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1.
Blood Adv ; 2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-39208369

RESUMO

Hematopoietic stem cells (HSCs) readily recover from acute stress, but persistent stress can reduce their viability and long-term potential. Here we show that the nuclear factor of activated T cells 5 (NFAT5), a transcription modulator of inflammatory responses, protects the HSC pool under stress. NFAT5 restrains HSC differentiation to multipotent progenitors (MPPs) after bone marrow transplantation and bone marrow ablation with ionizing radiation or chemotherapy. Correspondingly, NFAT5-deficient HSCs fail to support long-term reconstitution of hematopoietic progenitors and mature blood cells after serial transplant. Evidence from competitive transplant assays shows that these defects are HSC-intrinsic. NFAT5-deficient HSCs exhibit enhanced expression of type I interferon (IFN-I) response genes after transplant, and suppressing IFN-I-receptor prevents their exacerbated differentiation and cell death after reconstitution and improves long-term regeneration potential. Blockade of IFN-I receptor also prevented the overdifferentiation of NFAT5-deficient HSCs after bone marrow ablation. These findings show that long-term IFN-I responses to different hematopoietic stressors drive HSCs towards more differentiated progenitors, and that NFAT5 has an HSC-intrinsic role limiting IFN-I responses to preserve reconstitution potential. Our identification of cell-intrinsic mechanisms that strengthen the resistance of HSCs to stress could help to devise approaches to protect long-term stemness during the treatment of hematopoietic malignancies.

2.
Int J Mol Sci ; 25(14)2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39063034

RESUMO

Duchenne and Becker muscular dystrophies, caused by pathogenic variants in DMD, are the most common inherited neuromuscular conditions in childhood. These diseases follow an X-linked recessive inheritance pattern, and mainly males are affected. The most prevalent pathogenic variants in the DMD gene are copy number variants (CNVs), and most patients achieve their genetic diagnosis through Multiplex Ligation-dependent Probe Amplification (MLPA) or exome sequencing. Here, we investigated a female patient presenting with muscular dystrophy who remained genetically undiagnosed after MLPA and exome sequencing. RNA sequencing (RNAseq) from the patient's muscle biopsy identified an 85% reduction in DMD expression compared to 116 muscle samples included in the cohort. A de novo balanced translocation between chromosome 17 and the X chromosome (t(X;17)(p21.1;q23.2)) disrupting the DMD and BCAS3 genes was identified through trio whole genome sequencing (WGS). The combined analysis of RNAseq and WGS played a crucial role in the detection and characterisation of the disease-causing variant in this patient, who had been undiagnosed for over two decades. This case illustrates the diagnostic odyssey of female DMD patients with complex structural variants that are not detected by current panel or exome sequencing analysis.


Assuntos
Cromossomos Humanos X , Distrofina , Genômica , Distrofia Muscular de Duchenne , Translocação Genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/diagnóstico , Feminino , Distrofina/genética , Cromossomos Humanos X/genética , Genômica/métodos , Variações do Número de Cópias de DNA , Sequenciamento do Exoma , Transcriptoma/genética , Cromossomos Humanos Par 17/genética
3.
J Pathol ; 263(4-5): 482-495, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38872438

RESUMO

Liver fibrosis is the consequence of chronic liver injury in the presence of an inflammatory component. Although the main executors of this activation are known, the mechanisms that lead to the inflammatory process that mediates the production of pro-fibrotic factors are not well characterized. Epidermal growth factor receptor (EGFR) signaling in hepatocytes is essential for the regenerative processes of the liver; however, its potential role in regulating the fibrotic niche is not yet clear. Our group generated a mouse model that expresses an inactive truncated form of the EGFR specifically in hepatocytes (ΔEGFR mice). Here, we have analyzed the response of WT and ΔEGFR mice to chronic treatment with carbon tetrachloride (CCl4), which induces a pro-inflammatory and fibrotic process in the liver. The results indicated that the hallmarks of liver fibrosis were attenuated in CCl4-treated ΔEGFR mice when compared with CCl4-treated WT mice, coinciding with a faster resolution of the fibrotic process and ameliorated damage. The absence of EGFR activity in hepatocytes induced changes in the pattern of immune cells in the liver, with a notable increase in the population of M2 macrophages, more related to fibrosis resolution, as well as in the population of lymphocytes related to eradication of the damage. Transcriptome analysis of hepatocytes, and secretome studies of extracellular media from in vitro experiments, allowed us to elucidate the specific molecular mechanisms regulated by EGFR that mediate hepatocyte production of both pro-fibrotic and pro-inflammatory mediators; these have consequences for the deposition of extracellular matrix proteins, as well as for the immune microenvironment. Overall, our study uncovered novel mechanistic insights regarding EGFR kinase-dependent actions in hepatocytes that reveal its key role in chronic liver damage. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Tetracloreto de Carbono , Receptores ErbB , Hepatócitos , Transdução de Sinais , Animais , Receptores ErbB/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/patologia , Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Masculino , Comunicação Celular , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Transgênicos
4.
Cancers (Basel) ; 16(4)2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38398126

RESUMO

The aim of this study was to determine how TERTp mutations impact glioblastoma prognosis. MATERIALS AND METHODS: TERTp mutations were assessed in a retrospective cohort of 258 uniformly treated glioblastoma patients. RNA-sequencing and whole exome sequencing results were available in a subset of patients. RESULTS: Overall, there were no differences in outcomes between patients with mutated TERTp-wt or TERTp. However, we found significant differences according to the type of TERTp mutation. Progression-free survival (mPFS) was 9.1 months for those with the C250T mutation and 7 months for those with either the C228T mutation or TERTp-wt (p = 0.016). Overall survival (mOS) was 21.9 and 15 months, respectively (p = 0.026). This differential effect was more pronounced in patients with MGMTp methylation (mPFS: p = 0.008; mOS: p = 0.021). Multivariate analysis identified the C250T mutation as an independent prognostic factor for longer mOS (HR 0.69; p = 0.044). We found no differences according to TERTp mutation status in molecular alterations common in glioblastoma, nor in copy number variants in genes related to alternative lengthening of telomeres. Nevertheless, in the gene enrichment analysis adjusted for MGMTp methylation status, some Reactome gene sets were differentially enriched, suggesting that the C250T mutation may exert a lesser effect on telomeres or chromosomes. CONCLUSIONS: In our series, patients exhibiting the C250T mutation had a more favorable prognosis compared to those with either TERPp-wt or TERTp C228T mutations. Additionally, our findings suggest a reduced involvement of the C250T mutation in the underlying biological mechanisms related to telomeres.

5.
Cell Death Dis ; 14(12): 838, 2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38110339

RESUMO

Acute infection and chronic infection are the two most common fates of pathogenic virus infections. While several factors that contribute to these fates are described, the critical control points and the mechanisms that underlie infection fate regulation are incompletely understood. Using the acute and chronic lymphocytic choriomeningitis virus (LCMV) infection model of mice, we find that the early dynamic pattern of the IFN-I response is a differentiating trait between both infection fates. Acute-infected mice generate a 2-wave IFN-I response while chronic-infected mice generate only a 1-wave response. The underlying cause is a temporal difference in CD8 T cell-mediated killing of splenic marginal zone CD169+ macrophages. It occurs later in acute infection and thus enables CD169+ marginal zone macrophages to produce the 2nd IFN-I wave. This is required for subsequent immune events including induction of inflammatory macrophages, generation of effector CD8+ T cells and virus clearance. Importantly, these benefits come at a cost for the host in the form of spleen fibrosis. Due to an earlier marginal zone destruction, these ordered immune events are deregulated in chronic infection. Our findings demonstrate the critical importance of kinetically well-coordinated sequential immune events for acute infection control and highlights that it may come at a cost for the host organism.


Assuntos
Coriomeningite Linfocítica , Camundongos , Animais , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/fisiologia , Infecção Persistente , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos , Macrófagos/patologia
6.
Nat Commun ; 14(1): 7471, 2023 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-37978290

RESUMO

Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.


Assuntos
Macrófagos , Metformina , Humanos , Animais , Camundongos , Macrófagos/metabolismo , Células Mieloides , Inflamação/metabolismo , Metformina/farmacologia , Terapia de Imunossupressão
7.
JCI Insight ; 8(20)2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37870961

RESUMO

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.


Assuntos
Carcinoma , Neoplasias Colorretais , Proteínas Proto-Oncogênicas B-raf , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Animais , Humanos , Camundongos , Neoplasias Colorretais/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
8.
Mol Ther ; 31(8): 2507-2523, 2023 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-37143324

RESUMO

Age-related and chemotherapy-induced bone loss depends on cellular senescence and the cell secretory phenotype. However, the factors secreted in the senescent microenvironment that contribute to bone loss remain elusive. Here, we report a central role for the inflammatory alternative complement system in skeletal bone loss. Through transcriptomic analysis of bone samples, we identified complement factor D, a rate-limiting factor of the alternative pathway of complement, which is among the most responsive factors to chemotherapy or estrogen deficiency. We show that osteoblasts and osteocytes are major inducers of complement activation, while monocytes and osteoclasts are their primary targets. Genetic deletion of C5ar1, the receptor of the anaphylatoxin C5a, or treatment with a C5AR1 inhibitor reduced monocyte chemotaxis and osteoclast differentiation. Moreover, genetic deficiency or inhibition of C5AR1 partially prevented bone loss and osteoclastogenesis upon chemotherapy or ovariectomy. Altogether, these lines of evidence support the idea that inhibition of alternative complement pathways may have some therapeutic benefit in osteopenic disorders.


Assuntos
Osteoclastos , Osteogênese , Feminino , Animais , Osteoclastos/metabolismo , Osteogênese/genética , Osteoblastos/metabolismo , Monócitos/metabolismo , Complemento C5a/genética , Complemento C5a/metabolismo
9.
Front Immunol ; 14: 1149747, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37215143

RESUMO

Introduction: Tumor infiltrating lymphocytes (TILs) are known to be a prognostic and predictive biomarker in breast cancer, particularly in triple negative breast cancer (TNBC) patients. International guidelines have been proposed to evaluate them in the clinical setting as a continuous variable, without a clear defined cut-off. However, there are scenarios where the immune infiltration is heterogeneous that some areas of the patient's tumour have high numbers of TILs while other areas completely lack them. This spontaneous presentation of a heterogeneous immune infiltration could be a great opportunity to study why some tumours present TILs at diagnosis but others do not, while eliminating inter patient's differences. Methods: In this study, we have identified five TNBC patients that showed great TIL heterogeneity, with areas of low (≤5%) and high (≥50%) numbers of TILs in their surgical specimens. To evaluate immune infiltration heterogeneity, we performed and analyzed bulk RNA-sequencing in three independent triplicates from the high and low TIL areas of each patient. Results: Gene expression was homogeneous within the triplicates in each area but was remarkable different between TILs regions. These differences were not only due to the presence of TILs as there were other non-inflammatory genes and pathways differentially expressed between the two areas. Discussion: This highlights the importance of intratumour heterogeneity driving the immune infiltration, and not patient's characteristics like the HLA phenotype, germline DNA or immune repertoire.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Prognóstico , Linfócitos do Interstício Tumoral
10.
Cell Rep Med ; 4(4): 101006, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37044092

RESUMO

Elucidating the adaptive mechanisms that prevent host immune response in cancer will help predict efficacy of anti-programmed death-1 (PD1)/L1 therapies. Here, we study the cell-intrinsic response of lung cancer (LC) to interferon-γ (IFNγ), a cytokine that promotes immunoresponse and modulates programmed death-ligand 1 (PD-L1) levels. We report complete refractoriness to IFNγ in a subset of LCs as a result of JAK2 or IFNGR1 inactivation. A submaximal response affects another subset that shows constitutive low levels of IFNγ-stimulated genes (IγSGs) coupled with decreased H3K27ac (histone 3 acetylation at lysine 27) deposition and promoter hypermethylation and reduced IFN regulatory factor 1 (IRF1) recruitment to the DNA on IFNγ stimulation. Most of these are neuroendocrine small cell LCs (SCLCs) with oncogenic MYC/MYCL1/MYCN. The oncogenic activation of MYC in SCLC cells downregulates JAK2 and impairs IγSGs stimulation by IFNγ. MYC amplification tends to associate with a worse response to anti-PD1/L1 therapies. Hence alterations affecting the JAK/STAT pathway and MYC activation prevent stimulation by IFNγ and may predict anti-PD1/L1 efficacy in LC.


Assuntos
Interferon gama , Neoplasias Pulmonares , Humanos , Interferon gama/genética , Transdução de Sinais/genética , Antígeno B7-H1/genética , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo
11.
Cells ; 12(6)2023 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-36980184

RESUMO

Glioblastoma (GBM) is the most frequent primary malignant brain tumor and has a dismal prognosis. Unfortunately, despite the recent revolution of immune checkpoint inhibitors in many solid tumors, these have not shown a benefit in overall survival in GBM patients. Therefore, new potential treatment targets as well as diagnostic, prognostic, and/or predictive biomarkers are needed to improve outcomes in this population. The ß-galactoside binding protein Galectin-1 (Gal-1) is a protein with a wide range of pro-tumor functions such as proliferation, invasion, angiogenesis, and immune suppression. Here, we evaluated Gal-1 expression by immunohistochemistry in a homogenously treated cohort of GBM (the GLIOCAT project) and correlated its expression with clinical and molecular data. We observed that Gal-1 is a negative prognostic factor in GBM. Interestingly, we observed higher levels of Gal-1 expression in the mesenchymal/classical subtypes compared to the less aggressive proneural subtype. We also observed a Gal-1 expression correlation with immune suppressive signatures of CD4 T-cells and macrophages, as well as with several GBM established biomarkers, including SHC1, PD-L1, PAX2, MEOX2, YKL-40, TCIRG1, YWHAG, OLIG2, SOX2, Ki-67, and SOX11. Moreover, Gal-1 levels were significantly lower in grade 4 IDH-1 mutant astrocytomas, which have a better prognosis. Our results confirm the role of Gal-1 as a prognostic factor and also suggest its value as an immune-suppressive biomarker in GBM.


Assuntos
Astrocitoma , Glioblastoma , ATPases Vacuolares Próton-Translocadoras , Humanos , Galectina 1/genética , Galectina 1/metabolismo , Prognóstico , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioblastoma/metabolismo , Astrocitoma/metabolismo , Biomarcadores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas 14-3-3/metabolismo
12.
J Cachexia Sarcopenia Muscle ; 14(2): 964-977, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36860172

RESUMO

BACKGROUND: Inclusion body myositis (IBM) is an inflammatory myopathy clinically characterized by proximal and distal muscle weakness, with inflammatory infiltrates, rimmed vacuoles and mitochondrial changes in muscle histopathology. There is scarce knowledge on IBM aetiology, and non-established biomarkers or effective treatments are available, partly due to the lack of validated disease models. METHODS: We have performed transcriptomics and functional validation of IBM muscle pathological hallmarks in fibroblasts from IBM patients (n = 14) and healthy controls (n = 12), paired by age and sex. The results comprise an mRNA-seq, together with functional inflammatory, autophagy, mitochondrial and metabolic changes between patients and controls. RESULTS: Gene expression profile of IBM vs control fibroblasts revealed 778 differentially expressed genes (P-value adj < 0.05) related to inflammation, mitochondria, cell cycle regulation and metabolism. Functionally, an increased inflammatory profile was observed in IBM fibroblasts with higher supernatant cytokine secretion (three-fold increase). Autophagy was reduced considering basal protein mediators (18.4% reduced), time-course autophagosome formation (LC3BII 39% reduced, P-value < 0.05), and autophagosome microscopic evaluation. Mitochondria displayed reduced genetic content (by 33.9%, P-value < 0.05) and function (30.2%-decrease in respiration, 45.6%-decline in enzymatic activity (P-value < 0.001), 14.3%-higher oxidative stress, 135.2%-increased antioxidant defence (P-value < 0.05), 11.6%-reduced mitochondrial membrane potential (P-value < 0.05) and 42.8%-reduced mitochondrial elongation (P-value < 0.05)). In accordance, at the metabolite level, organic acid showed a 1.8-fold change increase, with conserved amino acid profile. Correlating to disease evolution, oxidative stress and inflammation emerge as potential markers of prognosis. CONCLUSIONS: These findings confirm the presence of molecular disturbances in peripheral tissues from IBM patients and prompt patients' derived fibroblasts as a promising disease model, which may eventually be exported to other neuromuscular disorders. We additionally identify new molecular players in IBM associated with disease progression, setting the path to deepen in disease aetiology, in the identification of novel biomarkers or in the standardization of biomimetic platforms to assay new therapeutic strategies for preclinical studies.


Assuntos
Miosite de Corpos de Inclusão , Miosite , Humanos , Miosite de Corpos de Inclusão/diagnóstico , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/metabolismo , Músculos/metabolismo , Inflamação/patologia , Biomarcadores/metabolismo
13.
Ann Clin Transl Neurol ; 10(3): 408-425, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36651622

RESUMO

OBJECTIVE: Mutations in ANXA11 cause amyotrophic lateral sclerosis (ALS) and have recently been identified as a cause of multisystem proteinopathy and adult-onset muscular dystrophy. These conditions are adult-onset diseases and result from the substitution of Aspartate 40 (Asp40) for an apolar residue in the intrinsically disordered domain (IDD) of ANXA11. Some ALS-related variants are known to affect ANXA11 IDD; however, the mechanism by which the myopathy occurs is unknown. METHODS: Genetic analysis was performed using WES-trio. For the study of variant pathogenicity, we used recombinant proteins, muscle biopsy, and fibroblasts. RESULTS: Here we describe an individual with severe and rapidly progressive childhood-onset oculopharyngeal muscular dystrophy who carries a new ANXA11 variant at position Asp40 (p.Asp40Ile; c.118_119delGAinsAT). p.Asp40Ile is predicted to enhance the aggregation propensity of ANXA11 to a greater extent than other changes affecting this residue. In vitro studies using recombinant ANXA11p.Asp40Ile showed abnormal phase separation and confirmed this variant is more aggregation-prone than the ALS-associated variant ANXA11p.Asp40Gly . The study of the patient's fibroblasts revealed defects in stress granules dynamics and clearance, and muscle histopathology showed a myopathic pattern with ANXA11 protein aggregates. Super-resolution imaging showed aggregates expressed as pearl strips or large complex structures in the sarcoplasm, and as layered subsarcolemmal chains probably reflecting ANXA11 multifunctionality. INTERPRETATION: We demonstrate common pathophysiology for disorders associated with ANXA11 Asp40 allelic variants. Clinical phenotypes may result from different deleterious impacts of variants upon ANXA11 stability against aggregation, and differential muscle or motor neuron dysfunction expressed as a temporal and tissue-specific continuum.


Assuntos
Esclerose Lateral Amiotrófica , Doenças Musculares , Humanos , Esclerose Lateral Amiotrófica/genética , Ácido Aspártico/genética , Neurônios Motores/metabolismo , Doenças Musculares/patologia , Mutação
14.
Brief Bioinform ; 24(1)2023 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-36611239

RESUMO

Analysis of the methylome of tumor cell-free deoxyribonucleic acid (DNA; cfDNA) has emerged as a powerful non-invasive technique for cancer subtyping and prognosis. However, its application is frequently hampered by the quality and total cfDNA yield. Here, we demonstrate the feasibility of very low-input cfDNA for whole-methylome and copy-number profiling studies using enzymatic conversion of unmethylated cysteines [enzymatic methyl-seq (EM-seq)] to better preserve DNA integrity. We created a model for predicting genomic subtyping and prognosis with high accuracy. We validated our tool by comparing whole-genome CpG sequencing with in situ cohorts generated with bisulfite conversion and array hybridization, demonstrating that, despite the different techniques and sample origins, information on cfDNA methylation is comparable with in situ cohorts. Our findings support use of liquid biopsy followed by EM-seq to assess methylome of cancer patients, enabling validation in external cohorts. This advance is particularly relevant for rare cancers like neuroblastomas where liquid-biopsy volume is restricted by ethical regulations in pediatric patients.


Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Criança , Epigenoma , Metilação de DNA , Genômica/métodos , Neoplasias/genética , DNA
15.
J Med Genet ; 60(6): 615-619, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36535754

RESUMO

BACKGROUND: Up to 7% of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) remain genetically undiagnosed after routine genetic testing. These patients are thought to carry deep intronic variants, structural variants or splicing alterations not detected through multiplex ligation-dependent probe amplification or exome sequencing. METHODS: RNA was extracted from seven muscle biopsy samples of patients with genetically undiagnosed DMD/BMD after routine genetic diagnosis. RT-PCR of the DMD gene was performed to detect the presence of alternative transcripts. Droplet digital PCR and whole-genome sequencing were also performed in some patients. RESULTS: We identified an alteration in the mRNA level in all the patients. We detected three pseudoexons in DMD caused by deep intronic variants, two of them not previously reported. We also identified a chromosomal rearrangement between Xp21.2 and 8p22. Furthermore, we detected three exon skipping events with unclear pathogenicity. CONCLUSION: These findings indicate that mRNA analysis of the DMD gene is a valuable tool to reach a precise genetic diagnosis in patients with a clinical and anatomopathological suspicion of dystrophinopathy that remain genetically undiagnosed after routine genetic testing.


Assuntos
Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofina/genética , RNA Mensageiro/genética , Mutação , Reação em Cadeia da Polimerase Multiplex
16.
Cancer Res ; 82(24): 4670-4679, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36222720

RESUMO

Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression. SIGNIFICANCE: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.


Assuntos
Antineoplásicos , Neoplasias da Mama , Imunoconjugados , Feminino , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Catepsina B/metabolismo , Linhagem Celular Tumoral , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais
17.
Int J Mol Sci ; 23(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36232299

RESUMO

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.


Assuntos
Miopatias Mitocondriais , Timidina Quinase/metabolismo , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/patologia , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Succinato Desidrogenase , Timidina Quinase/genética
18.
Sci Rep ; 12(1): 14439, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-36002559

RESUMO

RNA-Sequencing (RNA-Seq) can identify gene fusions in tumors, but not all these fusions have functional consequences. Using multiple data bases, we have performed an in silico analysis of fusions detected by RNA-Seq in tumor samples from 139 newly diagnosed glioblastoma patients to identify in-frame fusions with predictable oncogenic potential. Among 61 samples with fusions, there were 103 different fusions, involving 167 different genes, including 20 known oncogenes or tumor suppressor genes (TSGs), 16 associated with cancer but not oncogenes or TSGs, and 32 not associated with cancer but previously shown to be involved in fusions in gliomas. After selecting in-frame fusions able to produce a protein product and running Oncofuse, we identified 30 fusions with predictable oncogenic potential and classified them into four non-overlapping categories: six previously described in cancer; six involving an oncogene or TSG; four predicted by Oncofuse to have oncogenic potential; and 14 other in-frame fusions. Only 24 patients harbored one or more of these 30 fusions, and only two fusions were present in more than one patient: FGFR3::TACC3 and EGFR::SEPTIN14. This in silico study provides a good starting point for the identification of gene fusions with functional consequences in the pathogenesis or treatment of glioblastoma.


Assuntos
Glioblastoma , Glioma , Carcinogênese , Fusão Gênica , Glioblastoma/patologia , Glioma/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Fusão Oncogênica/genética , RNA-Seq
19.
EMBO Mol Med ; 14(7): e15619, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35695059

RESUMO

Low-flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3caH1047R activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K-driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K-driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human low-flow vascular malformations.


Assuntos
Fosfatidilinositol 3-Quinases , Malformações Vasculares , Aminopiridinas , Animais , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Células Endoteliais/metabolismo , Imidazóis , Camundongos , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Malformações Vasculares/genética , Malformações Vasculares/metabolismo , Malformações Vasculares/patologia
20.
J Invest Dermatol ; 142(5): 1391-1400.e15, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34687742

RESUMO

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLCγ1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLCγ1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKCθ) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKCθ-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKCθ blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKCθ target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKCθ) in 81 human mycosis fungoides samples. In summary, PKCθ can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies.


Assuntos
Linfoma Cutâneo de Células T , Micose Fungoide , Neoplasias Cutâneas , Animais , Embrião de Galinha , Linfoma Cutâneo de Células T/genética , Micose Fungoide/genética , Proteína Quinase C-theta/genética , Proteína Quinase C-theta/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias Cutâneas/genética
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