RESUMO
Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Neoplasias Encefálicas/genética , Estudos de Casos e Controles , Proliferação de Células , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Glioblastoma/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Transcriptoma , Células Tumorais Cultivadas , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: The aim of this study was to demonstrate the in vivo safety and antitumor effect of a novel recombinant vesicular stomatitis virus (VSV): G protein less (GLESS)-fusion-associated small transmembrane (FAST)-VSV. METHODS: Viral infection efficiency and cell proliferation were detected using an inverted fluorescence microscope and alarmaBlue assay, respectively. To evaluate the safety of the virus, different doses of GLESS-FAST-VSV and a positive control virus (VSV∆M51) were injected into normal F344 rats and C57BL/6 mice, and each animal's weight, survival time, and pathological changes were examined on the following day. To evaluate the efficacy of the virus, RG2 and GL261 cells were used to construct rat and mouse glioma models, respectively, via a stereotactic method. After multiple intratumoral injections of the virus, tumor growth (size) and the survival time of the animals were observed. RESULTS: In vitro experiments showed that GLESS-FAST-VSV could infect and kill brain tumor cells and had less toxic effects on normal cells. After direct injection of GLESS-FAST-VSV into the animal brains, all animals tolerated the virus well, and no animal death, encephalitis, or ventriculitis was observed. In contrast, all animals that received brain injections of VSV∆M51 in the brain died. Moreover, multiple injections of GLESS-FAST-VSV in brain tumors significantly prolonged the survival of normal-immunity animals harboring brain tumors. CONCLUSIONS: GLESS-FAST-VSV exhibited little neurotoxicity and could be injected directly into the tumor to effectively inhibit tumor growth and prolong the survival of normal-immunity animals, laying a theoretical foundation for the early application of such viruses in clinical trials.
Assuntos
Glioma , Estomatite Vesicular , Animais , Glioma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos F344 , Vírus da Estomatite Vesicular IndianaRESUMO
BACKGROUND: Gallbladder carcinoma (GBC) is highly lethal, and effective treatment will require synergistic anti-tumor management. The study is aimed at investigating the oncolytic value of myxoma virus (MYXV) infection against GBC and optimizing MYXV oncolytic efficiency. METHODS: We examined the permissiveness of GBC cell lines to MYXV infection and compared the effects of MYXV on cell viability among GBC and control permissive glioma cells in vitro and in vivo after MYXV + rapamycin (Rap) treatment, which is known to enhance cell permissiveness to MYXV by upregulating p-Akt levels. We also assessed MYXV + hyaluronan (HA) therapy efficiency by examinating Akt activation status, MMP-9 expression, cell viability, and collagen distribution. We further compared hydraulic conductivity, tumor area, and survival of tumor-bearing mice between the MYXV + Rap and MYXV + HA therapeutic regimens. RESULTS: MYXV + Rap treatment could considerably increase the oncolytic ability of MYXV against GBC cell lines in vitro but not against GBC xenografts in vivo. We found higher levels of collagen IV in GBC tumors than in glioma tumors. Diffusion analysis demonstrated that collagen IV could physically hinder MYXV intratumoral distribution. HA-CD44 interplay was found to activate the Akt signaling pathway, which increases oncolytic rates. HA was also found to enhance the MMP-9 secretion, which contributes to collagen IV degradation. CONCLUSIONS: Unlike MYXV + Rap, MYXV + HA therapy significantly enhanced the anti-tumor effects of MYXV in vivo and prolonged survival of GBC tumor-bearing mice. HA may optimize the oncolytic effects of MYXV on GBC via the HA-CD44 interaction which can promote viral infection and diffusion.
Assuntos
Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/terapia , Terapia Viral Oncolítica , Animais , Linhagem Celular Tumoral , Neoplasias da Vesícula Biliar/patologia , Neoplasias da Vesícula Biliar/virologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Hialurônico/administração & dosagem , Técnicas In Vitro , Camundongos , Myxoma virus/genética , Sirolimo/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oncolytic viruses constitute a promising therapy against malignant gliomas (MGs). However, virus-induced type I IFN greatly limits its clinical application. The kinase mammalian target of rapamycin (mTOR) stimulates type I IFN production via phosphorylation of its effector proteins, 4E-BPs and S6Ks. Here we show that mouse embryonic fibroblasts and mice lacking S6K1 and S6K2 are more susceptible to vesicular stomatitis virus (VSV) infection than their WT counterparts as a result of an impaired type I IFN response. We used this knowledge to employ a pharmacoviral approach to treat MGs. The highly specific inhibitor of mTOR rapamycin, in combination with an IFN-sensitive VSV-mutant strain (VSV(DeltaM51)), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSV(DeltaM51) selectively killed tumor, but not normal cells, in MG-bearing rats treated with rapamycin. These results demonstrate that reducing type I IFNs through inhibition of mTORC1 is an effective strategy to augment the therapeutic activity of VSV(DeltaM51).
Assuntos
Glioma/metabolismo , Glioma/terapia , Interferon Tipo I/biossíntese , Fatores de Transcrição/metabolismo , Estomatite Vesicular/metabolismo , Vesiculovirus/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Glioma/genética , Glioma/virologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Transplante de Neoplasias , Terapia Viral Oncolítica , Proteínas , Ratos , Ratos Endogâmicos F344 , Proteínas Quinases S6 Ribossômicas/deficiência , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Estomatite Vesicular/genética , Estomatite Vesicular/virologia , Vesiculovirus/genéticaRESUMO
Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Histonas/genética , Histonas/metabolismo , Glioblastoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Cromatina/metabolismo , Epigênese Genética , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismoRESUMO
The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target, suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias Encefálicas/metabolismo , Inibidores Enzimáticos/uso terapêutico , Glioma/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/terapia , Glioma/terapia , Humanos , Invasividade Neoplásica/fisiopatologia , Fatores de Crescimento Neural/metabolismo , Proteínas Recombinantes de FusãoRESUMO
The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered "cured" (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSV(ΔM51). Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).
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Neoplasias Encefálicas/terapia , Modelos Animais de Doenças , Glioma/terapia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Terapia Viral Oncolítica , Sirolimo/uso terapêutico , Vaccinia virus/genética , Animais , Antibióticos Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/genética , Terapia Combinada , Feminino , Vetores Genéticos/uso terapêutico , Glioma/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Endogâmicos F344 , Taxa de Sobrevida , Transgenes/fisiologia , Células Tumorais Cultivadas , Vacinas Sintéticas/uso terapêutico , Replicação ViralRESUMO
Central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT) is a rare, aggressive tumor that most often affects very young children. The common decisive molecular defect in AT/RT has been shown to be a single genetic alteration, i.e., the loss of hSNF5 gene that encodes for a subunit of the SWI/SNF complex that modulates chromatin remodeling activities. As a result, AT/RT cells display unregulated cell proliferation due to the dysfunction of an important epigenetic control. We have previously demonstrated the preclinical efficacy of the oncolytic double-deleted vaccinia virus (VVDD) against AT/RT. Here we report the establishment of a modified VVDD engineered to express wild type hSNF5 gene. We show that this reconstructed vaccinia virus retains comparable infectivity and in vitro cytotoxicity of the parent strain. However, in addition, hSNF5-arming of VVDD results in a decreased cell cycle S phase population and down-regulation of cyclin D1. These findings suggest that hSNF5-arming of VVDD may increase the efficacy in the treatment of AT/RT and validates, as a proof-of-concept, an experimental approach to enhance the effective use of novel modified oncolytic viruses in the treatment of tumors with loss of a tumor suppressor gene function.
Assuntos
Sistema Nervoso Central/patologia , Vírus Oncolíticos/metabolismo , Tumor Rabdoide/genética , Tumor Rabdoide/virologia , Proteína SMARCB1/metabolismo , Vaccinia virus/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary glial tumors of the central nervous system, most commonly glioblastoma multiforme (GBM), are aggressive lesions with a dismal prognosis. Despite identification and isolation of human brain tumor stem cells (BTSCs), characteristics that distinguish BTSCs from neural stem cells remain to be elucidated. We cultured cells isolated from gliomas, using the neurosphere culture system, to understand their growth requirements. Both CD133(+) and CD133(-) adult GBM BTSCs proliferated in the absence of exogenous mitogenic stimulation and gave rise to multipotent GBM spheres that were capable of self-renewal. Epidermal growth factor (EGF) and fibroblast growth factor-2 enhanced GBM BTSC survival, proliferation, and subsequent sphere size. Blockade of EGF receptor (EGFR) signaling reduced exogenous mitogen-independent GBM sphere growth. Implantation of as few as 10 exogenous mitogen-independent GBM BTSCs led to the formation of highly invasive intracranial tumors, which closely resembled human GBMs, in immunocompromised mice. These results demonstrate that exogenous mitogen independence, mediated in part through EGFR signaling, is one characteristic that distinguishes CD133(+) and CD133(-) GBM BTSCs from neural stem cells. This novel experimental system will permit the elucidation of additional constitutively activated mechanisms that promote GBM BTSC survival, self-renewal, and proliferation.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Mitógenos/farmacologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Adolescente , Adulto , Animais , Antígenos CD/metabolismo , Neoplasias Encefálicas/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Citometria de Fluxo , Glioblastoma/metabolismo , Glicoproteínas/metabolismo , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Peptídeos/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Adulto JovemRESUMO
The invasive nature of cancers in general, and malignant gliomas in particular, is a major clinical problem rendering tumors incurable by conventional therapies. Using a novel invasive glioma mouse model established by serial in vivo selection, we identified the p75 neurotrophin receptor (p75(NTR)) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75(NTR) dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75(NTR)-mediated invasion was neurotrophin dependent, resulting in the activation of downstream pathways and producing striking cytoskeletal changes of the invading cells. These results provide the first evidence for p75(NTR) as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target.
Assuntos
Glioma , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Feminino , Perfilação da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Fatores de Crescimento Neural/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Fator de Crescimento Neural/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.
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Neoplasias Encefálicas , Glioblastoma , Vírus Oncolíticos , Animais , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , PeptídeosRESUMO
Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Interleucina-33/metabolismo , Animais , Neoplasias Encefálicas/mortalidade , Carcinogênese , Núcleo Celular/metabolismo , Citocinas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioma/mortalidade , Humanos , Inflamação , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos SCID , Microglia , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/patologia , Microambiente Tumoral/imunologiaRESUMO
PURPOSE: Rhabdoid tumors are highly aggressive pediatric tumors that are usually refractory to available treatments. The purpose of this study was to evaluate the therapeutic potential of two oncolytic viruses, myxoma virus (MV) and an attenuated vesicular stomatitis virus (VSV(DeltaM51)), in experimental models of human rhabdoid tumor. EXPERIMENTAL DESIGN: Four human rhabdoid tumor cell lines were cultured in vitro and treated with live or inactivated control virus. Cytopathic effect, viral gene expression, infectious viral titers, and cell viability were examined at various time points after infection. To study viral oncolysis in vivo, human rhabdoid tumor cells were implanted s.c. in the hind flank or intracranially in CD-1 nude mice and treated with intratumoral (i.t.) or i.v. injections of live or UV-inactivated virus. Viral distribution and effects on tumor size and survival were assessed. RESULTS: All rhabdoid tumor cell lines tested in vitro were susceptible to productive lethal infections by MV and VSV(DeltaM51). I.t. injection of live MV or VSV(DeltaM51) dramatically reduced the size of s.c. rhabdoid tumor xenografts compared with control animals. I.v. administration of VSV(DeltaM51) or i.t. injection of MV prolonged the median survival of mice with brain xenografts compared with controls (VSV(DeltaM51): 25 days versus 21 days, log-rank test, P = 0.0036; MV: median survival not reached versus 21 days, log-rank test, P = 0.0007). Most of the MV-treated animals (4 of 6; 66.7%) were alive and apparently "cured" when the experiment was arbitrarily ended (>180 days). CONCLUSIONS: These results suggest that VSV(DeltaM51) and MV could be novel effective therapies against human rhabdoid tumor.
Assuntos
Myxoma virus/fisiologia , Terapia Viral Oncolítica/métodos , Tumor Rabdoide/terapia , Tumor Rabdoide/virologia , Vesiculovirus/fisiologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This corrects the article DOI: 10.1038/ncomms14278.
RESUMO
BACKGROUND: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. METHODS: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. RESULTS: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. CONCLUSION: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.
Assuntos
Benzimidazóis/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioma/patologia , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Gradação de Tumores , RNA Interferente Pequeno/genéticaRESUMO
Myxoma virus, a poxvirus previously considered rabbit specific, can replicate productively in a variety of human tumor cells in culture. The purpose of this study was to determine if there was efficacy or toxicities of this oncolytic virus against experimental models of human malignant gliomas in vitro, in vivo, and ex vivo in malignant glioma specimens. In vitro, the majority of glioma cell lines tested (7 of 8, 87.5%) were fully permissive for myxoma virus replication and killed by infection. In vivo, intracerebral (i.c.) myxoma virus inoculation was well tolerated and produced only minimal focal inflammatory changes at the site of viral inoculation. U87 and U251 orthotopic xenograft models were used to assess myxoma virus efficacy in vivo. A single intratumoral injection of myxoma virus dramatically prolonged median survival compared with treatment with UV-inactivated myxoma virus. Median survival was not reached in myxoma virus-treated groups versus 47.3 days (U87; P = 0.0002) and 50.7 days (U251; P = 0.0027) in UV-inactivated myxoma virus-treated groups. Most myxoma virus-treated animals (12 of 13, 92%) were alive and apparently "cured" when the experiment was finished (>130 days). Interestingly, we found a selective and long-lived myxoma virus infection in gliomas in vivo. This is the first demonstration of the oncolytic activity of myxoma virus in vivo. The nonpathogenic nature of myxoma virus outside of the rabbit host, its capacity to be genetically modified, its ability to produce a long-lived infection in human tumor cells, and the lack of preexisting antibodies in the human population suggest that myxoma virus may be an attractive oncolytic agent against human malignant glioma.
Assuntos
Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/virologia , Glioma/terapia , Glioma/virologia , Myxoma virus/fisiologia , Terapia Viral Oncolítica/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Malignant glioma still has a poor prognosis and remains incurable. Although temozolomide (TMZ) has demonstrated antitumor activity, its use recently has been halted because of some patients' resistance to this drug. New treatments are desperately needed. An oncolytic virus (virotherapy) is being developed as a novel cancer therapy. We have previously reported that recombinant Vesicular Stomatitis Virus (VSV-ΔM51) and double deleted Vaccinia Virus (vvDD) infected and killed glioma cell lines in vitro and prolonged survival in animal glioma models. As a proposed ex vivo test, the oncolytic potential of VSV-ΔM51 and vvDD in the established human brain tumor stem cells (BTSCs) and the differentiated cells from fresh brain tumor tissues in vitro were further investigated. METHODS: BTSCs from fresh surgical glioblastoma multiforme (GBM) specimens were isolated and cultured, and the characterization of BTSCs were tested. The sensitivity of BTSCs to TMZ and the susceptibility of TMZ resistant BTSCs and their differentiated cells to both oncolytic viruses were examined. RESULTS: The BTSC spheres cultured had all the characteristics of stem cells. The GFP-labeled VSV-ΔM51 and vvDD could infect TMZ resistant BTSCs and cause cytopathic effects. The VSV-ΔM51and vvDD inhibited the self-renewal activity of TMZ resistant BTSCs. And the VSV-ΔM51and vvDD also infected and caused cytopathic effects in differentiated BTSCs. CONCLUSION: VSV-ΔM51and vvDD could infect and kill both the TMZ resistant BTSCs and the differentiated compartments of GBMs in vitro, suggesting that they may be an effective treatment supplement for GBM therapy, particularly for TMZ resistant GBM patients.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Neoplásicas/patologia , Recombinação Genética/genética , Vaccinia virus/genética , Estomatite Vesicular/genética , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Temozolomida , Replicação ViralRESUMO
Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Interferon-alfa/imunologia , Interferon beta/imunologia , Tiazóis/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Antineoplásicos/síntese química , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/imunologia , Glioblastoma/genética , Glioblastoma/imunologia , Glioblastoma/mortalidade , Humanos , Imunidade Inata/efeitos dos fármacos , Memória Imunológica , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/imunologia , Interferon-alfa/genética , Interferon-alfa/farmacologia , Interferon beta/genética , Interferon beta/farmacologia , Camundongos , Poli I-C/farmacologia , Transdução de Sinais , Análise de Sobrevida , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Tiazóis/síntese química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Vesiculovirus/genética , Vesiculovirus/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Myxoma virus (MYXV) is a promising oncolytic agent and is highly effective against immortalized glioma cells but less effective against brain tumor initiating cells (BTICs), which are believed to mediate glioma development/recurrence. MYXV encodes various proteins to attenuate host cell apoptosis, including an antiapoptotic Bcl-2 homologue known as M011L. Such proteins may limit the ability of MYXV to kill BTICs, which have heightened resistance to apoptosis. We hypothesized that infecting BTICs with an M011L-deficient MYXV construct would overcome BTIC resistance to MYXV. METHODS: We used patient-derived BTICs to evaluate the efficacy of M011L knockout virus (vMyx-M011L-KO) versus wild-type MYXV (vMyx-WT) and characterized the mechanism of virus-induced cell death in vitro. To extend our findings in a novel immunocompetent animal model, we derived, cultured, and characterized a C57Bl/6J murine BTIC (mBTIC0309) from a spontaneous murine glioma and evaluated vMyx-M011L-KO efficacy with and without temozolomide (TMZ) in mBTIC0309-bearing mice. RESULTS: We demonstrated that vMyx-M011L-KO induces apoptosis in BTICs, dramatically increasing sensitivity to the virus. vMyx-WT failed to induce apoptosis as M011L protein prevented Bax activation and cytochrome c release. In vivo, intracranial implantation of mBTIC0309 generated tumors that closely recapitulated the pathological and molecular profile of human gliomas. Treatment of tumor-bearing mice with vMyx-M011L-KO significantly prolonged survival in immunocompetent-but not immunodeficient-mouse models, an effect that is significantly enhanced in combination with TMZ. CONCLUSIONS: Our data suggest that vMyx-M011L-KO is an effective, well-tolerated, proapoptotic oncolytic virus and a strong candidate for clinical translation.
RESUMO
PURPOSE: Glioblastoma is one of the most lethal cancers in humans, and with existing therapy, survival remains at 14.6 months. Current barriers to successful treatment include their infiltrative behavior, extensive tumor heterogeneity, and the presence of a stem-like population of cells, termed brain tumor-initiating cells (BTIC) that confer resistance to conventional therapies. EXPERIMENTAL DESIGN: To develop therapeutic strategies that target BTICs, we focused on a repurposing approach that explored already-marketed (clinically approved) drugs for therapeutic potential against patient-derived BTICs that encompass the genetic and phenotypic heterogeneity of glioblastoma observed clinically. RESULTS: Using a high-throughput in vitro drug screen, we found that montelukast, clioquinol, and disulfiram (DSF) were cytotoxic against a large panel of patient-derived BTICs. Of these compounds, disulfiram, an off-patent drug previously used to treat alcoholism, in the presence of a copper supplement, showed low nanomolar efficacy in BTICs including those resistant to temozolomide and the highly infiltrative quiescent stem-like population. Low dose DSF-Cu significantly augmented temozolomide activity in vitro, and importantly, prolonged in vivo survival in patient-derived BTIC models established from both newly diagnosed and recurrent tumors. Moreover, we found that in addition to acting as a potent proteasome inhibitor, DSF-Cu functionally impairs DNA repair pathways and enhances the effects of DNA alkylating agents and radiation. These observations suggest that DSF-Cu inhibits proteasome activity and augments the therapeutic effects of DNA-damaging agents (temozolomide and radiation). CONCLUSIONS: DSF-Cu should be considered as an adjuvant therapy for the treatment of patients with glioblastoma in both newly diagnosed and recurrent settings. Clin Cancer Res; 22(15); 3860-75. ©2016 AACR.