RESUMEN
2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vß13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27 weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3' region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.
Asunto(s)
Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Transgenes/genética , Animales , Southern Blotting , Cromosomas , Expresión Génica , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
BACKGROUND: Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option. METHODS: Using hormone-sensitive breast cancer cells stably transfected with the aromatase gene (MCF-7/AC-1), works presented herein describe the in vitro and in vivo antitumor efficacy of the following compounds: dalotuzumab (DALO; "MK-0646"; anti-IGF-1R antibody), ridaforolimus (RIDA; "MK-8669"; mTORC1 small molecule inhibitor) and letrozole ("LET", aromatase inhibitor). RESULTS: With the exception of MK-0646, all single agent and combination treatment arms effectively inhibited xenograft tumor growth, albeit to varying degrees. Correlative tissue analyses revealed MK-0646 alone and in combination with LET induced insulin receptor alpha A (InsR-A) isoform upregulation (both mRNA and protein expression), thereby further supporting a triple therapy approach. CONCLUSION: These data provide preclinical rationalization towards the combined triple therapy of LET plus MK-0646 plus MK-8669 as an efficacious anti-tumor strategy for ER-positive breast tumors.
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Anticuerpos Monoclonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Sinergismo Farmacológico , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Sirolimus/análogos & derivados , Animales , Anticuerpos Monoclonales Humanizados , Antígenos CD/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor de Insulina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Histone deacetylases (HDACs) play diverse roles in many diseases including cancer, sarcopenia, and Alzheimer's. Different isoforms of HDACs appear to play disparate roles in the cell and are associated with specific diseases; as such, a substantial effort has been made to develop isoform-selective HDAC inhibitors. Our group focused on developing HDAC1/HDAC2-specific inhibitors as a cancer therapeutic. In the course of characterizing the mechanism of inhibition of a novel HDAC1/2-selective inhibitor, it was determined that it did not exhibit classical Michaelis-Menten kinetic behavior; this result is in contrast to the seminal HDAC inhibitor SAHA. Enzymatic assays, along with a newly developed binding assay, were used to determine the rates of binding and the affinities of both the HDAC1/2-selective inhibitor and SAHA. The mechanism of action studies identified a potential conformational change required for optimal binding by the selective inhibitor. A model of this putative conformational change is proposed.
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Antineoplásicos/química , Benzoatos/química , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/química , Ácidos Hidroxámicos/química , Xantenos/química , Animales , Antineoplásicos/farmacología , Benzoatos/farmacología , Línea Celular Tumoral , Femenino , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Xenoinjertos , Histona Desacetilasa 1/química , Histona Desacetilasa 2/química , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Cinética , Ratones , Ratones Desnudos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Vorinostat , Xantenos/farmacologíaRESUMEN
Mice deficient in the DNA damage sensor P53 display normal T cell development but eventually succumb to thymic lymphomas. Here, we show that inactivation of the TCR beta gene enhancer (E beta) results in a block of T cell development at stages where recombination-activating genes (RAG) are expressed. Introduction of the E beta mutation into p53-/- mice dramatically accelerates the onset of lethal thymic lymphomas that harbor RAG-dependent aberrant rearrangements, chromosome 14 and 12 translocations, and amplification of the chromosomal region 9A1-A5.3. Phenotypic and genetic analyses suggest that lymphomas emerge through a normal thymocyte development pathway. These findings provide genetic evidence that block of lymphocyte development at stages with RAG endonuclease activity can provoke lymphomagenesis on a background with deficient DNA damage responses.
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Aberraciones Cromosómicas , Proteínas de Unión al ADN/metabolismo , Linfoma/genética , Linfoma/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Animales , Apoptosis , Línea Celular Tumoral , Daño del ADN , Proteínas de Unión al ADN/genética , Reordenamiento Génico de Linfocito T/genética , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Linfoma/inmunología , Linfoma/metabolismo , Ratones , Ratones Noqueados , Eliminación de Secuencia/genética , Cariotipificación Espectral , Linfocitos T/citología , Timo/citología , Timo/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The therapeutic effectiveness of oncolytic viruses (OVs) delivered intravenously is limited by the development of neutralizing antibody responses against the virus. To circumvent this limitation and to enable repeated systemic administration of OVs, here we develop Synthetic RNA viruses consisting of a viral RNA genome (vRNA) formulated within lipid nanoparticles. For two Synthetic RNA virus drug candidates, Seneca Valley virus (SVV) and Coxsackievirus A21, we demonstrate vRNA delivery and replication, virus assembly, spread and lysis of tumor cells leading to potent anti-tumor efficacy, even in the presence of OV neutralizing antibodies in the bloodstream. Synthetic-SVV replication in tumors promotes immune cell infiltration, remodeling of the tumor microenvironment, and enhances the activity of anti-PD-1 checkpoint inhibitor. In mouse and non-human primates, Synthetic-SVV is well tolerated reaching exposure well above the requirement for anti-tumor activity. Altogether, the Synthetic RNA virus platform provides an approach that enables repeat intravenous administration of viral immunotherapy.
Asunto(s)
Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Picornaviridae , Animales , Anticuerpos Neutralizantes , Inmunoterapia , Liposomas , Ratones , Nanopartículas , Neoplasias/terapia , Virus Oncolíticos/genética , ARN Viral/genética , Microambiente TumoralRESUMEN
ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell-derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen-specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti-PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).
Asunto(s)
Herpesvirus Humano 1/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Animales , Línea Celular Tumoral/trasplante , Terapia Combinada/métodos , Modelos Animales de Enfermedad , Femenino , Herpesvirus Humano 1/genética , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inyecciones Intralesiones , Ratones , MicroARNs/genética , MicroARNs/inmunología , Neoplasias/inmunología , Neoplasias/patología , Virus Oncolíticos/genética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Distribución Tisular , Transgenes/genética , Transgenes/inmunología , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/inmunología , Replicación Viral/genéticaRESUMEN
Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2-mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.
Asunto(s)
Linfocitos T CD8-positivos , Subtipo EP4 de Receptores de Prostaglandina E , Animales , Ciclooxigenasa 2 , Dinoprostona , Macrófagos , RatonesRESUMEN
Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. In vivo, this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1.
RESUMEN
We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.
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Antineoplásicos/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Inhibidores de Histona Desacetilasas , Organofosfonatos/farmacocinética , Proteínas Represoras/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasas/metabolismo , Ratones , Ratones Desnudos , Organofosfonatos/síntesis química , Organofosfonatos/química , Proteínas Represoras/metabolismo , Trasplante HeterólogoRESUMEN
To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the V beta 13 promoter was either deleted (P13(-/-)) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13(R/R)). In P13(-/-) mice, cleavage, rearrangement, and transcription of V beta 13, but not the flanking V beta gene segments, were significantly inhibited. In P13(R/R) mice, inhibition of V beta 13 rearrangement was less severe and was not associated with any apparent reduction in V beta 13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal V beta 13-recombination signal sequence junction and V beta 13 coding joint formation of both wild-type and mutant V beta 13 alleles. However, a low level of aberrant V beta 13 cleavage was consistently detected, especially in TCR transgenic P13(R/R) mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated V beta gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant V beta cleavages during allelic exclusion.
Asunto(s)
Alelos , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Regiones Promotoras Genéticas , Animales , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Mutación , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Recombinación Genética , Transcripción Genética , TransgenesRESUMEN
Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155-62. ©2016 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias Experimentales/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Ratones , Pirazoles/administración & dosificación , Pirazoles/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Pirimidinonas , Distribución Aleatoria , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Sirolimus/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Ácido Fólico/análogos & derivados , Expresión Génica , Alcaloides de la Vinca/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Análisis por Conglomerados , Biología Computacional/métodos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Receptores de Folato Anclados a GPI/antagonistas & inhibidores , Ácido Fólico/farmacología , Perfilación de la Expresión Génica , Humanos , Ratones , Platino (Metal)/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin-like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination. EXPERIMENTAL DESIGN: In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10-40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored. RESULTS: Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER(+)/high-proliferative breast cancer showed antitumor activity. CONCLUSIONS: Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER(+)/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379).
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Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Sirolimus/análogos & derivados , Adulto , Anciano , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Humanos , Persona de Mediana Edad , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inhibidores , Receptores de Somatomedina/inmunología , Receptores de Somatomedina/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/administración & dosificación , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.
RESUMEN
UNLABELLED: Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant. SIGNIFICANCE: NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antineoplásicos/farmacología , Carcinoma Adenoide Quístico/genética , Inhibidores de Proteasas/farmacología , Neoplasias de la Mama Triple Negativas/genética , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores , Carcinoma Adenoide Quístico/tratamiento farmacológico , Carcinoma Adenoide Quístico/metabolismo , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Óxidos S-Cíclicos/farmacología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Exoma , Femenino , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Genes myc , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Mutación , Pronóstico , Inhibidores de Proteasas/administración & dosificación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Notch/antagonistas & inhibidores , Receptores Notch/química , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Tiadiazoles/farmacología , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.
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Receptor Notch1/metabolismo , Animales , Línea Celular Tumoral , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Mutación , Receptor Notch1/genéticaRESUMEN
Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.
RESUMEN
Two genetically engineered, conditional mouse models of lung tumor formation, K-ras(LSL-G12D) and K-ras(LSL-G12D)/p53(LSL-R270H), are commonly used to model human lung cancer. Developed by Tyler Jacks and colleagues, these models have been invaluable to study in vivo lung cancer initiation and progression in a genetically and physiologically relevant context. However, heterogeneity, multiplicity and complexity of tumor formation in these models make it challenging to monitor tumor growth in vivo and have limited the application of these models in oncology drug discovery. Here, we describe a novel analytical method to quantitatively measure total lung tumor burden in live animals using micro-computed tomography imaging. Applying this methodology, we studied the kinetics of tumor development and response to targeted therapy in vivo in K-ras and K-ras/p53 mice. Consistent with previous reports, lung tumors in both models developed in a time- and dose (Cre recombinase)-dependent manner. Furthermore, the compound K-ras(LSL-G12D)/p53(LSL-R270H) mice developed tumors faster and more robustly than mice harboring a single K-ras(LSL-G12D) oncogene, as expected. Erlotinib, a small molecule inhibitor of the epidermal growth factor receptor, significantly inhibited tumor growth in K-ras(LSL-G12D)/p53(LSL-R270H) mice. These results demonstrate that this novel imaging technique can be used to monitor both tumor progression and response to treatment and therefore supports a broader application of these genetically engineered mouse models in oncology drug discovery and development.
Asunto(s)
Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/patología , Tomografía Computarizada de Haz Cónico/métodos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Carga Tumoral , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/tratamiento farmacológico , Adenoviridae/genética , Animales , Antineoplásicos/administración & dosificación , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Clorhidrato de Erlotinib , Femenino , Genes p53 , Genes ras , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Ratones Transgénicos , Neovascularización Patológica/diagnóstico por imagen , Quinazolinas/administración & dosificación , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genéticaRESUMEN
The recombination activating genes (RAG) play a central role in the generation of a diverse immune repertoire by mediating DNA recombination events at antigen receptor loci in developing B and T lymphocytes. However, inappropriate RAG activity throughout the genome has been implicated in a large variety of human and mouse lymphomas. Mechanisms by which RAG can provoke or perpetuate lymphoma include deregulation of certain genes by translocation to antigen receptor regulatory regions, the formation of chimeric oncogenes, inactivation of tumor suppressor or micro-RNA loci, or activation of oncogenes. Here we present the T cell receptor enhancer (Ebeta) deficient mouse as a tractable in vivo model system to study the role of RAG activity in the context of lymphoma development, and contrast our system with those of others. We posit a general hypothesis that virtually any mutation that impairs early lymphocyte development at stages when RAG is expressed can constitute a pro-carcinogenic event. Our model system provides a means to assess the roles of RAG activity in human lymphoid malignancies.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Linfoma/genética , Animales , Apoptosis , Ciclo Celular/fisiología , Proteínas de Unión al ADN/genética , Genes RAG-1 , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Genes bcl-2 , Genes p53 , Linfocitos/fisiología , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Transgénicos/metabolismo , Mutación , Transducción de SeñalRESUMEN
During RNA interference (RNAi), long dsRNA is processed to approximately 21 nt duplexes, short interfering RNAs (siRNAs), which silence genes through a mRNA degradation pathway. Small temporal RNAs (stRNAs) and micro-RNAs (miRNAs) are approximately 21 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. Here we report that synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules can target a gene for silencing, and the mechanism of silencing appears to be through mRNA degradation and not translational repression. The sequence and structural configuration of these RNAs are important, and even slight modification in structure can affect the silencing activity of the hairpins. Furthermore, these RNAs are active when expressed by DNA vectors containing polymerase III promoters, opening the possibility for new approaches in stable RNAi-based loss of function studies.