replating ability. scRNA-seq identified 18 cell clusters and pseudotime analysis revealed higher percentage of FPD cells differentiating towards monocytes, supporting enhanced myeloid differentiation. The pathway enrichment analysis of differentially expressed genes in HSC cluster revealed upregulation of inflammatory (TNFα via NF-kB) and pro-survival pathways (PI3K/AKT/mTOR). Accordingly, cytokine profiling revealed upregulation of numerous cytokines and chemokines in FPD compared to healthy samples including TNFα, CXCL8, CCL24, CCL2, and IL1β (&amp;gt;2.5 to 1000-fold, p&amp;lt;0.05), secreted by progenitors, monocytes, and MSCs. Further, under inflammatory stress mediated by TNFα, CXCL8, CCL24, CCL2, and IL1β, FPD HSPCs exhibited resistant to growth suppression and have increased myeloid colony formation. However, pharmacological inhibition of individual cytokines only partially rescued the differentiation defects, possibly due to compensatory effects of each cytokine. Phospho-flow analysis revealed that increased cytokine levels leads to the activation of PI3K/mTOR and JAK signaling that we also found upregulated using scRNA-seq analysis. Interestingly, inhibition of mTORi (rapamycin and AZD2014), PI3Ki (idelalisib), and JAKi (ruxolitinib) showed an increase in percentage of megakaryocytes (up to 2-fold, p&amp;lt;0.05) and a decrease in monocytes (up to 0.6-fold change, p&amp;lt;0.05). Also, inhibition of these pathways in FPD reduced cytokines levels. In line with human FPD data, in vivo treatment of Runx1 R188Q/+ mouse with ruxolitinib suppressed myeloid differentiation with no effects on megakaryocytes, while rapamycin resulted in significantly increased platelet activation compared to untreated mice (&amp;gt;1.7-fold, p&amp;lt;0.05) and a reduction of monocytes (&amp;lt;0.5-fold, p&amp;lt;0.05), suggesting mTOR inhibition might be an effective intervention strategy. Conclusion: FPD HSPCs manifest defective megakaryopoiesis with increased myelopoiesis. The elevated autocrine and paracrine inflammatory stress is an early event in FPD, driving myeloid differentiation. This inflammatory stress mediates activation of PI3K/mTOR and JAK signaling, leading to augmented myeloid differentiation. Consequently, the observed FPD phenotypes are the result of combined transcriptional changes and inflammatory stress. Targeting inflammatory and pro-survival signaling at an early stage might mitigate hematopoietic defects and delay disease evolution in FPD. Our results advance mechanistic understanding and hold significant clinical implications.

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence. To explore this further, HSCs isolated from 6-month-old SCD mice or age-matched controls were interrogated for senescence associated β-galactosidase (SA-β-gal) activity. On average 19.5% of SCD HSCs were SA-β-gal+ versus only 7.06% of HSCs isolated from controls (n=10-11/group, p=0.004). HSCs isolated from SCD mice also displayed elevated ROS, DNA damage, and expression of molecular markers of senescence (e.g. p21, BCLXL). Importantly, 23.14 ± 5.61% of Lin-CD34+CD38- bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) isolated from pediatric individuals with SCD were SA-β-gal+. In contrast, only 4.97 ± 2.97% of HSPCs were SA-β-gal+ in age-matched, non-SCD control individuals (n=8 control and n=5 SCD individuals, p=&amp;lt;0.0001). Further, 47.46 ± 17.49% of Lin-CD34+CD38-CD90+CD45RA- BM cells, which are highly enriched for human HSCs, were SA-β-gal+ in young individuals with SCD, relative to only 4.41 ± 2.37% in the BM of age-matched control individuals (n=4 control and n=5 SCD individuals, p=0.0019). We also detected elevated expression of p16, p21, and BCL2 in BM HSPCs isolated from pediatric individuals with SCD, relative to controls (n=6 control and n=9 SCD individuals). In total, these data reveal that SCD pathophysiology aberrantly invokes senescence programs and damages HSC function in mice and humans. Clearance of senescent HSCs from mouse BM has previously been shown to restore aging-related HSC dysfunction. We therefore tested if clearance of senescent HSCs from the BM could restore function to the SCD HSC pool. 2-month-old SCD and control mice were treated with two weekly cycles of 75 mg/kg/day of the senolytic, ABT-263, via oral gavage. 14 days after treatment, WBM was isolated and tested for HSC function and frequency. Senolytic therapy significantly increased HSC numbers in SCD mice BM, relative to vehicle-treated controls (n=5/group). Importantly, the in vivo hematopoietic repopulating activity of WBM from ABT-263-treated SCD mice was comparable to non-SCD controls, revealing a correction of our previously observed severe transplantation defect (n=12/group). In sum, these data reveal premature onset of senescence as a previously unappreciated phenotype of the HSC pool during SCD. This work provides the first proof-of-principle that treatment of individuals with SCD using senolytics may restore function to the HSC pool, and ongoing preclinical work aims to test if senolytic therapy enhances the yield and function of SCD HSCs mobilized for autologous therapy.

Detection and Prevention of ADAR1p150-Induced Hematopoietic Stem and Progenitor Cell Aging

Introduction: Over the last decade, ADAR1p150 has been linked by our research team and others to malignant progenitor reprogramming and therapeutic resistance in a broad array of malignancies. However, ADAR1p150's role in accelerated hematopoietic stem and progenitor cell (HSPC) aging in response to inflammatory cytokines had not been clearly elucidated. Here, we investigate ADAR1 splice isoform switching and the ADAR1p150 inhibitory effects of Rebecsinib on human HSPC function in normal humanized aged normal bone marrow (aNBM) HSPC mouse models. Methods: Whole transcriptome RNA sequencing (RNA-seq) was utilized with splice isoform and editome analysis to detect ADAR1 splice isoform switching and adenosine to inosine RNA editing. Rebecsinib (17S-FD-895), a pre-IND drug candidate, has shown promising potential as an inhibitor of ADAR1p150-mediated RNA editing ( Crews, Ma...Jamieson. Cell Stem Cell 2023), which is an important therapeutic target in patients with myelofibrosis or acute myeloid leukemia that overexpress ADAR1p150 compared with ADAR1p110. Human CD34 + cells, isolated from aged normal bone marrow (aNBM) from patients undergoing elective hip replacement surgery, were transduced with a lentiviral ADAR1-nano-luciferase reporter and transplanted into NSG-SGM3 adult mice that secrete human SCF, IL-3, and GM-CSF cytokines that support normal hematopoiesis. After engraftment confirmation peripheral blood human CD45 flow cytometric assessment and IVIS 200 system imaging of ADAR1-nano-luc-GFP reporter expressing HSPC engraftment, the mice were randomly divided into vehicle-treated and Rebecsinib-treated groups (15mg/kg, twice a week for two weeks). Peripheral blood, bone marrow, and spleen were collected upon treatment completion for analysis. Results: By HSPC RNA-seq, we detected an imbalance between ADAR1p150 and ADAR1p110 expression together with increased RNA editing and splicing alterations using whole transcriptome RNA sequencing (RNA-seq) of aged compared with young bone marrow hematopoietic stem and progenitor cells (HSPCs). However, the functional relevance of ADAR1 splice isoform switching had not been evaluated in human-aged normal bone marrow HSPC xenograft (PDX) mouse models nor had this cytokine-induced HSPC aging been reversed. Flow cytometric analysis and immunohistochemistry staining revealed that Rebecsinib treatment at a dose of 10mg/kg, twice a week for two weeks, effectively spared normal human immune cells (CD3+, CD14+, and CD19+ cells). Remarkably, Rebecsinib treatment not only preserved Hematopoietic Stem (CD34+CD38-) cells and Progenitor (CD34+CD38+) cells in peripheral blood, bone marrow, and spleen but also promoted their expansion. These findings demonstrate the potential of Rebecsinib to enhance the retention of HSPC populations in the bone marrow niche. Conclusions: Our pre-clinical experiments utilizing RNA-seq, a novel ADAR1-nano-luciferase-GFP reporter, and aNBM PDX mouse models provide compelling evidence supporting the potential of Rebecsinib to restore the balance of ADAR1p150:p110 ratios and engraftment in humanized aNBM HSPC mouse models. These findings highlight Rebecsinib as a promising therapeutic candidate for targeting leukemia and myelofibrosis while preserving essential HSPC populations.

Genomic Determinants of Therapy Response in ETV6::RUNX1 Leukemia

ETV6::RUNX1 leukemia is the second most common childhood B-cell acute lymphoblastic leukemia subtype. Although it has a low overall relapse risk, a significant proportion of relapses occur within this subtype due to its relatively high incidence. No consistent genomic biomarkers have been identified that predict therapy response beyond early therapy response as measured by minimal residual disease at the end of induction. In this study, our aim was to use multiomic data from 175 ETV6::RUNX1 leukemias treated according to the NOPHO ALL2000 and ALL2008 protocols to identify genomic features that predict therapy response at presentation. Cases were classified into three responder groups based on MRD at end-of-induction (EOI) therapy: fast responders with negative MRD, intermediate responders with positive but &amp;lt;0.1% MRD, and slow responders with &amp;gt;0.1% MRD. Response data was also collected at mid-induction (day 15) and end-of-consolidation (EOC), when available. Diagnostic and remission DNA were analyzed using array-based copy number data (n=142) or whole genome sequencing (WGS) (n=33), and diagnostic bulk RNA sequencing (n=51) was complemented with single cell multiomics (RNA, surface protein and VDJ sequencing) data from both healthy (n=8) and leukemic bone marrow cells upon chemotherapy (n=8). Overall, the frequency of somatic genetic changes was not significantly associated with treatment response suggesting that genetic diversification through elevated random mutagenesis events may not underlie the drug resistant phenotype. However, analysis of mutational signatures revealed that fast-responding leukemic blasts frequently exhibited the APOBEC mutational signature and had higher cell cycle activity (Figure 1A). Accordingly, we show that expression of A3B is highly specific to cycling cells also in healthy bone marrow hematopoietic progenitor cells, pro- and pre-B cells. We also examined RAG-mediated genetic instability that manifests in focal structural variations and has highest activity in ETV6::RUNX1leukemia (Figure 1B). We confirmed several recurrent RAG off-target sites, including significant enrichment of the known risk locus TBX1LR1 in slow responding cases. Strikingly, slow-responding leukemias had also frequent rearrangements in the immunoglobulin kappa genes and the kappa-deleting elements, despite their pro-B-like transcriptome state upon diagnosis. Based on the single cell multiomics profiles, we found that upon treatment the blast phenotype shifts towards an immature-B-like transcriptome, with concomitant expression of Ig light chains with productive sequence, thereby permitting activation of BCR signaling that provides a survival advantage. At CNV level, we found that the chromosomal alterations related to the translocation event associated with treatment response. Specifically, slow responders had more often the amplification of der(21)t(12;21) chromosome, impacting expression of USP5, TIGAR and SLC2A3, associated in earlier studies to chemoresistance and tumorigenesis. In comparison, fast responders were often missing a region of chromosome 12p with lower expression of several genes including oncogenic KRAS. In summary, combining multiomic and response data, we characterized genetic alterations and mutational processes that affect the treatment response in ETV6::RUNX1leukemia. Our findings highlight the genetic and transcriptomic heterogeneity of this common pediatric leukemia subtype and its implications for treatment sensitivity, paving the way for better classification of the disease at presentation.

In Vivo Delivery of RNA By Lipid Nanoparticles (LNP) to Hematopoietic Stem Cells and T Cells in Humanized Mice and Non-Human Primates

Genomic medicines have significant therapeutic potential to cure rare genetic diseases and cancer, but currently, most treatments rely on ex vivo cell editing, which is time consuming, costly, and suffers from logistic and manufacturing challenges. In contrast, in vivo delivery of genomic medicines would unlock new therapeutic areas and provide broader patient access. However, the success of in vivo delivery is dependent on the development of safe delivery vehicles capable of transporting the genomic medicines to their intended target tissues and cells. In recent years, lipid nanoparticles (LNPs) have emerged as versatile delivery solutions for RNA therapeutics but have seldom been used fo r in vivo delivery beyond liver and muscle tissue. Challenges in extrahepatic delivery of LNPs include adequate tissue-specific targeting to achieve therapeutic levels of genome editing while avoiding anti-drug immune responses. We have developed novel LNPs designed to deliver RNA payloads to either T cells or hematopoietic stem cells (HSCs) in vivo. Using our novel T-cell LNPs, we have demonstrated single-dose in vivo delivery of GFP mRNA in two humanized mouse models. In the spleen of humanized NSG mice (pre-engrafted with human HSCs 12-weeks prior to study), we achieved GFP expression in 24% of human CD3 + T cells. In another model, NSG mice infused with human T cells 14-days prior to study, GFP expression was observed in 76% of T cells in the spleen. Single-dose administration of this T-cell LNP in rhesus macaque resulted in 40% GFP expression in peripheral blood T cells. Furthermore, we did not observe any detectable GFP expression in human or rhesus B cells, suggesting this LNP system may be suitable for the development of in vivo CAR-T therapies for B cell malignancies. Separately, we treated humanized NBSGW mice (pre-engrafted with human HSCs for 12 weeks) with a single dose of our HSC LNPs delivering GFP mRNA. Expression of GFP was observed in approximately 90% of bone marrow hematopoietic stem and progenitor cells (HSPCs defined as Lin -CD34 +CD38 - cells) and approximately 95% of long-term bone marrow HSC-enriched population (HSCs defined as Lin -CD34 +CD38 -CD90 +CD45RA - cells). Single-dose intravenous administration of the same LNP in cynomolgus macaques led to GFP expression in 61% of HSPCs and 62% of HSCs in the bone marrow. Gene editing presents a greater challenge than reporter mRNA delivery due to the increased size and complexity of the RNA cargos. Toward this goal, we went on to demonstrate that our HSC LNP platform can deliver Cas9 mRNA, with a beta-2 microglobulin (B2M) sgRNA, to human HSCs in humanized NBSGW mice resulting in the knockout (KO) of the B2M gene through the introduction of insertion or deletion mutations. Likewise, following administration of a B2M editing T-cell LNP in humanized NSG mice, we demonstrated B2M KO in splenic human CD3 + T cells. Together, these in vivo delivery results indicate our LNP delivery platform is capable of efficiently delivering RNA payloads to different hematopoietic cell types in blood and tissues, providing a potential delivery platform for in vivo genome editing.

Inhibition of Topors Ubiquitin Ligase Augments the Efficacy of DNA Hypomethylating Agents through DNMT1 Stabilization

DNA hypomethylating agents (HMAs) are commonly used to treat myeloid malignancies including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, their therapeutic effects are not satisfactory. We performed CRISPR-Cas9 knockout (KO) screening to identify molecular targets that enhance the efficacy of HMAs. MDS-L and MOLM-13 cells expressing Cas9 were infected with an sgRNA lentiviral library containing 12,409 sgRNAs targeting 1,383 epigenetic factors and exposed to low-dose HMAs, decitabine (DAC) or azacitidine (AZA), for 14 days. The read counts of sgRNA against TOPORS which encodes a ubiquitin/SUMO E3 ligase, were reproducibly decreased during culture specifically in the presence of HMAs in both screening. To validate the screening results, we performed competitive growth assays by co-culturing parental cells and TOPORS-KO cells using MOLM-13, MDS-L, SKK-1, and SKM-1 cells. In all cell lines, GFP-positive TOPORS-KO cells decreased over time only in the presence of HMAs. We established single-cell clones of TOPORS-KO MOLM-13 and MDS-L cells using two different sgRNAs. The cell growth of all TOPORS-KO clones was dramatically suppressed only in the presence of DAC. Mice transplanted with TOPORS-KO cells showed significantly longer survival when treated with DAC. Next, we analyzed Topors-deficient mice and no significant changes were observed in the peripheral blood or in the percentage of bone marrow (BM) hematopoietic stem and progenitor cells. Topors−/− BM cells successfully restored hematopoiesis in lethally irradiated recipient mice at levels comparable to those of WT BM cells after transplantation, suggesting that Topors is dispensable for normal hematopoiesis. Topors−/−MLL/AF9 leukemic cells showed comparable growth in the absence of DAC but significantly inferior growth in the presence of DAC compared to WT MLL/AF9 leukemic cells. We transplanted them with support BM cells into lethally irradiated recipient mice and treated with DAC. Topors−/− leukemic cells significantly decreased during treatment compared with WT leukemic cells. Enhanced cytotoxic effects of TOPORS KO combined with HMAs was closely associated with increased apoptosis. Cell cycle analysis revealed that among TOPORS-KO cells, those in the G2/M phase accumulate and those in the S phase are reduced upon DAC treatment. RNA sequencing on cells treated with DAC showed that gene sets associated with chromosome segregation and mitosis are significantly downregulated in TOPORS-KO cells. Addback experiment revealed that TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. To identify TOPORS ubiquitination substrates, we performed second CRISPR-Cas9 screening with the same library used as the first screening, using TOPORS-KO MDS-L and MOLM-13 clone cells. Both screening showed that UHRF1-DNMT1 axis is implicated in the augmented sensitivity to DAC. Consistently, DNMT1 remained more stable in TOPORS-KO cells than WT cells after DAC exposure not only in human MDS/AML cell lines but also in murine leukemic cells. HMAs incorporated into DNA trap DNMT1 and form DNA-DNMT1 crosslinks, which causes apoptosis and mitotic defects. DNMT1-DNA crosslink formation triggers the prominent SUMOylation of DNMT1 followed by ubiquitination, which promotes the resolution of crosslinks via proteasomal degradation and cell fitness. Mass analysis of ubiquitinated peptides and immunoprecipitation with Nanobody against DNMT1 demonstrated that TOPORS loss causes accumulation of SUMOylated DNMT1 due to inefficient ubiquitination, suggesting that TOPORS works as SUMO-targeted ubiquitin ligase for DNMT1. In patient samples treated with AZA (n=23), treatment resistance significantly correlated with TOPORS expression levels. Furthermore, an ubiquitination inhibitor TAK-243 as well as a SUMOylation inhibitor TAK-981 showed synergistic effect with HMAs through DNMT1 stabilization. These results suggested that they are likely to be promising therapeutic agents in clinical practice. Our findings unveil a novel mechanism of resistance to HMAs and provide an attractive therapeutic strategy for myeloid malignancies that interferes with resolution of DNA-DNMT1 crosslinks by targeting DNMT1 post-translational modification.

CD72 is a pan-tumor antigen associated to pediatric acute leukemia.

In the development of novel immunotherapeutic approaches, the step of target identification is a challenging process, because it aims at identifying robust tumor-associated antigens (TAAs) specific for the pathological population and causing no off-target effects. Here we propose CD72 as a novel and robust TAA for pediatric acute leukemias. We provided an outline of CD72 expression assessed by flow cytometry on a variety of cancer cell lines and primary samples, including normal bone marrow (BM) samples and hematopoietic stem and progenitor cells. We analyzed CD 72 expression on a cohort of 495 pathological pediatric BM aspirates, including: 215 B-cell precursor acute lymphoblastic leukemias (BCP-ALL), 156 acute myeloid leukemias (AMLs), 88 T-lineage ALLs or lymphoblastic lymphomas with BM infiltration, 13 B-lineage lymphoblastic lymphomas with BM infiltration, 9 myelodysplastic syndromes with increased blasts (5%-9% blasts on BM: MDS-IB1) and 14 non-hematopoietic solid tumors infiltrating BM. Results showed that CD72 is highly expressed in almost all BCP-ALL and the majority of AML at diagnosis, including BCP-ALL cases characterized by CD19 loss. These findings support a potential role for advanced diagnostics and novel immunotherapy approaches, providing a pan-ALL and AML target.

Mycobacterium tuberculosis infection drives differential responses in the bone marrow hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) play a vital role in the host response to infection through the rapid and robust production of mature immune cells. These HSPC responses can be influenced, directly and indirectly, by pathogens as well. Infection with Mycobacterium tuberculosis (Mtb) can drive lymphopoiesis through modulation of type I interferon (IFN) signaling. We have previously found that the presence of a drug resistance (DR)-conferring mutation in Mtb drives altered host-pathogen interactions and heightened type I IFN production in vitro. But the impacts of this DR mutation on in vivo host responses to Mtb infection, particularly the hematopoietic compartment, remain unexplored. Using a mouse model, we show that, while drug-sensitive Mtb infection induces expansion of HSPC subsets and a skew toward lymphopoiesis, DR Mtb infection fails to induce an expansion of these subsets and an accumulation of mature granulocytes in the bone marrow. Using single-cell RNA sequencing, we show that the HSCs from DR Mtb-infected mice fail to upregulate pathways related to cytokine signaling across all profiled HSC subsets. Collectively, our studies report a novel finding of a chronic infection that fails to induce a potent hematopoietic response that can be further investigated to understand pathogen-host interaction at the level of hematopoiesis.

Does DNA methylation in the fetal brain leave an epigenetic memory in the blood?

Epigenetic memory is an emerging concept that refers to the process in which epigenetic changes occurring early-in life can lead to long-term programs of gene regulation in time and space. By leveraging neural network regression modeling of DNA methylation data in pigs, we show that specific methylations in the adult blood can reliably predict methylation changes that occurred in the fetal brain. Genes associated with these methylations represented known markers of specific cell types of blood including bone marrow hematopoietic progenitor cells, and ependymal and oligodendrocyte cells of brain. This suggested that methylation changes that occurred in the developing brain were maintained as an epigenetic memory in the blood through the adult life.

Early activation of inflammatory pathways in UBA1-mutated hematopoietic stem and progenitor cells in VEXAS

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a pleiotropic, severe autoinflammatory disease caused by somatic mutations in the ubiquitin-like modifier activating enzyme 1 (UBA1) gene. To elucidate VEXAS pathophysiology, we performed transcriptome sequencing of single bone marrow mononuclear cells and hematopoietic stem and progenitor cells (HSPCs) from VEXAS patients. HSPCs are biased toward myeloid (granulocytic) differentiation, and against lymphoid differentiation in VEXAS. Activation of multiple inflammatory pathways (interferons and tumor necrosis factor alpha) occurs ontogenically early in primitive hematopoietic cells and particularly in the myeloid lineage in VEXAS, and inflammation is prominent in UBA1-mutated cells. Dysregulation in protein degradation likely leads to higher stress response in VEXAS HSPCs, which positively correlates with inflammation. TCR usage is restricted and there are increased cytotoxicity and IFN-γ signaling in Tcells. In VEXAS syndrome, both aberrant inflammation and myeloid predominance appear intrinsic to hematopoietic stem cells mutated in UBA1.

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment

BackgroundInteractions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and...

O-GlcNAc glycosylation orchestrates fate decision and niche function of bone marrow stromal progenitors.

In mammals, interactions between the bone marrow (BM) stroma and hematopoietic progenitors contribute to bone-BM homeostasis. Perinatal bone growth and ossification provide a microenvironment for the transition to definitive hematopoiesis; however, mechanisms and interactions orchestrating the development of skeletal and hematopoietic systems remain largely unknown. Here, we establish intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) modification as a posttranslational switch that dictates the differentiation fate and niche function of early BM stromal cells (BMSCs). By modifying and activating RUNX2, O-GlcNAcylation promotes osteogenic differentiation of BMSCs and stromal IL-7 expression to support lymphopoiesis. In contrast, C/EBPβ-dependent marrow adipogenesis and expression of myelopoietic stem cell factor (SCF) is inhibited by O-GlcNAcylation. Ablating O-GlcNAc transferase (OGT) in BMSCs leads to impaired bone formation, increased marrow adiposity, as well as defective B-cell lymphopoiesis and myeloid overproduction in mice. Thus, the balance of osteogenic and adipogenic differentiation of BMSCs is determined by reciprocal O-GlcNAc regulation of transcription factors, which simultaneously shapes the hematopoietic niche.

EFFECTS OF TRAUMA PLASMA-DERIVED EXOSOMES ON HEMATOPOIETIC PROGENITOR CELLS.

Background: Severe trauma disrupts bone marrow function resulting in persistent anemia and immunosuppression. Exosomes are extracellular vesicles implicated in disease, cellular functions, and immunomodulation. The effects of trauma plasma-derived exosomes on bone marrow hematopoiesis are unstudied; we hypothesized that trauma plasma-derived exosomes suppress bone marrow hematopoietic progenitor cell (HPC) growth and contribute to increased inflammatory cytokines and HPC mobilization. Methods: Plasma was collected from a prospective, cohort study of trauma patients (n = 15) with hip and/or femur fractures and an ISS > 15 and elective total hip arthroplasty (THA) patients (n = 15). Exosomes were isolated from both groups using the Invitrogen Total Exosome Isolation Kit. Healthy bone marrow was cultured with 2% plasma, 50 μg, 100 μg, or 200 μg of exosomal protein and HPC (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units [CFU-GEMM], erythroid burst-forming units [BFU-E], and macrophage colony-forming units [CFU-GM]) growth assessed. After culturing healthy bone marrow stroma with 100 μg of exosomal protein, expression of cytokines and factors influencing HPC mobilization were assessed by qPCR. Differences were compared using the ANOVA, with significance defined as P < 0.05. Results: The only demographic difference was age; trauma patients were significantly younger than THA (mean 44 vs. 63 years). In vitro exposure to trauma plasma significantly decreased growth of all HPCs. In vitro exposure to 100 μg or 200 μg of trauma exosomal protein significantly decreased growth of BFU-E and CFU-GM, whereas 50 μg had no effect. Culture of trauma exosomal protein with bone marrow stromal cells resulted in increased expression of IFN-γ, IL-1α, TNF-α, G-CSF, CXCR4, SDF-1, and VCAM-1 in bone marrow stroma. Conclusions: Both plasma and plasma-derived exosomes from trauma patients adversely affect bone marrow function. Plasma-derived exosomes may contribute to altered hematopoiesis after severe trauma; analysis of exosomal content may improve our understanding of altered bone marrow function.

Cord Blood–Derived Endothelial Progenitor Cells Promote In Vivo Regeneration of Human Hematopoietic Bone Marrow

Victims of acute radiation exposure are susceptible to hematopoietic toxicity due to bone marrow damage and loss of mature blood elements. Here, we evaluated cord blood-derived endothelial progenitor cells (CB-EPCs) as a potential cellular therapy for mitigation of hematologic acute radiation syndrome. CB-EPCs express endothelial cell markers and maintain their growth characteristics beyond 10+ passages without diminishing their doubling capacity. Further, CB-EPCs can be cryopreserved in vapor-phase liquid nitrogen and easily recovered for propagation, making them an attractive nonimmunogenic cellular therapy for off-the-shelf use. Importantly, we show CB-EPCs have the capacity to potently expand adult human bone marrow hematopoietic progenitor cells both in vitro and in vivo. To demonstrate the role of CB-EPCs in promoting in vivo human immune reconstitution after irradiation, we employed a novel humanized mouse model established by transplant of CD34+ bone marrow cells from 9 unique adult organ donors into immunocompromised NSG-SGM3 mice. The response of the humanized immune system to ionizing irradiation was then tested by exposure to 1 Gy followed by subcutaneous treatment of CB-EPCs, Food and Drug Administration-approved growth factor pegfilgrastim (0.3 mg/kg), or saline. At day 7, total human bone marrow was decreased by 80% in irradiated controls. However, treatment with either growth factor pegfilgrastim or CB-EPCs increased recovery of total human bone marrow by 2.5-fold compared with saline. Notably, CB-EPCs also increased recovery of both human CD34+ progenitors by 5-fold and colony-forming capacity by 3-fold versus saline. Additionally, CB-EPCs promoted recovery of endogenous bone marrow endothelial cells as observed by both increased vessel area and length compared with saline. These findings indicate the feasibility of using humanized mice engrafted with adult bone marrow for radiation research and the development of CB-EPCs as an off-the-shelf cellular therapy for mitigation of hematologic acute radiation syndrome.

Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone.

Colony-stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 to stimulate osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Compared to other mesenchymal subpopulations, MALPs expressed Csf1 at a much higher level and this expression was further increased during aging. To investigate its role, we constructed MALP-deficient Csf1 CKO mice using AdipoqCre. These mice had increased femoral trabecular bone mass, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prrx1Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs synthesize Csf1 to control bone remodeling and hematopoiesis.

Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

Hem1 (hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass because of defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially because of decreased mitogen-activated protein kinase and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wildtype into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.