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1.
Nat Commun ; 13(1): 528, 2022 01 26.
Article in English | MEDLINE | ID: mdl-35082281

ABSTRACT

Neutrophils perform critical functions in the innate response to infection, including through the production of neutrophil extracellular traps (NETs) - web-like DNA structures which are extruded from neutrophils upon activation. Elevated levels of NETs have been linked to autoimmunity but this association is poorly understood. By contrast, IL-17 producing Th17 cells are a key player in various autoimmune diseases but are also crucial for immunity against fungal and bacterial infections. Here we show that NETs, through their protein component histones, directly activate T cells and specifically enhance Th17 cell differentiation. This modulatory role of neutrophils, NETs and their histones is mediated downstream of TLR2 in T cells, resulting in phosphorylation of STAT3. The innate stimulation of a specific adaptive immune cell subset provides an additional mechanism demonstrating a direct link between neutrophils, NETs and T cell autoimmunity.


Subject(s)
Cell Differentiation , Extracellular Traps/metabolism , Histones/metabolism , Neutrophils/metabolism , Th17 Cells/immunology , Toll-Like Receptor 2/metabolism , Adult , Autoimmunity , DNA/metabolism , Female , Humans , Immunity, Innate , Male , Young Adult
2.
Clin Cancer Res ; 27(15): 4338-4352, 2021 08 01.
Article in English | MEDLINE | ID: mdl-33994371

ABSTRACT

PURPOSE: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. EXPERIMENTAL DESIGN: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. RESULTS: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. CONCLUSIONS: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.


Subject(s)
Carbazoles/administration & dosage , Carbazoles/pharmacology , Chromatin/drug effects , Histone Deacetylase Inhibitors/administration & dosage , Histone Deacetylase Inhibitors/pharmacology , Neuroblastoma/drug therapy , Panobinostat/administration & dosage , Panobinostat/pharmacology , Animals , Drug Combinations , Drug Evaluation, Preclinical , Mice , Tumor Cells, Cultured
4.
Cell ; 184(5): 1330-1347.e13, 2021 03 04.
Article in English | MEDLINE | ID: mdl-33636130

ABSTRACT

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.


Subject(s)
Bone Resorption/pathology , Osteoclasts/pathology , RANK Ligand/metabolism , Animals , Apoptosis , Bone Resorption/metabolism , Cell Fusion , Cells, Cultured , Humans , Macrophages/cytology , Mice , Osteochondrodysplasias/drug therapy , Osteochondrodysplasias/genetics , Osteochondrodysplasias/metabolism , Osteochondrodysplasias/pathology , Osteoclasts/metabolism , Signal Transduction
5.
Cancer Res ; 81(3): 634-647, 2021 02 01.
Article in English | MEDLINE | ID: mdl-33218968

ABSTRACT

Bone marrow adipocytes (BMAd) have recently been implicated in accelerating bone metastatic cancers, such as acute myelogenous leukemia and breast cancer. Importantly, bone marrow adipose tissue (BMAT) expands with aging and obesity, two key risk factors in multiple myeloma disease prevalence, suggesting that BMAds may influence and be influenced by myeloma cells in the marrow. Here, we provide evidence that reciprocal interactions and cross-regulation of myeloma cells and BMAds play a role in multiple myeloma pathogenesis and treatment response. Bone marrow biopsies from patients with multiple myeloma revealed significant loss of BMAT with myeloma cell infiltration of the marrow, whereas BMAT was restored after treatment for multiple myeloma. Myeloma cells reduced BMAT in different preclinical murine models of multiple myeloma and in vitro using myeloma cell-adipocyte cocultures. In addition, multiple myeloma cells altered adipocyte gene expression and cytokine secretory profiles, which were also associated with bioenergetic changes and induction of a senescent-like phenotype. In vivo, senescence markers were also increased in the bone marrow of tumor-burdened mice. BMAds, in turn, provided resistance to dexamethasone-induced cell-cycle arrest and apoptosis, illuminating a new possible driver of myeloma cell evolution in a drug-resistant clone. Our findings reveal that bidirectional interactions between BMAds and myeloma cells have significant implications for the pathogenesis and treatment of multiple myeloma. Targeting senescence in the BMAd or other bone marrow cells may represent a novel therapeutic approach for treatment of multiple myeloma. SIGNIFICANCE: This study changes the foundational understanding of how cancer cells hijack the bone marrow microenvironment and demonstrates that tumor cells induce senescence and metabolic changes in adipocytes, potentially driving new therapeutic directions.


Subject(s)
Adipocytes/pathology , Adipose Tissue/pathology , Bone Marrow Cells/pathology , Cellular Senescence , Multiple Myeloma/pathology , 3T3 Cells , Adipocytes/metabolism , Adipocytes/physiology , Aging/pathology , Animals , Antineoplastic Agents, Hormonal/pharmacology , Apoptosis/drug effects , Biopsy , Bone Marrow/drug effects , Bone Marrow/pathology , Cell Communication/physiology , Cell Cycle/drug effects , Coculture Techniques , Cohort Studies , Cytokines/metabolism , Dexamethasone/pharmacology , Disease Progression , Drug Resistance, Neoplasm , Female , Gene Expression , Humans , Mice , Mice, Inbred C57BL , Mice, SCID , Multiple Myeloma/drug therapy , Multiple Myeloma/etiology , Obesity/pathology , Phenotype
6.
Blood ; 134(1): 30-43, 2019 07 04.
Article in English | MEDLINE | ID: mdl-31023703

ABSTRACT

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.


Subject(s)
Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Recurrence, Local/genetics , Neoplastic Stem Cells/pathology , Stem Cell Niche/genetics , Animals , Humans , Mice , Neoplasm Recurrence, Local/pathology , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Transcriptome , Axl Receptor Tyrosine Kinase
7.
J Cell Physiol ; 233(2): 1156-1167, 2018 Feb.
Article in English | MEDLINE | ID: mdl-28460416

ABSTRACT

The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.


Subject(s)
Adipocytes/metabolism , Adipogenesis , Adipose Tissue/metabolism , Bone Marrow Cells/metabolism , Glycoproteins/metabolism , Mesenchymal Stem Cells/metabolism , Osteocytes/metabolism , 3T3-L1 Cells , Adaptor Proteins, Signal Transducing , Adipose Tissue/cytology , Adiposity , Animals , Culture Media, Conditioned/metabolism , Glycoproteins/deficiency , Glycoproteins/genetics , Intercellular Signaling Peptides and Proteins , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Paracrine Communication , Phenotype , Stem Cell Niche , Wnt Signaling Pathway
8.
Oncotarget ; 8(40): 68047-68058, 2017 Sep 15.
Article in English | MEDLINE | ID: mdl-28978095

ABSTRACT

Melphalan is a cytotoxic chemotherapy used to treat patients with multiple myeloma (MM). Bone resorption by osteoclasts, by remodeling the bone surface, can reactivate dormant MM cells held in the endosteal niche to promote tumor development. Dormant MM cells can be reactivated after melphalan treatment; however, it is unclear whether melphalan treatment increases osteoclast formation to modify the endosteal niche. Melphalan treatment of mice for 14 days decreased bone volume and the endosteal bone surface, and this was associated with increases in osteoclast numbers. Bone marrow cells (BMC) from melphalan-treated mice formed more osteoclasts than BMCs from vehicle-treated mice, suggesting that osteoclast progenitors were increased. Melphalan also increased osteoclast formation in BMCs and RAW264.7 cells in vitro, which was prevented with the cell stress response (CSR) inhibitor KNK437. Melphalan also increased expression of the osteoclast regulator the microphthalmia-associated transcription factor (MITF), but not nuclear factor of activated T cells 1 (NFATc1). Melphalan increased expression of MITF-dependent cell fusion factors, dendritic cell-specific transmembrane protein (Dc-stamp) and osteoclast-stimulatory transmembrane protein (Oc-stamp) and increased cell fusion. Expression of osteoclast stimulator receptor activator of NFκB ligand (RANKL) was unaffected by melphalan treatment. These data suggest that melphalan stimulates osteoclast formation by increasing osteoclast progenitor recruitment and differentiation in a CSR-dependent manner. Melphalan-induced osteoclast formation is associated with bone loss and reduced endosteal bone surface. As well as affecting bone structure this may contribute to dormant tumor cell activation, which has implications for how melphalan is used to treat patients with MM.

9.
Blood ; 129(26): 3452-3464, 2017 06 29.
Article in English | MEDLINE | ID: mdl-28515094

ABSTRACT

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.


Subject(s)
Bone Density/drug effects , Bone Morphogenetic Proteins/antagonists & inhibitors , Fractures, Bone/prevention & control , Osteocytes/chemistry , Osteogenesis/drug effects , Adaptor Proteins, Signal Transducing , Animals , Antibodies/pharmacology , Antibodies/therapeutic use , Bone Morphogenetic Proteins/immunology , Cell Line, Tumor , Diphosphonates/therapeutic use , Genetic Markers/immunology , Humans , Imidazoles/therapeutic use , Mice , Multiple Myeloma/complications , Tumor Cells, Cultured , Zoledronic Acid
10.
Nat Commun ; 6: 8983, 2015 Dec 03.
Article in English | MEDLINE | ID: mdl-26632274

ABSTRACT

Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse.


Subject(s)
Bone Remodeling/physiology , Multiple Myeloma/metabolism , Osteoclasts/physiology , Aged , Aged, 80 and over , Animals , Cell Line, Tumor , Coculture Techniques , Female , Humans , Male , Mice , Mice, Inbred Strains , Middle Aged , Osteoblasts/physiology
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