RESUMO
Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.
Assuntos
Antígeno B7-H1 , Vesículas Extracelulares , Linfoma de Células B , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Vesículas Extracelulares/metabolismo , Linfoma/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Macrófagos/metabolismo , Camundongos , Neoplasias/metabolismoRESUMO
Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.
Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptor Notch1/fisiologia , Animais , Evolução Clonal , Progressão da Doença , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Genes p53 , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Linfócitos do Interstício Tumoral/imunologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Fosfoproteínas/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/fisiologia , Transcriptoma , Microambiente Tumoral , Proteína Supressora de Tumor p53/fisiologia , Regulação para CimaRESUMO
T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic mature T-cell malignancy. It typically presents with exponentially rising lymphocyte counts, splenomegaly, and bone marrow infiltration. Effective treatment options are scarce and a better understanding of TPLL's pathogenesis is desirable. Activation of the TCL1 proto-oncogene and loss-of-function perturbations of the tumor suppressor ATM are TPLL's genomic hallmarks. The leukemic cell reveals a phenotype of active T-cell receptor (TCR) signaling and aberrant DNA damage responses. Regulatory networks based on the profile of microRNA (miR) have not been described for T-PLL. In a combined approach of small-RNA and transcriptome sequencing in 46 clinically and moleculary well-characterized T-PLL, we identified a global T-PLL-specific miR expression profile that involves 34 significantly deregulated miR species. This pattern strikingly resembled miR-ome signatures of TCR-activated T cells. By integrating these T-PLL miR profiles with transcriptome data, we uncovered regulatory networks associated with cell survival signaling and DNA damage response pathways. Despite a miR-ome that discerned leukemic from normal T cells, there were also robust subsets of T-PLL defined by a small set of specific miR. Most prominently, miR-141 and the miR- 200c-cluster separated cases into two major subgroups. Furthermore, increased expression of miR-223-3p as well as reduced expression of miR-21 and the miR-29 cluster were associated with more activated Tcell phenotypes and more aggressive disease presentations. Based on the implicated pathobiological role of these miR deregulations, targeting strategies around their effectors appear worth pursuing. We also established a combinatorial miR-based overall survival score for T-PLL (miROS-T-PLL), that might improve current clinical stratifications.
Assuntos
Leucemia Prolinfocítica de Células T , MicroRNAs , Dano ao DNA , Humanos , Leucemia Prolinfocítica de Células T/tratamento farmacológico , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/patologia , Ativação Linfocitária , MicroRNAs/genética , Linfócitos TRESUMO
The maize smut fungus Ustilago maydis is a model organism for elucidating host colonization strategies of biotrophic fungi. Here, we performed an in depth transcriptional profiling of the entire plant-associated development of U. maydis wild-type strains. In our analysis, we focused on fungal metabolism, nutritional strategies, secreted effectors, and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to stages on the plant surface, establishment of biotrophy, and induction of tumors. These modules are likely the key determinants for U. maydis virulence. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We show that oligopeptide transporters likely involved in nitrogen assimilation are important virulence factors. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. We demonstrate a crucial role for leaf tumor formation and effector gene expression for one of these transcription factors.
Assuntos
Proteínas Fúngicas/genética , Doenças das Plantas/microbiologia , Transcriptoma , Ustilago/genética , Fatores de Virulência/genética , Zea mays/microbiologia , Biomassa , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Nitrogênio/metabolismo , Tumores de Planta/microbiologia , Análise de Sequência de RNA , Fatores de Transcrição/genética , Ustilago/crescimento & desenvolvimento , Ustilago/patogenicidade , Ustilago/fisiologia , Virulência/genéticaRESUMO
T-cell prolymphocytic leukemia (T-PLL) is an aggressive tumor with leukemic presentation of mature T-lymphocytes. Here, we aimed at characterizing the initial events in the molecular pathogenesis of T-PLL and particularly, at determining the point in T-cell differentiation when the hallmark oncogenic events, that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) occur. To this end, we mined whole genome and transcriptome sequencing data of 17 and 11 T-PLL cases, respectively. Mapping of the 14q32.1 locus breakpoints identified only TCL1A, which was moreover significantly overexpressed in T-PLL as compared to benign CD4+ and CD8+ T-cells, as the only common oncogenic target of aberrations. In cases with t(14;14), the breakpoints mapped telomeric and in cases with inv(14) centromeric or in the 3'-untranslated region of TCL1A. Regarding the T-cell receptor alpha (TRA) locus-TCL1A breakpoint junctions, all 17 breakpoints involved recombination signal sequences and 15 junctions contained nontemplated (N-) nucleotides. All T-PLL cases studied carried in-frame TRA rearrangements on the intact allele, which skewed significantly toward usage of distal/central TRAV/TRAJ gene segments as compared to the illegitimate TRA rearrangements. Our findings suggest that the oncogenic TRA-TCL1A/MTCP1 rearrangements in T-PLL occur during opening of the TRA locus, that is, during the progression from CD4+ immature single positive to early double positive thymocyte stage, just before physiologic TCL1A expression is silenced. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Thereafter, it switches off RAG and DNTT expression in line with the mature T-cell phenotype at presentation of T-PLL.
Assuntos
Rearranjo Gênico , Predisposição Genética para Doença , Leucemia Prolinfocítica de Células T/genética , Receptores de Antígenos de Linfócitos T/genética , Transcriptoma , Sequenciamento Completo do Genoma , Alelos , Aberrações Cromossômicas , Estudo de Associação Genômica Ampla , Humanos , Leucemia Prolinfocítica de Células T/diagnóstico , Proteínas de Fusão Oncogênica/genética , FenótipoRESUMO
RHOA is a member of the Rho family of GTPases that are involved in fundamental cellular processes including cell adhesion, migration, and proliferation. RHOA can stimulate the formation of stress fibers and focal adhesions and is a key regulator of actomyosin dynamics in various tissues. In a Genematcher-facilitated collaboration, we were able to identify four unrelated individuals with a specific phenotype characterized by hypopigmented areas of the skin, dental anomalies, body asymmetry, and limb length discrepancy due to hemihypotrophy of one half of the body, as well as brain magnetic resonance imaging (MRI) anomalies. Using whole-exome and ultra-deep amplicon sequencing and comparing genomic data of affected and unaffected areas of the skin, we discovered that all four individuals carried the identical RHOA missense variant, c.139G>A; p.Glu47Lys, in a postzygotic state. Molecular modeling and in silico analysis of the affected p.Glu47Lys residue in RHOA indicated that this exchange is predicted to specifically alter the interaction of RHOA with its downstream effectors containing a PKN-type binding domain and thereby disrupts its ability to activate signaling. Our findings indicate that the recurrent postzygotic RHOA missense variant p.Glu47Lys causes a specific mosaic disorder in humans.
Assuntos
Alelos , Códon , Estudos de Associação Genética , Variação Genética , Placa Neural/metabolismo , Fenótipo , Proteína rhoA de Ligação ao GTP/genética , Adolescente , Adulto , Encéfalo/anormalidades , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Humanos , Imageamento por Ressonância Magnética , Modelos Moleculares , Placa Neural/anormalidades , Placa Neural/embriologia , Conformação Proteica , Relação Estrutura-Atividade , Adulto Jovem , Proteína rhoA de Ligação ao GTP/químicaRESUMO
Patient-derived xenograft (PDX) models have become an important asset in translational cancer research. However, to provide a robust preclinical platform, PDXs need to accommodate the tumor heterogeneity that is observed in patients. Colorectal cancer (CRC) can be stratified into four consensus molecular subtypes (CMS) with distinct biological and clinical features. Surprisingly, using a set of CRC patients, we revealed the partial representation of tumor heterogeneity in PDX models. The epithelial subtypes, the largest subgroups of CRC subtype, were very ineffective in establishing PDXs, indicating the need for further optimization to develop an effective personalized therapeutic approach to CRC. Moreover, we showed that tumor cell proliferation was associated with successful PDX establishment and able to distinguish patient with poor clinical outcomes within CMS2 group.
Assuntos
Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Xenoenxertos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Humanos , CamundongosRESUMO
Preclinical studies have suggested that platelets influence the host response during sepsis. We sought to assess the association of admission thrombocytopenia with the presentation, outcome, and host response in patients with sepsis. Nine hundred thirty-one consecutive sepsis patients were stratified according to platelet counts (very low <50 × 10(9)/L, intermediate-low 50 × 10(9) to 99 × 10(9)/L, low 100 × 10(9) to 149 × 10(9)/L, or normal 150 × 10(9) to 399 × 10(9)/L) on admission to the intensive care unit. Sepsis patients with platelet counts <50 × 10(9)/L and 50 × 10(9) to 99 × 10(9)/L presented with higher Acute Physiology and Chronic Health Evaluation scores and more shock. Both levels of thrombocytopenia were independently associated with increased 30-day mortality (hazard ratios with 95% confidence intervals 2.00 [1.32-3.05] and 1.72 [1.22-2.44], respectively). To account for baseline differences besides platelet counts, propensity matching was performed, after which the association between thrombocytopenia and the host response was tested, as evaluated by measuring 17 plasma biomarkers indicative of activation and/or dysregulation of pathways implicated in sepsis pathogenesis and by whole genome blood leukocyte expression profiling. In the propensity matched cohort, platelet counts < 50 × 10(9)/L were associated with increased cytokine levels and enhanced endothelial cell activation. All thrombocytopenic groups showed evidence of impaired vascular integrity, whereas coagulation activation was similar between groups. Blood microarray analysis revealed a distinct gene expression pattern in sepsis patients with <50 × 10(9)/L platelets, showing reduced signaling in leukocyte adhesion and diapedesis and increased complement signaling. These data show that admission thrombocytopenia is associated with enhanced mortality and a more disturbed host response during sepsis independent of disease severity, thereby providing clinical validity to animal studies on the role of platelets in severe infection.
Assuntos
Estado Terminal , Citocinas/sangue , Sepse/sangue , Sepse/complicações , Trombocitopenia/complicações , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Estado Terminal/mortalidade , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Prognóstico , Sepse/diagnóstico , Sepse/mortalidade , Análise de Sobrevida , Trombocitopenia/sangue , Trombocitopenia/diagnóstico , Trombocitopenia/mortalidadeRESUMO
OBJECTIVE: To determine the association of gender with the presentation, outcome, and host response in critically ill patients with sepsis. DESIGN AND SETTING: A prospective observational cohort study in the ICU of two tertiary hospitals between January 2011 and January 2014. PATIENTS: All consecutive critically ill patients admitted with sepsis, involving 1,815 admissions (1,533 patients). INTERVENTIONS: The host response was evaluated on ICU admission by measuring 19 plasma biomarkers reflecting organ systems implicated in sepsis pathogenesis (1,205 admissions) and by applying genome-wide blood gene expression profiling (582 admissions). MEASUREMENTS AND MAIN RESULTS: Sepsis patients admitted to the ICU were more frequently males (61.0%; p < 0.0001 vs females). Baseline characteristics were not different between genders. Urosepsis was more common in females; endocarditis and mediastinitis in men. Disease severity was similar throughout ICU stay. Mortality was similar up to 1 year after ICU admission, and gender was not associated with 90-day mortality in multivariate analyses in a variety of subgroups. Although plasma proteome analyses (including systemic inflammatory and cytokine responses, and activation of coagulation) were largely similar between genders, females showed enhanced endothelial cell activation; this difference was virtually absent in patients more than 55 years old. More than 80% of the leukocyte blood gene expression response was similar in male and female patients. CONCLUSIONS: The host response and outcome in male and female sepsis patients requiring ICU admission are largely similar.
Assuntos
Estado Terminal , Mediadores da Inflamação/imunologia , Sepse/imunologia , Adulto , Fatores Etários , Idoso , Biomarcadores , Citocinas/imunologia , Células Endoteliais/metabolismo , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Escores de Disfunção Orgânica , Estudos Prospectivos , Sepse/sangue , Sepse/mortalidade , Fatores Sexuais , Centros de Atenção TerciáriaRESUMO
RATIONALE: Preclinical studies suggest that hospitalized patients are susceptible to infections caused by nosocomial respiratory pathogens at least in part because of immune suppression caused by the condition for which they were admitted. OBJECTIVES: We aimed to characterize the systemic host response in hospital-acquired pneumonia (HAP) when compared with community-acquired pneumonia (CAP). METHODS: We performed a prospective study in two intensive care units (ICUs) in 453 patients with HAP (n = 222) or CAP (n = 231). Immune responses were determined on ICU admission by measuring 19 plasma biomarkers reflecting organ systems implicated in infection pathogenesis (in 192 patients with HAP and 183 patients with CAP) and by applying genome-wide blood gene expression profiling (in 111 patients with HAP and 110 patients with CAP). MEASUREMENTS AND MAIN RESULTS: Patients with HAP and CAP presented with similar disease severities and mortality rates did not differ up to 1 year after admission. Plasma proteome analysis revealed largely similar responses, including systemic inflammatory and cytokine responses, and activation of coagulation and the vascular endothelium. The blood leukocyte genomic response was greater than 75% common in patients with HAP and CAP, comprising proinflammatory, antiinflammatory, T-cell signaling, and metabolic pathway gene sets. Patients with HAP showed overexpression of genes involved in cell-cell junction remodeling, adhesion, and diapedesis, which corresponded with lower plasma levels of matrix metalloproteinase-8 and soluble E-selectin. In addition, patients with HAP demonstrated underexpression of a type-I interferon signaling gene signature. CONCLUSIONS: Patients with HAP and CAP present with a largely similar host response at ICU admission.
Assuntos
Infecções Comunitárias Adquiridas/imunologia , Infecção Hospitalar/imunologia , Pneumonia/imunologia , Estado Terminal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
RATIONALE: Community-acquired pneumonia (CAP) accounts for a major proportion of intensive care unit (ICU) admissions for respiratory failure and sepsis. Diagnostic uncertainty complicates case management, which may delay appropriate cause-specific treatment. OBJECTIVES: To characterize the blood genomic response in patients with suspected CAP and identify a candidate biomarker for the rapid diagnosis of CAP on ICU admission. METHODS: The study comprised two cohorts of consecutively enrolled patients treated for suspected CAP on ICU admission. Patients were designated CAP (cases) and no-CAP patients (control subjects) by post hoc assessment. The first (discovery) cohort (101 CAP and 33 no-CAP patients) was enrolled between January 2011 and July 2012; the second (validation) cohort (70 CAP and 30 no-CAP patients) between July 2012 and June 2013. Blood was collected within 24 hours of ICU admission. MEASUREMENTS AND MAIN RESULTS: Blood microarray analysis of CAP and no-CAP patients revealed shared and distinct gene expression patterns. A 78-gene signature was defined for CAP, from which a FAIM3:PLAC8 gene expression ratio was derived with area under curve of 0.845 (95% confidence interval, 0.764-0.917) and positive and negative predictive values of 83% and 81%, respectively. Robustness of the FAIM3:PLAC8 ratio was ascertained by quantitative polymerase chain reaction in the validation cohort. The FAIM3:PLAC8 ratio outperformed plasma procalcitonin and IL-8 and IL-6 in discriminating between CAP and no-CAP patients. CONCLUSIONS: CAP and no-CAP patients presented shared and distinct blood genomic responses. We propose the FAIM3:PLAC8 ratio as a candidate biomarker to assist in the rapid diagnosis of CAP on ICU admission. Clinical trial registered with www.clinicaltrials.gov (NCT 01905033).
Assuntos
Proteínas Reguladoras de Apoptose/genética , Biomarcadores/sangue , Infecções Comunitárias Adquiridas/diagnóstico , Pneumonia/diagnóstico , Proteínas/genética , Idoso , Calcitonina/sangue , Peptídeo Relacionado com Gene de Calcitonina , Feminino , Perfilação da Expressão Gênica , Humanos , Unidades de Terapia Intensiva , Interleucina-6/sangue , Interleucina-8/sangue , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Análise Serial de TecidosRESUMO
BACKGROUND: Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. METHODS: Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. RESULTS: We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. CONCLUSIONS: The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general.
Assuntos
Síndrome de Bartter , Humanos , Haplótipos , Alelos , Genoma Humano , Canais de Cloreto/genéticaRESUMO
T-cell prolymphocytic leukemia (T-PLL) is a chemotherapy-refractory T-cell malignancy with limited therapeutic options and a poor prognosis. Current disease concepts implicate TCL1A oncogene-mediated enhanced T-cell receptor (TCR) signaling and aberrant DNA repair as central perturbed pathways. We discovered that recurrent gains on chromosome 8q more frequently involve the argonaute RISC catalytic component 2 (AGO2) gene than the adjacent MYC locus as the affected minimally amplified genomic region. AGO2 has been understood as a protumorigenic key regulator of miRNA (miR) processing. Here, in primary tumor material and cell line models, AGO2 overrepresentation associated (i) with higher disease burden, (ii) with enhanced in vitro viability and growth of leukemic T cells, and (iii) with miR-omes and transcriptomes that highlight altered survival signaling, abrogated cell-cycle control, and defective DNA damage responses. However, AGO2 elicited also immediate, rather non-RNA-mediated, effects in leukemic T cells. Systems of genetically modulated AGO2 revealed that it enhances TCR signaling, particularly at the level of ZAP70, PLCγ1, and LAT kinase phosphoactivation. In global mass spectrometric analyses, AGO2 interacted with a unique set of partners in a TCR-stimulated context, including the TCR kinases LCK and ZAP70, forming membranous protein complexes. Models of their three-dimensional structure also suggested that AGO2 undergoes posttranscriptional modifications by ZAP70. This novel TCR-associated noncanonical function of AGO2 represents, in addition to TCL1A-mediated TCR signal augmentation, another enhancer mechanism of this important deregulated growth pathway in T-PLL. These findings further emphasize TCR signaling intermediates as candidates for therapeutic targeting. SIGNIFICANCE: The identification of AGO2-mediated activation of oncogenic T cells through signal amplifying protein-protein interactions advances the understanding of leukemogenic AGO2 functions and underlines the role of aberrant TCR signaling in T-PLL.
Assuntos
Leucemia Prolinfocítica de Células T , MicroRNAs , Humanos , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fosforilação , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Linfócitos T/metabolismoRESUMO
The extent of non-coding RNA alterations in patients with sepsis and their relationship to clinical characteristics, soluble mediators of the host response to infection, as well as an advocated in vivo model of acute systemic inflammation is unknown. Here we obtained whole blood from 156 patients with sepsis and 82 healthy subjects among whom eight were challenged with lipopolysaccharide in a clinically controlled setting (human endotoxemia). Via next-generation microarray analysis of leukocyte RNA we found that long non-coding RNA and, to a lesser extent, small non-coding RNA were significantly altered in sepsis relative to health. Long non-coding RNA expression, but not small non-coding RNA, was largely recapitulated in human endotoxemia. Integrating RNA profiles and plasma protein levels revealed known as well as previously unobserved pathways, including non-sensory olfactory receptor activity. We provide a benchmark dissection of the blood leukocyte 'regulome' that can facilitate prioritization of future functional studies.
Assuntos
Estado Terminal , Leucócitos/metabolismo , RNA não Traduzido/metabolismo , Sepse/metabolismo , Idoso , Estudos de Casos e Controles , Endotoxemia/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , TranscriptomaRESUMO
Single-cell transcriptomics have revolutionized our understanding of the cell composition of tumors and allowed us to identify new subtypes of cells. Despite rapid technological advancements, single-cell analysis remains resource-intense hampering the scalability that is required to profile a sufficient number of samples for clinical associations. Therefore, more scalable approaches are needed to understand the contribution of individual cell types to the development and treatment response of solid tumors such as esophageal adenocarcinoma where comprehensive genomic studies have only led to a small number of targeted therapies. Due to the limited treatment options and late diagnosis, esophageal adenocarcinoma has a poor prognosis. Understanding the interaction between and dysfunction of individual cell populations provides an opportunity for the development of new interventions. In an attempt to address the technological and clinical needs, we developed a protocol for the separation of esophageal carcinoma tissue into leukocytes (CD45+), epithelial cells (EpCAM+), and fibroblasts (two out of PDGFRα, CD90, anti-fibroblast) by fluorescence-activated cell sorting and subsequent RNA sequencing. We confirm successful separation of the three cell populations by mapping their transcriptomic profiles to reference cell lineage expression data. Gene-level analysis further supports the isolation of individual cell populations with high expression of CD3, CD4, CD8, CD19, and CD20 for leukocytes, CDH1 and MUC1 for epithelial cells, and FAP, SMA, COL1A1, and COL3A1 for fibroblasts. As a proof of concept, we profiled tumor samples of nine patients and explored expression differences in the three cell populations between tumor and normal tissue. Interestingly, we found that angiogenesis-related genes were upregulated in fibroblasts isolated from tumors compared with normal tissue. Overall, we suggest our protocol as a complementary and more scalable approach compared with single-cell RNA sequencing to investigate associations between clinical parameters and transcriptomic alterations of specific cell populations in esophageal adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Análise de Célula Única , Transcriptoma/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa/patologia , Análise de Componente PrincipalRESUMO
The host immune response is characterized by a complex interplay of signal-specific cellular transcriptional responses. The magnitude of the immune response is dependent on the strength of the external stimulus. Knowledge on leukocyte transcriptional responses altered in response to different stimulus dosages in man is lacking. Here, we sought to identify leukocyte transcriptional signatures dependent on LPS dose in humans. Healthy human volunteers were administered 1 ng/kg (n = 7), 2 ng/kg (n = 6), or 4 ng/kg (n = 7) LPS intravenously. Blood was collected before (pre-LPS) and 4 h after LPS administration. Total RNA was analyzed by microarrays and generalized linear models. Pathway analysis was performed by using Ingenuity pathway analysis. Leukocyte transcriptomes altered per LPS dosage were predominantly shared, with 47% common signatures relative to pre-LPS. A univariate linear model identified a set of 3736 genes that exhibited a dependency on differing LPS dosages. Neutrophil, monocyte, and lymphocyte counts explained 38.9% of the variance in the LPS dose-dependent gene set. A multivariate linear model including leukocyte composition delineated a set of 295 genes with a dependency on LPS dose. Evaluation of the 295 gene signature in patients with sepsis due to abdominal infections showed significant correlations. Promoter regions of the LPS dose gene set were enriched for YY1, EGR1, ELK1, GABPA, KLF4, and REL transcription factor binding sites. Intravenous injection of 1, 2, or 4 ng/kg LPS was accompanied by both shared and distinct leukocyte transcriptional alterations. These data may assist in assessing the severity of the insult in patients with abdominal sepsis.
Assuntos
Endotoxemia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos , Lipopolissacarídeos/toxicidade , Transcrição Gênica/efeitos dos fármacos , Adulto , Relação Dose-Resposta Imunológica , Endotoxemia/induzido quimicamente , Endotoxemia/imunologia , Endotoxemia/patologia , Regulação da Expressão Gênica/imunologia , Humanos , Fator 4 Semelhante a Kruppel , Leucócitos/imunologia , Leucócitos/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica/imunologiaRESUMO
Colorectal cancer (CRC) is a highly heterogeneous disease both from a molecular and clinical perspective. Several distinct molecular entities, such as microsatellite instability (MSI), have been defined that make up biologically distinct subgroups with their own clinical course. Recent data indicated that CRC can be best segregated into four groups called consensus molecular subtypes (CMS1-4), each of which has a unique biology and gene expression pattern. In order to develop improved, subtype-specific therapies and to gain insight into the molecular wiring and origin of these subtypes, reliable models are needed. This study was designed to determine the heterogeneity and identify the presence of CMSs in a large panel of CRC cell lines, primary cultures and patient-derived xenografts (PDX). We provide a repository encompassing this heterogeneity and moreover describe that a large part of the models can be robustly assigned to one of the four CMSs, independent of the stromal contribution. We subsequently validate our CMS stratification by functional analysis which for instance shows mesenchymal enrichment in CMS4 and metabolic dysregulation in CMS3. Finally, we observe a clear difference in sensitivity to chemotherapy-induced apoptosis, specifically between CMS2 and CMS4. This relates to the in vivo efficacy of chemotherapy, which delays outgrowth of CMS2, but not CMS4 xenografts. Combined our data indicate that molecular subtypes are faithfully modelled in CRC cell cultures and PDXs, representing tumour cell intrinsic and stable features. This repository provides researchers with a platform to study CRC using the existing heterogeneity.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Modelos Biológicos , Neoplasias Experimentais/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fluoruracila/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Oxaliplatina/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The heterogeneous nature of colorectal cancer (CRC) complicates prognosis and is suggested to be a determining factor in the efficacy of adjuvant therapy for individual patients. Based on gene expression profiling, CRC is currently classified into four consensus molecular subtypes (CMSs), characterized by specific biological programs, thus suggesting the existence of unifying developmental drivers for each CMS Using human organoid cultures, we investigated the role of such developmental drivers at the premalignant stage of distinct CRC subtypes and found that TGFß plays an important role in the development of the mesenchymal CMS4, which is of special interest due to its association with dismal prognosis. We show that in tubular adenomas (TAs), which progress to classical CRCs, the dominating response to TGFß is death by apoptosis. By contrast, induction of a mesenchymal phenotype upon TGFß treatment prevails in a genetically engineered organoid culture carrying a BRAF(V) (600E) mutation, constituting a model system for sessile serrated adenomas (SSAs). Our data indicate that TGFß signaling is already active in SSA precursor lesions and that TGFß is a critical cue for directing SSAs to the mesenchymal, poor-prognosis CMS4 of CRC.
Assuntos
Adenoma/patologia , Carcinogênese , Neoplasias Colorretais/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Humanos , OrganoidesRESUMO
HIV patients have an increased risk to develop sepsis and HIV infection affects several components of the immune system involved in sepsis pathogenesis. We hypothesized that HIV infection might aggrevate the aberrant immune response during sepsis, so we aimed to determine the impact of HIV infection on the genomic host response to sepsis. We compared whole blood leukocyte gene expression profiles among sepsis patients with or without HIV co-infection in the intensive care unit (ICU) and validated our findings in a cohort of patients admitted to the same ICUs in a different time frame. To examine the influence of HIV infection per se, we also determined the expression of genes of interest in a cohort of asymptomatic HIV patients. We identified a predominantly common host response in sepsis patients with or without HIV co-infection. HIV positive sepsis patients in both ICU cohorts showed overexpression of genes involved in granzyme signaling (GZMA, GZMB), cytotoxic T-cell signaling (CD8A, CD8B) and T-cell inhibitory signaling (LAG3), compared to HIV negative patients. Enhanced expression of CD8A, CD8B and LAG3 was also unmasked in asymptomatic HIV patients. Plasma levels of granzymes in sepsis patients were largely below detection limit, without differences according to HIV status. These results demonstrate that sepsis is characterized by a massive common response with few differences between HIV positive and HIV negative sepsis patients. Observed differences in granzyme signaling, cytotoxic T-cell signaling and T-cell inhibitory signaling appear to be changes commonly observed in asymptomatic HIV patients which persist during sepsis.