RESUMO
Adipocytes occupy 70% of the cellular volume within the bone marrow (BM) wherein multiple myeloma (MM) originates and resides. However, the nature of the interaction between MM cells and adipocytes remains unclear. Cancer-associated adipocytes support tumor cells through various mechanisms, including metabolic reprogramming of cancer cells. We hypothesized that metabolic interactions mediate the dependence of MM cells on BM adipocytes. Here we show that BM aspirates from precursor states of MM, including monoclonal gammopathy of undetermined significance and smoldering MM, exhibit significant upregulation of adipogenic commitment compared with healthy donors. In vitro coculture assays revealed an adipocyte-induced increase in MM cell proliferation in monoclonal gammopathy of undetermined significance/smoldering MM compared with newly diagnosed MM. Using murine MM cell/BM adipocyte coculture assays, we describe MM-induced lipolysis in adipocytes via activation of the lipolysis pathway. Upregulation of fatty acid transporters 1 and 4 on MM cells mediated the uptake of secreted free fatty acids (FFAs) by adjacent MM cells. The effect of FFAs on MM cells was dose dependent and revealed increased proliferation at lower concentrations vs induction of lipotoxicity at higher concentrations. Lipotoxicity occurred via the ferroptosis pathway. Exogenous treatment with arachidonic acid, a very-long-chain FFA, in a murine plasmacytoma model displayed a reduction in tumor burden. Taken together, our data reveal a novel pathway involving MM cell-induced lipolysis in BM adipocytes and suggest prevention of FFA uptake by MM cells as a potential target for myeloma therapeutics.
Assuntos
Adipócitos/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Lipólise , Mieloma Múltiplo/metabolismo , Adipócitos/citologia , Adipócitos/patologia , Animais , Linhagem Celular , Técnicas de Cocultura , Humanos , Masculino , Camundongos SCID , Mieloma Múltiplo/patologia , Células Tumorais CultivadasRESUMO
PURPOSE OF REVIEW: Bone disease is a defining characteristic of multiple myeloma (MM) and the major cause of morbidity. It manifests as lytic lesions or osteopenia and is often associated with severe pain, pathological fracture, spinal cord compression, vertebral collapse, and hypercalcemia. Here, we have reviewed recent data on understanding its biology and treatment. RECENT FINDINGS: The imbalance between bone regeneration and bone resorption underlies the pathogenesis of osteolytic bone disease. Increased osteoclast proliferation and activity accompanied by inhibition of bone-forming osteoblasts leads to progressive bone loss and lytic lesions. Although tremendous progress has been made, MM remains an incurable disease. Novel agents targeting bone disease are under investigation with the goal of not only preventing bone loss and improving bone quality but also harnessing MM tumor growth. Current data illustrate that the interactions between MM cells and the tumor-bone microenvironment contribute to the bone disease and continued MM progression. A better understanding of this microenvironment is critical for novel therapeutic treatments of both MM and associated bone disease.
Assuntos
Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/metabolismo , Hipercalcemia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/metabolismo , Osteólise/metabolismo , Conservadores da Densidade Óssea/uso terapêutico , Doenças Ósseas Metabólicas/tratamento farmacológico , Doenças Ósseas Metabólicas/etiologia , Dor do Câncer/etiologia , Difosfonatos/uso terapêutico , Fraturas Espontâneas/etiologia , Humanos , Hipercalcemia/etiologia , Mieloma Múltiplo/complicações , Mieloma Múltiplo/tratamento farmacológico , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteólise/tratamento farmacológico , Osteólise/etiologia , Compressão da Medula Espinal/etiologia , Fraturas da Coluna Vertebral/etiologia , Microambiente TumoralRESUMO
Pericytes are perivascular cells localized to capillaries that promote vessel maturation, and their absence can contribute to vessel loss. Whether impaired endothelial-pericyte interaction contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Using 3G5-specific, immunoglobulin G-coated magnetic beads, we isolated pericytes from the lungs of healthy subjects and PAH patients, followed by lineage validation. PAH pericytes seeded with healthy pulmonary microvascular endothelial cells failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those with healthy pericytes. After the demonstration of abnormal polarization toward endothelium via live-imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. PAH pericytes had reduced expression of frizzled 7 (Fzd7) and cdc42, genes crucial for Wnt/PCP activation. With simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes in vitro and in a murine model of angiogenesis, motility and polarization toward pulmonary microvascular endothelial cells were reduced, whereas with restoration of both genes in PAH pericytes, endothelial-pericyte association was improved, with larger vascular networks. These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis are regulated by Wnt/PCP activation, which can be targeted to prevent vessel loss in PAH.
Assuntos
Polaridade Celular , Hipertensão Pulmonar/fisiopatologia , Pulmão/fisiopatologia , Neovascularização Patológica , Pericitos/citologia , Proteínas Wnt/metabolismo , Adolescente , Adulto , Animais , Movimento Celular , Criança , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/citologia , Feminino , Receptores Frizzled , Técnicas de Silenciamento de Genes , Humanos , Hipertensão Pulmonar/metabolismo , Imunoglobulina G/química , Pulmão/irrigação sanguínea , Magnetismo , Masculino , Camundongos , Camundongos SCID , Microcirculação , Pessoa de Meia-Idade , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSFneutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Mielopoese , Osteócitos/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Células da Medula Óssea/metabolismo , Proliferação de Células , Células Cultivadas , Microambiente Celular/genética , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Células Mieloides/metabolismo , Osteócitos/citologia , Osteócitos/ultraestrutura , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/metabolismoRESUMO
The bone marrow cavity is essential for the proper development of the hematopoietic system. In the last few decades, it has become clear that mesenchymal stem/progenitor cells as well as cells of the osteoblast lineage, besides maintaining bone homeostasis, are also fundamental regulators of bone marrow hematopoiesis. Several studies have demonstrated the direct involvement of mesenchymal and osteoblast lineage cells in the maintenance and regulation of supportive microenvironments necessary for quiescence, self-renewal and differentiation of hematopoietic stem cells. In addition, specific niches have also been identified within the bone marrow for maturing hematopoietic cells. Here we will review recent findings that have highlighted the roles of mesenchymal progenitors and cells of the osteoblast lineage in regulating distinct stages of hematopoiesis.
Assuntos
Diferenciação Celular/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Linfócitos B , Medula Óssea , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/fisiologiaRESUMO
BACKGROUND: Obesity is an established, modifiable risk factor of multiple myeloma (MM); yet, no lifestyle interventions are routinely recommended for patients with overweight or obesity with MM precursor conditions. Prolonged nightly fasting is a simple, practical dietary regimen supported by research, suggesting that the synchronization of feeding-fasting timing with sleep-wake cycles favorably affects metabolic pathways implicated in MM. We describe the design and rationale of a randomized controlled pilot trial evaluating the efficacy of a regular, prolonged nighttime fasting schedule among individuals with overweight or obesity at high risk for developing MM or a related lymphoid malignancy. OBJECTIVE: We aim to investigate the effects of 4-month prolonged nightly fasting on body composition and tumor biomarkers among individuals with overweight or obesity with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or smoldering Waldenström macroglobulinemia (SWM). METHODS: Individuals with MGUS, SMM, or SWM aged ≥18 years and a BMI of ≥25 kg/m2 are randomized to either a 14-hour nighttime fasting intervention or a healthy lifestyle education control group. Participants' baseline diet and lifestyle patterns are characterized through two 24-hour dietary recalls: questionnaires querying demographic, comorbidity, lifestyle, and quality-of-life information; and wrist actigraphy measurements for 7 days. Fasting intervention participants are supported through one-on-one telephone counseling by a health coach and automated SMS text messaging to support fasting goals. Primary end points of body composition, including visceral and subcutaneous fat (by dual-energy x-ray absorptiometry); bone marrow adiposity (by bone marrow histology); and tumor biomarkers, specifically M-proteins and serum free light-chain concentrations (by gel-based and serum free light-chain assays), are assessed at baseline and after the 4-month study period; changes therein from baseline are evaluated using a repeated measures mixed-effects model that accounts for the correlation between baseline and follow-up measures and is generally robust to missing data. Feasibility is assessed as participant retention (percent dropout in each arm) and percentage of days participants achieved a ≥14-hour fast. RESULTS: The PROlonged nightly FASTing (PROFAST) study was funded in June 2022. Participant recruitment commenced in April 2023. As of July 2023, six participants consented to the study. The study is expected to be completed by April 2024, and data analysis and results are expected to be published in the first quarter of 2025. CONCLUSIONS: PROFAST serves as an important first step in exploring the premise that prolonged nightly fasting is a strategy to control obesity and obesity-related mechanisms of myelomagenesis. In evaluating the feasibility and impact of prolonged nightly fasting on body composition, bone marrow adipose tissue, and biomarkers of tumor burden, this pilot study may generate hypotheses regarding metabolic mechanisms underlying MM development and ultimately inform clinical and public health strategies for MM prevention. TRIAL REGISTRATION: ClinicalTrials.gov NCT05565638; http://clinicaltrials.gov/ct2/show/NCT05565638. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51368.
RESUMO
A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4(+)CD25(+)FOXP3(+) natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS(-/-) mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS(-/-) nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS(-/-) nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.
Assuntos
Autoimunidade/imunologia , Fatores de Transcrição Forkhead/imunologia , Tolerância Imunológica/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Actinas/metabolismo , Animais , Diferenciação Celular/imunologia , Polaridade Celular/imunologia , Imunofluorescência , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estatísticas não Paramétricas , Fator de Crescimento Transformador beta/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.
Assuntos
Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese Imperfeita/metabolismo , Adipogenia/efeitos dos fármacos , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Autofagia/efeitos dos fármacos , Western Blotting , Ácidos Borônicos/farmacologia , Bortezomib , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese Imperfeita/patologia , Pirazinas/farmacologiaRESUMO
The regulatory effects of the immune system on the skeleton during homeostasis and activation have been appreciated for years. In the past decade it has become evident that bone tissue can also regulate immune cell development. In the bone marrow, the differentiation of hematopoietic progenitors requires specific microenvironments, called "niches," provided by various subsets of stromal cells, many of which are of mesenchymal origin. Among these stromal cell populations, cells of the osteoblast lineage serve a supportive function in the maintenance of normal hematopoiesis, and B lymphopoiesis in particular. Within the osteoblast lineage, distinct differentiation stages exert differential regulatory effects on hematopoietic development. In this review we will highlight the critical role of osteoblast progenitors in the perivascular B lymphocyte niche.
Assuntos
Linfócitos B/citologia , Medula Óssea/patologia , Osteoblastos/citologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Linhagem da Célula , Células-Tronco Hematopoéticas/citologia , Homeostase , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Transdução de Sinais , Células Estromais/citologiaRESUMO
The overweight/obesity epidemic is a serious public health concern that affects >40% of adults globally and increases the risk of numerous chronic diseases, such as type 2 diabetes, heart disease, and various cancers. Multiple myeloma (MM) is a lymphohematopoietic cancer caused by the uncontrolled clonal expansion of plasma cells. Recent studies have shown that obesity is a risk factor not only for MM but also monoclonal gammopathy of undetermined significance (MGUS), a precursor disease state of MM. Furthermore, obesity may promote the transition from MGUS to MM. Thus, in this review, we summarize the epidemiological evidence regarding the role of obesity in MM and MGUS, discuss the biologic mechanisms that drive these disease processes, and detail the obesity-targeted pharmacologic and lifestyle interventions that may reduce the risk of progression from MGUS to MM.
Assuntos
Diabetes Mellitus Tipo 2 , Gamopatia Monoclonal de Significância Indeterminada , Mieloma Múltiplo , Adulto , Humanos , Mieloma Múltiplo/epidemiologia , Mieloma Múltiplo/etiologia , Gamopatia Monoclonal de Significância Indeterminada/epidemiologia , Gamopatia Monoclonal de Significância Indeterminada/etiologia , Obesidade/complicações , Obesidade/epidemiologia , Fatores de RiscoRESUMO
Osteoblasts and their progenitors play an important role in the support of hematopoiesis within the bone marrow (BM) microenvironment. We have previously reported that parathyroid hormone receptor (PTH1R) signaling in osteoprogenitors is required for normal B cell precursor differentiation, and for trafficking of maturing B cells out of the BM. Cells of the osteoblast lineage have been implicated in the regulation of several other hematopoietic cell populations, but the effects of PTH1R signaling in osteoprogenitors on other maturing hematopoietic populations have not been investigated. Here we report that numbers of maturing myeloid, T cell, and erythroid populations were increased in the BM of mice lacking PTH1R in Osx-expressing osteoprogenitors (PTH1R-OsxKO mice; knockout [KO]). This increase in maturing hematopoietic populations was not associated with an increase in progenitor populations or proliferation. The spleens of PTH1R-OsxKO mice were small with decreased numbers of all hematopoietic populations, suggesting that trafficking of mature hematopoietic populations between BM and spleen is impaired in the absence of PTH1R in osteoprogenitors. RNA sequencing (RNAseq) of osteoprogenitors and their descendants in bone and BM revealed increased expression of vascular cell adhesion protein 1 (VCAM-1) and C-X-C motif chemokine ligand 12 (CXCL12), factors that are involved in trafficking of several hematopoietic populations. © 2022 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Osteoblastos , Receptor Tipo 1 de Hormônio Paratireóideo , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Osteoblastos/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Transdução de SinaisRESUMO
Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.
Assuntos
Envelhecimento/fisiologia , Transplante de Medula Óssea/métodos , Pesquisa Fetal , Osteogênese Imperfeita/prevenção & controle , Osteogênese Imperfeita/terapia , Útero/fisiologia , Animais , Células da Medula Óssea/citologia , Colágeno/metabolismo , Modelos Animais de Doenças , Espaço Extracelular/química , Feminino , Técnicas de Introdução de Genes , Genes Dominantes , Sobrevivência de Enxerto , Camundongos , Camundongos Transgênicos , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Fenótipo , Gravidez , Análise Espectral Raman , Taxa de Sobrevida , Doadores de TecidosRESUMO
MYC upregulation is associated with multidrug refractory disease in patients with multiple myeloma (MM). We, isolated patient-derived MM cells with high MYC expression and discovered that NCOR2 was down-regulated in these cells. NCOR2 is a transcriptional coregulatory protein and its role in MM remains unknown. To define the role of NCOR2 in MM, we created NCOR2 knockout human myeloma cell lines and demonstrated that NCOR2 knockout led to high MYC expression. Furthermore, NCOR2 knockout conferred resistance to pomalidomide, BET and HDAC inhibitors, independent of Cereblon (CRBN), indicating high MYC expression as a cause of multidrug resistance. Moreover, NCOR2 interacted with the nucleosome remodeling and deacetylase (NuRD) complex and repressed the expression of CD180 by directly binding to its promoter and inducing MYC expression. Next, we generated lenalidomide-resistant and pomalidomide-resistant human myeloma cell lines. Whole-exome sequencing revealed that these cell lines acquired the same exonic mutations of NCOR2. These cell lines showed NCOR2 downregulation and MYC upregulation independent of CRBN and demonstrated resistance to BET and HDAC inhibitors. Our findings reveal a novel CRBN independent molecular mechanism associated with drug resistance. Low NCOR2 expression can serve as a potential biomarker for drug resistance and needs further validation in larger prospective studies.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Correpressor 2 de Receptor Nuclear/genética , Proteínas Proto-Oncogênicas c-myc/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Técnicas de Inativação de Genes , Inibidores de Histona Desacetilases/farmacologia , Humanos , Talidomida/análogos & derivados , Talidomida/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.
Assuntos
Terapia Genética/efeitos adversos , Terapia Genética/métodos , Síndrome de Wiskott-Aldrich/terapia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Western Blotting , Feminino , Imunofenotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Reação em Cadeia da Polimerase , Linfócitos T/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD(+) B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1.
Assuntos
Linfócitos B/citologia , Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Animais , Anticorpos Neutralizantes/química , Apoptose , Osso e Ossos/metabolismo , Diferenciação Celular , Quimiocina CXCL12/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Imuno-Histoquímica , Interleucina-7/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteócitos/citologia , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Microtomografia por Raio-XAssuntos
Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Compostos Macrocíclicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Microambiente Celular , Resistencia a Medicamentos Antineoplásicos , HumanosRESUMO
Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Biallelic mutations in the TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, are responsible for more than one half of ARO patients. However, a few patients with monoallelic mutations have been described, raising the possibility of a dominant-like TCIRG1-dependent osteopetrosis, of a digenic disease, or of peculiar mutations difficult to detect with standard methods. We describe here a novel genomic deletion in the TCIRG1 gene explaining why, in some patients, mutations in only one allele have previously been found. The analysis of a proband from a consanguineous Turkish family allowed us to define the deletion boundaries encompassing introns 10 and 13 and occurring within AluSx repeat sequences, suggesting Alu-mediated homologous recombination as a mechanism. An identical genomic deletion at the heterozygous level was found in four unrelated Italian families in whom only a single mutated allele has previously been found. TCIRG1 haplotype analysis in these five families suggests a possible common ancestral origin for this large deletion. In summary, we describe the identification of a novel genomic deletion in the TCIRG1 gene that is of clinical relevance, especially in prenatal diagnosis.