RESUMO
BACKGROUND: Twenty percent of children with hepatoblastoma (HB) have lung metastasis at diagnosis. Treatment protocols recommend surgical removal of chemotherapy-refractory lung nodules, however no chronological order is established. As hepatectomy is followed by release of growth factors, it has been proposed that partial hepatectomy (PH) could boost local or distant residual tumor growth. METHODS: To evaluate the impact of PH on distant tumor growth, PH was performed in mice subcutaneously implanted with a HB patient-derived xenograft (PDX). The influence of PH on tumor growth at primary site was assessed by performing PH concomitantly to HB PDXs orthotopic implantation. RESULTS: Subcutaneously implanted HB PDX failed to show any influence of hepatectomy on tumor growth. Instead, intrahepatic tumor growth of one of the 4 HB PDXs implanted orthotopically was clearly enhanced. Cells derived from the hepatectomy-sensitive HB PDX exposed to hepatic growth factor (HGF) showed increased proliferation rate compared to cells derived from a hepatectomy-insensitive model, suggesting that the HGF/MET pathway could be one of the effectors of the crosstalk between liver regeneration and HB growth. CONCLUSION: These results suggest that hepatectomy can contribute to HB growth in some patients, further studies will be necessary to identify biomarkers predictive of patient risk of PH-induced HB recurrence. IMPACT: Key message: Cytokines and growth factors secreted following partial hepatectomy can contribute to intrahepatic tumor growth in some hepatoblastoma models. What does it add to the existing literature: It is the first article about the impact of liver regeneration induced by partial hepatectomy on hepatoblastoma local or distant tumoral growth in nude mice. What is the impact: It is important to identify the secreted factors that enhance tumor growth and to define biomarkers predictive of patient risk of partial hepatectomy-induced hepatoblastoma recurrence.
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The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.
Assuntos
Mutação com Ganho de Função , Doenças Hematológicas/imunologia , Plasmócitos/imunologia , Receptores CXCR4/imunologia , Transdução de Sinais/imunologia , Animais , Doenças Hematológicas/genética , Humanos , Camundongos , Camundongos Transgênicos , Receptores CXCR4/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologiaRESUMO
Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.
Assuntos
Proliferação de Células , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Canais de Cálcio Tipo L/metabolismo , Di-Hidropiridinas/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Células-Tronco Mesenquimais/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
A rare location, t(6;11)(q27;q23) (MLL-AF6), is associated with poor outcome in childhood acute myeloid leukemia (AML). The described mechanism by which MLL-AF6, through constitutive self-association and in cooperation with DOT-1L, activates aberrant gene expression does not explain the biological differences existing between t(6;11)-rearranged and other MLL-positive patients nor their different clinical outcome. Here, we show that AF6 is expressed in the cytoplasm of healthy bone marrow cells and controls rat sarcoma viral oncogene (RAS)-guanosine triphosphate (GTP) levels. By contrast, in MLL-AF6-rearranged cells, AF6 is found localized in the nucleus, leading to aberrant activation of RAS and of its downstream targets. Silencing MLL-AF6, we restored AF6 localization in the cytoplasm, thus mediating significant reduction of RAS-GTP levels and of cell clonogenic potential. The rescue of RAS-GTP levels after MLL-AF6 and AF6 co-silencing confirmed that MLL-AF6 oncoprotein potentiates the activity of the RAS pathway through retention of AF6 within the nucleus. Exposure of MLL-AF6-rearranged AML blasts to tipifarnib, a RAS inhibitor, leads to cell autophagy and apoptosis, thus supporting RAS targeting as a novel potential therapeutic strategy in patients carrying t(6;11). Altogether, these data point to a novel role of the MLL-AF6 chimera and show that its gene partner, AF6, is crucial in AML development.
Assuntos
Núcleo Celular/metabolismo , Cinesinas/metabolismo , Leucemia Mieloide , Proteína de Leucina Linfoide-Mieloide/metabolismo , Miosinas/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Criança , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 6 , Inativação Gênica , Humanos , Cinesinas/genética , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Miosinas/genética , Proteínas de Fusão Oncogênica/genética , Transporte Proteico , Ativação Transcricional , Translocação GenéticaAssuntos
Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Leucemia Mieloide Aguda/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Translocação Genética , Adolescente , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/terapia , Masculino , MicroRNAs/genética , Prognóstico , RNA Neoplásico/genética , Recidiva , Estudos RetrospectivosRESUMO
WHIM Syndrome is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report a decrease in bone mineral density in 25% of WHIM patients and bone defects leading to osteoporosis in a WHIM mouse model. Imbalanced bone tissue is observed in mutant mice combining reduced osteoprogenitor cells and increased osteoclast numbers. Mechanistically, impaired CXCR4 desensitization disrupts cell cycle progression and osteogenic commitment of skeletal stromal/stem cells, while increasing their pro-osteoclastogenic capacities. Impaired osteogenic differentiation is evidenced in primary bone marrow stromal cells from WHIM patients. In mice, chronic treatment with the CXCR4 antagonist AMD3100 normalizes in vitro osteogenic fate of mutant skeletal stromal/stem cells and reverses in vivo the loss of skeletal cells, demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of skeletal stromal/stem cells. Our study provides mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of skeletal cells and the balance between bone formation and resorption.
Assuntos
Síndromes de Imunodeficiência , Osteoporose , Doenças da Imunodeficiência Primária , Receptores CXCR4 , Animais , Camundongos , Síndromes de Imunodeficiência/genética , Mutação , Osteogênese/genética , Osteoporose/genética , Doenças da Imunodeficiência Primária/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , HumanosRESUMO
Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolytic activity. Here we show that patients with MDS clones harbouring mutations in the TET2 gene are characterised by phenotypic defects in their circulating NK cells. Remarkably, NK cells and MDS clones from the same patient share the TET2 genotype, and the NK cells are characterised by increased methylation of genomic DNA and reduced expression of Killer Immunoglobulin-like receptors (KIR), perforin, and TNF-α. In vitro inhibition of TET2 in NK cells of healthy donors reduces their cytotoxicity, supporting its critical role in NK cell function. Conversely, NK cells from patients treated with azacytidine (#NCT02985190; https://clinicaltrials.gov/ ) show increased KIR and cytolytic protein expression, and IFN-γ production. Altogether, our findings show that, in addition to their oncogenic consequences in the myeloid cell subsets, TET2 mutations contribute to repressing NK-cell function in MDS patients.
Assuntos
Dioxigenases , Síndromes Mielodisplásicas , Humanos , Metilação , Síndromes Mielodisplásicas/metabolismo , Células Matadoras Naturais , Azacitidina/farmacologia , Receptores KIR/genética , Mutação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismoRESUMO
Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality.
Assuntos
Antineoplásicos , Neoplasias da Mama , Neoplasias da Mama/metabolismo , Feminino , Humanos , Imunidade Inata , Células Matadoras Naturais/metabolismo , Linfonodos/patologia , Microambiente TumoralRESUMO
Mesenchymal stromal cells (MSC) are a rare, heterogeneous and multipotent population that can be isolated from several tissues. MSC were originally discovered in the bone marrow and studied for their capacity to maintain hematopoietic cells. We will describe here methods to isolate, culture, and bank MSC from human bone marrow. Then, characterization protocols by flow cytometry, clonogenic assays and doubling time evaluation will be developed. Finally, in vitro MSC culture and differentiation into osteoblasts, adipocytes, and chondrocytes will be explained. Thus, this chapter will detail all bases to work on MSC with consensus and clear methods and protocols.
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Diferenciação Celular , Proliferação de Células , Células-Tronco Mesenquimais/fisiologia , Adipogenia , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Separação Celular , Células Cultivadas , Condrogênese , Criopreservação , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese , FenótipoRESUMO
Patients with acute myeloid leukemia (AML) carrying high-risk genetic lesions or high residual disease levels after therapy are particularly exposed to the risk of relapse. Here, we identified the long non-coding RNA CDK6-AS1 able to cluster an AML subgroup with peculiar gene signatures linked to hematopoietic cell differentiation and mitochondrial dynamics. CDK6-AS1 silencing triggered hematopoietic commitment in healthy CD34+ cells, whereas in AML cells the pathological undifferentiated state was rescued. This latter phenomenon derived from RUNX1 transcriptional control, responsible for the stemness of hematopoietic precursors and for the block of differentiation in AML. By CDK6-AS1 silencing in vitro, AML mitochondrial mass decreased with augmented pharmacological sensitivity to mitochondria-targeting drugs. In vivo, the combination of tigecycline and cytarabine reduced leukemia progression in the AML-PDX model with high CDK6-AS1 levels, supporting the concept of a mitochondrial vulnerability. Together, these findings uncover CDK6-AS1 as crucial in myeloid differentiation and mitochondrial mass regulation.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Criança , Pré-Escolar , Citarabina/uso terapêutico , Monitoramento de Medicamentos , Etoposídeo/uso terapêutico , Feminino , Humanos , Idarubicina/uso terapêutico , Quimioterapia de Indução/métodos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Masculino , Mutação , Neoplasia Residual , Proteína 1 Parceira de Translocação de RUNX1 , Análise de Sobrevida , Translocação GenéticaRESUMO
The generation of a potent humoral immune response by B cells relies on the integration of signals induced by the B cell receptor, toll-like receptors and both negative and positive co-receptors. Several reports also suggest that integrin signaling plays an important role in this process. How integrin signaling is regulated in B cells is however still partially understood. Integrin activity and function are controlled by several mechanisms including regulation by molecular adaptors of the paxillin family. In B cells, Leupaxin (Lpxn) is the most expressed member of the family and in vitro studies suggest that it could dampen BCR signaling. Here, we report that Lpxn expression is increased in germinal center B cells compared to naïve B cells. Moreover, Lpxn deficiency leads to decreased B cell differentiation into plasma cells in vitro. However, Lpxn seems dispensable for the generation of a potent B cell immune response in vivo. Altogether our results suggest that Lpxn is dispensable for T-dependent and T-independent B cell immune responses.
Assuntos
Linfócitos B/imunologia , Moléculas de Adesão Celular/metabolismo , Centro Germinativo/imunologia , Plasmócitos/imunologia , Fatores de Transcrição/metabolismo , Animais , Formação de Anticorpos , Moléculas de Adesão Celular/genética , Diferenciação Celular , Células Cultivadas , Imunidade Humoral , Ativação Linfocitária , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The somatic translocation t(8;21)(q22;q22)/RUNX1-RUNX1T1 is one of the most frequent rearrangements found in children with standard-risk acute myeloid leukemia (AML). Despite the favorable prognostic role of this aberration, we recently observed a higher than expected frequency of relapse. Here, we employed an integrated high-throughput approach aimed at identifying new biological features predicting relapse among 34 t(8;21)-rearranged patients. We found that the DNA methylation status of patients who suffered from relapse was peculiarly different from that of children maintaining complete remission. The epigenetic signature, made up of 337 differentially methylated regions, was then integrated with gene and protein expression profiles, leading to a network, where cell-to-cell adhesion and cell-motility pathways were found to be aberrantly activated in relapsed patients. We identified most of these factors as RUNX1-RUNX1T1 targets, with Ras Homolog Family Member (RHOB) overexpression being the core of this network. We documented how RHOB re-organized the actin cytoskeleton through its downstream ROCK-LIMK-COFILIN axis: this increases blast adhesion by stress fiber formation, and reduces mitochondrial apoptotic cell death after chemotherapy treatment. Altogether, our data show an epigenetic heterogeneity within t(8;21)-rearranged AML patients at diagnosis able to influence the program of the chimeric transcript, promoting blast re-emergence and progression to relapse.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Epigenômica , Heterogeneidade Genética , Leucemia Mieloide Aguda/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Translocação Genética , Proteína rhoB de Ligação ao GTP/metabolismo , Adolescente , Crise Blástica/patologia , Adesão Celular/genética , Movimento Celular/genética , Criança , Pré-Escolar , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Citoesqueleto/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Recidiva , RiscoAssuntos
Doenças Autoimunes/patologia , Inflamação/patologia , Leucemia Mielomonocítica Crônica/patologia , Mutação , Síndromes Mielodisplásicas/patologia , Enzimas Ativadoras de Ubiquitina/genética , Idoso , Doenças Autoimunes/complicações , Doenças Autoimunes/genética , Estudos de Casos e Controles , Humanos , Inflamação/complicações , Inflamação/genética , Leucemia Mielomonocítica Crônica/complicações , Leucemia Mielomonocítica Crônica/genética , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/genética , Prevalência , Prognóstico , Estudos RetrospectivosRESUMO
The genomic landscape of children with acute myeloid leukemia (AML) who do not carry any cytogenetic abnormality (CN-AML) is particularly heterogeneous and challenging, being characterized by different clinical outcomes. To provide new genetic insights into this AML subset, we analyzed through RNA-seq 13 pediatric CN-AML cases, corroborating our findings in an independent cohort of 168 AML patients enrolled in the AIEOP AML 2002/01 study. We identified a chimeric transcript involving NUP98 and PHF23, resulting from a cryptic t(11;17)(p15;p13) translocation, demonstrating, for the first time, that NUP98-PHF23 is a novel recurrent (2.6%) abnormality in pediatric CN-AML.