Differentiation Of Hematopoietic Stem Cells Research Articles

Single Cell Analyses Indicate SRSF2 and TET2 Co-Mutation Results in Skewed Haematopoiesis in Myelodysplastic Syndromes

We used single-cell ATAC-seq (scATAC-seq) to study how synchronous SRSF2 and TET2 co-mutations result in skewed myelopoiesis compared with SRSF2 mutation only in subjects with myelodysplastic neoplasms (MDNs). We identified 621 differentially expressed genes in comparing of transcriptome sequencing data of bone marrow mononuclear cells ( p < 0.01; |log2FC| > 2; Figure 1 A). Up-regulated genes in the co-mutation cohort involved those expressed in pluripotent haematopoietic stem cells (HSCs), cytokine receptor interactions and cancer-related signaling pathways ( Figure 1 B). We extended our observation by interrogating data from the GSE58831 public dataset where we identified 18 similar pathways in subjects with SRSF2 and TET2 co-mutations ( Figure 1 C ). We then used scATAC-seq to verify regulatory networks with genes and transcription factors. 49,532 cells had high-quality scATAC-seq profiles and passed stringent quality filtering with a median of 12,858 fragments per cell 39% of which mapped to peaks. 16 cell types were annotated according to cell marker genes and aligned with the public single cell RNA-seq gene expression matrix of haematopoietic cell types ( Figure 1 D-E). Next, we compared transcription factor (TF) and cis-elements active during differentiation of HSC to myelopoiesis betweenthe SRSF2 and TET2 co-mutation and SRSF2 mutation-only cohorts. In the SRSF2 and TET2 co-mutation cohort we found GATA4, RUNX1, SNAI1, REL and JDP2 favour myeloid differentiation and in NF-KB related inflammatory responses. Related gene activity correlated with high activity of inflammatory cytokines such as BCL2A1, EGR1, IL-1β, and NLRP3( Figure 1 F). SRSF2 and TET2 co-mutations result in early clonal dominance at the haematopoietic stem cell stage. 19,836 peaks were accessible in SRSF2 and TET2 co-mutated samples and annotated for putative functions by GREAT ontology enrichment analysis ( Figure 1 G-H). More accessibility peaks in SRSF2 and TET2 co-mutation were restricted to cancer-related process (reactive oxygen species biosynthetic process, regulation of phagocytosis, intrinsic apoptotic signaling pathway, mRNA catabolism) and cytokine related pathways (IL-1 mediated signaling pathway) compared with SRSF2 mutation-only samples. Next, we focused on the IL-1-mediated signaling pathway, a regulator of HSC function. We isolated umbilical cord blood HSCs (CD34+) monitored their expansion in liquid culture with or without (IL-1A or IL-1B (25 ng/ml). HSCs cultured with IL-1A or IL-1B differentiated and expanded significantly faster than untreated HSCs over 7-day period ( Figure 1 I). To directly address the effects of IL-1 on HSC differentiation we used colony formation assays in methylcellulose ± IL-1. Strikingly, IL-1-treated HSCs produced almost exclusively myeloid-committed granulocyte/macrophage (GM)-type colonies containing abundant macrophages (CFU-GM), a higher proportion of immature multi-lineage granulocyte/ erythrocyte /macrophage/megakaryocyte (CFU-GEMM)-type colonies containing mostly immature myeloblasts and a lower proportion of burst-forming unit-erythroid (BFU-E). IL-1-treated HSCs also had decreased re-placing capacity ( Figure 1 J-K). Next, we constructed a SRSF2 P95H single mutation and SRSF2 P95H mutation with TET2 knockout cell lines. We found that co-mutant cells had significantly increased NLRP3 and IL-1β expression in LPS (1 ug/ml) and ATP (5 mM) treated cultures whereas no change was detected IL-1α expression ( Figure 1 L-M). In conclusion, our data preliminary indicate SRSF2 and TET2 co-mutated causes increased NLRP3-mediated IL-1β secretion resulting in skewed differentiation of HSCs in persons with MDNs.

A Study on Heterogeneity and Early Response to Chemotherapy in Pediatric ETV6-RUNX1 Positive Acute Lymphoblastic Leukemia By RNA-Seq Expression Profile

Objective ETV6-RUNX1 positive ALL is the most common type in childhood acute lymphoblastic leukemia(ALL), and it is also a type with good prognosis. However, there are still recurrent or refractory cases in this type, and there is still heterogeneity among individuals.In this research, we investigate the heterogeneity of ETV6-RUNX1 positive ALL in children by RNA-seq expression profile, and to explore the early response to chemotherapy and microenvironment characteristics of different clusters, so as to provide evidences for the exploration of genetic high-risk factors and clinical personalized diagnosis and treatment of such children. Method From September 2019 to June 2021, 72 children with newly diagnosed ETV6-RUNX1 positive ALL in standard risk group were enrolled and treated with CCLG-ALL-2018 chemotherapy protocol, which were all detected by RNA-seq technology. The sequencing results were analyzed by cluster analysis and bioinformatics analysis. We explored the early response to chemotherapy and microenvironment characteristics of the different clusters. Result ETV6-RUNX1 positive ALL were divided into three groups according to the RNA-seq expression profile including 16 cases (22.2%) in ER1 group, 35 cases (48.6%) in ER2 group and 21 cases (29.2%) in ER3 group. The number of patients with co-negative minimal residual disease (MRD) by flow cytometry (MRD-FCM) and next-generation sequencing (MRD-Gene) on the 15th day of chemotherapy in ER1 and ER3 groups was significantly different (3/16 vs. 13/21, P=0.0178), while there were 17 cases (17/35) with co-negative MRD in ER2 group after fifteen-day chemotherapy. It showed that the proportion of NK cells ( P=0.056) and CD8 + T cells ( P=0.047) in peripheral blood in ER1 group were both higher than those in ER3 group. Compared with ER3 group, ER1 group showed low expression of RPL41 and high expression of MRPS28 and SERPIND1, which were mainly involved in stress, proliferation and drug resistance of cells and the differentiation of hematopoietic stem cell. In addition, ER1 group showed lower expression of ribosome-related genes and higher activation of mTOR pathway than ER3 group. Conclusion The heterogeneity in gene expression profile were presented in children with ETV6-RUNX1 positive ALL. Compared with ER3 group, patients in ER1 group may have a poorer early response to chemotherapy with a relatively higher risk due to the stronger proliferation and drug resistance of leukemic cells probably. For patients in ER1 group, even if MRD is negative on the 15th day of themotherapy, it may be necessary to strengthen the observation of early response and consider the treatment for escalated risk in time.

Luspatercept Modulates Inflammation in the Bone Marrow, Restores Effective Erythropoiesis/Hematopoiesis, and Provides Sustained Clinical Benefit Versus Epoetin Alfa (EA): Biomarker Analysis from the Phase 3 COMMANDS Study

Introduction :Anemia is common in patients (pts) with myelodysplastic syndromes (MDS) due to ineffective erythropoiesis, characterized by increased dysplastic erythroid precursors (EPs) and abortive differentiation in the bone marrow (BM). Insufficient red blood cells (RBCs) lead to chronic anemia and heart failure due to oxygen deprivation. Luspatercept is approved to treat anemia after erythropoiesis-stimulating agent (ESA) failure in pts with transfusion-dependent, lower-risk MDS (LR-MDS). In the phase 3 COMMANDS trial (NCT03682536), 58.5% of ESA-naive luspatercept treated pts with LR-MDS experienced RBC transfusion independence for ≥ 12 wk with concurrent mean hemoglobin (Hb) increase ≥ 1.5 g/dL during wk 1-24, vs 31.2% of EA-treated pts. Aims :To differentiate the mechanism of action of luspatercept from EA and determine its correlation with clinical benefit in the COMMANDS study. Methods :Cytomorphology assessments were performed on BM aspirates at baseline, wk 24 and 48. Complete blood count, Hb, and reticulocytes (retics) were measured in peripheral blood. BM mononuclear cells (BMMC) were subjected to bulk RNA-seq. Serum cytokine analysis was performed at screening and wk 24 using Rules-Based Medicine's Human Inflammation MAP ® v.1.1 and a custom Human MAP ELISA panel. Results :In ITT population, EPs, retics, and Hb were increased vs baseline in the luspatercept arm at wk 24 ( P < 0.001) and wk 48 ( P < 0.005). EPs, retics, and Hb were increased in the EA arm at wk 24 ( P < 0.01), but this was not sustained to wk 48. Subgroup analysis showed similar trends for ring sideroblast (RS)+ pts in both arms. While RS− pts in the EA arm showed similar trends to the ITT population, luspatercept RS− pts showed trends of EP expansion post-wk 24 only. Retics increased significantly in RS− luspatercept pts at wk 24 ( P < 0.005). Hb increased more significantly at wk 24 with EA ( P < 0.003) but at wk 48 with luspatercept ( P < 0.005).Gene set enrichment analysis of BMMC revealed enrichment of early, mid, and late EP genes at baseline favored response to luspatercept, but enrichment of mid and late EP genes was unfavorable for EA (Figure A). Significant downregulation of REACTOME IL-1 signaling (normalized enrichment score [NES] = −2.27; adjusted [Adj] P < 0.0001 [vs EA = not significant (ns)]), BIOCARTA IL-6 pathway (NES = −1.99; Adj P = 0.0025 [vs EA = ns]) and IFNα response (NES: −2.26; Adj P < 0.0001 [vs EA = ns]) was observed with luspatercept. BIOCARTA IL-1R pathway was more significantly reduced with luspatercept (NES = −1.78; Adj P = 0.0073) compared with EA (NES = −1.79; Adj P = 0.0172), as was IL-10 signaling (luspatercept NES = −2.39; Adj P < 0.001 vs EA NES = −1.82; Adj P = 0.0125). TGF-β, apoptosis, and spliceosome pathways down regulation was also observed with luspatercept. At wk 24, luspatercept responders had a median 640 pg/mL decrease ( P = 0.0035) of N-terminal pro-brain natriuretic peptide (NT-proBNP), produced by the heart, compared with a median 360 pg/mL decrease in EA arm responders ( P = 0.37). At wk 24, non-responders had a median increase of 775 pg/mL ( P = 0.0032) in the EA arm and 31.5 pg/mL ( P = 0.95) in the luspatercept arm. Conclusions : Multiplatform analysis of primary BM samples from a phase 3 trial of luspatercept revealed novel response-associated biomarkers. Compared with EA, luspatercept led to sustained increase of EPs and retics over 48 wk with a concomitant Hb increase. RS− pts showed Hb accumulation peaked at wk 24 in the EA arm. Conversely, luspatercept in RS− pts significantly increased retics at wk 24 and EPs past wk 24 with concomitant peaking of Hb at wk 48, suggesting gradual and sustained clinical benefit. Unlike EA, which plays a role in early differentiation of hematopoietic stem cells (HSCs), luspatercept acts on different erythroid stages, leading to EP expansion and maturation. Furthermore, downregulation of IL-1, IL-1R, IFNα, IL-6 pro-inflammatory (Figure B), and IL-10 anti-inflammatory signaling with luspatercept suggests modulation of the BM inflammatory environment in MDS to restore HSC function for normal hematopoiesis (Wang J, et al. Front Immunol 2022;13:951937). Additionally, decreased or static levels of NT-proBNP with luspatercept suggest a positive effect in mitigating cardiac damage. These novel insights obtained from analysis of a large controlled clinical trial mechanistically differentiate the superior clinical benefit of luspatercept from EA in treating anemia due to MDS.

From in silico to in Vitro to In Vivo: Increasing Megakaryocytes to Treat Thrombocytopenia

Hematopoietic stem cells undergo a multi-step differentiation program through several precursor stages to give rise to megakaryocytes (Mk), which ultimately release platelets. Thrombocytopenia can manifest in various clinical settings and can adversely affect a patient's quality of life through easy bruising and risk of bleeding. An example is chemotherapy-induced thrombocytopenia (CIT) which occurs when myelosuppressive chemotherapy is given, especially with certain agents such as platinum analogs and cyclophosphamide. We used a single-cell RNA-sequencing approach to study the differentiation of hematopoietic stem cells to Mk progenitors (MkP) and delineated the trajectory that defines the transition of cells to the megakaryocytic lineage. Using our proprietary platform which pairs new approaches in computation and machine learning in drug discovery, we predicted small molecule interventions that could modulate the hematopoietic lineage direction to increase the differentiation towards MkP and subsequent increases in platelet counts. Using an in vitro 5-day CD34-based Mk differentiation assay we demonstrated that some of our predicted interventions increased generation of MkP from stem cells. Analysis of early MkP (CD34-CD235a-CD71+CD41b+CD42b-) and late MkP (CD34-CD235a-CD71-CD41b+CD42b+) by flow cytometry elucidated differential mechanisms of action such as induction of early or late MKP or both. We performed an 8-day study in healthy mice, which revealed that a selected set of compounds increased MkPs in vivo, consistent with our predicted cell behavior and in vitro findings. To evaluate a possible application for this MkP and platelet increases we tested the efficacy of one of our hit compounds CP3 in a CIT mouse model using Carboplatin. In this model, we recorded a >70 % reduction in platelet counts on day 9 post carboplatin administration, with platelet numbers recovering on day 13 and return to basal levels by day 21. To assess the efficacy of CP3, mice dosed first with this compound were challenged with carboplatin. The primary endpoint of this study was the ability of the compound to rescue mice from nadir of thrombocytopenia and accelerate recovery to baseline. Prior to carboplatin dosing, 7 days of CP3 dosing showed a dose dependent increase in platelet counts confirming our initial studies. However, CP3 dosing did not rescue platelet nadir or accelerate recovery to baseline in the CIT model. This study demonstrated the capacity of CP3 to increase platelets in a healthy BM environment but not under conditions of myelosuppression as seen in the CIT mouse model. Taken together, these findings highlight the benefits of our platform to predict compounds that modulate lineage differentiation towards MkP, and a resultant increase in platelet numbers in an animal model.

Potentiality of Bone Marrow-Derived Mesenchymal Stromal Cells (BM-MSCs) to Differentiate into the Osteogenic Lineage in in Vitro Model of Tissue Regeneration

Introduction:The mesenchymal stromal cells (MSCs) have gained considerable popularity owing to the vast possibilities and lack of ethical constraints. MSCs serve as multipotent progenitors for several niche components in the bone marrow (BM) and play an essential role in the quiescence, proliferation, and differentiation of hematopoietic stem cells. Recently, MSCs from BM (BM-MSCs) represent an attractive cell source for tissue engineering, thanks to their immunomodulatory property, self-renewing and high proliferative capability. BM-MSCs have multi-lineages (adipogenesis, chondrogenesis, osteogenesis) potential and in combination with biomaterials, well support cell-attachments and stimulate extracellular matrix synthesis. For these reasons, BM-MSCs have a great potential for regenerative medicine applications. The present research aims to evaluate the ability of BM-MSCs to proliferate and differentiate toward the osteogenic lineage into innovative three-dimensional bioresorbable scaffold based on gelatin-chitosan hydrogel with a poly(lactic acid) lattice structure (PLA-CH) for regenerative medicine applications in the presence of fetal bovine serum (FBS) or human platelet lysate (HPL) with or without the osteogenic medium (OM). Methods: BM-MSCs were expanded to the passages 3 or 4 and seeded into the PLA-CH, in dry state, at a cellular density of 7x10 5 cells/scaffolds in growth medium (GM). For inducing osteogenic differentiation, cells/scaffolds constructs were cultured in 24-well plates for 4 weeks with OM consisting of a high-glucose DMEM supplemented with 10% FBS or 5% HPL, 10 −7M Dex, 25 mg/ml l-ascorbic acid, and 3mM NaH 2PO 4. For histomorphological analysis at optical microscope, scaffolds were embedded in paraffin using an automatic processor Donatello series 2 (Diapath S.p.A., Bergamo, Italy). Serial paraffin sections (5 µm thick) of each sample were cut, deparaffinized, and rehydrated, according to standard procedures and stained with hematoxylin-eosin stain for general morphology and Von Kossa (Bio-Optica, Milan, Italy) for calcium deposition. Immunohistochemistry for the osteogenic marker Osteocalcin (OSC) (Santa Cruz Biotechnologies, USA) was also performed. For Scanning Electron Microscopy (SEM) (EVO LS-10 manufactured by ZEISS) observation, samples have been progressively dehydrated through immersion in alcohol solutions. The local changes in elemental composition were investigated using the Energy Dispersive X-ray (EDX) analyzer. Results: The results showed that BM-MSCs have higher affinity to attachment, migrate and differentiate toward the osteogenic lineage inside the novel scaffold PLA-CH both with HPL and FBS as supplement in the culture media. The use of HPL for cell expansion offers the possibility to obtain a safer cellular products without xenogenic contaminants. Cells develop a large spreading area with elongated fibroblast-like morphology preferentially inside the hydrogel pores, showing great affinity with the scaffold. This is confirmed also by histomorphological analyses at optical microscope, showing homogeneous colonization of cells inside the hydrogel pores (figure 1a). The results showed the presence of calcium deposits in the mash of the scaffold with Von Kossa stain (figure 1b), moreover cells appeared well integrated with a positivity for OSC showing a clear differentiation state as osteoblasts (figure 1c). Calcium and phosphorous were detected with SEM-EDX on the samples incubated in osteogenic medium (figure 1 d,e). Conclusion: MSCs, derived from bone marrow, associated with scaffold PLA-CH proved to be biocompatible and promising for personalized medicine. In fact, BM-MSCs were able to growth, colonize and osteo-differentiate throughout the hydrogel, demonstrating their potential application for bone regenerative medicine. Figure 1. Histomorphological analysis at the optical microscope of PLA-CH with BM-hMSCs in the GM at 100 m m ( a), Von Kossa stain showing calcium deposit distribution on PLA-CH with BM-hMSCs in the OM at 30 m m ( b), immunohistochemistry for OCN with BM-hMSCs differentiated to osteoblast in the OM at 10 m m ( c). Evaluation of calcium (Ca) and phosphorous (P) with SEM-EDX of PLA-CH with BM-hMSCs in OM day 28 ( d,e).

TIF1γ Counteracts Ferroptosis to Drive Erythroid Progenitor Differentiation

Understanding in-vivo mechanisms of hematopoiesis is critical for developing directed blood differentiation approaches to treat blood disorders such as leukemias. Zebrafish moonshine ( mon) mutant embryos defective for transcriptional intermediary factor 1 gamma ( tif1γ), a conserved transcription elongation and chromatin factor, lack red blood cells due to a block in hematopoietic stem cell differentiation along the erythroid lineage. We recently showed that TIF1γ plays a critical role in mitochondrial metabolism, including maintaining adequate coenzyme Q levels. Through a chemical suppressor screen for the mon mutant, we identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase to promote erythroid differentiation in mon mutants due to its functional link to the electron transport chain. In agreement, our in-vivo metabolomics analyses identified nucleotide metabolism as the most significantly altered processes in mon mutants, with elevated levels of uridine monophosphate and low levels of N-carbamoyl-L-aspartate. Interestingly, imbalances in nucleotide metabolism similar to those in mon mutants have also been reported in the presence of active ferroptosis, a pathway of programmed cell death due to iron-dependent lipid peroxidation. Through transcriptome profiling upon tif1γ loss of function in zebrafish embryos at the onset of hematopoiesis, we uncovered an expression signature indicative of activated ferroptosis. gpx4 and fsp1, inhibitors of ferroptosis acting in parallel pathways, were both downregulated, and chac1 and ptgs2, an enhancer and marker of ferroptosis, respectively, were upregulated. In addition, parallel genome-wide expression and chromatin immunoprecipitation analyses identified anti-ferroptotic genes as direct TIF1γ targets in human cells. In support of these results, we found mon mutants to exhibit increased levels of lipid peroxidation. Functionally, tif1γ loss of function synergizes with loss of gpx4 in blocking erythroid differentiation. These results demonstrate a tight coordination of nucleotide metabolism, mitochondrial respiration, and the regulation of lipid peroxidation as a key function of tif1γ-dependent transcription that drives cell fate decisions in the early erythroid lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases.

Identification of Cellular Compositions in Different Microenvironments and Their Potential Impacts on Hematopoietic Stem Cells HSCs Using Single-Cell RNA Sequencing with Systematical Confirmation.

Hematopoietic stem cells (HSCs) are stem cells that can differentiate into various blood cells and have long-term self-renewal capacity. At present, HSC transplantation is an effective therapeutic means for many malignant hematological diseases, such as aplastic hematological diseases and autoimmune diseases. The hematopoietic microenvironment affects the proliferation, differentiation, and homeostasis of HSCs. The regulatory effect of the hematopoietic microenvironment on HSCs is complex and has not been thoroughly studied yet. In this study, we focused on mononuclear cells (MNCs), which provided an important microenvironment for HSCs and established a methodological system for identifying cellular composition by means of multiple technologies and methods. First, single-cell RNA sequencing (scRNA-seq) technology was used to investigate the cellular composition of cells originating from different microenvironments during different stages of hematopoiesis, including mouse fetal liver mononuclear cells (FL-MNCs), bone marrow mononuclear cells (BM-MNCs), and in vitro-cultured fetal liver stromal cells. Second, bioinformatics analysis showed a higher proportion and stronger proliferation of the HSCs in FL-MNCs than those in BM-MNCs. On the other hand, macrophages in in vitro-cultured fetal liver stromal cells were enriched to about 76%. Differential gene expression analysis and Gene Ontology (GO) functional enrichment analysis demonstrated that fetal liver macrophages have strong cell migration and actin skeleton formation capabilities, allowing them to participate in the hematopoietic homeostasis through endocytosis and exocytosis. Last, various validation experiments such as quantitative real-time PCR (qRT-PCR), ELISA, and confocal image assays were performed on randomly selected target genes or proteins secreted by fetal liver macrophages to further demonstrate the potential relationship between HSCs and the cells inhabiting their microenvironment. This system, which integrates multiple methods, could be used to better understand the fate of these specific cells by determining regulation mechanism of both HSCs and macrophages and could also be extended to studies in other cellular models.

A Dual Role of SOX11 Expression in the Formation of T-Cell Acute Lymphoblastic Leukemia in Mouse Models

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Intensified chemotherapy and bone marrow transplants have improved the survival of T-ALL patients. However, around 20% of patients still suffer from primary resistant or relapsed T-ALL. Therefore, improving our knowledge of T-ALL biology and developing precision oncology therapeutics is essential for further progress in T-ALL treatment. SRY-related HMG-box 11 (SOX11) is a transcription factor that regulates the development and survival of multiple embryonic tissues and is crucial for the nervous system. SOX11 is expressed in a multitude of tumor types, where it has been associated with either increased or decreased cell proliferation, metastatic potential, and with either good or bad prognosis, depending on cancer type. Despite being absent in the T-cell lineage, SOX11 is expressed in a subset of T-ALLs, where it plays an unknown role. Based on having a malignantly restricted expression, we hypothesize that SOX11 might be oncogenic in T-ALL. Aim: Identify the role of aberrant SOX11 expression in (pre)leukemic T-cells. Methods: Using a conditional Rosa26-SOX11 overexpression mouse model, we generated mice with T-cell specific (using Lck-cre) SOX11 expression (SOX11 Lck). To assess the self-renewal potential of SOX11 Lck thymocytes, we performed thymic transplantations on sublethally irradiated mice expressing a different allele of CD45. To study SOX11 in a leukemic context, we crossed the SOX11 Lck mice with two established T-ALL mouse models that are either driven by the overexpression of Lmo2 (SOX11 Lck-Lmo2 CD2) or by the loss of the tumor suppressor Pten (SOX11/Pten Lck). An aging cohort of these T-ALL models was followed, and tumor samples were collected. Thymi of 8-weeks old mice were used to study the impact of SOX11 in a pre-leukemic context, and T-cell tumors arising in these models were also used for RNA-sequencing. Using a retroviral strategy, we overexpressed SOX11 in human cord blood stem cells, which we differentiated towards the T-cell lineage ex vivo by co-culturing them on OP9 stromal cells expressing Delta-like Notch ligand 4. Furthermore, we transduced Jurkat cells with a SOX11 expression vector, or with an empty vector, and injected the cells into immunocompromised mice to assess the effect of SOX11 on tumor growth in vivo. Results: In SOX11 Lck mice, we found a significant increase in the immature double-negative 3 (DN3) thymic fraction, with a concomitant decrease in the amount of more mature double-positive CD4 +CD8 + (DP) thymocytes (Fig. A). A possible explanation for this phenotype would be the establishment of a stem-like self-renewal program similar to the one driven by Lmo2. However, by performing thymic transplantations into sublethally irradiated mice, we concluded that SOX11 does not confer self-renewal properties to thymocytes. Additionally, we aged a cohort of SOX11 Lck mice and did not observe formation of T-cell malignancies up to 500 days of age. Given that the expression of SOX11 does not suffice to produce T-cell tumors, we generated the SOX11 Lck-Lmo2 CD2 and SOX11/Pten Lck T-ALL mouse models. In SOX11 Lck-Lmo2 CD2 mice, we found a synergistic effect of the co-expression of both genes on the expansion of DN3 thymocytes (Fig. A), which led to accelerated leukemia formation (Fig. B). By sequencing the RNA of pre-leukemic and leukemic DN3 cells, we observed that SOX11 is mostly regulating cell adhesion, morphology, and cell-cell interactions to promote this oncogenic phenotype in Lmo2-driven T-ALL. On the other hand, SOX11/Pten Lck mice suffered from delayed leukemogenesis (Fig. B), and this tumor suppressor role might be associated with the decrease in DP cells observed in the pre-leukemic stage. Lastly, we set out to validate these findings in a human context. The ex vivo differentiation of human hematopoietic stem cells toward the T-cell lineage showed that SOX11 strongly accelerates early commitment to double-negative T-cell precursors. Also, we observed that Jurkat T-ALL cells engraft faster in immunocompromised mice when expressing SOX11. Conclusions: By generating multiple models of SOX11 expression in the T-cell lineage, we showed that SOX11 impacts T-cell differentiation, both in mice and humans, and that it can promote or hinder T-ALL formation depending on the oncogenic driver.

A Phase 2 Study of Canakinumab in Patients with Lower-Risk Myelodysplastic Syndromes or Chronic Myelomonocytic Leukemia

Background Myelodysplastic syndromes (MDS) lie on a continuum of myeloid neoplastic progression between clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Interleukin 1 beta (IL-1β)-induced inflammasome activation is a mechanism of cancer initiation and progression. Given that IL-1β upregulation induces aberrant myeloid differentiation of hematopoietic stem and progenitor cells (HSPCs), we hypothesize that IL-1β plays a critical role in MDS pathogenesis, and targeting IL-1β signaling will overcome aberrant MDS HSPCs' myeloid skewing. Here, we report final results of a phase 2 study evaluating the safety and efficacy of canakinumab, an IL-1β inhibitor, in patients with lower-risk MDS or chronic myelomonocytic leukemia (CMML). Methods We conducted a single center, open-label phase 2 trial at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS/CMML who have previously been treated were included in the study. Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. The primary objective was to determine the clinical activity of canakinumab in MDS/CMML patients. Response was assessed using the modified International Working Group 2006 criteria for MDS (Cheson et al. 2006). Secondary objectives were to evaluate the safety profile including rate of transfusion independence (TI), duration of response (DoR), progression-free survival (PFS), and overall survival (OS). All patients who received 1 dose of study drug were included in the analyses. This study is registered with ClinicalTrials.gov (NCT04239157). Findings Between August 2020 and May 2023, 27 patients were enrolled in the phase 2 portion of this study, and 2 patients failed screening. The median age was 74 years with 15 (60%) male patients; 20 patients (80%) were post-hypomethylating agent failure with a median number of prior lines of therapy of 2 (1-5). Transfusion dependency was observed in 24 patients (96%) prior to canakinumab initiation. Fourteen (56%) patients showed normal karyotype. IPSS-R stratification revealed an intermediate-2 risk in 12 (48%) patients and a high risk in one (4%). The most common mutations were SF3B1 (40%), TET2 (32%), DNMT3A (28%), and RUNX1 (24%). IPSS -M risk score was calculated for 24 cases showing one (4%) very low (VL), four (16%) low (L), 14 (16%) moderate low (ML), eight (32%) moderate high (MH), five (20%) high (H), and two (8%) very high (VH) risk categories. Canakinumab was well tolerated and no drug-related toxicities were observed. One death due to sepsis, which was deemed not treatment-related, occurred on the study drug. Out of 23 evaluable patients, the overall response rate was 17·4%, with erythroid and platelet hematological improvement (HI-E and HI-P, respectively) confirmed in 3 (13.0%) patients and 1 (4.3%) patient, respectively. Thirteen patients had stable disease (56·5%) and 6 (26·1%) progressed during therapy, 1 of which transformed to AML (Fig. 1). TI was achieved in 3 patients (median DoR 8·53 months (95% CI 0·41-16·1) and 2 of them maintained TI for over 12 months. With a median follow-up time of 22·6 months (95% CI 15·0-29·4), median OS was 17·3 months (95% CI 14·3-not estimable). We performed separate univariate analyses to evaluate any associations between the IPSS-M and OS/PFS. The median OS in patients with higher-risk MDS by IPSS-M (MH, H, VH) was 15·0 months vs 29·4 months in the lower-risk disease by IPSS-M (VL, L, ML) group (p=0·12). Interestingly, statistically significant findings were observed with 1-year PFS when stratifying patients into higher vs lower risk MDS by IPSS-M (64·3% vs 100·0%, respectively; p=0·022, Fig. 2). Conclusion In this cohort of MDS patients who had experienced multiple lines of prior therapy and exhibited high-molecular-complexity, canakinumab showed limited efficacy (HI 17.3%). Nevertheless, canakinumab showed a good safety profile and yielded sustained long-term responses in patients with single somatic driver mutation in TET2 or DNMT3A. This suggests that clonal complexity, and therefore disease burden, may be a determining factor in response to canakinumab. Therefore, we have amended the protocol of a phase 1/2 clinical trial evaluating the safety and activity of canakinumab in clonal cytopenia of unknown significance (CCUS) and low-molecular complexity MDS patients. Our results will clarify the role of IL-1β signaling in MDS initiation and progression.

High Frequency of RUNX1 Mutation in Acute Myeloid Leukemia at Puerto Rico

Acute Myeloid Leukemia (AML) is the most common form of acute leukemia in adults, with a median age at diagnosis of 68 years. Recurrent genomic abnormalities are established as diagnostic, prognostic markers, and play an essential role in leukemogenesis. For instance, loss-of-function mutations of the runt-related transcription factor-1 (RUNX1) have been associated with distinct clinical and genetic features and overall adverse risk in adult AML. Lack of a specific targeted therapy and resistance to standard intensive chemotherapy likely accounts for the poorer outcome observed in AML expressing RUNX1. Additionally, germline RUNX1 mutations result in the well-described autosomal-dominant familial platelet disorder with predisposition to hematologic malignancies. Previuos studies has identified mutant RUNX1 in 5-10% of patients at a median age of 68 years old. The objective of our retrospective study was to identify the incidence and characteristics of RUNX1 mutations in our population. RUNX1 plays an important role in the regulation and differentiation of normal hematopoietic stem cells. Therefore, intragenic mutation of RUNX1 leads to impaired hematopoiesis and ultimately leukemia. The study examined newly diagnosed (ND) patients-AML with RUNX1 mutation by NGS; that were at least 18 years of age or older with cytogenetic and molecular analysis in a period of 4 years (2019-2022). According to the Puerto Rico Cancer Registry from 2015 to 2019 a total of 551 cases of AML were reported. All data regarding demographic, bone marrow biopsy, cytogenetics, and molecular markers were obtained from electronic medical record at San Juan City Hospital with institutional review board approval in acordance with the Declaration of Helsinki. The San Juan City Hospital is the major referral center on the island for initial evaluation and management of AML. A total of 157 patients with ND AML were identified during the study period. 58% female and 42% male with a median age of 57 years old at time of diagnosis. Compared to an expected ~5-10% incidence of RUNX1 mutations, our study population revealed the presence of RUNX1 in 24% of our AML patients, with a median age less than 60 years old. More specificially within the 37 patients with RUNX1 mutations, 46% female and 54% male with a median age of 56 years old with an average variable allele frequency(VAF) of 40%. Complex cytogenetics was identified in 41% of RUNX1 cases. Co-occuring mutation of RUNX1 with TP53 was identified in 19% of patients. Our study showed a high expression of RUNX1 in Puerto Rican ND-AML. The median age at diagnosis and the average VAF suggest the possibility of a germline RUNX1 in our population. Previous studies in medical literature have identified a correlation for germline RUNX1 in patients diagnosed before 60 years old and with a VAF above 40%. Future studies with larger sample size and longer follow-up may confirm if there is a germline predisposition in our patients for RUNX1.

Establishment of Human Bone Marrow-on-Chip As a Preclinical Model to Evaluate Drug-Induced Toxicities and Myelofibrosis Patient-Specific Pathophysiology

Hematopoiesis takes place in the bone marrow (BM) and is supported by a complex cellular and molecular network in the BM microenvironment. The inaccessibility of living human BM hampers the study of its pathophysiology under stress induced by drugs, radiation, or genetic mutations. Commonly used models of the human BM microenvironment rely on mouse models and 2D or 3D static tissue cultures. Although existing in vitro hematopoiesis models have led to advances in the field, they fail to recapitulate the biology of cellular interactions, cell extrinsic factors, and complex functions of living human BM hematopoiesis. This has also limited our understanding of human BM biology and the influence of BM niche cells in BM defects in diseases such as myeloproliferative neoplasms (MPNs) and leukemias. Consequently, there is an unmet need to develop a preclinical human BM hematopoiesis model that supports growth and differentiation of multilineage hematopoietic stem cells to enable evaluation of investigational compounds for myeloerythroid toxicity and efficacy. Microphysiologic systems (MPS), such as organ-on-chips, emulate the complex interplay between intrinsic and extrinsic cell factors in the BM microenvironment, the 3D tissue architecture, and are more clinically relevant compared with static co-culture approaches (PMID: 31988457; PMID: 37359774). Here we present the development of a human BM-on-chip MPS model to recapitulate both healthy and MPN disease and evaluate the alterations in the BM niche environment. We utilized a commercially available organ-on-chip microfluidic platform (Emulate, Boston, MA) to develop a living human BM with a functional hematopoietic niche in vitro by perfusion with culture medium. This fluidic-controlled modular chip comprises an osteogenic apical channel separated from a vascular basal channel by a semiporous membrane, which allows for media perfusion, cellular crosstalk, and intercellular signaling. The osteogenic apical channel is filled with a fibrin gel that allows the co-culture of all critical cells of the BM niche, including osteoblasts, endothelial cells, mesenchymal stem cells, hematopoietic stem and progenitor cells. The parallel basal channel is lined by human vascular endothelium and is perfused with culture medium optimized to allow survival and development of functional hematopoietic lineage niche cell populations. This unidirectional fluidic MPS model allows for collection of cell effluents for evaluation of cell-extrinsic factors such as cytokines, extracellular vesicles, and metabolites. The modular and accessible nature of this system provides an advantage to investigate the effects of the microenvironment and drug candidates individually on the osteogenic and vascular compartments. This human BM-on-chip has been tested with cells from several healthy and myelofibrosis donors. Utilizing imaging, flow cytometry, cytokine profiling, and transcriptomics, we demonstrate that the vascularized human BM-on-chip recapitulates the living human hematopoietic niche, with BM relevant cytokine levels, retains hematopoietic stem and progenitor cells in in vivo-like proportions for over 3 weeks in culture, allows for the differentiation of BM niche lineage cell populations similar to human BM aspirate, and captures human donor variabilities. We also show that BM-on-chips derived from MPN patients recapitulate the hematopoietic defects to understand the biology and disease response in myelofibrosis. Our robust experimental workflow and bioinformatics platform will be utilized to evaluate candidate compounds for BM myelotoxicities, drug mechanisms of action, and biomarkers in a multifactorial manner to support preclinical programs. This biomimetic platform offers a new approach for analysis of drug responses and toxicities in BM as well as for study of hematopoiesis and hematologic diseases such as myelofibrosis.

CPT1a Controls the Function of Long-Term Hematopoietic Stem Cells By Regulating the Balance between Fatty Acid Oxidation and Oxphos in Mitochondria

Hematopoietic stem cells (HSCs) rely on the balance in place of quiescence and self-renewal to sustain stem cell potential and differentiation to generate mature blood cells. Appropriate regulation of cellular and mitochondrial metabolic processes such as glycolysis, OXPHOS, fatty acid metabolism, and amino acid metabolism are required for HSC maintenance. However, the mechanisms by which cellular and mitochondrial metabolisms regulate HSC quiescence, proliferation, and differentiation remain to be fully understood. Fatty acid oxidation (FAO) is a metabolic process involving the breakdown of fatty acids into acetyl-CoA, which is then transported into mitochondria by carnitine palmitoyltransferase 1a (CPT1a). It has been reported that inhibiting FAO using non-specific inhibitors of CPT1a impairs HSC maintenance. Interestingly, CPT1a forms a complex with the Fanconi anemia complementation group D2 (FANCD2) protein (T Zhang et al., Scientific Reports, 2017). Given that Fanconi anemia is associated with HSC defects, this CPT1a complex may play a critical role in maintaining fatty acid dependent mitochondrial metabolism that mediates HSC dormancy and stemness. However, the exact role, in HSCs, of mitochondrial CPT1a, the rate-limiting enzyme in FAO, remains unknown. Here, we find that CPT1a is highly enriched in hematopoietic stem cells. Further, we investigated the role of CPT1a in hematopoiesis utilizing a CPT1aconditional knockout in mice driven by Vav1-Cre. The CPT1a knockout ( Cpt1aΔ/Δ) displays profound HSC defects, including loss of HSC self-renewal and accelerated differentiation. Mechanistically, we find that loss of Cpt1a results in increased ATP production, mitochondrial membrane potential (Δφm), and mitochondrial ROS accumulation in long-term HSCs (LT-HSCs), as well as defective LT-HSC function indicated by bone marrow transplantation and 5-FU genotoxic stress assays. Furthermore, utilizing mitochondrial complex formation assays and measurements of mitochondrial complex activity, BM cells of Cpt1aΔ/Δ mice display enhanced mitochondrial complex formation and up-regulation of mitochondrial complex activity. Our results suggest that Cpt1a regulates FAO transport and that loss of Cpt1a deregulates mitochondrial metabolisms including oxidative phosphorylation (OXPHOS). Taken together, our study suggests that CPT1a plays a key role in LT-HSC maintenance by controlling the balance between FAO and OXPHOS in mitochondria. Because of the noncanonical relationship between CPT1a and FANCD2 for the regulation of mitochondrial metabolism, correction of fatty acid metabolism and OXPHOS may present a novel therapeutic avenue for Fanconi anemia.

Efficient and Stable Makassar Base Editing in Nonhuman Primates

Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin chain. This nucleotide can be modified using adenine base editors (ABEs) to convert the defective SCD β-globin gene (HBB S) into the non-pathogenic Makassar β-globin (HBB M) variant. Proof-of-concept for this editing strategy in human and mouse hematopoietic stem cells (HSCs) has been previously demonstrated by transplantation after ex vivo modification as well as by in vivo adenoviral editing followed by selection. Here, we evaluated the feasibility, long-term efficiency, and safety of autologous ex vivo Makassar base editing in the preclinical nonhuman primate (NHP) large animal model to pave the way towards clinical applications in humans. As NHPs don't carry the sickle mutation (position A7 in the protospacer), bystander editing at position A9 was monitored as a surrogate. NHP CD34 + hematopoietic stem and progenitor cells (HSPCs) were enriched, CD34 +CD90 + HSCs FACS-purified as previously reported to increase the editing efficiency of short- and long-term engrafting cells, primed over-night, and ABE8e-NRCH base editor mRNA was delivered by electroporation. Base-edited HSCs were cultured overnight and then combined with unmodified CD34 +CD90 - cells and infused into animals after myeloablative total body irradiation. The infusion product was quality controlled by flow-cytometry, next generation sequencing (NGS), and assessing the mono-/bi-allelic base editing efficiency on a single cell level from colony-forming cell (CFC) assays. Animals were followed for up to 200 days analyzing the editing efficiency in peripheral blood (PB) white blood cells (WBCs), FACS-purified lineages from the PB, bone marrow (BM) WBCs, as well as FACS-purified BM HSPCs. Comprehensive bioinformatics analysis was performed on the generated NGS data to determine the frequency of editing over time as well as the on- and off-target editing within the protospacer at all four target locations. Finally, chromosomal integrity after base editing was confirmed performing Kromatid karyotyping assays on gene-edited BM-derived HSPCs. Ex vivo base editing of CD34 +CD90 + was highly reproducible at the Makassar locus reaching more than 60% efficiency within bulk HSCs. No impact of base editing on the myeloid and erythroid differentiation of HSCs was seen in CFC assays. Clonal analysis of colonies revealed that more than 90% of HSCs in the infusion product were edited with > 20% of cells showing biallelic editing. Gene-edited HSCs combined with unmodified progenitors rapidly engrafted in myeloablated animals without any noticeable delay in the recovery of neutrophils, red blood cells, as well as platelets and multilineage reconstitution was established within 3-6 months. Most importantly, stable editing of >20% was observed in PB WBCs and across all lineages throughout the entire follow-up. Similarly, the BM stem cell compartment fully recovered to baseline within 6-month post-transplant. Engrafted HSPCs demonstrated normal erythro-myeloid colony-formation potential. Assessment of editing in colonies showed persistence of HSCs with mono- and bi-allelic Makassar editing 6-month post-transplant. Closely matching the editing efficiency in the PB, all BM lineages, HSPC subsets, and colonies demonstrated >20% editing efficiency confirming successful editing of long-term persisting multipotent HSCs. Finally, comprehensive assessment of NGS data from all tissues in conjunction with the Kromatid assay revealed no unwanted off-target effects or chromosomal rearrangements. Editing was primarily observed at positions A9 and A12 confirming highly specific on-target editing. In summary, we demonstrate efficient, persistent, and safe base editing of NHP HSCs for the treatment of SCD. The editing efficiency of the bystander adenine in NHP HSCs is almost identical to the previously observed editing in human patient HSCs to generate the Makassar HBB. Base edited HSCs showed no impairment in homing, long-term engraftment, or multilineage differentiation in the NHP. These findings have major implications for the application of base editing in patients with hematological disease and disorders.

Early Molecular Steps in Human Bone Marrow Stem Cell Differentiation Trajectories

Understanding the molecular signatures of human hematopoietic stem/progenitor cells (HSCPs) and gene network changes leading to differentiation induction are of utmost importance to achieve therapeutic hematopoietic stem cell (HSC) expansion. The identification of surface markers has improved the prospective isolation of human bone marrow HSCs over the last decades, but the isolated population still remains very heterogeneous. Single cell sequencing approaches have the potential to unbiased assort stem cells along their differentiation stages and trajectories, and to decipher molecular markers in differentiation hierarchies. However, whole transcriptome single cell sequencing often suffers from inadequate detection of low-expressed genes with increased drop-outs. To address this limitation and to provide sensitive mRNA quantification in human HSPCs, we developed a selected targeted gene panel (600 genes) in combination with 50 protein markers to cover most relevant genes/proteins of early HSPC differentiation and cell cycle status. We employed single cell CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) based multi-omic profiling to >60.000 high-quality FACS-enriched human bone marrow HSPCs (CD34 + and CD34 +CD38 - cells) from 15 healthy donors representing young, mid and old age groups. Here, we demonstrate the most comprehensive molecular map of early human HSC differentiation and provide evidence of early branching points into lineage trajectories across different age groups using Slingshot pseudotime analysis. Our data demonstrate that the HSC branched into two main lineages, with the first branching point giving rise to megakaryocytic and erythroid progenitors (MKP/ERP), followed by lymphoid and myeloid progenitors (LMP/MDP). As expected, the most immature HSCs were defined by high expression of CD34, CD90, CRHBP, HOPX, HLF, MLLT3. However, we observed a number of hitherto undescribed HSC markers. The MKP/ERP lineage was defined by GATA1, TFRC, CD36, MPL and VWF expression. Furthermore, we found CD274, the Programmed Cell Death receptor ligand 1, to be selectively expressed in the MKP cluster. Re-clustering and trajectory analysis of the most immature CD34 +CD38 -/low cells confirmed early lineage commitment, suggesting that an MKP/ERP development does not include myeloid or lymphoid progenitors. A similar pattern was observed for all age groups. Interestingly, we observed increased expression of novel marker genes ( HLA-E, DLK1, ADGRG6, ADGRG1, MMRN1) and proteins (CD137 and CD273) in the most immature HSCs. Besides a gradual upregulation of CD38 along differentiation, differentiated cells showed lower CD45 levels. Furthermore, MKP/ERP committed cells showed a downregulation of SPINK2. TradeSeq analysis defined the continuous single cell expression changes in genes and markers upon early differentiation and lineage commitment, allowing the detection of decision-making gene networks. Our data adds further evidence to the revised model of hematopoiesis that challenges the dogma of a common myeloid progenitor giving rise to erythroid, megakaryocytic and myeloid cells. Furthermore, we see a structured hierarchical organization with a sequential loss of lineage potential and not an early commitment of uni-lineage restricted precursors. We report on the upregulation of ADGRG6, ADGRG1, DLK1, HLA-E, MMRN1 CD137 and CD273 in the most immature HSCs. Molecular and functional characterization of CD273 hi and low HSCs are presented in an adjacent report by our group at this meeting (Schmachtel et al.). In conclusion, we provide a valuable resource for the continuous regulation of genes and surface proteins along early differentiation of very immature human HSCs.

Human Hematopoietic Stem Cells Have Altered Mitochondrial Activity after Stem Cell Transplantation

Hematopoietic stem cells (HSCs) give rise to the entire blood system. With their high regenerative potential, they are sometimes the only curative option for many malignancies. The success of HSCT depends on both the quantity of HSCs and on their ability to produce life-long blood. However, their regenerative potential is not unlimited. Transplanted HSCs sustain injury that alters their subsequent ability to generate blood cells. This leads to ineffective hematopoiesis and can ultimately lead to graft failure. Injury to HSCs is also a risk factor for secondary neoplasms, including leukemia. Our goal is to elucidate the mechanisms behind post-transplant HSC decline. Mitochondria are critically important for HSC function (Filippi Exp Hematol 2021). During regeneration of the bone marrow, HSCs become activated and undergo mitochondrial reprogramming, including reorganization of the mitochondrial network and enhanced mitochondrial activity (Filippi et al Blood 2019, Ito et al Nat Rev Mol Cell Biol 2014). However, after transplantation, HSCs accumulate mitochondria that are abnormal in organization and activity. These abnormal mitochondria lead to decline of HSC function and ineffective hematopoiesis (Hinge et al Cell Stem Cell 2020). While these findings have been replicated in murine models, it has not been studied within the human system. Our hypothesis is that after transplantation, human HSCs have altered mitochondria leading to ineffective hematopoiesis. We examined changes in HSC function using post-transplant samples of patient's bone marrow and a xenotransplant system (human CD34+ peripheral blood stem cells transplanted into sublethally irradiated immunodeficient NOD/SCID/IL-2 receptor-γ null mice). We first measured mitochondrial membrane potential using tetramethyl rhodamine ethyl ester (TMRE) dye and analyzed via flow cytometry. Three to six months after transplant in the xenotransplant model, we found that the mitochondrial membrane potential of huCD34+CD38-CD90+ HSCs was similar to the non-transplanted HSCs. However, huCD34+CD38+ progenitors exhibited a drastic 3-fold increase in mitochondrial membrane potential in comparison to the non-transplanted, huCD34+CD38+ progenitor cells. The mitochondrial membrane potential of CD34+CD38+ progenitors from transplanted bone marrow patient samples was also higher than non-transplanted CD34+CD38+bone marrow control cells. To understand if alterations in mitochondrial activity affect HSC differentiation, we placed xenotransplanted HSCs into in vitro culture. Post-transplanted huCD34+ cells failed to expand within 6 days of in vitro culture (serum-free culture with self-renewing cytokines, SCF+TPO+FLT3) compared to huCD34+ control. In subsequent differentiation assays, post-transplanted huCD34+ cultures gave rise to myeloid cells only. This contrasts with control huCD34+ cultures that produced myeloid, erythroid, and megakaryocytic cells. Interestingly, if the post-transplanted HSCs were given serum and cytokines for differentiation after only 3 days in self-renewing conditions, the cultured cells expanded similarly to non-transplanted HSCs and generated myeloid, erythroid, and megakaryocytic cells. This suggests that transplanted HSCs have decreased proliferation and differentiation ability in comparison to non-transplanted HSCs and respond differently to cytokines in vitro. This could account for ineffective hematopoiesis observed in some post-transplant patients. In conclusion, this study indicates that both mitochondrial activity and in-vitro growth change in HSCs after transplantation, despite re-establishing quiescence. This indicates that human hematopoietic stem and progenitor cells incur injury after transplantation. These findings are clinically important as the changes in mitochondrial activity in HSCs may play a role in post-transplant hematopoietic stem cell decline. Future single cell RNA sequencing analyses will provide mechanistic information regarding post-transplant changes in metabolism and potential targets for improving HSC function in transplant patients.

Identification of Acute Myeloid Leukemia Cell Surface Therapeutic Targets Using Single Cell RNA Sequencing Supported By Surface Proteomics

Background: Acute myeloid leukemia (AML) comprises diverse genomic subgroups and remains hard to treat in most patients. Despite breakthroughs in the therapeutic arsenal in recent years, clinical usage of therapeutic antibodies or chimeric antigen receptor T (CAR-T) cells has been lagging in contrast to other hematological malignancies. In fact, CD33 represents the only antibody-based strategy approved for this disease to date, highlighting the need to identify new promising targets. AML cells span a wide range of aberrant myeloid differentiation programs, complexifying the identification, by bulk genomics, of targets expressed in the most immature leukemic cells. Aims and Methods: To identify the expression landscape of surface proteins in immature leukemic cells, we performed single-cell RNA sequencing (scRNA-seq, 10x 3' Reagent Kits) of primary human AML cells from 20 specimens of the Leucegene cohort enriched in intermediate and adverse genetic backgrounds ( KMT2A-rearranged n=5, chromosome 5 and/or 7 deletions (abn5/7, n=5) complex karyotype (n=4), NPM1/DNMT3A/FLT3-ITD triple-mutant (n=3) and others (n=3)). A Random Forest classifier was developed to unbiasedly classify AML cells into distinct differentiation stages using normal bone marrow-derived scRNA-seq data from the Human Cell Atlas (HCA) consortium. Genes were scored based on their probability of coding for proteins expressed at the cell surface using the SPAT algorithm developed by our group (https://doi.org/10.1101/2023.07.07.547075), retaining high score ones. To validate surface expression, we concomitantly analyzed the surface proteome (hereafter named surfaceome) of 100 primary human AML samples from the Leucegene cohort, including all 20 samples profiled by scRNA-seq. Results: After quality control, we profiled and characterized 103 690 high quality cells (mean of 5185 cells/sample). We trained a Random Forest classifier to annotate cells in a two step process, first identifying plasma cells based on a restricted list of genes abundantly expressed in these cells and subsequently assigning the remaining cells to one of 33 cell types. We performed a five-fold cross validation of the model and subsequently determined the accuracy of our classifier to be 92% on the test subset of the HCA data. Applied to our AML cell collection, a total of 35 053 cells (34%) were unbiasedly classified as Hematopoietic Stem Cell (HSC)-like, corresponding to the most phenotypically immature leukemic cells in each patient sample (ranging from 4 to 74 %). Accordingly, HSC-like AML cells preferentially express genes associated with normal HSCs, such as CD34, FAM30A, and SPINK2, and globally lack expression of mature lineages defining genes, further validating our classifier. The proportion of HSC-like cells varied among AML subgroups, and was lowest in KMT2A-r AML (median 19%) and highest in abn5/7 samples (46%). Integration of our AML atlas using Harmony algorithm preserved differentiation hierarchies across samples, with most cell types, including HSC-like cells, occupying a defined area in the low dimensional embedding. To identify new surface antigens specifically expressed in immature leukemic cells, we compared the high (≥8) SPAT score gene expression profile of AML HSC-like cells with that of normal HSC cells (HCA), and identified 60 genes significantly overexpressed in AML immature cells. Of those, 39 genes were also detected at the protein level by the surfaceome analysis, supporting their predicted expression at the cell surface in AML samples. 59% of these 39 genes (n=23) were detected in over 80% of the specimens analyzed by the surfaceome, and thus are nearly universally expressed in our AML cohort. To identify targets of therapies that could be repurposed, we next evaluated the relevance of our findings by querying the Thera-SAbDab database. Most interestingly, 8 of the 39 AML specific HSC markers are targeted by therapeutic antibodies FDA-approved or in clinical trials for the treatment of AML (n=4, IL3RA, FLT3, CD37 and TNFRSF10B) or other indications (n = 4). Conclusion Our genetically diverse AML single-cell atlas, supported by mass spectrometry, enables the identification of both subset-specific and pan-AML surface protein genes. These represent potential targets for antibody based strategy development or therapy repurposing in AML.

Bone Marrow as a Therapeutic Target for Type 2 Diabetes Complications.

Type 2 diabetes mellitus (T2DM) is a world epidemic with a high prevalence and mortality. The origin of macro and microvascular complications associated with T2DM is complex and new mechanisms to explain their development are emerging. The changes induced by T2DM in the microenvironment of bone marrow (BM) alter the expansion and differentiation of stem cells and have been related to the development of micro and macrovascular diseases. Alterations in the differentiation and function of hematopoietic, endothelial, and mesenchymal stem cells in T2DM patients reduced the mobility of BM stem cells to the circulation and some immature, dysfunctional, or inflammatory cells pass to the blood (mobilopathy). Consequently, tissue repair is impaired, and the tissue damage caused by hyperglycemia, oxidative stress, and inflammation is increased. These alterations can contribute to diabetic complications, decreasing the quality of life, and increasing mortality. The modulation of the bone marrow microenvironment may be a therapeutic target for treating T2DM and its complications. This article analyses the changes induced in BM and their impact on the development of cardiovascular and kidney complications in T2DM. Also, different therapeutic strategies to restore the bone marrow microenvironment and function through the modulation of oxidative stress, inflammation, and adipogenicity are discussed, considering bone marrow as a novel potential therapeutic target to treat vascular complications of diabetes.

Aggf1 Specifies Hemangioblasts at the Top of Regulatory Hierarchy via Npas4l and mTOR-S6K-Emp2-ERK Signaling.

Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins. Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown. These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.

Vitisin A, as a Type of Pyranoanthocyanin, Suppresses Inflammation by Restricting Hematopoietic Stem Cell Differentiation toward Monocytes in Bone Marrow.

Hematopoietic stem and progenitor cells (HSPCs) could be differentiated into mature myeloid and lymphoid cells, maintaining the requirements of immune cells. Atherosclerosis and ulcerative colitis (UC) drive HSPC homeostasis destruction, which triggers expansive HSPC proliferation and Ly6Chi monocyte production, contributing to aggravated inflammation. Vitisin A belongs to the anthocyanin derivatives with excellent stability and bioactivity in vitro. However, there is no report about the anti-inflammation of Vitisin A via reprogramming HSPC differentiation toward monocytes. In this study, we found that Vitisin A presents anti-inflammatory ability during the development of atherosclerosis and UC by depressing Ly6Chi monocyte production from bone marrow. This performance depended on restricted HSPC differentiation, which suggested that Vitisin A participated in monocyte generation and carried out the immunomodulation. Together, Vitisin A ameliorates inflammation during atherosclerosis and UC via the suppressed differentiation of HSPCs toward monocytes, which could be considered an ideal functional component with immunomodulatory effects.

Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing

Aspartyl-tRNA synthetase 2 (Dars2) is involved in the regulation of mitochondrial protein synthesis and tissue-specific mitochondrial unfolded protein response (UPRmt). The role of Dars2 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) is unknown. Here, we show that knockout (KO) of Dars2 significantly impairs the maintenance of hematopoietic stem and progenitor cells (HSPCs) without involving its tRNA synthetase activity. Dars2 KO results in significantly reduced expression of Srsf2/3/6 and impairs multiple events of mRNA alternative splicing (AS). Dars2 directly localizes to Srsf3-labeled spliceosomes in HSPCs and regulates the stability of Srsf3. Dars2-deficient HSPCs exhibit aberrant AS of mTOR and Slc22a17. Dars2 KO greatly suppresses the levels of labile ferrous iron and iron-sulfur cluster-containing proteins, which dampens mitochondrial metabolic activity and DNA damage repair pathways in HSPCs. Our study reveals that Dars2 plays a crucial role in the iron-sulfur metabolism and maintenance of HSPCs by modulating RNA splicing.