Myeloid Progenitor Cells Research Articles

Exploring molecular disparities of H-type vasculature endothelial cells in osteonecrosis of the femoral head through single-cell analysis

ObjectiveRecent studies highlight the role of H-type vasculature in bone regeneration. This study, based on single-cell RNA sequencing (scRNA-seq), aims to explore the changes in H-type vasculature endothelial cells (H_ECs) in osteonecrosis of the femoral head (ONFH) and hip osteoarthritis (HOA), focusing on the death modes such as ferroptosis, pyroptosis, and parthanatos.MethodsWe re-analyzed the scRNA-seq data of femoral head samples publicly available in 2022. This study selected nine femoral head samples (3 each from HOA, ONFH stage 3 A, and ONFH stage 4). CD31 + EMCN + endothelial cells were classified as H_ECs. Molecular differences were assessed using Gene Ontology and KEGG analysis. Hypoxia, ferroptosis, pyroptosis, and parthanatos indices were calculated, and transcription factors were predicted using SENIC. Cell communication was analyzed with CellChat.ResultsAfter integrating the 9 samples, 14 cell types were identified: B cells, Mesenchymal stem cells, Osteoblasts, Endothelial cells, Monocytes, T cells, NK cells, Fibroblasts, Macrophages, Common myeloid progenitors, Chondrocytes, Myelocytes, Osteoclasts, and Pericytes. The number of endothelial cells and H_ECs decreased with necrosis severity. H_ECs showed higher angiogenic capacity but lower stress resistance compared to other endothelial cells. Angiogenic capacity decreased in necrotic samples, accompanied by an elevation in inflammation levels. The hypoxia index was higher, with ferroptosis increased in stage 3 A and parthanatos in stages 3 A and 4. No change was observed in pyroptosis. Cell communication analysis revealed downregulation of SLIT3-ROBO4 signaling during necrosis.ConclusionH_ECs show molecular differences compared to other endothelial cells. Ferroptosis and parthanatos contribute to the demise of H_ECs in ONFH, with pericytes and fibroblasts supporting H_EC angiogenesis.

Norchelerythrine from Corydalis incisa (Thunb.) Pers. promotes differentiation and apoptosis by activating DNA damage response in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most prevalent form of leukemia in adults. The cornerstone of first‑line chemotherapy for AML has poor survival rates, underscoring the urgent need for development of novel therapeutic agents. Differentiation therapy targets the blockade of differentiation in myeloid progenitor cells. The present study screened 100 plant extracts native to South Korea to search for those with differentiation‑inducing activity in AML. Differentiation‑inducing activity was assessed by measuring CD11b expression using fluorescence activated cell sorting. Of these, Corydalis incisa (Thunb.) Pers. (CIP) exhibited the highest efficacy. CIP induced myeloid differentiation, decreased viability and increased cell apoptosis and cell cycle arrest in HL‑60, U937 and THP‑1 cells. Furthermore, ultra‑performance liquid chromatography‑quadrupole time‑of‑flight mass spectrometry identified norchelerythrine as the primary anti‑leukemic compound in CIP. Norchelerythrine induced differentiation and promoted cell cycle arrest and apoptosis, mirroring the tumor‑suppressive effects of CIP, and notably decreased cell viability in patients with various genetic abnormalities. The present mechanistic study showed that norchelerythrine stimulated reactive oxygen species generation, leading to activation of DNA damage signaling and upregulation of p21cip1, a cyclin‑dependent kinase inhibitor. Overall, norchelerythrine isolated from CIP may be a novel therapeutic option in AML.

Nature-inspired platform nanotechnology for RNA delivery to myeloid cells and their bone marrow progenitors.

Nucleic acid therapeutics are used for silencing, expressing or editing genes in vivo. However, their systemic stability and targeted delivery to bone marrow resident cells remains a challenge. In this study we present a nanotechnology platform based on natural lipoproteins, designed for delivering small interfering RNA (siRNA), antisense oligonucleotides and messenger RNA to myeloid cells and haematopoietic stem and progenitor cells in the bone marrow. We developed a prototype apolipoprotein nanoparticle (aNP) that stably incorporates siRNA into its core. We then created a comprehensive library of aNP formulations and extensively characterized their physicochemical properties and in vitro performance. From this library, we selected eight representative aNP-siRNA formulations and evaluated their ability to silence lysosomal-associated membrane protein 1 (Lamp1) expression in immune cell subsets in mice after intravenous administration. Using the most effective aNP identified from the screening process, we tested the platform's potential for therapeutic gene silencing in a syngeneic murine tumour model. We also demonstrated the aNP platform's suitability for splice-switching with antisense oligonucleotides and for protein production with messenger RNA by myeloid progenitor cells in the bone marrow. Our data indicate that the aNP platform holds translational potential for delivering various types of nucleic acid therapeutics to myeloid cells and their progenitors.

KLF4 enhances transplantation-induced hematopoiesis by inhibiting TLRs and non-canonical NFκB signaling at a steady state.

The transcription factor Krüppel-like factor 4 (KLF4) acts as a transcriptional activator and repressor. KLF4 plays a role in various cellular processes, including the dedifferentiation of somatic cells into induced pluripotent stem cells. Although it has been shown to enhance self-renewal in embryonic and leukemia stem cells, its role in adult hematopoietic stem cells (HSC) remains underexplored. We demonstrate that conditional deletion of the Klf4 gene in hematopoietic cells led to an increased frequency of immunophenotypic hematopoietic stem cells (HSCs) in the bone marrow, along with a normal distribution of lymphoid and myeloid progenitor cells. Non-competitive bone marrow transplants showed normal engraftment and multi-lineage reconstitution, except for monocytes and T cells. However, the loss of KLF4 hindered hematological reconstitution in competitive serial bone marrow transplants, highlighting a critical role for KLF4 in stress-induced hematopoiesis. Transcriptome analysis revealed an upregulation of NFκB2 and toll-like receptors (e.g., TLR4) in Klf4-null HSCs during homeostasis. Flow cytometry and immunoblot analysis confirmed the increased cell surface expression of TLR4 and the activation of NFκB2 in HSCs under homeostatic conditions, whereas NFκB2 expression drops after radiation compared to steady-state levels. Our findings suggest that the constitutive activation of the TLR4-NFκB2 pathway inhibits the ability of HSCs to regenerate blood after transplantation in cytoablated bone marrow.

Abstract TP350: The Effect of Type 2 Diabetes on the Transcriptome of Immune Cells in the Bone Marrow in Response to Ischemic Stroke

Type 2 diabetes mellitus (T2DM) induces chronic inflammation, which can worsen the secondary injury following ischemic stroke. Yet, whether and how T2DM-induced inflammation affects the production of bone marrow-derived immune cells and their transcriptome remains largely unexplored. Hypothesis: T2DM biases the immune system to a proinflammatory phenotype in the bone marrow, increases the transmigration of myeloid cells and compounds the secondary injury caused by stroke. Methods: We induced ischemic stroke by distal occlusion of the middle cerebral artery (MCAO) in T2DM mouse model db/db mice and normoglycemic control db/+ mice. Three days post-stroke, bone marrow (BM) cells and tissue were subjected to single-cell RNA sequencing (scRNAseq) and GeoMx digital spatial profiling (DSP). Myeloid cell populations were quantified by Fluorescent cell activated sorting (FACS). We used the NicheNet tool to analyze the cell-cell communication between BM populations. Results: We detected an increased frequency of hematopoietic precursor and myeloid progenitor cells in the BM of T2DM mice compared to control mice via FACS, although CD11b (+) cells were significantly increased only in control and not in T2DM mice after stroke. GeoMx DSP found that the upregulation of cd47, s100a9, cd177, pecam1 genes in both CD115 and Ly6G expressing cells were more pronounced in T2DM BM compared to control BM after stroke. Pathway enrichment analysis pointed to an increased activation of signals for myeloid cell activation and migration in T2DM stroke BM compared to control stroke BM, relative to their respective non-stroke counterpart. NicheNet integrated with single-cell RNA sequencing data corroborated with a differential increase of ccr2 and ccr3 interactions with a number of chemokine ligands in the BM of T2DM stroke mice compared to control stroke mice. Conclusion: our data suggest that T2DM enhances myeloid cell migration and chemotaxis after stroke. Ongoing studies will identify the tissue compartments to which increased myeloid cells transmigrate in T2DM mice after stroke and the effect on stroke outcomes.

Evaluating immune cell abundance across hepatocellular carcinoma disease stages I-III: Findings from TCGA analysis.

621 Background: Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is the sixth most prevalent cancer globally and a leading cause of cancer-related mortality. The tumor microenvironment (TME) in HCC, characterized by complex interactions between immune and tumor cells, significantly influences tumor progression and immune suppression. Understanding the immune landscape across the different stages of HCC can inform prognosis and therapeutic strategies. Methods: We analyzed RNA seq data from 333 HCC patients (disease stages I-III), from The Cancer Genome Atlas (TCGA), using the immune deconvolution algorithms CIBERSORT and xCell. Differential gene expression analysis was performed using the DESeq2 package in R. Immune cell abundance across various HCC stages was imputed, and survival analyses was conducted using Kaplan-Meier curves and log-rank tests. Results: Of the 19,514 genes analyzed, 2,774 were up-regulated and 947 were down-regulated in stage II and III in comparison to stage I. CIBERSORT and xCell analyses revealed significant variability in the immune cell composition between HCC stages. Notable differences were observed in the T cells (CD4 and CD8 subsets), B cells, plasma cells, and stromal components. ANOVA and post hoc tests identified significant stage-specific differences in several immune cell types. For the survival analysis, we categorized patients into low and high groups based on median cell abundances. Overall survival showed no statistically significant differences among the various groups for common myeloid and lymphoid progenitor cells. In contrast, disease-free survival demonstrated statistically significant differences across the groups for both cell types. Conclusions: This study emphasizes the tumor microenvironment's pivotal role in HCC, revealing that immune cell composition significantly influences disease-free survival, despite limited effects on overall survival. These findings advocate for further exploration into targeted immunotherapies and the development of predictive models that integrate immune landscape characteristics, ultimately aiming to enhance patient outcomes in HCC. Cell type xCell Cibersort Mean SD Mean SD T cell CD4 naive 0.00466 0.01994 1.31e-5 1.69e-4 T cell CD8 0.01368 0.03253 0.03161 0.04947 Eosinophil 3.96e-5 3.41e-4 1.67e-4 8.69e-4 Macrophage 0.02323 0.02441 0.02924 0.04345 Macrophage.M1 0.00914 0.01526 0.01806 0.02054 Macrophage.M2 0.02303 0.01697 0.05439 0.04815 B cell memory 0.00196 0.01418 0.00107 0.00733 B cell naive 0.00163 0.01034 0.01319 0.02148 Neutrophil 2.14e-4 0.00137 0.00112 0.00539 Immune score 0.04621 0.06817 - - Table presents various cell type abundances for each algorithm used.

Integrated Genomics Reveal Potential Resistance Mechanisms of PANoptosis-Associated Genes in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is marked by the proliferation of abnormal myeloid progenitor cells in the bone marrow and blood, leading to low cure rates despite new drug approvals from 2017 to 2018. Current therapies often fail due to the emergence of drug resistance mechanisms, such as those involving anti-apoptotic pathways and immune evasion, highlighting an urgent need for novel approaches to overcome these limitations. Programmed cell death (PCD) is crucial for tissue homeostasis, with PANoptosis-a form of PCD integrating pyroptosis, apoptosis, and necroptosis-recently identified. This process, regulated by the PANoptosome complex, could be key to overcoming AML drug resistance. Targeting multiple PCD pathways simultaneously may prove more effective than single-target therapies. Research suggests that disrupting anti-apoptotic mechanisms, such as those involving Bcl-2, can enhance drug sensitivity in AML. This study hypothesizes that PANoptosis-associated resistance genes (PARGs) play a critical role in AML drug resistance by modulating immune responses and offers a multi-faceted approach to tackle this challenge. Using RNA sequencing data from the Cancer Genome Atlas and Gene Expression Omnibus databases, we performed differential expression analysis to identify significantly dysregulated PARGs in AML. Regression analysis identified prognostic PARGs, bridging a key gap in understanding how these genes contribute to treatment resistance. We then verified their expression in AML cell lines and cell samples treated with cytarabine using RT-qPCR. Hierarchical clustering revealed distinct PARG expression patterns, and functional enrichment analysis highlighted their involvement in immune-related pathways. The combination of bioinformatics and experimental validation underscores how these genes may mediate immune modulation in drug resistance, providing a robust framework for further study. Our findings suggest that PARGs contribute to AML resistance by modulating immune responses and provide potential targets for therapeutic intervention. This study highlights the potential of targeting PARGs to improve treatment outcomes in AML. By analyzing the expression changes of these genes in response to standard clinical treatments, we provide a framework for developing multi-target therapeutic strategies that simultaneously disrupt multiple programmed cell death pathways. Such an approach directly addresses the limitations of current treatments by offering a method to enhance drug sensitivity and mitigate resistance, potentially improving survival rates. Our findings underscore the importance of a comprehensive understanding of PCD mechanisms and pave the way for innovative treatments that could significantly impact AML management.

Progenitor effect in the spleen drives early recovery via universal hematopoietic cell inflation.

Hematopoietic stem cells (HSCs) possess the capacity to regenerate the entire hematopoietic system. However, the precise HSC dynamics in the early post-transplantation phase remain an enigma. Clinically, the initial hematopoiesis in the post-transplantation period is critical, necessitating strategies to accelerate hematopoietic recovery. Here, we uncovered the spatiotemporal dynamics of early active hematopoiesis, "hematopoietic cell inflation," using a highly sensitive invivo imaging system. Hematopoietic cell inflation occurs in three peaks in the spleen after transplantation, with common myeloid progenitors (CMPs), notably characterized by HSC-like signatures, playing a central role. Leveraging these findings, we developed expanded CMPs (exCMPs), which exhibit a gene expression pattern that selectively proliferates in the spleen and promotes hematopoietic expansion. Moreover, universal exCMPs supported early hematopoiesis in allogeneic transplantation. Human universal exCMPs have the potential to be a viable therapeutic enhancement for all HSC transplant patients.

Exploring the comorbidity mechanisms between atherosclerosis and hashimoto's thyroiditis based on microarray and single-cell sequencing analysis.

Atherosclerosis (AS) is a chronic vascular disease characterized by inflammation of the arterial wall and the formation of cholesterol plaques. Hashimoto's thyroiditis (HT) is an autoimmune disorder marked by chronic inflammation and destruction of thyroid tissue. Although previous studies have identified common risk factors between AS and HT, the specific etiology and pathogenic mechanisms underlying these associations remain unclear. We obtained relevant datasets for AS and HT from the Gene Expression Omnibus (GEO). By employing the Limma package, we pinpointed common differentially expressed genes (DEGs) and discerned co-expression modules linked to AS and HT via Weighted Gene Co-expression Network Analysis (WGCNA). We elucidated gene functions and regulatory networks across various biological scenarios through enrichment and pathway analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Core genes were identified using Cytoscape software and further validated with external datasets. We also conducted immune infiltration analysis on these core genes utilizing the CIBERSORT method. Lastly, Single-cell analysis was instrumental in uncovering common diagnostic markers. Based on differential analysis and WGCNA, we identified 119 candidate genes within the cohorts for AS and HT. KEGG and GO enrichment analyses indicate that these genes are significantly involved in antigen processing and presentation, along with various immune-inflammatory pathways. Two pivotal genes, PTPRC and TYROBP, were identified using five algorithms from the cytoHubba plugin. Validation through external datasets confirmed their substantial diagnostic value for AS and HT. Moreover, the results of Gene Set Enrichment Analysis (GSEA) indicated that these core genes are significantly enriched in various receptor interactions and signaling pathways. Immune infiltration analysis revealed a strong association of lymphocytes and macrophages with the pathogenesis of AS and HT. Single-cell analysis demonstrated predominant expression of the core genes in macrophages, monocytes, T cells and Common Myeloid Progenitor (CMP). This study proposes that an aberrant immune response might represent a shared pathogenic mechanism in AS and HT. The genes PTPRC and TYROBP are identified as critical potential biomarkers and therapeutic targets for these comorbid conditions. Furthermore, the core genes and their interactions with immune cells could serve as promising targets for future diagnostic and therapeutic strategies.

Early differentiation of committed erythroid cells defined by miR-144/451 expression.

Before committing to an erythroid cell lineage, hematopoietic stem cells differentiate along a myeloid cell pathway to generate megakaryocyte-erythroid biopotential progenitor cells in bone marrow. Recent studies suggest that erythroid progenitors (EryPs) could be generated at the level of common myeloid progenitors (CMPs). However, due to a lack of suitable markers, little is known about the early differentiation of these committed EryP cells during CMP development. Herein, using miR-144/451-eGFP knock-in mice, we found that early differentiation of committed erythroid cells could be defined by miR-144/451 expression within CMPs. Single-cell RNA sequencing showed that miR-144/451+ progenitors had obvious differentiation characteristics of erythroid lineage cells and diverged from megakaryocyte and other myeloid cell lineages. These progenitors exclusively gave rise to erythroid cells, both in vitro and in vivo, and the commitment to an erythroid cell lineage was accompanied by loss of CD53 expression. Our findings will facilitate further understanding of the molecular mechanisms governing erythroid development and support the identification of therapeutic targets for diseases related to erythrocyte development.

NR2F6 regulates stem cell hematopoiesis and myelopoiesis in mice.

Nuclear receptors regulate hematopoietic stem cells (HSCs) and peripheral immune cells in mice and humans. The nuclear orphan receptor NR2F6 (EAR-2) has been shown to control murine hematopoiesis. Still, detailed analysis of the distinct stem cell, myeloid, and lymphoid progenitors in the bone marrow in a genetic loss of function model remains pending. In this study, we found that adult germline Nr2f6-deficient mice contained increased percentages of total long-term and short-term HSCs, as well as a subpopulation within the lineage-biased multipotent progenitor (MPP3) cells. The loss of NR2F6 thus led to an increase in the percentage of LSK+ cells. Following the differentiation from the common myeloid progenitors (CMP), the granulocyte-monocyte progenitors (GMP) were decreased, while monocyte-dendritic progenitors (MDP) were increased in Nr2f6-deficient bone marrow. Within the pre-conventional dendritic progenitors (pre-cDCs), the subpopulation of pre-cDC2s was reduced in the bone marrow of Nr2f6-deficient mice. We did not observe differences in the development of common lymphoid progenitor populations. Our findings contrast previous studies but underscore the role of NR2F6 in regulating gene expression levels during mouse bone marrow hematopoiesis and myelopoiesis.

Dual ASXL1 and CSF3R mutations drive myeloid biased stem cell expansion and enhance neutrophil differentiation.

Mutations in the epigenetic regulator Additional Sex Combs-Like 1 (ASXL1) are frequently observed in chronic neutrophilic leukemia (CNL). CNL is a myeloproliferative neoplasm (MPN) driven by activating mutations in the Colony Stimulating Factor 3 Receptor (CSF3R), which cause excessive neutrophil production. Despite the high rates of co-occurrence, the interplay between ASXL1 and CSF3R mutations in hematopoiesis and leukemia remains poorly understood. Here, we present a new mouse model with both Asxl1Y588X and Csf3rT621I mutations, which recapitulates features of human MPNs. Csf3r-mutant mice exhibit an age-associated depletion of hematopoietic stem cells, which is tempered by adding of Asxl1Y588X. This combination of mutations causes an expansion of myeloid-biased long-term hematopoietic stem cells. As the mice age, they develop neutrophilia, but leukemia is rare, suggesting additional mutations may be required for transformation. Using models of myeloid differentiation, we find that Asxl1 truncation enhances CSF3RT618I-driven neutrophil differentiation, activating inflammatory pathways associated with mature myeloid cell production. Moreover, cells with both mutations have increased H3K4me1 at neutrophil-associated enhancers. Mutant ASXL1 is known to decrease the genome-wide abundance of the repressive histone mark H2AK119ub. While we see the expected decrease in H2AK119ub in Asxl1-mutant cells, this effect is reversed when CSF3R is also mutated, suggesting a complex interplay between these mutations in regulating chromatin dynamics during hematopoiesis. Our findings highlight context-dependent effects of ASXL1 mutation in myeloid disorders and provide insights into the mechanisms underlying neutrophil differentiation in ASXL1 and CSF3R dual mutant MPN.

Menin inhibitors in the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the (oligo)clonal expansion of myeloid progenitor cells. Despite advances in treatment, AML remains challenging to cure, particularly in patients with specific genetic abnormalities. Menin inhibitors have emerged as a promising therapeutic approach, targeting key genetic drivers of AML, such as KMT2A (lysine methyl transferase 2A) rearrangements and NPM1 mutations. Here, we review the clinical value of menin inhibitors, highlighting their mechanism of action, efficacy, safety, and potential to transform AML treatment.

Targeting protein tyrosine phosphatase non-receptor type 6 (PTPN6) as a therapeutic strategy in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of myeloid progenitor cells. Despite advancements in treatment, the prognosis for AML patients remains poor, highlighting the need for novel therapeutic targets. Protein Tyrosine Phosphatase Non-Receptor Type 6 (PTPN6), also known as SHP-1, is a critical regulator of hematopoietic cell signaling and has been implicated in various leukemias. This study investigates the therapeutic potential of targeting PTPN6 in AML. We employed both in vitro and in vivo models to evaluate the effects of PTPN6 inhibition on AML cell proliferation, apoptosis, and differentiation. Our results demonstrate that PTPN6 inhibition leads to a significant reduction in AML cell viability, induces apoptosis, and promotes differentiation of leukemic cells into mature myeloid cells. Mechanistic studies revealed that PTPN6 inhibition disrupts key signaling pathways involved in AML pathogenesis, including the JAK/STAT and PI3K/AKT pathways. Furthermore, the combination of PTPN6 inhibitors with standard chemotherapeutic agents exhibited a synergistic effect, enhancing the overall therapeutic efficacy. These findings suggest that PTPN6 is a promising therapeutic target in AML and warrants further investigation into the development of PTPN6 inhibitors for clinical application in AML treatment.

Evi1 governs Kdm6b-mediated histone demethylation to regulate the Laptm4b-driven mTOR pathway in hematopoietic progenitor cells.

Ecotropic viral integration site 1 (EVI1/MECOM) is frequently upregulated in myeloid malignancies. Here, we present an Evi1-transgenic mouse model with inducible expression in hematopoietic stem/progenitor cells (HSPCs). Upon induction of Evi1 expression, mice displayed anemia, thrombocytopenia, lymphopenia, and erythroid and megakaryocyte dysplasia with a significant expansion of committed myeloid progenitor cells, resembling human myelodysplastic syndrome/myeloproliferative neoplasm-like (MDS/MPN-like) disease. Evi1 overexpression prompted HSPCs to exit quiescence and accelerated their proliferation, leading to expansion of committed myeloid progenitors while inhibiting lymphopoiesis. Analysis of global gene expression and Evi1 binding site profiling in HSPCs revealed that Evi1 directly upregulated lysine demethylase 6b (Kdm6b). Subsequently, Kdm6b-mediated H3K27me3 demethylation resulted in activation of various genes, including Laptm4b. Interestingly, KDM6B and LAPTM4B are positively correlated with EVI1 expression in patients with MDS. The EVI1/KDM6B/H3K27me3/LAPTM4B signaling pathway was also identified in EVI1hi human leukemia cell lines. We found that hyperactivation of the LAPTM4B-driven mTOR pathway was crucial for the growth of EVI1hi leukemia cells. Knockdown of Laptm4b partially rescued Evi1-induced abnormal hematopoiesis in vivo. Thus, our study establishes a mouse model to investigate EVI1hi myeloid malignancies, demonstrating the significance of the EVI1-mediated KDM6B/H3K27me3/LAPTM4B signaling axis in their maintenance.

Siwu decoction mitigates radiation-induced immune senescence by attenuating hematopoietic damage

BackgroundTo investigate the long term effects of ionizing radiation (IR) on hematopoietic stem/progenitor cells (HSPCs), immune tissues and cells, and the effects of Siwu decoction (SWD) on immune senescence mice.MethodsC57BL/6 J mice were exposed to 6.0 Gy 60Co γ irradiation. After 8-weeks of IR, SWD (5, 10, 20 g/kg/d) was administered for 30 days. The changes of HSPCs in bone marrow (BM) and T, B type lymphocyte and natural killer (NK) cells in spleen were detected by flow cytometry. The changes of peripheral blood cells were also examined. Hematoxylin–eosin staining were used to detect the pathological lesions of hippocampus, spleen and thymus tissues. Absolute mouse telomere length quantification qPCR assay kit was used to measure the telomere length of BM cells. The expression of factors associated with inflammation and aging such as p16, β-galactosidase in spleen, thymus and BM was determined.ResultsAdministration of SWD could increase the proportion of LSK (Lin−, Sca-1 + , c-Kit−), multipotent progenitor cells and multipotent progenitor cells and decrease the proportion of common myeloid progenitors and granulocyte–macrophage progenitors in BM. The proportion of B cells and NK cells in spleen and the content of white blood cells, red blood cells, hemoglobin, lymphocytes and eosinophils in peripheral blood were increased, at the same time, the proportion of neutrophils and monocytes was reduced by SWD. The pathological lesions of hippocampus, spleen and thymus were improved. The expression of p16 and β-galactosidase in spleen, thymus and BM was reduced and shortening of the telomere of BM cells was inhibited after administration. In addition, SWD could reduce the content of Janus activated kinase (JAK) 1, JAK2 and signal transducer and activator of transcription 3 (STAT3) in BM and spleen.ConclusionsSWD could slow down IR-induced immune senescence by improving hematopoietic and immunologic injury. SWD might reduce the inflammation level of BM hematopoietic microenvironment by acting on JAK/STAT signaling pathway, while the immune damage of mice was improved by affecting the differentiation of HSPCs. The remission of hematopoietic and immunologic senescence was further demonstrated at the overall level.Graphical

The epigenetic role of EZH2 in acute myeloid leukemia.

Acute myeloid leukemia (AML), a malignant disease of the bone marrow, is characterized by the clonal expansion of myeloid progenitor cells and a block in differentiation. The high heterogeneity of AML significantly impedes the development of effective treatment strategies. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), regulates the expression of downstream target genes through the trimethylation of lysine 27 on histone 3 (H3K27me3). Increasing evidence suggests that the dysregulation of EZH2 expression in various cancers is closely associated with tumorigenesis. In the review, we examine the role of EZH2 in AML, highlighting its crucial involvement in regulating stemness, proliferation, differentiation, immune response, drug resistance and recurrence. Furthermore, we summarize the application of EZH2 inhibitors in AML treatment and discuss their potential in combination with other therapeutic modalities. Therefore, targeting EZH2 may represent a novel and promising strategy for the treatment of AML.

Parallel Immunotranscriptomic and Immunohistochemistry Enhance Cluster Discrimination in Acute Myelogenous Leukemia

Background: Acute myeloid leukemia (AML) demonstrates cell differentiation stages emphasizing the role for hematopoietic maturation arrest during leukemogenesis. Indeed, less differentiated AML stages retain enhanced sensitivity to hypomethylating agents (HMA) plus BCL-2 inhibitor, a phenomenon less observed in monocytic skewed cluster, for which menin inhibitors represent promising intervention. Deconvolution of bulk AML transcriptome allows for better understanding of disease pathogenesis. However, the role of “Immunotranscriptome analysis [i.e. CD34, CD117, HLADR, CD33, CD38, CD11b, BCL2 Gene Expression (GE)]” in identifying maturation arrest remains uncharacterized. In this study, we seek to harmonize marrow immunohistochemistry (IHC) and bulk AML immunotranscriptomic to better discriminate cluster designation. Methods: After IRB approval, 49 AML cases were included for analysis. 6 clusters were designed based on marrow IHC [i.e., Hematopoietic Stem Cell (HSC)-like, Multipotent Progenitor (MPP)-like, Common Myeloid Progenitor (CMP)-like, Granulocytic Monocytic (GMP)-like, Monocyte Progenitor (MP)-like, Granulocyte Progenitor (GP)-like]. Differential immunotranscriptomic for individual GE [i.e. CD34, CD33, CD117 etc.] was examined in our “founder” IHC cohort. Next, discrimination analysis [DA] to detect cluster “membership” was obtained by integrating individual Immunotranscriptome expression into categorical IHC clustering. Descriptive statistics, ANOVA and discrimination procedure were performed with SAS software. Results: Mean age was 65 years (range, 25-92). 22(51%) of cases were male. 19(44%), 22(51%), 2(5%), and 1(2%) were white, African Ancestry (AA), Hispanic and Asians. ELN22 risk was available in 5/49(10.2%), 11/49 (22.4%), 33/49 (67.3%) of favorable, intermediate and adverse groups. 7/49(14.2%), 3/49(6.1%), 10/49(20.4%), 7/49(14.2%), 16/49(33%), and 6/49(12.2%) were HSC, GMP, MP, GP, CMP and MPP-like. CD34; p=0.003, HLA-DR; p=0.01, CD11b; p=0.03 were differentially expressed among clusters. “Stringent” MDS mutations (mut) [i.e. splicing, ASXL1, cohesin, BCOR etc.] were observed in higher proportion of CMP-like cluster (60% vs 21.8%, p=0.01). 7/13 (88%) vs 1/13(12%) of cases with and without MP-like cluster definition harbored KM2TA abnormality, p=<0.0001. By adding GE to our “founder” IHC clustering, 6/6 (100%, p=<0.0001) of pt were allocated in HSC-like disease, 3/3 (100%, p=0.0001) to GMP-like, 6/9 (66.3%. p=0.33) to MP-like, 6/6 (100%, p=<0.0001) GP-like, 2/13 (15.3%) CMP-like and 3/3 (100%, p=0.0001) MPP-like. Conclusions: Our analysis demonstrates that leukemia clusters can be identified based on IHC. GE facilitates robust cluster “membership” for all groups except MP-like and CMP-like. While KM2TA and MDS like mutations were observed significantly higher in MP-like and CMP-like diseases, it is possible that complex epigenetic rewiring [i.e. GE changes induced by KM2TA and MDS mutations] alter Immunotranscriptome ability to enhance cluster designation. Validation of our founder cohort findings is needed.

Construction of a Myeloid Gene Signature Revealing Novel Therapeutic Targets for Myeloid Malignancies

FH, SL and BG equal contributors Myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML) exhibit shared hallmarks of hyperinflammation and aberrant clonal expansion. Chronic MPNs exhibit a propensity for transformation to secondary AML (sAML), which imparts a dismal prognosis with limited treatment options. However, the pathogenesis of leukemia transition for MPN and the bidirectional relationship between MPN and AML remain incompletely understood. In this study, we investigated commonly-featured genes and pathways of these two diseases to explore novel therapeutic strategies. To dissect the causal association between MPN and AML, we first performed two-sample bidirectional mendelian randomization (MR) analysis which revealed that MPN predicts the risk of AML across multiple statistical methods with consistent directionality and significance. Heterogeneity, pleiotropy, or outlier effects were ruled out by sensitivity analyses with no reverse causality from AML to MPN. To identify a potential myeloid signature, we estimated causal effects of 2,940 plasma proteins on MPN via a two-sample MR framework and identified 55 candidates significantly associated with increased risk of MPN (P < 0.05, OR > 1). Utilizing data from the BEAT-AML cohort, we further screened the prognostic value of these candidates using Cox and log-rank test survival analyses and identified a 4-gene myeloid signature comprising MPO, CDCP1, CRISP3 and DXCR, with each conferring prognostic significance individually. Our scRNA-seq results confirmed enrichment of myeloid signature genes in myeloid progenitor cells, with further elevation in MPN. Of note, expression of myeloid signature genes was induced by Jak2V617F and MPLW515L in Ba/F3 cells. We then investigated the combined prognostic significance of these four genes by constructing a risk score via LASSO regression modeling and divided patients into high- and low-risk groups accordingly. Cox analysis validated the risk score as an independent prognostic factor (HR: 3.01 (1.2 - 7.6), P = 0.019) and Kaplan-Meier survival analysis demonstrated that low-risk AML patients had a significantly better survival than high-risk AML patients with median survival of 21.9 months vs. 11.9 months (P < 0.0001). The prediction accuracy of the myeloid signature was successfully validated in the TCGA-LAML cohort. The risk score significantly correlated with several key clinical parameters, including age, platelet count, ELN2017 staging, monocyte percentage and bone marrow cellularity. To interrogate the underlying mechanism of the myeloid signature, we performed gene set enrichment analysis (GSEA) and observed enrichment of several inflammatory pathways in high-risk AML patients. A similar enrichment of inflammatory pathways in MPN samples compared to healthy donors observed in our previous scRNA-seq results reinforced hyperinflammation as a shared etiology of myeloid malignancies. To explore potential therapeutic strategies, we performed in silico drug sensitivity screening which predicted specific vulnerability of high-risk AML to compounds targeting PI3K/AKT/mTOR pathway signaling. Activation of PI3K/AKT/mTOR signaling in both MPN and AML samples observed across multiple single cell and bulk RNA-seq datasets further prompted us to evaluate targeted therapies against this pathway in myeloid malignancies. MPN and AML cells exhibited sensitivity to mTOR inhibitors as indicated by suppression of cellular proliferation, metabolism, colony formation, and inflammatory cytokine secretion in conjunction with induction of apoptosis. We further evaluated the therapeutic effects of mTOR inhibition in vivo via administration of omipalisib, a potent dual PI3K/mTOR inhibitor, to JAK2V617F knock-in mice. Omipalisib treatment significantly ameliorated features of myeloid malignancies including splenomegaly and leukocytosis which were both significantly reduced following 4 weeks of treatment, without affecting body weight. In summary, our MR analyses reveal that MPN predicts the risk of AML and enabled the construction of a novel myeloid signature which risk stratified AML patients across two different cohorts. We further demonstrate shared activation of PI3K/AKT/mTOR pathway across myeloid malignancies, with in vitro and in vivo data providing a rationale for therapeutic targeting of PI3K/AKT/mTOR pathway in these diseases.

Targeting Co-Regulatory Feedback Loop of MYC and GSPT1 By MYC/GSPT1 Dual Degradation Is Synthetic Lethal in Combination with Ven/Aza in TP53 Mutant Acute Myeloid Leukemia Stem and Progenitor Cells

Targeting Co-Regulatory Feedback Loop of MYC and GSPT1 By MYC/GSPT1 Dual Degradation Is Synthetic Lethal in Combination with Ven/Aza in TP53 Mutant Acute Myeloid Leukemia Stem and Progenitor Cells