RESUMO
Despite the success of poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory double-stranded RNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T-cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi-resistant ovarian tumor growth in vivo, and promotes antitumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.
RESUMO
Despite the success of Poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers Euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory dsRNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi resistant ovarian tumor growth in vivo, and promotes anti-tumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.
RESUMO
Poly ADP-ribose polymerase inhibitors (PARPi) are first-line maintenance therapy for ovarian cancer and an alternative therapy for several other cancer types. However, PARPi-resistance is rising and there is currently an unmet need to combat PARPi-resistant tumors. Here, we created an immunocompetent, PARPi-resistant mouse model to test the efficacy of combinatory PARPi and euchromatic histone methyltransferase 1/2 inhibitor (EHMTi) in the treatment of PARPi-resistant ovarian cancer. We discovered that inhibition of EHMT1/2 resensitizes cells to PARPi. Markedly, we show that single EHMTi and combinatory EHMTi/PARPi significantly reduced PARPi-resistant tumor burden and that this reduction is partially dependent on CD8 T cells. Altogether, our results show a low-toxicity drug that effectively treats PARPi-resistant ovarian cancer in an immune-dependent manner, supporting its entry into clinical development and potential incorporation of immunotherapy. Implications: Targeting the epigenome of therapy-resistant ovarian cancer induces an anti-tumor response mediated in part through an anti-tumor immune response.
RESUMO
Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Fígado/virologia , Replicação Viral , 1-Fosfatidilinositol 4-Quinase/química , Antivirais/farmacologia , Ligação Competitiva , Linhagem Celular , Inativação Gênica , Genótipo , Humanos , Concentração Inibidora 50 , Espectrometria de Massas/métodos , Proteômica/métodos , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologiaRESUMO
Assessing the functional significance of novel putative oncogenes remains a significant challenge given the limitations of current loss-of-function tools. Here, we describe a method that employs TALEN or CRISPR/Cas9-mediated knock-in of inducible degron tags (Degron-KI) that provides a versatile approach for the functional characterization of novel cancer genes and addresses many of the shortcomings of current tools. The Degron-KI system allows for highly specific, inducible, and allele-targeted inhibition of endogenous protein function, and the ability to titrate protein depletion with this system is able to better mimic pharmacologic inhibition compared with RNAi or genetic knockout approaches. The Degron-KI system was able to faithfully recapitulate the effects of pharmacologic EZH2 and PI3Kα inhibitors in cancer cell lines. The application of this system to the study of a poorly understood putative oncogene, SF3B1, provided the first causal link between SF3B1 hotspot mutations and splicing alterations. Surprisingly, we found that SF3B1-mutant cells are not dependent upon the mutated allele for in vitro growth, but instead depend upon the function of the remaining wild-type alleles. Collectively, these results demonstrate the broad utility of the Degron-KI system for the functional characterization of cancer genes.
Assuntos
Genes Neoplásicos , Neoplasias/genética , Proliferação de Células , Células HCT116 , Humanos , Mutação , Fosfoproteínas/genética , Estabilidade Proteica , Proteólise , Fatores de Processamento de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genéticaRESUMO
Epigenetic dysregulation is an emerging hallmark of cancers. We developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancers. When applied to 115 lines from the Cancer Cell Line Encyclopedia, this approach identified distinct molecular chromatin signatures. One signature was characterized by increased histone 3 lysine 36 (H3K36) dimethylation, exhibited by several lines harboring translocations in NSD2, which encodes a methyltransferase. A previously unknown NSD2 p.Glu1099Lys (p.E1099K) variant was identified in nontranslocated acute lymphoblastic leukemia (ALL) cell lines sharing this signature. Ectopic expression of the variant induced a chromatin signature characteristic of NSD2 hyperactivation and promoted transformation. NSD2 knockdown selectively inhibited the proliferation of NSD2-mutant lines and impaired the in vivo growth of an NSD2-mutant ALL xenograft. Sequencing analysis of >1,000 pediatric cancer genomes identified the NSD2 p.E1099K alteration in 14% of t(12;21) ETV6-RUNX1-containing ALLs. These findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes.
Assuntos
Cromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Repressoras/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Criança , Feminino , Predisposição Genética para Doença , Variação Genética , Humanos , Camundongos , Camundongos SCID , Células NIH 3T3 , Transplante de Neoplasias , Análise de Sequência de DNA , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule. METHODS: Two hexahistidine-tagged activin receptor-like kinases were expressed in E. coli, purified, and bound to nickel beads. A fluorescent kinase ligand (F-KL) that binds to the ATP-binding site of these kinases with nanomolar affinity was developed. Binding of F-KL with kinase on the bead made the beads bright, and inhibitors decreased the brightness. RESULTS: A test panel of 17 nonfluorescent kinase inhibitors, spanning two orders of magnitude affinity for the kinases, gave K(d) values for the kinases that correlated well with a fluorescence polarization assay. Results were obtained for the kinases in duplex, using an autosampler to send beads from a 96-well plate to a flow cytometer in a format suitable for high throughput screening. CONCLUSIONS: Inhibitors of kinases can be measured in duplex in a high throughput format by flow cytometry, if a suitable fluorescent ligand is available.
Assuntos
Ligação Competitiva , Citometria de Fluxo/métodos , Microesferas , Inibidores de Proteínas Quinases/análise , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Trifosfato de Adenosina/farmacologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/metabolismo , Humanos , Ligantes , Níquel/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Cytomegalovirus (CMV) is transmitted by transfusion of infected blood products and can cause serious diseases in specific risk groups. CMV can be present in infected blood as cell-free virus (CFV), cell-associated actively replicating virus (CAV), and cell-associated latent virus (LV). STUDY DESIGN AND METHODS: In vitro models for all three infectious forms of CMV and virus detection assays based on both tissue culture and polymerase chain reaction (PCR) were developed. The utility of the CMV model systems and assays were tested by validation studies of a novel pathogen inactivation agent, PEN110, for red blood cells. RESULTS: Reproducible high titers of CFV and CAV were obtained by optimized tissue culture techniques for CMV-infected MRC-5 cells. An LV model was obtained with CMV-infected THP-1 cells and reactivation of virus replication by phorbol ester treatment. The model systems showed that PEN110 treatment is effective against all three forms of CMV as measured by tissue culture-based infectivity assays and a long-range PCR method specific for detection of damage to CMV viral DNA. CONCLUSION: This study describes model systems to the relevant forms of CMV in blood and detection assays that can be used to evaluate the efficacy of viral inactivation agents.
Assuntos
Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Poliaminas/farmacologia , Inativação de Vírus/efeitos dos fármacos , Bioensaio , Linhagem Celular , Citomegalovirus/fisiologia , Infecções por Citomegalovirus , DNA Viral/análise , Estudos de Avaliação como Assunto , Fibroblastos/virologia , Humanos , Técnicas In Vitro , Pulmão/citologia , Pulmão/embriologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Latência Viral , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: The residual risk of HIV infection after HIV screening tests in combination with the risk of new emerging pathogens entering the blood supply has sparked research on the development of a technology for reduction of pathogens in RBCs. STUDY DESIGN AND METHODS: HIV-1 was treated with PEN110 (INACTINE) and analyzed for the kinetics of virus reduction in RBC, the effect of PEN110 on nucleic acids, the integrity of the virus morphology and viral proteins, and the ability of the virus to bind HIV cell receptors and enter susceptible cells. RESULTS: PEN110 effectively reduced HIV-1 to the limit of detection for a reduction factor of at least 5.57 log 50 percent tissue culture infectious dose per bulk test. The PEN110-treated virions maintained their morphology, protein integrity, and functionality. However, the PEN110-treated HIV-1 RNA genome was neither functional to serve as a template for RT-PCR amplification of about 1 kb nor able to support viral DNA synthesis in cell culture. CONCLUSION: These results suggest that PEN110 inactivates HIV-1 by targeting the viral nucleic acid.