Efferocytosis by bone marrow mesenchymal stromal cells disrupts osteoblastic differentiation via mitochondrial remodeling.

The efficient clearance of dead and dying cells, efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs and on their function. To investigate, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including bone marrow remodeling and bone loss in the setting of aging, cancer and other diseases.

An RNA-targeting CRISPR-Cas13d system alleviates disease-related phenotypes in Huntington's disease models.

Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Since the reduction of pathogenic mutant HTT messenger RNA is therapeutic, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d-CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patients with HD and induced pluripotent stem cell-derived neurons. We show that intrastriatal delivery of Cas13d-CAGEX via an adeno-associated viral vector selectively reduces mutant HTT mRNA and protein levels in the striatum of heterozygous zQ175 mice, a model of HD. This also led to improved motor coordination, attenuated striatal atrophy and reduction of mutant HTT protein aggregates. These phenotypic improvements lasted for at least eight months without adverse effects and with minimal off-target transcriptomic effects. Taken together, we demonstrate proof of principle of an RNA-targeting CRISPR-Cas13d system as a therapeutic approach for HD, a strategy with implications for the treatment of other dominantly inherited disorders.

Mitochondrial Uncoupling Induces Epigenome Remodeling and Promotes Differentiation in Neuroblastoma.

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the NAD+/NADH and pyruvate/lactate ratios and also the α-ketoglutarate/2-hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and ß-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and ß-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma. SIGNIFICANCE: Targeting cancer metabolism using the mitochondrial uncoupler niclosamide ethanolamine leads to methylome reprogramming and differentiation in neuroblastoma, providing a therapeutic opportunity to reverse the Warburg effect and suppress tumor growth. See related commentary by Byrne and Bell, p.167.

Monitoring and Identifying Insulin-Like Growth Factor 1 Variants by Liquid Chromatography-High-Resolution Mass Spectrometry in a Clinical Laboratory.

Protein and peptide hormones often exist as sequence variants with different molecular mass. Monitoring these variants of different molecular mass by mass spectrometry using mass-to-charge (m/z) ratio that is indicative of the wild type may lead to inaccurate quantitative results. However, liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based techniques can capture these differences and provide an opportunity to resolve, or partially resolve, variant complexity. In this chapter, we describe a general approach for monitoring a set of peptide variants with similar m/z ratios and isotopic envelopes, but different in amino acid sequences. As an example, we use insulin-like growth factor-1 (IGF-1) to demonstrate a DNA database-guided approach to monitor protein variants by LC-HRMS in a clinical laboratory. The workflow is automated and therefore avoids manual calculations that are prone to human error. The method can also monitor multiple IGF-1 variants and discover new ones. It can also provide a profile of a patient's IGF-1 status and be used to explore genotype-phenotype relationships in IGF-1 variants.

What is the role of the microenvironment in MDS?

Treating myelodysplastic syndromes (MDS) remains challenging. Hematopoiesis occurs within a heterogeneous, complex and dynamic microenvironment, and a multiplicity of mutations in hematopoietic stem and progenitor cells (HSPCs) lead to MDS. But is there a role for the microenvironment? Here we review experimental and conceptual arguments that support a role for the microenvironment, provide evidence for the disruption of the microenvironment in MDS, and explore microenvironmental signals that may provide a targetable and conserved vulnerability in MDS that transcend genetic heterogeneity.

Aged marrow macrophages expand platelet-biased hematopoietic stem cells via Interleukin1B.

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mφs also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mφs, were markedly increased in aged mice, consistent with functional defects in Mφ phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mφs induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mφs and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.

EVI1 overexpression reprograms hematopoiesis via upregulation of Spi1 transcription.

Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement. We show that EVI1 overexpression causes global distortion of hematopoiesis, with suppression of erythropoiesis and lymphopoiesis, and marked premalignant expansion of myelopoiesis that eventually results in leukemic transformation. We show that myeloid skewing is dependent on DNA binding by EVI1, which upregulates Spi1, encoding master myeloid regulator PU.1. We show that EVI1 binds to the -14 kb upstream regulatory element (-14kbURE) at Spi1; knockdown of Spi1 dampens the myeloid skewing. Furthermore, deletion of the -14kbURE at Spi1 abrogates the effects of EVI1 on hematopoietic stem cells. These findings support a novel mechanism of leukemogenesis through EVI1 overexpression.

The microenvironment in myelodysplastic syndromes: Niche-mediated disease initiation and progression.

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoietic stem and progenitor cells and represent the most common cause of acquired marrow failure. Hallmarked by ineffective hematopoiesis, dysplastic marrow, and risk of transformation to acute leukemia, MDS remains a poorly treated disease. Although identification of hematopoietic aberrations in human MDS has contributed significantly to our understanding of MDS pathogenesis, evidence now identify the bone marrow microenvironment (BMME) as another key contributor to disease initiation and progression. With improved understanding of the BMME, we are beginning to refine the role of the hematopoietic niche in MDS. Despite genetic diversity in MDS, interaction between MDS and the BMME appears to be a common disease feature and therefore represents an appealing therapeutic target. Further understanding of the interdependent relationship between MDS and its niche is needed to delineate the mechanisms underlying hematopoietic failure and how the microenvironment can be targeted clinically. This review provides an overview of data from human MDS and murine models supporting a role for BMME dysfunction at several steps of disease pathogenesis. Although no models or human studies so far have combined all of these findings, we review current data identifying BMME involvement in each step of MDS pathogenesis organized to reflect the chronology of BMME contribution as the normal hematopoietic system becomes myelodysplastic and MDS progresses to marrow failure and transformation. Although microenvironmental heterogeneity and dysfunction certainly add complexity to this syndrome, data are already demonstrating that targeting microenvironmental signals may represent novel therapeutic strategies for MDS treatment.

Targeting of the bone marrow microenvironment improves outcome in a murine model of myelodysplastic syndrome.

In vitro evidence suggests that the bone marrow microenvironment (BMME) is altered in myelodysplastic syndromes (MDSs). Here, we study the BMME in MDS in vivo using a transgenic murine model of MDS with hematopoietic expression of the translocation product NUP98-HOXD13 (NHD13). This model exhibits a prolonged period of cytopenias prior to transformation to leukemia and is therefore ideal to interrogate the role of the BMME in MDS. In this model, hematopoietic stem and progenitor cells (HSPCs) were decreased in NHD13 mice by flow cytometric analysis. The reduction in the total phenotypic HSPC pool in NHD13 mice was confirmed functionally with transplantation assays. Marrow microenvironmental cellular components of the NHD13 BMME were found to be abnormal, including increases in endothelial cells and in dysfunctional mesenchymal and osteoblastic populations, whereas megakaryocytes were decreased. Both CC chemokine ligand 3 and vascular endothelial growth factor, previously shown to be increased in human MDS, were increased in NHD13 mice. To assess whether the BMME contributes to disease progression in NHD13 mice, we performed transplantation of NHD13 marrow into NHD13 mice or their wild-type (WT) littermates. WT recipients as compared with NHD13 recipients of NHD13 marrow had a lower rate of the combined outcome of progression to leukemia and death. Moreover, hematopoietic function was superior in a WT BMME as compared with an NHD13 BMME. Our data therefore demonstrate a contributory role of the BMME to disease progression in MDS and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function and overall survival.

Different associations of CD45 isoforms with STAT3, PKC and ERK regulate IL-6-induced proliferation in myeloma.

In response to interleukin 6 (IL-6) stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT)3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK), and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing) the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC) and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC/NF-κB pathways.