Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation.

Nucleoporins, the components of nuclear pore complexes (NPCs), can play cell type- and tissue-specific functions. Yet, the physiological roles and mechanisms of action for most NPC components have not yet been established. We report that Nup358, a nucleoporin linked to several myeloid disorders, is required for the developmental progression of early myeloid progenitors. We found that Nup358 ablation in mice results in the loss of myeloid-committed progenitors and mature myeloid cells and the accumulation of myeloid-primed multipotent progenitors (MPPs) in bone marrow. Accumulated MPPs in Nup358 knockout mice are greatly restricted to megakaryocyte/erythrocyte-biased MPP2, which fail to progress into committed myeloid progenitors. Mechanistically, we found that Nup358 is required for histone deacetylase 3 (HDAC3) nuclear import and function in MPP2 cells and established that this nucleoporin regulates HDAC3 nuclear translocation in a SUMOylation-independent manner. Our study identifies a critical function for Nup358 in myeloid-primed MPP2 differentiation and uncovers an unexpected role for NPCs in the early steps of myelopoiesis.

GM-CSF receptor expression determines opposing innate memory phenotypes at different stages of myelopoiesis.

ABSTRACT: Inflammatory responses must be tightly coordinated with the activation of emergency myelopoiesis to produce potent myeloid cells that fight infection without causing excessive host damage. Here, we show that granulocyte-macrophage colony-stimulating factor (GM-CSF) programs myeloid-committed progenitors to produce trained macrophages (increased cytokine response), but programs the upstream noncommitted LKS+ progenitors (defined as Lin- c-Kit+ Sca-1+ cells) to produce tolerized macrophages (decreased cytokine response). In myeloid progenitors, GM-CSF strongly activates signal transducer and activator of transcription 5 (STAT5), Ras-Raf-extracellular signal regulated kinase (ERK), and Akt-mTOR signaling pathways, which are essential to establish a training program, whereas in LKS+ progenitors, GM-CSF induces NF-κB translocation to the nucleus to establish a tolerization program. These differences arise from higher GM-CSF receptor expression in myeloid progenitors compared with LKS+ cells. We demonstrate that ß-catenin regulation of NF-κB nuclear translocation is central in this process. In myeloid progenitors, glycogen synthase kinase 3 (GSK3) inactivation by strong ERK and phosphatidylinositol 3 kinase (PI3K)-Akt signaling increases cytoplasmic ß-catenin levels to block NF-κB nuclear translocation. In contrast, when ERK and PI3K-Akt signaling are weak, active GSK3 causes a decrease in ß-catenin, allowing NF-κB nuclear translocation in LKS+ progenitors. Finally, GM-CSF-induced LKS+ tolerization takes place in several murine models of trained immunity and in human CD34+ CD38- progenitors. Our study reveals that in addition to activating myelopoiesis, GM-CSF also programs early and immediate myeloid progenitors to produce opposing immune memory phenotypes. We propose that the inflammatory response from immediate myeloid progenitors may be balanced by the tolerized phenotype of early progenitors, thus providing a mechanism for appropriate resolution of inflammation and protection against a prolonged cytokine storm.

Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non-small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34- cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia. SIGNIFICANCE: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth.

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

Sensitivity to targeted UBA1 inhibition in a myeloid cell line model of VEXAS syndrome.

Somatic UBA1 mutations in hematopoietic cells are a hallmark of Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, which is a late-onset inflammatory disease associated with bone marrow failure and high mortality. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous mutations affecting methionine-41 (M41), leading to the expression of UBA1M41T, UBA1M41V, or UBA1M41L and globally reduced protein polyubiquitination. Here, we used CRISPR-Cas9 to engineer isogenic 32D mouse myeloid cell lines expressing hemizygous Uba1WT or Uba1M41L from the endogenous locus. Consistent with prior analyses of patients with VEXAS syndrome samples, hemizygous Uba1M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform, reduced polyubiquitination, abnormal cytoplasmic vacuoles, and increased production of interleukin-1ß and inflammatory chemokines. Vacuoles in Uba1M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Uba1M41L cells were more sensitive to the UBA1 inhibitor TAK243. TAK243 treatment promoted apoptosis in Uba1M41L cells and led to preferential loss of Uba1M41L cells in competition assays with Uba1WT cells. Knock-in of a TAK243-binding mutation, Uba1A580S, conferred TAK243 resistance. In addition, overexpression of catalytically active UBA1b in Uba1M41L cells restored polyubiquitination and increased TAK243 resistance. Altogether, these data indicate that loss of UBA1b underlies a key biochemical phenotype associated with VEXAS syndrome and renders cells with reduced UBA1 activity vulnerable to targeted UBA1 inhibition. Our Uba1M41L knock-in cell line is a useful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.

Single-cell RNA sequencing-guided fate-mapping toolkit delineates the contribution of yolk sac erythro-myeloid progenitors.

Erythro-myeloid progenitors of the yolk sac that originates during early embryo development has been suggested to generate tissue-resident macrophage, mast cell, and even endothelial cell populations from fetal to adult stages. However, the heterogeneity of erythro-myeloid progenitors (EMPs) is not well characterized. Here, we adapt single-cell RNA sequencing to dissect the heterogeneity of EMPs and establish several fate-mapping tools for each EMP subset to trace the contributions of different EMP subsets. We identify two primitive and one definitive EMP subsets from the yolk sac. In addition, we find that primitive EMPs are decoupled from definitive EMPs. Furthermore, we confirm that primitive and definitive EMPs give rise to microglia and other tissue-resident macrophages, respectively. In contrast, only Kit+ Csf1r- primitive EMPs generate endothelial cells transiently during early embryo development. Overall, our results delineate the contribution of yolk sac EMPs more clearly based on the single-cell RNA sequencing (scRNA-seq)-guided fate-mapping toolkit.

Associations of myeloid cells with cellular and humoral responses following vaccinations in patients with neuroimmunological diseases.

Disease-modifying therapies (DMTs) are widely used in neuroimmunological diseases such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). Although these treatments are known to predispose patients to infections and affect their responses to vaccination, little is known about the impact of DMTs on the myeloid cell compartment. In this study, we use mass cytometry to examine DMT-associated changes in the innate immune system in untreated and treated patients with MS (n = 39) or NMOSD (n = 23). We also investigated the association between changes in myeloid cell phenotypes and longitudinal responsiveness to homologous primary, secondary, and tertiary SARS-CoV-2 mRNA vaccinations. Multiple DMT-associated myeloid cell clusters, in particular CD64+HLADRlow granulocytes, showed significant correlations with B and T cell responses induced by vaccination. Our findings suggest the potential role of myeloid cells in cellular and humoral responses following vaccination in DMT-treated patients with neuroimmunological diseases.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma.

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory T cells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated T cell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

Mercury chloride activates the IFNγ-IRF1 signaling in myeloid progenitors and promotes monopoiesis in mice.

Inorganic mercury (Hg2+) is a highly toxic heavy metal in the environment. To date, the impacts of Hg2+ on the development of monocytes, or monopoiesis, have not been fully addressed. The aim of the present study was to investigate the impact of Hg2+ on monopoiesis. In this study, we treated B10.S mice and DBA/2 mice with 10 µM or 50 µM HgCl2 via drinking water for 4 wk, and we then evaluated the development of monocytes. Treatment with 50 µM HgCl2, but not 10 µM HgCl2, increased the number of monocytes in the blood, spleen and bone marrow (BM) of B10.S mice. Accordingly, treatment with 50 µM HgCl2, but not 10 µM HgCl2, increased the number of common myeloid progenitors (CMP) and granulocyte-macrophage progenitors (GMP) in the BM. Functional analyses indicated that treatment with 50 µM HgCl2 promoted the differentiation of CMP and GMP to monocytes in the BM of B10.S mice. Mechanistically, treatment with 50 µM HgCl2 induced the production of IFNγ, which activated the Jak1/3-STAT1/3-IRF1 signaling in CMP and GMP and enhanced their differentiation potential for monocytes in the BM, thus likely leading to increased number of mature monocytes in B10.S mice. Moreover, the increased monopoiesis by Hg2+ was associated with the increased inflammatory status in B10.S mice. In contrast, treatment with 50 µM HgCl2 did not impact the monopoiesis in DBA/2 mice. Our study reveals the impact of Hg on the development of monocytes.

Secondary bile acids function through the vitamin D receptor in myeloid progenitors to promote myelopoiesis.

Metabolic products of the microbiota can alter hematopoiesis. However, the contribution and site of action of bile acids is poorly understood. Here, we demonstrate that the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), increase bone marrow myelopoiesis. Treatment of bone marrow cells with DCA and LCA preferentially expanded immunophenotypic and functional colony-forming unit-granulocyte and macrophage (CFU-GM) granulocyte-monocyte progenitors (GMPs). DCA treatment of sorted hematopoietic stem and progenitor cells (HSPCs) increased CFU-GMs, indicating that direct exposure of HSPCs to DCA sufficed to increase GMPs. The vitamin D receptor (VDR) was required for the DCA-induced increase in CFU-GMs and GMPs. Single-cell RNA sequencing revealed that DCA significantly upregulated genes associated with myeloid differentiation and proliferation in GMPs. The action of DCA on HSPCs to expand GMPs in a VDR-dependent manner suggests microbiome-host interactions could directly affect bone marrow hematopoiesis and potentially the severity of infectious and inflammatory disease.

The Derivation and Use of HoxB8-Driven Conditionally Immortalized Macrophages.

The use of Hox-driven conditionally immortalized immune cells has significantly increased in biomedical research over the past 15 years. HoxB8-driven conditionally immortalized myeloid progenitor cells maintain their ability to differentiate into functional macrophages. There are multiple benefits to this conditional immortalization strategy including the ability for unlimited propagation, genetic mutability, primary-like immune cells (macrophages, dendritic cells, and granulocytes) on demand, derivation from variety of mouse strains, and simple cryopreservation and reconstitution. In this chapter, we will discuss how to derive and use these HoxB8-conditionally immortalized myeloid progenitor cells.

Limited plasticity of monocyte fate and function associated with epigenetic scripting at the level of progenitors.

Myeloid cell heterogeneity is known, but whether it is cell-intrinsic or environmentally-directed remains unclear. Here, an inducible/reversible system pausing myeloid differentiation allowed the definition of clone-specific functions that clustered monocytes into subsets with distinctive molecular features. These subsets were orthogonal to the classical/nonclassical categorization and had inherent, restricted characteristics that did not shift under homeostasis, after irradiation, or with infectious stress. Rather, their functional fate was constrained by chromatin accessibility established at or before the granulocyte-monocyte or monocyte-dendritic progenitor level. Subsets of primary monocytes had differential ability to control distinct infectious agents in vivo. Therefore, monocytes are a heterogeneous population of functionally restricted subtypes defined by the epigenome of their progenitors that are differentially selected by physiologic challenges with limited plasticity to transition from one subset to another.

Proteasome inhibitor bortezomib stabilizes and activates p53 in hematopoietic stem/progenitors and double-negative T cells in vivo.

We have previously shown that proteasome inhibitor bortezomib stabilizes p53 in stem and progenitor cells within gastrointestinal tissues. Here, we characterize the effect of bortezomib treatment on primary and secondary lymphoid tissues in mice. We find that bortezomib stabilizes p53 in significant fractions of hematopoietic stem and progenitor cells in the bone marrow, including common lymphoid and myeloid progenitors, granulocyte-monocyte progenitors, and dendritic cell progenitors. The stabilization of p53 is also observed in multipotent progenitors and hematopoietic stem cells, albeit at lower frequencies. In the thymus, bortezomib stabilizes p53 in CD4-CD8- T cells. Although there is less p53 stabilization in secondary lymphoid organs, cells in the germinal center of the spleen and Peyer's patch accumulate p53 in response to bortezomib. Bortezomib induces the upregulation of p53 target genes and p53 dependent/independent apoptosis in the bone marrow and thymus, suggesting that cells in these organs are robustly affected by proteasome inhibition. Comparative analysis of cell percentages in the bone marrow indicates expanded stem and multipotent progenitor pools in p53R172H mutant mice compared with p53 wild-type mice, suggesting a critical role for p53 in regulating the development and maturation of hematopoietic cells in the bone marrow. We propose that progenitors along the hematopoietic differentiation pathway express relatively high levels of p53 protein, which under steady-state conditions is constantly degraded by Mdm2 E3 ligase; however, these cells rapidly respond to stress to regulate stem cell renewal and consequently maintain the genomic integrity of hematopoietic stem/progenitor cell populations.

Characterization and CRISPR/Cas9-mediated genetic manipulation of neutrophils derived from Hoxb8-ER-immortalized myeloid progenitors.

Neutrophils represent a first line of defense against a wide variety of microbial pathogens. Transduction with an estrogen receptor-Hoxb8 transcription factor fusion construct conditionally immortalizes myeloid progenitor cells (NeutPro) capable of differentiation into neutrophils. This system has been very useful for generating large numbers of murine neutrophils for in vitro and in vivo studies. However, some questions remain as to how closely neutrophils derived from these immortalized progenitors reflect primary neutrophils. Here we describe our experience with NeutPro-derived neutrophils as it relates to our studies of Yersinia pestis pathogenesis. NeutPro neutrophils have circular or multilobed nuclei, similar to primary bone marrow neutrophils. Differentiation of neutrophils from NeutPro cells leads to increased expression of CD11b, GR1, CD62L, and Ly6G. However, the NeutPro neutrophils expressed lower levels of Ly6G than bone marrow neutrophils. NeutPro neutrophils produced reactive oxygen species at slightly lower levels than bone marrow neutrophils, and the 2 cell types phagocytosed and killed Y. pestis in vitro to a similar degree. To further demonstrate their utility, we used a nonviral method for nuclear delivery of CRISPR/Cas9 guide RNA complexes to delete genes of interest in NeutPro cells. In summary, we have found these cells to be morphologically and functionally equivalent to primary neutrophils and useful for in vitro assays related to studies of bacterial pathogenesis.

Analyzing mitochondrial respiration of human induced pluripotent stem cell-derived myeloid progenitors using Seahorse technology.

Mitochondrial metabolism is critical in hematopoietic stem cell maintenance and differentiation. Here, we present a step-by-step protocol to efficiently differentiate human induced pluripotent stem cells into myeloid progenitors by a robust feeder- and serum-free system. Furthermore, we provide a protocol to subsequently assess mitochondrial function in iPSC-derived myeloid progenitors. We comprehensively describe a protocol to analyze and to quantify key parameters of mitochondrial respiration of iPSC-derived myeloid progenitors by the Seahorse XFe96 Analyzer. Additionally, our protocol includes extensive troubleshooting suggestions. For complete details on the use and execution of this protocol, please refer to Fan et al. (2022).1.

The flow cytometry myeloid progenitor count: A reproducible parameter for diagnosis and prognosis of myelodysplastic syndromes.

BACKGROUND: The bone marrow blast count is central to the diagnosis and monitoring of myelodysplastic syndromes (MDS). It is an independent risk factor for worse prognosis whether based on the morphology blast count or the flow cytometry (FC) myeloid progenitor (MyP) count. It is a principal population in FC MDS analysis also because once defined; it provides significant contributions to the overall FC MDS score. METHODS: We elected to investigate inter-analyst agreement for the most fundamental parameter of the FC MDS diagnostic score: the MyP count. A common gating strategy was agreed and used by seven cytometrists for blind analysis of 34 routine bone marrows sent for MDS work-up. Additionally, we compared the results with a computational approach. RESULTS: Concordance was excellent: Intraclass correlation was 0.993 whether measuring %MyP of total cells or CD45+ cells, and no significant difference was observed between files from different centers or for samples with abnormal MyP phenotypes. Computational and manual results were similar. Applying the common strategy to individual laboratories' control cohorts produced similar MyP reference ranges across centers. CONCLUSION: The FC MyP count offers a reliable diagnostic and prognostic measurement in MDS. The use of manual and computational approaches side by side may allow for optimizing both strategies. Considering its known prognostic power, the MyP count could be considered a useful and reliable addition to existing prognostic scoring systems.

TRAPPC1 is essential for the maintenance and differentiation of common myeloid progenitors in mice.

Myeloid cell development in bone marrow is essential for the maintenance of peripheral immune homeostasis. However, the role of intracellular protein trafficking pathways during myeloid cell differentiation is currently unknown. By mining bioinformatics data, we identify trafficking protein particle complex subunit 1 (TRAPPC1) as continuously upregulated during myeloid cell development. Using inducible ER-TRAPPC1 knockout mice and bone marrow chimeric mouse models, we demonstrate that TRAPPC1 deficiency causes severe monocyte and neutrophil defects, accompanied by a selective decrease in common myeloid progenitors (CMPs) and subsequent cell subsets in bone marrow. TRAPPC1-deleted CMPs differentiate poorly into monocytes and neutrophils in vivo and in vitro, in addition to exhibiting enhanced endoplasmic reticulum stress and apoptosis via a Ca2+ -mitochondria-dependent pathway. Cell cycle arrest and senescence of TRAPPC1-deleted CMPs are mediated by the activation of pancreatic endoplasmic reticulum kinase and the upregulation of cyclin-dependent kinase inhibitor p21. This study reveals the essential role of TRAPPC1 in the maintenance and differentiation of CMPs and highlights the significance of protein processing and trafficking processes in myeloid cell development.

Seasonal plasticity in immunocompetent cytokines (IL-2, IL-6, and TNF-α), myeloid progenitor cell (CFU-GM) proliferation, and LPS-induced oxido-inflammatory aberrations in a tropical rodent Funambulus pennanti: role of melatonin.

In seasonal breeders, photoperiods regulate the levels of circulatory melatonin, a well-known immunomodulator and an antioxidant. Melatonin is known to play a complex physiological role in maintaining the immune homeostasis by affecting cytokine production in immunocompetent cells. In this study, we have quantified seasonal and temporal variations in immunocompetent cytokines-IL-2, IL-6, and TNF-α-and circulatory corticosterone along with in- vitro proliferation of bone marrow-derived granulocyte macrophage-colony forming unit (CFU-GM) progenitor cells of a tropical seasonal breeder Funambulus pennanti (northern palm squirrel). Transient variations in antioxidant status of seasonal breeders might be due to the fluctuations associated with immunity and inflammation. Further, to establish a direct immunomodulatory effect of photoperiod, we recorded the LPS-induced oxidative and inflammatory responses of squirrels by housing them in artificial photoperiodic chambers mimicking summer and winter seasons respectively. We observed a marked variation in cytokines level, melatonin, and corticosterone , and CFU-GM cell proliferation during summer and winter seasons. High Peripheral melatonin levels directly correlated with cytokine IL-2 levels, and inversely correlated with TNF-α, and circulatory corticosterone level. LPS-challenged squirrels housed in short photoperiod (10L:14D; equivalent to winter days) showed a marked reduction in the components of the inflammatory cascade, CRP, TNF-α, IL-6, NOx, NF-κB, Cox-2, and PGES, with an overall improvement in antioxidant status when compared to squirrels maintained under a long photoperiod (16L:8D; equivalent to summer days). Our results underline the impact of seasonality, photoperiod, and melatonin in maintaining an intrinsic redox-immune homeostasis which helps the animal to withstand environmental stresses.