RESUMO
Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/patologia , COVID-19/virologia , Senescência Celular/efeitos dos fármacos , Terapia de Alvo Molecular , SARS-CoV-2/patogenicidade , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , COVID-19/complicações , Linhagem Celular , Cricetinae , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Quercetina/farmacologia , Quercetina/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Trombose/complicações , Trombose/imunologia , Trombose/metabolismoRESUMO
Aberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor ß (TGF-ß)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-programmed cell death 1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.
Assuntos
Imunidade Adaptativa , Proteínas Adaptadoras de Sinalização CARD/genética , Senescência Celular/fisiologia , Guanilato Ciclase/genética , Linfoma Difuso de Grandes Células B/imunologia , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Neoplasias/genética , Linfócitos T Citotóxicos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígenos CD79/genética , Linhagem Celular Tumoral , Quimiotaxia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Genes myc , Humanos , Inibidores de Checkpoint Imunológico , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/terapia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , NF-kappa B/genética , NF-kappa B/metabolismo , Mutação Puntual , Proteína 2 Ligante de Morte Celular Programada 1/antagonistas & inibidores , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , TranscriptomaRESUMO
Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2-inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células Cultivadas , Técnicas de Cocultura , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaAssuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasia Residual/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Resultado do TratamentoRESUMO
The mechanisms underlying T-ALL relapse remain essentially unknown. Multilevel-omics in 38 matched pairs of initial and relapsed T-ALL revealed 18 (47%) type-1 (defined by being derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). In both types of relapse, we observed known and novel drivers of multidrug resistance including MDR1 and MVP, NT5C2 and JAK-STAT activators. Patients with type-1 relapses were specifically characterized by IL7R upregulation. In remarkable contrast, type-2 relapses demonstrated (1) enrichment of constitutional cancer predisposition gene mutations, (2) divergent genetic and epigenetic remodeling, and (3) enrichment of somatic hypermutator phenotypes, related to BLM, BUB1B/PMS2 and TP53 mutations. T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. In sum, our analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Criança , Evolução Clonal/genética , Humanos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , RecidivaRESUMO
Structural variation (SV), involving deletions, duplications, inversions and translocations of DNA segments, is a major source of genetic variability in somatic cells and can dysregulate cancer-related pathways. However, discovering somatic SVs in single cells has been challenging, with copy-number-neutral and complex variants typically escaping detection. Here we describe single-cell tri-channel processing (scTRIP), a computational framework that integrates read depth, template strand and haplotype phase to comprehensively discover SVs in individual cells. We surveyed SV landscapes of 565 single cells, including transformed epithelial cells and patient-derived leukemic samples, to discover abundant SV classes, including inversions, translocations and complex DNA rearrangements. Analysis of the leukemic samples revealed four times more somatic SVs than cytogenetic karyotyping, submicroscopic copy-number alterations, oncogenic copy-neutral rearrangements and a subclonal chromothripsis event. Advancing current methods, single-cell tri-channel processing can directly measure SV mutational processes in individual cells, such as breakage-fusion-bridge cycles, facilitating studies of clonal evolution, genetic mosaicism and SV formation mechanisms, which could improve disease classification for precision medicine.
Assuntos
Biologia Computacional/métodos , Variação Estrutural do Genoma , Leucemia/genética , Análise de Célula Única/métodos , Linhagem Celular , Cromotripsia , Evolução Clonal , Rearranjo Gênico , Humanos , Mutação INDEL , Inversão de Sequência , Translocação GenéticaRESUMO
We aimed at identifying the developmental stage at which leukemic cells of pediatric T-ALLs are arrested and at defining leukemogenic mechanisms based on ATAC-Seq. Chromatin accessibility maps of seven developmental stages of human healthy T cells revealed progressive chromatin condensation during T-cell maturation. Developmental stages were distinguished by 2,823 signature chromatin regions with 95% accuracy. Open chromatin surrounding SAE1 was identified to best distinguish thymic developmental stages suggesting a potential role of SUMOylation in T-cell development. Deconvolution using signature regions revealed that T-ALLs, including those with mature immunophenotypes, resemble the most immature populations, which was confirmed by TF-binding motif profiles. We integrated ATAC-Seq and RNA-Seq and found DAB1, a gene not related to leukemia previously, to be overexpressed, abnormally spliced and hyper-accessible in T-ALLs. DAB1-negative patients formed a distinct subgroup with particularly immature chromatin profiles and hyper-accessible binding sites for SPI1 (PU.1), a TF crucial for normal T-cell maturation. In conclusion, our analyses of chromatin accessibility and TF-binding motifs showed that pediatric T-ALL cells are most similar to immature thymic precursors, indicating an early developmental arrest.
Assuntos
Células Precursoras de Linfócitos T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Criança , Cromatina , Humanos , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Ligação ProteicaRESUMO
Cancer cells display DNA hypermethylation at specific CpG islands in comparison to their normal healthy counterparts, but the mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. Here, we show that CpG island methylation in human T-cell acute lymphoblastic leukemia (T-ALL) mainly occurs at promoters of Polycomb Repressor Complex 2 (PRC2) target genes that are not expressed in normal or malignant T-cells and which display a reciprocal association with H3K27me3 binding. In addition, we revealed that this aberrant methylation profile reflects the epigenetic history of T-ALL and is established already in pre-leukemic, self-renewing thymocytes that precede T-ALL development. Finally, we unexpectedly uncover that this age-related CpG island hypermethylation signature in T-ALL is completely resistant to the FDA-approved hypomethylating agent Decitabine. Altogether, we here provide conceptual evidence for the involvement of a pre-leukemic phase characterized by self-renewing thymocytes in the pathogenesis of human T-ALL.
Assuntos
Envelhecimento , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Timócitos , Ilhas de CpG/genética , Metilação de DNA/genética , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genéticaRESUMO
We compared 24 primary pediatric T-cell acute lymphoblastic leukemias (T-ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient-derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome-wide chromatin accessibility (ATAC-Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T-ALL-derived PDX models.