RESUMO
TP 53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Apoptose , Células-Tronco/metabolismo , Linhagem Celular TumoralRESUMO
Lung cancer is currently the leading cause of cancer-related deaths worldwide. Significant improvements in lung cancer therapeutics have relied on a better understanding of lung cancer immunity and the development of novel immunotherapies, as best exemplified by the introduction of PD-1/PD-L1-based therapies. However, this improvement is limited to lung cancer patients who respond to anti-PD-1 immunotherapy. Further improvements in immunotherapy may benefit from a better understanding of innate immune response mechanisms in the lung. Toll-like receptors (TLRs) are a key component of the innate immune response and mediate the early recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). TLR signaling modulates the tumor microenvironment from "cold" to "hot" leading to immune sensitization of tumor cells to treatments and improved patient prognosis. In addition, TLR signaling activates the adaptive immune response to improve the response to cancer immunotherapy through the regulation of anti-tumor T cell activity. This review will highlight recent progress in our understanding of the role of TLRs in lung cancer immunity and immunotherapy.
Assuntos
Neoplasias Pulmonares , Receptores Toll-Like , Humanos , Neoplasias Pulmonares/terapia , Imunidade Inata , Imunidade Adaptativa , Fatores Imunológicos , Moléculas com Motivos Associados a Patógenos , Imunoterapia , Microambiente TumoralRESUMO
Ten-eleven Translocation (TET) dioxygenases mediated DNA methylation oxidation plays an important role in regulating the embryonic stem cells (ESCs) differentiation. Herein, we utilized a CRISPR/Cas9 based genome editing method to generate single, double, and triple Tet-deficient mouse ESCs (mESCs) and differentiated these cells toward cardiac progenitors. By using emerald green fluorescent protein (GFP; emGFP) expression under the control of Nkx2.5 promoter as marker for cardiac progenitor cells, we discovered that Tet1 and Tet2 depletion significantly impaired mESC-to-cardiac progenitor differentiation. Single-cell RNA-seq analysis further revealed that Tet deletion resulted in the accumulation of mesoderm progenitors to hamper cardiac differentiation. Re-expression of the Tet1 catalytic domain (Tet1CD) rescued the differentiation defect in Tet-triple knockout mESCs. Dead Cas9 (dCas9)-Tet1CD mediated loci-specific epigenome editing at the Hand1 loci validated the direct involvement of Tet-mediated epigenetic modifications in transcriptional regulation during cardiac differentiation. Our study establishes that Tet-mediated epigenetic remodeling is essential for maintaining proper transcriptional outputs to safeguard mESC-to-cardiac progenitor differentiation.
Assuntos
Células-Tronco Embrionárias Murinas , Proteínas Proto-Oncogênicas , Animais , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Inactivation of tumor-infiltrating lymphocytes (TIL) is one of the mechanisms mitigating antitumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the antitumor activity of TILs with an efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-sequencing analysis suggested that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-sequencing analysis showed that Tet2 ablation reshapes chromatin accessibility and favors binding of transcription factors geared toward CD8+ T-cell activation. Furthermore, the ETS family of transcription factors contributed to augmented CD8+ T-cell function following Tet2 depletion. Overall, our study establishes that Tet2 constitutes one of the epigenetic barriers that account for dysfunction of TILs and that Tet2 inactivation could promote antitumor immunity to suppress tumor growth. SIGNIFICANCE: This study suggests that ablation of TET2+ from TILs could promote their antitumor function by reshaping chromatin accessibility for key transcription factors and enhancing the transcription of genes essential for antitumor activity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA/deficiência , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Proteínas Proto-Oncogênicas/deficiência , Transferência Adotiva/métodos , Animais , Cromatina/metabolismo , Desmetilação do DNA , Proteínas de Ligação a DNA/genética , Dioxigenases , Modelos Animais de Doenças , Epigênese Genética , Deleção de Genes , Inativação Gênica , Inibidores de Checkpoint Imunológico/uso terapêutico , MAP Quinase Quinase Quinases , Melanoma Experimental/metabolismo , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Perforina/metabolismo , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A non-immunogenic tumor microenvironment (TME) is a significant barrier to immune checkpoint blockade (ICB) response. The impact of Polybromo-1 (PBRM1) on TME and response to ICB in renal cell carcinoma (RCC) remains to be resolved. Here we show that PBRM1/Pbrm1 deficiency reduces the binding of brahma-related gene 1 (BRG1) to the IFNγ receptor 2 (Ifngr2) promoter, decreasing STAT1 phosphorylation and the subsequent expression of IFNγ target genes. An analysis of 3 independent patient cohorts and of murine pre-clinical models reveals that PBRM1 loss is associated with a less immunogenic TME and upregulated angiogenesis. Pbrm1 deficient Renca subcutaneous tumors in mice are more resistance to ICB, and a retrospective analysis of the IMmotion150 RCC study also suggests that PBRM1 mutation reduces benefit from ICB. Our study sheds light on the influence of PBRM1 mutations on IFNγ-STAT1 signaling and TME, and can inform additional preclinical and clinical studies in RCC.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/microbiologia , Fatores de Transcrição/metabolismo , Animais , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Carcinoma de Células Renais/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutação , Fosforilação , Fator de Transcrição STAT1/metabolismo , Análise Serial de Tecidos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of ß-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of ß-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and ß-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.
Assuntos
Neoplasias Colorretais/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus gallolyticus subspecies gallolyticus/fisiologia , Animais , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Humanos , Camundongos , Transdução de Sinais , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The eosinophil is a multifunctional granulocyte best known for providing host defense against parasites. Paradoxically, eosinophils are also implicated in the pathogenesis of allergic inflammation, asthma, and hypereosinophilic syndromes. Emerging evidence also supports the potential for harnessing the cytotoxic power of eosinophils and redirecting it to kill solid tumors. Central to eosinophil physiology is interleukin-5 (IL-5) and its receptor (IL-5R) which is composed of a ligand-specific alpha chain (IL-5Rα) and the common beta chain (ßc). Eosinophil activation can lead to their degranulation, resulting in rapid release of an arsenal of tissue-destructive proinflammatory mediators and cytotoxic proteins that can be both beneficial and detrimental to the host. This review discusses eosinophil immunobiology and therapeutic strategies for targeting of IL-5 and IL-5R, as well as the potential for harnessing eosinophil cytotoxicity as a tumoricide.
Assuntos
Citotoxicidade Imunológica , Eosinófilos/imunologia , Síndrome Hipereosinofílica/tratamento farmacológico , Hipersensibilidade/tratamento farmacológico , Interleucina-5/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Receptores de Interleucina-5/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/tratamento farmacológico , Síndrome de Churg-Strauss/tratamento farmacológico , Ensaios Clínicos como Assunto , Dermatite Atópica/tratamento farmacológico , Humanos , Síndrome Hipereosinofílica/imunologia , Hipersensibilidade/imunologia , Inflamação/tratamento farmacológico , Pólipos Nasais/tratamento farmacológico , Neoplasias/imunologia , Oligonucleotídeos Fosforotioatos/uso terapêuticoRESUMO
Pulmonary alveolar proteinosis (PAP) is a rare lung disorder in which surfactant-derived lipoproteins accumulate excessively within pulmonary alveoli, causing severe respiratory distress. The importance of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of PAP has been confirmed in humans and mice, wherein GM-CSF signaling is required for pulmonary alveolar macrophage catabolism of surfactant. PAP is caused by disruption of GM-CSF signaling in these cells, and is usually caused by neutralizing autoantibodies to GM-CSF or is secondary to other underlying diseases. Rarely, genetic defects in surfactant proteins or the common beta chain for the GM-CSF receptor (GM-CSFR) are causal. Using a combination of cellular, molecular, and genomic approaches, we provide the first evidence that PAP can result from a genetic deficiency of the GM-CSFR alpha chain, encoded in the X-chromosome pseudoautosomal region 1.
Assuntos
Cromossomos Humanos X/genética , Proteinose Alveolar Pulmonar/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Animais , Antígeno CD11b/metabolismo , Pré-Escolar , Éxons , Feminino , Genótipo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Camundongos , Monócitos/citologia , Monócitos/metabolismo , Surfactantes Pulmonares/metabolismo , Transdução de Sinais/fisiologia , Síndrome de TurnerRESUMO
Eosinophils are critically dependent on IL-5 for their activation, differentiation, survival, and augmentation of cytotoxic activity. We previously showed that the cytoplasmic domain of the hematopoietic receptor, betac, which is shared by IL-5, IL-3, and GM-CSF, is directly ubiquitinated and degraded by the proteasomes in a JAK2-dependent manner. However, studies describing the spatial distribution, endocytic regulation, and trafficking of betac-sharing receptors in human eosinophils are currently lacking. Using deconvolution microscopy and biochemical methods, we clearly demonstrate that IL-5Rs reside in and are internalized by clathrin- and lipid raft-dependent endocytic pathways. Microscopy analyses in TF1 cells and human eosinophils revealed significant colocalization of betac, IL-5Ralpha, and Cy3-labeled IL-5 with transferrin- (clathrin) and cholera toxin-B- (lipid raft) positive vesicles. Moreover, whereas internalized IL-5Rs were detected in both clathrin- and lipid raft-positive vesicles, biochemical data revealed that tyrosine phosphorylated, ubiquitinated, and proteasome-degraded IL-5Rs partitioned to the soluble, nonraft fractions (clathrin-containing). Lastly, we show that optimal IL-5-induced signaling requires entry of activated IL-5Rs into the intracellular compartment, as coimmunoprecipitation of key signaling molecules with the IL-5R was completely blocked when either endocytic pathway was inhibited. These data provide the first evidence that IL-5Rs segregate and traffic into two distinct plasma membrane compartments, and they further establish that IL-5R endocytosis regulates signaling both positively and negatively.
Assuntos
Endocitose/fisiologia , Eosinófilos/fisiologia , Receptores de Interleucina-5/fisiologia , Transferrina/fisiologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Toxina da Cólera/farmacologia , Clatrina/fisiologia , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Humanos , Interleucina-5/farmacologia , Leucemia Eritroblástica Aguda , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transferrina/farmacologiaRESUMO
IL-5, IL-3, and GM-CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific alpha chain and a shared signaling chain, betac. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the betac-sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL-5 receptor (IL-5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for betac ubiquitination and proteasome degradation but only partially required for IL-5R internalization. Furthermore, we demonstrate the direct ubiquitination of the betac cytoplasmic domain and identify lysine residues 566 and 603 as sites of betac ubiquitination. Lastly, we show that ubiquitination of the betac cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL-5R internalization. We propose an updated working model of IL-5R down-regulation, whereby IL-5 ligation of its receptor activates JAK2/1 kinases, resulting in betac tyrosine phosphorylation, ubiquitination, and IL-5R internalization. Once inside the cell, proteasomes degrade the betac cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL-5R down-regulation.
Assuntos
Regulação para Baixo , Janus Quinases/metabolismo , Receptores de Interleucina-5/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular , Células Cultivadas , Citoplasma/metabolismo , Endocitose , Inibidores Enzimáticos/farmacologia , Eosinófilos/enzimologia , Eosinófilos/metabolismo , Eosinófilos/fisiologia , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/fisiologia , Lisina/química , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Tirfostinas/farmacologiaRESUMO
IL-3, IL-5, and GM-CSF are related hematopoietic cytoines that are important for allergic inflammation. The receptors for human IL-5, IL-3, and GM-CSF are members of the hematopoietin receptor superfamily and are comprised of a cytokine-specific alpha chain and the common beta chain that is shared among these cytokines for signaling. Each of these cytokines contributes to the differentiation and function of leukocyte subpopulations and have clinical importance in protective immunity and in the pathophysiology of a spectrum of immunologic diseases that are as diverse as allergy and asthma, pulmonary alveolar proteinosis, neurodegenerative diseases, and malignancies. Delineating the biology of these cytokines is enabling the development of new strategies for diagnosing and treating these diseases and modulating immune responses.