RESUMO
The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8+ T cells in the tumor microenvironment can be significantly enhanced.
Assuntos
Vacinas Anticâncer/química , Peptídeos/metabolismo , Células A549 , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Chlorocebus aethiops , Herpesvirus Humano 1/metabolismo , Humanos , Melanoma/imunologia , Melanoma/terapia , Camundongos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Peptídeos/imunologia , Vaccinia virus/metabolismo , Células Vero , Proteínas do Envelope Viral/metabolismoRESUMO
Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 8/genética , Estresse Fisiológico/genética , Replicação Viral , Linhagem Celular Tumoral , Replicação do DNA , Humanos , RNA Interferente Pequeno/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Ativação Viral/fisiologia , Latência Viral/genética , Replicação Viral/genéticaRESUMO
The successful use of expanded tumor-infiltrating lymphocytes (TIL) in adoptive TIL therapies has been reported, but the effects of the TIL expansion, immunophenotype, function, and T cell receptor (TCR) repertoire of the infused products relative to the tumor microenvironment (TME) are not well understood. In this study, we analyzed the tumor samples (n = 58) from treatment-naïve patients with renal cell carcinoma (RCC), "pre-rapidly expanded" TILs (pre-REP TIL, n = 15) and "rapidly expanded" TILs (REP TIL, n = 25) according to a clinical-grade TIL production protocol, with single-cell RNA (scRNA)+TCRαß-seq (TCRαß sequencing), TCRß-sequencing (TCRß-seq), and flow cytometry. REP TILs encompassed a greater abundance of CD4+ than CD8+ T cells, with increased LAG-3 and low PD-1 expressions in both CD4+ and CD8+ T cell compartments compared with the pre-REP TIL and tumor T cells. The REP protocol preferentially expanded small clones of the CD4+ phenotype (CD4, IL7R, KLRB1) in the TME, indicating that the largest exhausted T cell clones in the tumor do not expand during the expansion protocol. In addition, by generating a catalog of RCC-associated TCR motifs from >1,000 scRNA+TCRαß-seq and TCRß-seq RCC, healthy and other cancer sample cohorts, we quantified the RCC-associated TCRs from the expansion protocol. Unlike the low-remaining amount of anti-viral TCRs throughout the expansion, the quantity of the RCC-associated TCRs was high in the tumors and pre-REP TILs but decreased in the REP TILs. Our results provide an in-depth understanding of the origin, phenotype, and TCR specificity of RCC TIL products, paving the way for a more rationalized production of TILs. Significance: TILs are a heterogenous group of immune cells that recognize and attack the tumor, thus are utilized in various clinical trials. In our study, we explored the TILs in patients with kidney cancer by expanding the TILs using a clinical-grade protocol, as well as observed their characteristics and ability to recognize the tumor using in-depth experimental and computational tools.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Linfócitos do Interstício Tumoral , Carcinoma de Células Renais/genética , Linfócitos T CD8-Positivos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Microambiente TumoralRESUMO
BackgroundRelatlimab plus nivolumab (anti-lymphocyte-activation gene 3 plus anti-programmed death 1 [anti-LAG-3+anti-PD-1]) has been approved by the FDA as a first-line therapy for stage III/IV melanoma, but its detailed effect on the immune system is unknown.MethodsWe evaluated blood samples from 40 immunotherapy-naive or prior immunotherapy-refractory patients with metastatic melanoma treated with anti-LAG-3+anti-PD-1 in a phase I trial using single-cell RNA and T cell receptor sequencing (scRNA+TCRαß-Seq) combined with other multiomics profiling.ResultsThe highest LAG3 expression was noted in NK cells, Tregs, and CD8+ T cells, and these cell populations underwent the most significant changes during the treatment. Adaptive NK cells were enriched in responders and underwent profound transcriptomic changes during the therapy, resulting in an active phenotype. LAG3+ Tregs expanded, but based on the transcriptome profile, became metabolically silent during the treatment. Last, higher baseline TCR clonality was observed in responding patients, and their expanding CD8+ T cell clones gained a more cytotoxic and NK-like phenotype.ConclusionAnti-LAG-3+anti-PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8+ T cells.Trial registrationClinicalTrials.gov (NCT01968109)FundingCancer Foundation Finland, Sigrid Juselius Foundation, Signe and Ane Gyllenberg Foundation, Relander Foundation, State funding for university-level health research in Finland, a Helsinki Institute of Life Sciences Fellow grant, Academy of Finland (grant numbers 314442, 311081, 335432, and 335436), and an investigator-initiated research grant from BMS.
Assuntos
Antineoplásicos , Melanoma , Humanos , Receptor de Morte Celular Programada 1 , Melanoma/tratamento farmacológico , Melanoma/genética , Nivolumabe/uso terapêutico , Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos , Receptores de Antígenos de Linfócitos T/metabolismo , Melanoma Maligno CutâneoRESUMO
BACKGROUND: Prostate and seminal vesicle are two similar hormone responsive human organs that differ dramatically in their cancer incidence. DNA damage response (DDR) is required for maintenance of genomic integrity. METHODS: In this study we investigated the DDR and cell cycle checkpoint activation of these organs using orthotopic cultures of human surgery-derived tissues and primary cultures of isolated prostate and seminal vesicle cells. RESULTS: We find that the activation of ATM signaling pathway by ionizing radiation (IR) was comparable in both tissues. Previously, we have shown that the prostate secretory cells express low levels of histone variant H2AX and phosphorylated H2AX (γH2AX) after IR. Here we demonstrate that H2AX levels are low also in the secretory seminal vesicle cells suggesting that this is a common phenotype of postmitotic cells. We consequently established primary epithelial cell cultures from both organs to compare their DDR. Interestingly, contrary to human prostate epithelial cells (HPEC), primary seminal vesicle epithelial cells (HSVEC) displayed effective cell cycle checkpoints after IR and expressed higher levels of Wee1A checkpoint kinase. Furthermore, HSVEC but not HPEC cells were able to activate p53 and to induce p21 cell cycle inhibitor. DISCUSSION: Our results show that during replication, the checkpoint enforcement is more proficient in the seminal vesicle than in the prostate epithelium cells. This indicates a more stringent enforcement of DDR in replicating seminal vesicle epithelial cells, and suggests that epithelial regeneration combined with sub-optimal checkpoint responses may contribute to high frequency of genetic lesions in the prostate epithelium.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Dano ao DNA/genética , Células Epiteliais/fisiologia , Próstata/fisiologia , Glândulas Seminais/fisiologia , Células Cultivadas , Células Epiteliais/patologia , Epitélio/patologia , Epitélio/fisiologia , Humanos , Masculino , Próstata/patologia , Glândulas Seminais/patologiaRESUMO
Nucleophosmin (NPM, B23) is an abundant nucleolar phosphoprotein involved in ribosome biogenesis, and interacts with tumor suppressor proteins p53 and Rb. Here we show that NPM is a UV damage response protein that undergoes nucleoplasmic redistribution and regulates p53 and HDM2 levels and their interaction. By utilizing RNAi approaches and analyses of endogenous and ectopically expressed proteins, we demonstrate that NPM binds HDM2 and acts as a negative regulator of p53-HDM2 interaction. Viral stress, enforced by expression of Kaposi's sarcoma virus K cyclin, causes NPM redistribution, K cyclin-NPM association, and p53 stabilization by dissociation of HDM2-p53 complexes. The results demonstrate novel associations of HDM2 and K cyclin with NPM and implicate NPM as a crucial controller of p53 through inhibition of HDM2.
Assuntos
Nucléolo Celular/metabolismo , Ciclinas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Nucléolo Celular/efeitos da radiação , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Glutationa Transferase/metabolismo , Humanos , Camundongos , Nucleofosmina , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Testes de Precipitina , Biossíntese de Proteínas , Transporte Proteico , Proteínas Proto-Oncogênicas c-mdm2 , RNA Interferente Pequeno/farmacologia , Proteína SUMO-1/metabolismo , Proteína Supressora de Tumor p53/química , Raios Ultravioleta/efeitos adversos , Dedos de ZincoRESUMO
p27Kip1 (p27) tumour suppressor protein is regulated by multiple mechanisms including its turnover, localization and complex formation with its key targets, cyclin-dependent kinases (CDK) and cyclins. We have earlier shown that p27 exists in cells in a form that lacks cyclin/CDK interactions (hence non-CDK, p27(NCDK)) but the nature of p27(NCDK) has remained unresolved. Here we demonstrate that the epitope recognized by the p27(NCDK)-specific antibody resides in the p27 CDK-interaction domain and that p27(NCDK) is regulated by the balance of CDK inhibitors and cyclin-CDK complexes. We find that signalling by cellular growth promoting pathways, like phosphoinositol 3-kinase (PI3K) and specifically Akt/PKB kinase, inversely correlates with p27(NCDK) levels whereas total p27 levels are unaffected. p27(NCDK), but not total p27, is increased by cellular perturbations such as hyperosmotic and metabolic stress and activation of AMP-activated protein kinase (AMPK). By using AMPK catalytic subunit proficient and deficient cells we further demonstrate that the AMPK pathway governs p27(NCDK) responses to metabolic stress and PI3K inhibition. These results indicate that p27(NCDK) is a sensitive marker for both cell stress and proliferation over and above p27 and is regulated by Akt/PKB and AMPK pathways.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Biomarcadores/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico , Proteínas Quinases Ativadas por AMP/genética , Sequência de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Ribonucleotídeos/metabolismoRESUMO
Knowledge of clinically targetable tumor antigens is becoming vital for broader design and utility of therapeutic cancer vaccines. This information is obtained reliably by directly interrogating the MHC-I presented peptide ligands, the immunopeptidome, with state-of-the-art mass spectrometry. Our manuscript describes direct identification of novel tumor antigens for an aggressive triple-negative breast cancer model. Immunopeptidome profiling revealed 2481 unique antigens, among them a novel ERV antigen originating from an endogenous retrovirus element. The clinical benefit and tumor control potential of the identified tumor antigens and ERV antigen were studied in a preclinical model using two vaccine platforms and therapeutic settings. Prominent control of established tumors was achieved using an oncolytic adenovirus platform designed for flexible and specific tumor targeting, namely PeptiCRAd. Our study presents a pipeline integrating immunopeptidome analysis-driven antigen discovery with a therapeutic cancer vaccine platform for improved personalized oncolytic immunotherapy.
RESUMO
Molecular mimicry is one of the leading mechanisms by which infectious agents can induce autoimmunity. Whether a similar mechanism triggers an antitumor immune response is unexplored, and the role of antiviral T cells infiltrating the tumor has remained anecdotal. To address these questions, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we demonstrated that, in mice, preexisting immunity toward specific viral epitopes enhanced the efficacy of cancer immunotherapy via molecular mimicry in different settings. To understand whether this mechanism could partly explain immunotherapy responsiveness in humans, we analyzed a cohort of patients with melanoma undergoing anti-PD1 treatment who had a high IgG titer for cytomegalovirus (CMV). In this cohort of patients, we showed that high levels of CMV-specific antibodies were associated with prolonged progression-free survival and found that, in some cases, peripheral blood mononuclear cells (PBMC) could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell receptor sequencing revealed expansion of the same CD8+ T-cell clones when PBMCs were expanded with tumor or homologous viral peptides. In conclusion, we have demonstrated that preexisting immunity and molecular mimicry could influence the response to immunotherapies. In addition, we have developed a free online tool that can identify tumor antigens and neoantigens highly similar to pathogen antigens to exploit molecular mimicry and cross-reactive T cells in cancer vaccine development.
Assuntos
Imunidade/imunologia , Imunoterapia/métodos , Melanoma/imunologia , Mimetismo Molecular/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , CamundongosRESUMO
Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.
Assuntos
Antígenos de Histocompatibilidade Classe I , Microfluídica , Antígenos de Neoplasias , Humanos , Ligantes , PeptídeosRESUMO
Because of the high coverage of international vaccination programs, most people worldwide have been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T-cell repertoire. Although CD8+ antitumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4+ T-cell help is also required for an optimal antitumor immune response to occur. Hence, we investigated whether the pathogen-related CD4+ T-cell memory populations could be reengaged to support the CTLs, converting a weak primary antitumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of an oncolytic adenovirus coated with MHC-I-restricted tumor-specific peptides and developed it further by introducing pathogen-specific MHC-II-restricted peptides. Mice preimmunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid- and tumor-specific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced antitumor efficacy and induced TT-specific, CD40 ligand-expressing CD4+ T helper cells and maturation of antigen-presenting cells. Importantly, this approach could be extended to naturally occurring tumor peptides (both tumor-associated antigens and neoantigens), as well as to other pathogens beyond tetanus, highlighting the usefulness of this technique to take full advantage of CD4+ memory T-cell repertoires when designing immunotherapeutic treatment regimens. Finally, the antitumor effect was even more prominent when combined with the immune checkpoint inhibitor anti-PD-1, strengthening the rationale behind combination therapy with oncolytic viruses. SIGNIFICANCE: These findings establish a novel technology that enhances oncolytic cancer immunotherapy by capitalizing on pre-acquired immunity to pathogens to convert a weak antitumor immune response into a much stronger one.
Assuntos
Vacinas Anticâncer/administração & dosagem , Vacina contra Difteria, Tétano e Coqueluche/administração & dosagem , Memória Imunológica , Imunoterapia/métodos , Melanoma Experimental/terapia , Vacina Antipólio de Vírus Inativado/administração & dosagem , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral/transplante , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Vacina Antipólio de Vírus Inativado/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologiaRESUMO
Messenger RNA (mRNA) and microRNA (miRNA)-based therapeutics have become attractive alternatives to DNA-based therapeutics due to recent advances in manufacture, scalability and cost. Also, RNA-based therapeutics are considered safe since there are no risk of inducing genomic changes as well as the potential adverse effects would be only temporary due to the transient nature of RNA-based therapeutics. However, efficient in vivo delivery of RNA-based therapeutics remains a challenge. We have developed a delivery platform for RNA-based therapeutics by exploiting the physicochemical properties of enveloped viruses. By physically attaching cationic liposome/RNA complexes onto the viral envelope of vaccinia virus, we were able to deliver mRNA, self-replicating RNA as well as miRNA inside target cells. Also, we showed that this platform, called viRNA platform, can efficiently deliver functional miRNA mimics into B16.OVA tumour in vivo.
Assuntos
Neoplasias da Mama/terapia , Sistemas de Liberação de Medicamentos , Terapia Genética , Melanoma Experimental/terapia , MicroRNAs/administração & dosagem , RNA Mensageiro/metabolismo , Vaccinia virus/genética , Células A549 , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , RNA Mensageiro/genéticaRESUMO
Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.
Assuntos
Vacinas Anticâncer/administração & dosagem , Imunoterapia/métodos , Neoplasias/terapia , Vírus Oncolíticos/imunologia , Vacinação/métodos , Adenoviridae/imunologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/imunologia , Linhagem Celular Tumoral/transplante , Membrana Celular/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intralesionais , Camundongos , Nanopartículas/administração & dosagem , Neoplasias/imunologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activating gene mutations, gene amplifications and overexpressed proteins may be useful as targets for novel therapies. Alterations at chromosome locus 4q12 are associated with gliomas and the region harbors the receptor tyrosine kinase gene KIT, which is frequently amplified in gliomas, and also overexpressed in a subset of gliomas. KIT and its ligand stem cell factor are widely expressed in embryonic and adult mouse brain, and they play a role in many signal transduction pathways involved in cellular proliferation, differentiation and cancer cell metastasis. However, the function of KIT in gliomagenesis or disease progression remains unresolved as well as its role in neural and brain tumor development. In this study, we utilized lentivirus-mediated gene transfer to deliver the KIT gene into mouse astrocytes. The growth properties of KIT overexpressing cells were analyzed using several in vitro functional assays. The effect of receptor tyrosine kinase inhibitor imatinib on astrocyte growth was also investigated. Our results indicate that overexpression of KIT in mouse astrocytes promotes cell proliferation, and the increased proliferation is partly inhibited by imatinib treatment. Furthermore, KIT overexpression induces phenotypic changes in the cells suggesting that KIT may play a role in astrocyte growth regulation.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Glioma/enzimologia , Glioma/patologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-kit/biossíntese , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/fisiologia , Astrócitos/patologia , Benzamidas , Processos de Crescimento Celular , Ativação Enzimática , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Mesilato de Imatinib , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Células-Tronco Neoplásicas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , TransfecçãoRESUMO
Inhibition of RNA polymerase I (Pol I) is a promising strategy for modern cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme, but how this exceptional response is enforced is not known. Here, we define key elements requisite for the response. We show that Pol I preinitiation factors and polymerase subunits (e.g., RPA135) are required for BMH-21-mediated degradation of RPA194. We further find that Pol I inhibition and induced degradation by BMH-21 are conserved in yeast. Genetic analyses demonstrate that mutations that induce transcription elongation defects in Pol I result in hypersensitivity to BMH-21. Using a fully reconstituted Pol I transcription assay, we show that BMH-21 directly impairs transcription elongation by Pol I, resulting in long-lived polymerase pausing. These studies define a conserved regulatory checkpoint that monitors Pol I transcription and is activated by therapeutic intervention.
Assuntos
Inibidores Enzimáticos/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , RNA Polimerase I/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Estabilidade Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/genética , RNA Interferente Pequeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
In two brothers born to consanguineous parents, we identified an unusual neurological disease that manifested with ataxia, psychomotor retardation, cerebellar and cerebral atrophy, and leukodystrophy. Via linkage analysis and exome sequencing, we identified homozygous c.2801C>T (p.(Ser934Leu)) in POLR1A (encoding RPA194, largest subunit of RNA polymerase I) and c.511C>T (p.(Arg171Trp)) in OSBPL11 (encoding oxysterol-binding protein-like protein 11). Although in silico analysis, histopathologic evidence and functional verification indicated that both variants were deleterious, segregation with the patient phenotype established that the POLR1A defect underlies the disease, as a clinically unaffected sister also was homozygous for the OSBPL11 variant. Decreased nucleolar RPA194 was observed in the skin fibroblasts of only the affected brothers, whereas intracellular cholesterol accumulation was observed in the skin biopsies of the patients and the sister homozygous for the OSBPL11 variant. Our findings provide the first report showing a complex leukodystrophy associated with POLR1A. Variants in three other RNA polymerase subunits, POLR1C, POLR3A and POLR3B, are known to cause recessive leukodystrophy similar to the disease afflicting the present family but with a later onset. Of those, POLR1C is also implicated in a mandibulofacial dysostosis syndrome without leukodystrophy as POLR1A is. This syndrome is absent in the family we present.
Assuntos
Ataxia/genética , RNA Polimerases Dirigidas por DNA/genética , Deficiências do Desenvolvimento/genética , Leucoencefalopatias/genética , Mutação de Sentido Incorreto , Adulto , Ataxia/diagnóstico , Células Cultivadas , Criança , Colesterol/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Deficiências do Desenvolvimento/diagnóstico , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Leucoencefalopatias/diagnóstico , Masculino , Linhagem , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Irmãos , SíndromeRESUMO
Using a bio-oligo pull-down DNA-binding assay we investigated the binding capacity of endogenous, DNA damage-induced p53 in human diploid fibroblasts to several p53-responsive elements (REs) present in p53-regulated genes. During the course of p53 accumulation, we observed a decrease in p53 binding to the GADD45 but not to the p21(WAF1/CIP1) RE. Using mutated GADD45 sequences we show that this change is dependent on the presence of cytosines at position 3 in RE pentamers and on the p53 redox state. Site-directed mutagenesis experiments demonstrated that Cys277 (a residue directly contacting base 3 in a RE pentamer) is critical for differential regulation of GADD45 in DNA-damaged cells. These data represent a novel mechanism for differential affinity of p53 to distinct REs.
Assuntos
Cisteína/metabolismo , Dano ao DNA , DNA/metabolismo , Elementos de Resposta/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Substituição de Aminoácidos/genética , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Cisteína/química , DNA/química , DNA/genética , Fibroblastos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Mutação/genética , Oxirredução , Ligação Proteica/efeitos da radiação , Proteínas/genética , Radiação Ionizante , Especificidade por Substrato , Termodinâmica , Timina/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Raios Ultravioleta , Proteínas GADD45RESUMO
RNA polymerase I (Pol I) is a dedicated polymerase that transcribes the 45S ribosomal (r) RNA precursor. The 45S rRNA precursor is subsequently processed into the mature 5.8S, 18S, and 28S rRNAs and assembled into ribosomes in the nucleolus. Pol I activity is commonly deregulated in human cancers. On the basis of the discovery of lead molecule BMH-21, a series of pyridoquinazolinecarboxamides have been evaluated as inhibitors of Pol I and activators of the destruction of RPA194, the Pol I large catalytic subunit protein. Structure-activity relationships in assays of nucleolar stress and cell viability demonstrate key pharmacophores and their physicochemical properties required for potent activation of Pol I stress and cytotoxicity. This work identifies a set of bioactive compounds that potently cause RPA194 degradation that function in a tightly constrained chemical space. This work has yielded novel derivatives that contribute to the development of Pol I inhibitory cancer therapeutic strategies.
Assuntos
Amidas/síntese química , Antineoplásicos/síntese química , Piridinas/síntese química , Quinazolinas/síntese química , RNA Polimerase I/antagonistas & inibidores , Amidas/química , Amidas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Técnicas In Vitro , Microssomos Hepáticos/metabolismo , Piridinas/química , Piridinas/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
We define the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which are present at a high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , RNA Polimerase I/efeitos dos fármacos , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9- and BMH-22-mediated nucleolar stress was detected in ex vivo-cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting.