RESUMO
Introduction: Humans with gain-of-function (GOF) mutations in STAT1 (Signal Transducer and Activator of Transcription 1), a potent immune regulator, experience frequent infections. About one-third, especially those with DNA-binding domain (DBD) mutations such as T385M, also develop autoimmunity, sometimes accompanied by increases in T-helper 1 (Th1) and T-follicular helper (Tfh) CD4 effector T cells, resembling those that differentiate following infection-induced STAT1 signaling. However, environmental and molecular mechanisms contributing to autoimmunity in STAT1 GOF patients are not defined. Methods: We generated Stat1T385M/+ mutant mice to model the immune impacts of STAT1 DBD GOF under specific-pathogen free (SPF) conditions. Results: Stat1T385M/+ lymphocytes had more total Stat1 at baseline and also higher amounts of IFNg-induced pStat1. Young mutants exhibited expansion of Tfh-like cells, while older mutants developed autoimmunity accompanied by increased Tfh-like cells, B cell activation and germinal center (GC) formation. Mutant females exhibited these immune changes sooner and more robustly than males, identifying significant sex effects of Stat1T385M-induced immune dysregulation. Single cell RNA-Seq (scRNA-Seq) analysis revealed that Stat1T385M activated transcription of GC-associated programs in both B and T cells. However, it had the strongest transcriptional impact on T cells, promoting aberrant CD4 T cell activation and imparting both Tfh-like and Th1-like effector programs. Discussion: Collectively, these data demonstrate that in the absence of overt infection, Stat1T385M disrupted naïve CD4 T cell homeostasis and promoted expansion and differentiation of abnormal Tfh/Th1-like helper and GC-like B cells, eventually leading to sex-biased autoimmunity, suggesting a model for STAT1 GOF-induced immune dysregulation and autoimmune sequelae in humans.
Assuntos
Autoimunidade , Linfócitos T CD4-Positivos , Masculino , Feminino , Humanos , Animais , Camundongos , Autoimunidade/genética , Mutação com Ganho de Função , Mutação , Linfócitos T Auxiliares-Indutores , Fator de Transcrição STAT1/genéticaRESUMO
Mutations in IDH1, IDH2, and TET2 are recurrently observed in myeloid neoplasms. IDH1 and IDH2 encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). Oncogenic IDH1/2 mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms, IDH1, IDH2, and TET2 mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172K and Tet2 loss-of-function have divergent consequences on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in Idh2R172K cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of IDH1/2 and TET2 mutations, providing support for the optimization of genotype-specific therapies.
Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Isocitrato Desidrogenase , Células-Tronco , Animais , Camundongos , Dioxigenases/genética , Proteínas de Ligação a DNA/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mutação , Neoplasias , Células-Tronco/metabolismoRESUMO
Central nervous system (CNS) dissemination of B-precursor acute lymphoblastic leukemia (B-ALL) has poor prognosis and remains a therapeutic challenge. Here we performed targeted DNA sequencing as well as transcriptional and proteomic profiling of paired leukemia-infiltrating cells in the bone marrow (BM) and CNS of xenografts. Genes governing mRNA translation were upregulated in CNS leukemia, and subclonal genetic profiling confirmed this in both BM-concordant and BM-discordant CNS mutational populations. CNS leukemia cells were exquisitely sensitive to the translation inhibitor omacetaxine mepesuccinate, which reduced xenograft leptomeningeal disease burden. Proteomics demonstrated greater abundance of secreted proteins in CNS-infiltrating cells, including complement component 3 (C3), and drug targeting of C3 influenced CNS disease in xenografts. CNS-infiltrating cells also exhibited selection for stemness traits and metabolic reprogramming. Overall, our study identifies targeting of mRNA translation as a potential therapeutic approach for B-ALL leptomeningeal disease. SIGNIFICANCE: Cancer metastases are often driven by distinct subclones with unique biological properties. Here we show that in B-ALL CNS disease, the leptomeningeal environment selects for cells with unique functional dependencies. Pharmacologic inhibition of mRNA translation signaling treats CNS disease and offers a new therapeutic approach for this condition.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Doenças do Sistema Nervoso Central , Neoplasias do Sistema Nervoso Central , Neoplasias Meníngeas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/patologia , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Humanos , Neoplasias Meníngeas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Biossíntese de Proteínas/genética , ProteômicaRESUMO
In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit.
Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Cromatina/metabolismo , Análise por Conglomerados , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Nucleofosmina , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Reprodutibilidade dos Testes , Análise de SobrevidaRESUMO
Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 T cell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 T cell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.
Assuntos
Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Mycobacterium tuberculosis/fisiologia , Neutrófilos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Tuberculose/imunologia , Imunidade Adaptativa , Animais , Doença Crônica , Coinfecção , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Acute lymphoblastic leukemia (ALL) dissemination to the central nervous system (CNS) is a challenging clinical problem whose underlying mechanisms are poorly understood. Here, we show that primary human ALL samples injected into the femora of immunodeficient mice migrate to the skull and vertebral bone marrow and provoke bone lesions that enable passage into the subarachnoid space. Treatment of leukemia xenografted mice with a biologic antagonist of receptor activator of nuclear factor κB ligand (RANKL) blocks this entry route. In addition to erosion of cranial and vertebral bone, samples from individuals with B-ALL also penetrate the blood-cerebrospinal fluid barrier of recipient mice. Co-administration of C-X-C chemokine receptor 4 (CXCR4) and RANKL antagonists attenuate both identified routes of entry. Our findings suggest that targeted RANKL and CXCR4 pathway inhibitors could attenuate routes of leukemia blast CNS invasion and provide benefit for B-ALL-affected individuals.
Assuntos
Sistema Nervoso Central/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Animais , Crise Blástica/patologia , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/metabolismo , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos Endogâmicos NOD , Modelos Biológicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Invasividade Neoplásica , Osteoprotegerina/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras B/líquido cefalorraquidiano , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Ligante RANK/antagonistas & inibidores , Ligante RANK/metabolismo , Receptores CXCR4/metabolismo , Coluna Vertebral/patologia , Espaço Subaracnóideo/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype-phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients. SIGNIFICANCE: Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy.See related commentary by Wisdom and Kirsch p. 5459.
Assuntos
DNA Polimerase II , Neoplasias , Animais , DNA Polimerase II/genética , Humanos , Inibidores de Checkpoint Imunológico , Camundongos , Mutação , Neoplasias/genética , Proteínas de Ligação a Poli-ADP-Ribose/genéticaRESUMO
Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL-driven bone destruction, we examined the p53 -/-; Rag2 -/-; Prkdcscid/scid triple mutant (TM) mice and p53 -/-; Prkdcscid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed Rankl, encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD.Prkdcscid/scidIl2rgtm1Wjl /SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin-Fc (rOPG-Fc) protected the bone from B-ALL-induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL-mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Ligante RANK , Animais , Linfócitos B , Humanos , Camundongos , Camundongos Endogâmicos NOD , OsteoclastosRESUMO
Many pathogens subvert intestinal immunity to persist within the gastrointestinal tract (GIT); yet, the underlying mechanisms that enable sanctuary specifically in this reservoir are unclear. Using mass cytometry and network analysis, we demonstrate that chronic LCMV infection of the GIT leads to dysregulated microbial composition, a cascade of metabolic alterations, increased susceptibility to GI disease, and a system-wide recalibration of immune composition that defines viral persistence. Chronic infection led to outgrowth of activated Tbet-expressing T reg cell populations unique to the GIT and the rapid erosion of pathogen-specific CD8 tissue-resident memory T cells. Mechanistically, T reg cells and coinhibitory receptors maintained long-term viral sanctuary within the GIT, and their targeting reactivated T cells and eliminated this viral reservoir. Thus, our data provide a high-dimensional definition of the mechanisms of immune regulation that chronic viruses implement to exploit the unique microenvironment of the GIT and identify T reg cells as key modulators of viral persistence in the intestinal tract.
Assuntos
Trato Gastrointestinal/imunologia , Trato Gastrointestinal/virologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Animais , Efeito Espectador , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , Colite/complicações , Colite/virologia , Disbiose/complicações , Disbiose/virologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Regulação da Expressão Gênica , Ativação Linfocitária/imunologia , Depleção Linfocítica , Coriomeningite Linfocítica/genética , Camundongos Endogâmicos C57BL , Fenótipo , Linfócitos T Reguladores/imunologia , Transcriptoma/genéticaRESUMO
Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. SIGNIFICANCE: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.
Assuntos
Leucemia Mieloide Aguda/genética , Células Clonais , Feminino , Humanos , Masculino , RecidivaRESUMO
Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials.
RESUMO
Mass cytometers are time-of-flight (TOF) mass spectrometer-coupled flow cytometers (known as CyTOFs) that quantify the abundance of metal-tagged antibodies (Abs) or other cellular probes within single cell suspensions or laser-ablated tissue sections. While many strategies exist for covalently crosslinking to proteins, the Fluidigm MaxPar® process is currently the most widely used and involves first loading a metal-chelating polymer with an elementally and isotopically enriched metal. Once the chelation sites have been filled, a maleimide moiety on the polymer is reacted with the free thiol groups on the partially reduced monoclonal immunoglobulin G (IgG) Ab to form an irreversible covalent bond. Here we describe modifications to the Fluidigm MaxPar® protocol that increase the quantity of Ab per reaction up to 150 µg, introduce an initial Ab quality control step, utilize metal labels not included in the Fluidigm catalog, and provide an option to perform two reactions in one centrifugal filter.
Assuntos
Anticorpos Monoclonais/química , Células/citologia , Citometria de Fluxo/métodos , Imunoconjugados/química , Imunofenotipagem/métodos , Espectrometria de Massas/métodos , Metais/química , Quelantes/química , Humanos , Imunoglobulina G/imunologia , Polímeros/químicaRESUMO
CD8+ T cell exhaustion impedes control of chronic viral infection; yet how new T cell responses are mounted during chronic infection is unclear. Unlike T cells primed at the onset of infection that rapidly differentiate into effectors and exhaust, we demonstrate that virus-specific CD8+ T cells primed after establishment of chronic LCMV infection preferentially generate memory-like transcription factor TCF1+ cells that were transcriptionally and proteomically distinct, less exhausted, and more responsive to immunotherapy. Mechanistically, adaptations of antigen-presenting cells and diminished T cell signaling intensity promoted differentiation of the memory-like subset at the expense of rapid effector cell differentiation, which was now highly dependent on IL-21-mediated CD4+ T cell help for its functional generation. Chronic viral infection similarly redirected de novo differentiation of tumor-specific CD8+ T cells, ultimately preventing cancer control. Thus, targeting these T cell stimulatory pathways could enable strategies to control chronic infection, tumors, and enhance immunotherapeutic efficacy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Imunidade/imunologia , Memória Imunológica/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Diferenciação Celular/genética , Doença Crônica , Perfilação da Expressão Gênica/métodos , Imunidade/genética , Memória Imunológica/genética , Imunoterapia , Coriomeningite Linfocítica/terapia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Proteômica/métodos , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/imunologia , Fator 1 de Transcrição de Linfócitos T/metabolismoRESUMO
CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.
Assuntos
Caderinas/fisiologia , Carcinoma Lobular/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/patologia , Mutação , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma Lobular/imunologia , Carcinoma Lobular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Invasividade Neoplásica , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Transcriptoma , Proteínas de Sinalização YAP , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Purpose: Regulatory T (Treg) cells expressing the transcription factor FOXP3 are essential for the maintenance of immunologic self-tolerance but play a detrimental role in most cancers due to their ability to suppress antitumor immunity. The phenotype of human circulating Treg cells has been extensively studied, but less is known about tumor-infiltrating Treg cells. We studied the phenotype and function of tumor-infiltrating Treg cells in ovarian cancer and melanoma to identify potential Treg cell-associated molecules that can be targeted by tumor immunotherapies.Experimental Design: The phenotype of intratumoral and circulating Treg cells was analyzed by multicolor flow cytometry, mass cytometry, RNA-seq, and functional assays.Results: Treg cells isolated from ovarian tumors displayed a distinct cell surface phenotype with increased expression of a number of receptors associated with TCR engagement, including PD-1, 4-1BB, and ICOS. Higher PD-1 and 4-1BB expression was associated with increased responsiveness to further TCR stimulation and increased suppressive capacity, respectively. Transcriptomic and mass cytometry analyses revealed the presence of Treg cell subpopulations and further supported a highly activated state specifically in ovarian tumors. In comparison, Treg cells infiltrating melanomas displayed lower FOXP3, PD-1, 4-1BB, and ICOS expression and were less potent suppressors of CD8 T-cell proliferation.Conclusions: The highly activated phenotype of ovarian tumor-infiltrating Treg cells may be a key component of an immunosuppressive tumor microenvironment. Receptors that are expressed by tumor-infiltrating Treg cells could be exploited for the design of novel combination tumor immunotherapies. Clin Cancer Res; 24(22); 5685-96. ©2018 AACR.
Assuntos
Ativação Linfocitária/imunologia , Melanoma/imunologia , Neoplasias Ovarianas/imunologia , Linfócitos T Reguladores/imunologia , Biomarcadores , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Melanoma/genética , Neoplasias Ovarianas/genética , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , TranscriptomaRESUMO
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4wt overexpression as murine myoblasts expressing FGFR4V550E undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4V550E were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4V550E tumor-derived cell and human RMS cell viability. FGFR4V550E-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC50 values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/administração & dosagem , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Rabdomiossarcoma/tratamento farmacológico , Sulfonamidas/administração & dosagem , Animais , Área Sob a Curva , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Mutação , Transplante de Neoplasias , Fosforilação/efeitos dos fármacos , Piridazinas , Quinolinas/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Interleukin-7 receptor (IL-7R) signaling is critical for multiple stages of T-cell development, but a role in the establishment of the mature thymic architecture needed for T-cell development and thymocyte selection has not been established. Crosstalk signals between developing thymocytes and thymic epithelial cell (TEC) precursors are critical for their differentiation into cortical TECs (cTECs) and medullary TECs (mTECs). In addition, mTEC-derived factors have been implicated in the recruitment of thymic dendritic cells (DCs) and intrathymic DC development. We therefore examined corticomedullary structure and DC populations in the thymus of Il7r-/- mice. Analysis of TEC phenotype and spatial organization revealed a striking shift in the mTEC to cTEC ratio, accompanied by disorganized corticomedullary structure. Several of the thymic subsets known to have DC potential were nearly absent, accompanied by reductions in DC cell numbers. We also examined chemokine expression in the Il7r-/- thymus, and found a significant decrease in mTEC-derived CCR7 ligand expression, and high levels of cTEC-derived chemokines, including CCL25 and CXCL12. Although splenic DCs were similarly affected, bone marrow (BM) precursors capable of giving rise to DCs were unperturbed. Finally, BM chimeras showed that there was no intrinsic need for IL-7R signaling in the development or recruitment of thymic DCs, but that the provision of wild-type progenitors enhanced reconstitution of thymic DCs from Il7r-/- progenitors. Our results are therefore supportive of a model in which Il7r-dependent cells are required to set up the microenvironments that allow accumulation of thymic DCs.
Assuntos
Células Dendríticas/fisiologia , Células Epiteliais/fisiologia , Receptores de Interleucina-7/metabolismo , Linfócitos T/fisiologia , Timo/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Microambiente Celular , Quimiocina CXCL12/metabolismo , Quimiocinas CC/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR7/metabolismo , Receptores de Interleucina-7/genéticaRESUMO
In this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPA(bi)) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Resistencia a Medicamentos Antineoplásicos/genética , Janus Quinases/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fator Estimulador de Colônias/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Medicina de Precisão , Transcriptoma , Células Tumorais Cultivadas , Adulto JovemRESUMO
Acquisition of the arterial and haemogenic endothelium fates concurrently occur in the aorta-gonad-mesonephros (AGM) region prior to haematopoietic stem cell (HSC) generation. The arterial programme depends on Dll4 and the haemogenic endothelium/HSC on Jag1-mediated Notch1 signalling. How Notch1 distinguishes and executes these different programmes in response to particular ligands is poorly understood. By using two Notch1 activation trap mouse models with different sensitivity, here we show that arterial endothelial cells and HSCs originate from distinct precursors, characterized by different Notch1 signal strengths. Microarray analysis on AGM subpopulations demonstrates that the Jag1 ligand stimulates low Notch strength, inhibits the endothelial programme and is permissive for HSC specification. In the absence of Jag1, endothelial cells experience high Dll4-induced Notch activity and select the endothelial programme, thus precluding HSC formation. Interference with the Dll4 signal by ligand-specific blocking antibodies is sufficient to inhibit the endothelial programme and favour specification of the haematopoietic lineage.