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Patient-derived xenografts (PDX) model human intra- and intertumoral heterogeneity in the context of the intact tissue of immunocompromised mice. Histologic imaging via hematoxylin and eosin (H&E) staining is routinely performed on PDX samples, which could be harnessed for computational analysis. Prior studies of large clinical H&E image repositories have shown that deep learning analysis can identify intercellular and morphologic signals correlated with disease phenotype and therapeutic response. In this study, we developed an extensive, pan-cancer repository of >1,000 PDX and paired parental tumor H&E images. These images, curated from the PDX Development and Trial Centers Research Network Consortium, had a range of associated genomic and transcriptomic data, clinical metadata, pathologic assessments of cell composition, and, in several cases, detailed pathologic annotations of neoplastic, stromal, and necrotic regions. The amenability of these images to deep learning was highlighted through three applications: (i) development of a classifier for neoplastic, stromal, and necrotic regions; (ii) development of a predictor of xenograft-transplant lymphoproliferative disorder; and (iii) application of a published predictor of microsatellite instability. Together, this PDX Development and Trial Centers Research Network image repository provides a valuable resource for controlled digital pathology analysis, both for the evaluation of technical issues and for the development of computational image-based methods that make clinical predictions based on PDX treatment studies. Significance: A pan-cancer repository of >1,000 patient-derived xenograft hematoxylin and eosin-stained images will facilitate cancer biology investigations through histopathologic analysis and contributes important model system data that expand existing human histology repositories.
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Aprendizaje Profundo , Neoplasias , Humanos , Animales , Ratones , Neoplasias/genética , Neoplasias/patología , Neoplasias/diagnóstico por imagen , Genómica/métodos , Xenoinjertos , Ensayos Antitumor por Modelo de Xenoinjerto , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/patología , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Introduction: Wilms Tumor (WT), or nephroblastoma, is the most common pediatric kidney cancer. Most WTs display a "favorable" triphasic histology, in which the tumor is comprised of blastemal, stromal, and epithelial cell types. Blastemal predominance after neoadjuvant chemotherapy or diffuse anaplasia ("unfavorable" histology; 5-8%) portend a worse prognosis. Blastema likely provide the putative cancer stem cells (CSCs), which retain molecular and histologic features characteristic of nephron progenitor cells (NPCs), within WTs. NPCs arise in the metanephric mesenchyme (MM) and populate the cap mesenchyme (CM) in the developing kidney. WT blastemal cells, like NPCs, similarly express markers, SIX2 and CITED1. Tumor xenotransplantation is currently the only dependable method to propagate tumor tissue for research or therapeutic screening, since efforts to culture tumors in vitro as monolayers have invariably failed. Therefore, a critical need exists to propagate WT stem cells rapidly and efficiently for high-throughput, real-time drug screening. Methods: Previously, our lab developed niche conditions that support the propagation of murine NPCs in culture. Applying similar conditions to WTs, we assessed our ability to maintain key NPC "stemness" markers, SIX2, NCAM, and YAP1, and CSC marker ALDHI in cells from five distinct untreated patient tumors. Results: Accordingly, our culture conditions maintained the expression of these markers in cultured WT cells through multiple passages of rapidly dividing cells. Discussion: These findings suggest that our culture conditions sustain the WT blastemal population, as previously shown for normal NPCs. As a result, we have developed new WT cell lines and a multi-passage in vitro model for studying the blastemal lineage/CSCs in WTs. Furthermore, this system supports growth of heterogeneous WT cells, upon which potential drug therapies could be tested for efficacy and resistance.
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Role of DNA damage and demethylation on anticancer activity of DNA methyltransferase inhibitors (DNMTi) remains undefined. We report the effects of DNMT1 gene deletion/disruption (DNMT1-/-) on anticancer activity of a class of DNMTi in vitro, in vivo and in human cancers. The gene deletion markedly attenuated cytotoxicity and growth inhibition mediated by decitabine, azacitidine and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) in colon and breast cancer cells. The drugs induced DNA damage that concurred with DNMT1 inhibition, subsequent G2/M cell-cycle arrest and apoptosis, and upregulated p21 in DNMT1+/+ versus DNMT1-/- status, with aza-T-dCyd the most potent. Tumor growth and DNMT1 were significantly inhibited, and p21 was upmodulated in mice bearing HCT116 DNMT1+/+ xenograft and bladder PDX tumors. DNMT1 gene deletion occurred in ~ 9% human colon cancers and other cancer types at varying degrees. Decitabine and azacitidine demethylated CDKN2A/CDKN2B genes in DNMT1+/+ and DNMT1-/- conditions and increased histone-H3 acetylation with re-expression of p16INK4A/p15INK4B in DNMT1-/- state. Thus, DNMT1 deletion confers resistance to DNMTi, and their anti-cancer activity is determined by DNA damage effects. Patients with DNMT1 gene deletions may not respond to DNMTi treatment.
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Azacitidina , ADN (Citosina-5-)-Metiltransferasas , Humanos , Ratones , Animales , Decitabina/farmacología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , Azacitidina/farmacología , Daño del ADN , Desmetilación , ADN , Metilación de ADN , Línea Celular TumoralRESUMEN
BACKGROUND: Several new generation CDK4/6 inhibitors have been developed and approved for breast cancer therapy in combination with endocrine therapeutics. Application of these inhibitors either alone or in combination in other solid tumors has been proposed, but no imaging biomarkers of response have been reported in non-breast cancer animal models. The purpose of this study was to evaluate 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) Positron Emission Tomography (PET) as in vivo biomarker of response to palbociclib in a non-breast cancer model. METHODS: Twenty-four NSG mice bearing patient derived xenografts (PDX) of a well-characterized bladder tumor were randomized into 4 treatment groups: vehicle (n = 6); palbociclib (n = 6); temozolomide (n = 6); and palbociclib plus temozolomide (n = 6) and treated with two cycles of therapy or vehicle. Tumor uptake of [18F]FLT was determined by micro-PET/CT at baseline, 3 days, and 9 days post initiation of therapy. Following the second cycle of therapy, the mice were maintained until their tumors reached a size requiring humane termination. RESULTS: [18F]FLT uptake decreased significantly in the palbociclib and combination arms (p = 0.0423 and 0.0106 respectively at day 3 and 0.0012 and 0.0031 at day 9) with stable tumor volume. In the temozolomide arm [18F]FLT uptake increased with day 9 uptake significantly different than baseline (p = 0.0418) and progressive tumor growth was observed during the treatment phase. All groups exhibited progressive disease after day 22, 10 days following cessation of therapy. CONCLUSION: Significant decreases in [18F]FLT uptake as early as three days post initiation of therapy with palbociclib, alone or in combination with temozolomide, in this bladder cancer model correlates with an absence of tumor growth during therapy that persists until day 18 for the palbociclib group and day 22 for the combination group (6 days and 10 days) following cessation of therapy. These results support early modulation of [18F]FLT as an in vivo biomarker predictive of palbociclib therapy response in a non-breast cancer model.
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Didesoxinucleósidos , Neoplasias de la Vejiga Urinaria , Animales , Biomarcadores , Línea Celular Tumoral , Didesoxinucleósidos/metabolismo , Humanos , Ratones , Piperazinas , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Piridinas , Temozolomida/uso terapéutico , Timidina , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Preclinical studies provide valuable data in the early development of novel drugs for patients with cancer. Many cancer treatment regimens now utilize multiple agents with different targets to delay the emergence of drug-resistant tumor cells, and experimental agents are often evaluated in combination with FDA-approved drugs. The Biological Testing Branch (BTB) of the U.S. NCI has evaluated more than 70 FDA-approved oncology drugs to date in human xenograft models. Here, we report the first release of a publicly available, downloadable spreadsheet, ROADMAPS (Responses to Oncology Agents and Dosing in Models to Aid Preclinical Studies, dtp.cancer.gov/databases_tools/roadmaps.htm), that provides data filterable by agent, dose, dosing schedule, route of administration, tumor models tested, responses, host mouse strain, maximum weight loss, drug-related deaths, and vehicle formulation for preclinical experiments conducted by the BTB. Data from 70 different single targeted and cytotoxic agents and 140 different xenograft models were included. Multiple xenograft models were tested in immunocompromised mice for many cancer histologies, with lung cancer as the most broadly tested (24 models). Many of the dose levels and schedules used in these experiments were comparable with those tolerated in humans. Targeted and cytotoxic single agents were included. The online spreadsheet will be updated periodically as additional agent/dose/model combinations are evaluated. ROADMAPS is intended to serve as a publicly available resource for the research community to inform the design of clinically relevant, tolerable single and combinatorial regimens in preclinical mouse models. SIGNIFICANCE: ROADMAPS includes data that can be used to identify tolerable dosing regimens with activity against a variety of human tumors in different mouse strains, providing a resource for planning preclinical studies.
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Antineoplásicos , Neoplasias , Animales , Antineoplásicos/efectos adversos , Humanos , Ratones , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The therapeutic efficacy of temozolomide (TMZ) is hindered by inherent and acquired resistance. Biomarkers such as MGMT expression and MMR proficiency are used as predictors of response. However, not all MGMTlow/-ve/MMRproficient patients benefit from TMZ treatment, indicating a need for additional patient selection criteria. We explored the role of ATR in mediating TMZ resistance and whether ATR inhibitors (ATRi) could reverse this resistance in multiple cancer lines. We observed that only 31% of MGMTlow/-ve/MMRproficient patient-derived and established cancer lines are sensitive to TMZ at clinically relevant concentrations. TMZ treatment resulted in DNA damage signaling in both sensitive and resistant lines, but prolonged G2/M arrest and cell death were exclusive to sensitive models. Inhibition of ATR but not ATM, sensitized the majority of resistant models to TMZ and resulted in measurable DNA damage and persistent growth inhibition. Also, compromised homologous recombination (HR) via RAD51 or BRCA1 loss only conferred sensitivity to TMZ when combined with an ATRi. Furthermore, low REV3L mRNA expression correlated with sensitivity to the TMZ and ATRi combination in vitro and in vivo. This suggests that HR defects and low REV3L levels could be useful selection criteria for enhanced clinical efficacy of an ATRi plus TMZ combination.
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AIM: We developed a generic high-performance liquid chromatography mass spectrometry approach for quantitation of small molecule compounds without availability of isotopically labelled standard. METHODS: The assay utilized 50 µL of plasma and offers 8 potential internal standards (IS): acetaminophen, veliparib, busulfan, neratinib, erlotinib, abiraterone, bicalutamide, and paclitaxel. Preparation consisted of acetonitrile protein precipitation and aqueous dilution in a 96 well-plate format. Chromatographic separation was achieved with a Kinetex C18 reverse phase (2.6 µm, 2 mm x 50 mm) column and a gradient of 0.1 % formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an AB SCIEX4000QTRAP with electrospray, positive-mode ionization. Performance of the generic approach was evaluated with seven drugs (LMP744, olaparib, cabozantinib, triapine, ixabepilone, berzosertib, eribulin) for which validated assays were available. RESULTS: The 8 IS covered a range of polarity, size, and ionization; eluted over the range of chromatographic retention times; were quantitatively extracted; and suffered limited matrix effects. The generic approach proved to be linear for test drugs evaluated over at least 3 orders of magnitude starting at 1-10 ng/mL, with extension of assay ranges with analyte isotopologue MRM channels. At a bias of less than 16 % and precision within 15 %, the assay performance was acceptable. CONCLUSION: The generic approach has become a useful tool to further define the pharmacology of drugs studied in our laboratory and may be utilized as described, or as starting point to develop drug-specific assays with more extensive performance characterization.
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Preparaciones Farmacéuticas , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Técnicas de Dilución del Indicador , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Challenges remain on the selection of patients who potentially respond to a class of drugs that target epigenetics for cancer treatment. This study aims to investigate TET2/DNMT3A mutations and antitumor activity of a novel epigenetic agent in multiple human cancer cell lines and animal models. METHODS: Seventeen cancer cell lines and multiple xenograft models bearing representative human solid tumors were subjected to 4'-thio-2'-deoxycytidine (T-dCyd) or control treatment. Gene mutations in cell lines were examined by whole exome and/or Sanger sequencing. Specific gene expression was measured in cells and xenograft tumor samples by Western blotting and immunohistochemistry. TET2/DNMT3A mutation status in 47,571 human tumor samples was analyzed at cBioPortal for Cancer Genomics. RESULTS: Cell survival was significantly inhibited by T-dCyd in breast BT549, lung NCI-H23, melanoma SKMEL5 and renal ACHN cancer lines harboring deleterious TET2 and nonsynonymous DNMT3A mutations compared to 13 lines without such mutation pattern (P = 0.007). The treatment upregulated p21 and induced cell cycle arrest in NCI-H23 cells, and dramatically inhibited their xenograft tumor growth versus wildtype models. T-dCyd administrations led to a significant p21 increase and near eradication of tumor cells in the double-mutant xenografts by histological evaluation. TET2/DNMT3A was co-mutated in human lung, breast, skin and kidney cancers and frequently in angioimmunoblastic and peripheral T cell lymphomas and several types of leukemia. CONCLUSIONS: Cell and animal models with concurrent mutations in TET2 and DNMT3A were sensitive to T-dCyd treatment. The mutations were detectable in human solid tumors and frequently occur in some hematological malignancies.
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ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Unión al ADN/genética , Desoxicitidina/análogos & derivados , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Tionucleósidos/farmacología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/metabolismo , Desoxicitidina/farmacología , Dioxigenasas , Femenino , Células HCT116 , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia. We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2H-1λ2-quinoline-2,5(6H)-dione screening hit against the R132H and R132C mutant forms of isocitrate dehydrogenase (IDH1). Systematic SAR efforts produced a series of potent pyrid-2-one mIDH1 inhibitors, including the atropisomer (+)-119 (NCATS-SM5637, NSC 791985). In an engineered mIDH1-U87-xenograft mouse model, after a single oral dose of 30 mg/kg, 16 h post dose, between 16 and 48 h, (+)-119 showed higher tumoral concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).
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Inhibidores Enzimáticos/química , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Piridonas/química , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Glicina/análogos & derivados , Glicina/uso terapéutico , Semivida , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Ratones , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Mutagénesis Sitio-Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Piridinas/uso terapéutico , Piridonas/metabolismo , Piridonas/uso terapéutico , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this article, 5-aza-4'-thio-2'-ß-fluoro-2'-deoxycytidine (F-aza-T-dCyd, NSC801845), a novel cytidine analog, is first disclosed and compared with T-dCyd, F-T-dCyd, and aza-T-dCyd in cell culture and mouse xenograft studies in HCT-116 human colon carcinoma, OVCAR3 human ovarian carcinoma, NCI-H23 human NSCLC carcinoma, HL-60 human leukemia, and the PDX BL0382 bladder carcinoma. In three of five xenograft lines (HCT-116, HL-60, and BL-0382), F-aza-T-dCyd was more efficacious than aza-T-dCyd. Comparable activity was observed for these two agents against the NCI-H23 and OVCAR3 xenografts. In the HCT-116 study, F-aza-T-dCyd [10 mg/kg intraperitoneal (i.p.), QDx5 for four cycles], produced complete regression of the tumors in all mice with a response that proved durable beyond postimplant day 150 (129 days after the last dose). Similarly, complete tumor regression was observed in the HL-60 leukemia xenograft when mice were dosed with F-aza-T-dCyd (10 mg/kg i.p., QDx5 for three cycles). In the PDX BL-0382 bladder study, both oral and i.p. dosing of F-aza-T-dCyd (8 mg/kg QDx5 for three cycles) produced regressions that showed tumor regrowth beginning 13 days after dosing. These findings indicate that further development of F-aza-T-dCyd (NSC801845) is warranted. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/20/4/625/F1.large.jpg.
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Citidina/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Técnicas de Cultivo de Célula , Citidina/farmacología , Femenino , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ras/Raf/MEK/ERK (MAPK) and PI3K/AKT signaling pathways influence several cell functions involved in oncogenesis, making them attractive drug targets. We describe a novel multiplex immunoassay to quantitate isoform-specific phosphorylation of proteins in the PI3K/AKT and MAPK pathways as a tool to assess pharmacodynamic changes. Isoform-specific assays measuring total protein and site-specific phosphorylation levels of ERK1/2, MEK1/2, AKT1/2/3, and rpS6 were developed on the Luminex platform with validated antibody reagents. The multiplex assay demonstrated satisfactory analytic performance. Fit-for-purpose validation was performed with xenograft models treated with selected agents. In PC3 and HCC70 xenograft tumors, the PI3Kß inhibitor AZD8186 suppressed phosphorylation of AKT1, AKT2, and rpS6 for 4 to 7 hours post single dose, but levels returned to baseline by 24 hours. AKT3 phosphorylation was suppressed in PC3 xenografts at all doses tested, but only at the highest dose in HCC70. The AKT inhibitor MK-2206 reduced AKT1/2/3 phosphorylation in SW620 xenograft tumors 2 to 4 hours postdose, and the MEK inhibitor selumetinib reduced MEK1/2 and ERK1/2 phosphorylation by up to 50% and >90%, respectively. Clinical utility was demonstrated by analyzing biopsies from untreated patients with plexiform neurofibromas enrolled in a clinical trial of selumetinib (NCT02407405). These biopsies showed MEK and ERK phosphorylation levels sufficient for measuring up to 90% inhibition, and low AKT and rpS6 phosphorylation. This validated multiplex immunoassay demonstrates the degree and duration of phosphorylation modulation for three distinct classes of drugs targeting the PI3K/AKT and MAPK pathways.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones Desnudos , Fosforilación , Isoformas de Proteínas , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mouse kidney parvovirus (MKPV), also known as murine chapparvovirus (MuCPV), is an emerging, highly infectious agent that has been isolated from laboratory and wild mouse populations. In immunocompromised mice, MKPV produces severe chronic interstitial nephropathy and renal failure within 4 to 5 months of infection. However, the course of disease, severity of histologic lesions, and viral shedding are uncertain for immunocompetent mice. We evaluated MKPV infections in CD-1 and Swiss Webster mice, 2 immunocompetent stocks of mice. MKPV-positive CD-1 mice (n = 30) were identified at approximately 8 weeks of age by fecal PCR (polymerase chain reaction) and were subsequently housed individually for clinical observation and diagnostic sampling. Cage swabs, fecal pellets, urine, and blood were evaluated by PCR at 100 and 128 days following the initial positive test, which identified that 28 of 30 were persistently infected and 24 of these were viremic at 100 days. Histologic lesions associated with MKPV in CD-1 (n = 31) and Swiss mice (n = 11) included lymphoplasmacytic tubulointerstitial nephritis with tubular degeneration. Inclusion bodies were rare; however, intralesional MKPV mRNA was consistently detected via in situ hybridization within tubular epithelial cells of the renal cortex and within collecting duct lumina. In immunocompetent CD-1 mice, MKPV infection resulted in persistent shedding of virus for up to 10 months and a mild tubulointerstitial nephritis, raising concerns that this virus could produce study variations in immunocompetent models. Intranuclear inclusions were not a consistent feature of MKPV infection in immunocompetent mice.
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Nefritis Intersticial , Infecciones por Parvoviridae , Parvovirinae , Enfermedades de los Roedores , Animales , Riñón , Ratones , Ratones Endogámicos , Nefritis Intersticial/veterinaria , Infecciones por Parvoviridae/veterinaria , Parvovirinae/patogenicidadRESUMEN
Malignant peripheral nerve sheath tumors (MPNST) frequently overexpress eukaryotic initiation factor 4F components, and the eIF4A inhibitor silvestrol potently suppresses MPNST growth. However, silvestrol has suboptimal drug-like properties, including a bulky structure, poor oral bioavailability (<2%), sensitivity to MDR1 efflux, and pulmonary toxicity in dogs. We compared ten silvestrol-related rocaglates lacking the dioxanyl ring and found that didesmethylrocaglamide (DDR) and rocaglamide (Roc) had growth-inhibitory activity comparable with silvestrol. Structure-activity relationship analysis revealed that the dioxanyl ring present in silvestrol was dispensable for, but may enhance, cytotoxicity. Both DDR and Roc arrested MPNST cells at G2-M, increased the sub-G1 population, induced cleavage of caspases and PARP, and elevated the levels of the DNA-damage response marker γH2A.X, while decreasing the expression of AKT and ERK1/2, consistent with translation inhibition. Unlike silvestrol, DDR and Roc were not sensitive to MDR1 inhibition. Pharmacokinetic analysis confirmed that Roc had 50% oral bioavailability. Importantly, Roc, when administered intraperitoneally or orally, showed potent antitumor effects in an orthotopic MPNST mouse model and did not induce pulmonary toxicity in dogs as found with silvestrol. Treated tumors displayed degenerative changes and had more cleaved caspase-3-positive cells, indicative of increased apoptosis. Furthermore, Roc effectively suppressed the growth of osteosarcoma, Ewing sarcoma, and rhabdomyosarcoma cells and patient-derived xenografts. Both Roc- and DDR-treated sarcoma cells showed decreased levels of multiple oncogenic kinases, including insulin-like growth factor-1 receptor. The more favorable drug-like properties of DDR and Roc and the potent antitumor activity of Roc suggest that these rocaglamides could become viable treatments for MPNST and other sarcomas.
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Antineoplásicos Fitogénicos/farmacología , Benzofuranos/química , Benzofuranos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neurofibrosarcoma/tratamiento farmacológico , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Aglaia/química , Animales , Apoptosis , Caspasa 3/metabolismo , Ciclo Celular , Proliferación Celular , Humanos , Ratones , Neurofibrosarcoma/metabolismo , Neurofibrosarcoma/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The significance of the phenotypic plasticity afforded by epithelial-mesenchymal transition (EMT) for cancer progression and drug resistance remains to be fully elucidated in the clinic. We evaluated epithelial-mesenchymal phenotypic characteristics across a range of tumor histologies using a validated, high-resolution digital microscopic immunofluorescence assay (IFA) that incorporates ß-catenin detection and cellular morphology to delineate carcinoma cells from stromal fibroblasts and that quantitates the individual and colocalized expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (V) at subcellular resolution ("EMT-IFA"). We report the discovery of ß-catenin+ cancer cells that coexpress E-cadherin and vimentin in core-needle biopsies from patients with various advanced metastatic carcinomas, wherein these cells are transitioning between strongly epithelial and strongly mesenchymal-like phenotypes. Treatment of carcinoma models with anticancer drugs that differ in their mechanism of action (the tyrosine kinase inhibitor pazopanib in MKN45 gastric carcinoma xenografts and the combination of tubulin-targeting agent paclitaxel with the BCR-ABL inhibitor nilotinib in MDA-MB-468 breast cancer xenografts) caused changes in the tumor epithelial-mesenchymal character. Moreover, the appearance of partial EMT or mesenchymal-like carcinoma cells in MDA-MB-468 tumors treated with the paclitaxel-nilotinib combination resulted in upregulation of cancer stem cell (CSC) markers and susceptibility to FAK inhibitor. A metastatic prostate cancer patient treated with the PARP inhibitor talazoparib exhibited similar CSC marker upregulation. Therefore, the phenotypic plasticity conferred on carcinoma cells by EMT allows for rapid adaptation to cytotoxic or molecularly targeted therapy and could create a form of acquired drug resistance that is transient in nature. SIGNIFICANCE: Despite the role of EMT in metastasis and drug resistance, no standardized assessment of EMT phenotypic heterogeneity in human carcinomas exists; the EMT-IFA allows for clinical monitoring of tumor adaptation to therapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma/tratamiento farmacológico , Plasticidad de la Célula/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Células Madre Neoplásicas/patología , Animales , Antígenos CD/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/metabolismo , Biopsia con Aguja Gruesa , Cadherinas/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Indazoles , Masculino , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Vimentina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
BACKGROUND: Spontaneously metastatic xenograft models of cancer are infrequent and the few that exist are resource intensive. In xenografts, caliper measurements can be used to determine primary tumor burden and response to therapy but in metastatic disease models determination of the presence of metastatic disease, metastatic burden, and response to therapy are difficult, often requiring serial necropsy. In this study we characterized the development of visceral metastases in a patient derived xenograft model (PDXM) using in vivo imaging. RESULTS: We identified and characterized the previously unreported development of spontaneous liver and bone metastasis in a known patient derived xenograft, bladder xenograft BL0293F, developed by Jackson Laboratories and the University of California at Davis and available from the National Cancer Institute Patient-Derived Models Repository [1]. Among FDG-PET/CT, contrast-enhanced MRI and non-contrast MRI, non-contrast T2w MRI was the most effective and efficient imaging technique. On non-contrast T2 weighted MRI, hepatic metastases were observed in over 70% of animals at 52 days post tumor implantation without resection of the xenograft and in 100% of animals at day 52 following resection of the xenograft. In a group of animals receiving one cycle of effective chemotherapy, no animals demonstrated metastasis by imaging, confirming the utility of this model for therapy evaluation. There was good agreement between pathologic grade and extent of involvement observed on MRI T2w imaging. CONCLUSION: PDX BL0293F is a reliable visceral organ (liver) metastatic model with high penetrance in both non-aggravated and post excisional situations, providing a reliable window for therapy intervention prior to required excision of the xenograft. The imaging characteristics of this model are highly favorable for non-clinical research studies of metastatic disease when used in conjunction with non-contrast T2 weighted MRI.
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Imagenología Tridimensional , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Neoplasias de la Vejiga Urinaria/patología , Animales , Femenino , Humanos , Neoplasias Hepáticas/secundario , Imagen por Resonancia Magnética , Ratones Endogámicos NOD , Metástasis de la Neoplasia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The preclinical antitumor agent RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan, NSC 652287), an analog of the natural product α-terthiophene, failed during the development phase due to acute pulmonary toxicity in animal models. A series of synthetic modifications to RITA's heterocyclic scaffold resulted in activity ranging from broadly cytotoxic to highly selective. In the NCI 60-cell line screen, these "hyperselective" agents (e.g., imatinib) are rare. A selectivity index (SI) was developed to quantify this desirable feature, which is 20 for imatinib, whereas RITA's SI is only 0.10. One of the described hyperselective RITA analogs (SI = 7.9) completely lost activity in the presence of a known SULT1A1 inhibitor. These results, coupled with previous evidence that RITA is a SULT1A1 substrate, suggest that carbinol modification by a sulfate leaving group and subsequent formation of a reactive carbocation may explain RITA's broad cytotoxicity. Although SULT1A1 expression is required for susceptibility, hyperselective analogs exhibited reduced association of activity with SULT1A1 mRNA expression compared with RITA, apparently requiring some additional target(s). In support of this hypothesis, there is a strong correlation (P < 0.01, r = 0.95) between quantum mechanically calculated energy barriers for carbocation formation from sulfonated analogs and SI, indicating that hyperselective RITA analogs generate reactive carbocations less readily after sulfate activation. Importantly, narrowing the cytotoxicity profile of RITA did not eliminate its analogs' in vivo antitumor activity, as several new hyperselective agents, NSC 773097 (1), 773392 (2), and 782846 (6), displayed impressive activity against A498 xenografts in mice.
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Antineoplásicos/farmacología , Furanos/farmacología , Animales , Antineoplásicos/química , Arilsulfotransferasa/genética , Arilsulfotransferasa/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Furanos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Overexpression of myeloid cell leukemia-1 (Mcl-1) in cancers correlates with high tumor grade and poor survival. Additionally, Mcl-1 drives intrinsic and acquired resistance to many cancer therapeutics, including B cell lymphoma 2 family inhibitors, proteasome inhibitors, and antitubulins. Therefore, Mcl-1 inhibition could serve as a strategy to target cancers that require Mcl-1 to evade apoptosis. Herein, we describe the use of structure-based design to discover a novel compound (42) that robustly and specifically inhibits Mcl-1 in cell culture and animal xenograft models. Compound 42 binds to Mcl-1 with picomolar affinity and inhibited growth of Mcl-1-dependent tumor cell lines in the nanomolar range. Compound 42 also inhibited the growth of hematological and triple negative breast cancer xenografts at well-tolerated doses. These findings highlight the use of structure-based design to identify small molecule Mcl-1 inhibitors and support the use of 42 as a potential treatment strategy to block Mcl-1 activity and induce apoptosis in Mcl-1-dependent cancers.
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Antineoplásicos/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Azepinas/química , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Simulación de Dinámica Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We sought to examine the pharmacodynamic activation of the DNA damage response (DDR) pathway in tumors following anticancer treatment for confirmation of target engagement. EXPERIMENTAL DESIGN: We evaluated the time course and spatial activation of 3 protein biomarkers of DNA damage recognition and repair (γH2AX, pS343-Nbs1, and Rad51) simultaneously in a quantitative multiplex immunofluorescence assay (IFA) to assess DDR pathway activation in tumor tissues following exposure to DNA-damaging agents. RESULTS: Because of inherent biological variability, baseline DDR biomarker levels were evaluated in a colorectal cancer microarray to establish clinically relevant thresholds for pharmacodynamic activation. Xenograft-bearing mice and clinical colorectal tumor biopsies obtained from subjects exposed to DNA-damaging therapeutic regimens demonstrated marked intratumor heterogeneity in the timing and extent of DDR biomarker activation due, in part, to the cell-cycle dependency of DNA damage biomarker expression. CONCLUSIONS: We have demonstrated the clinical utility of this DDR multiplex IFA in preclinical models and clinical specimens following exposure to multiple classes of cytotoxic agents, DNA repair protein inhibitors, and molecularly targeted agents, in both homologous recombination-proficient and -deficient contexts. Levels exceeding 4% nuclear area positive (NAP) γH2AX, 4% NAP pS343-Nbs1, and 5% cells with ≥5 Rad51 nuclear foci indicate a DDR activation response to treatment in human colorectal cancer tissue. Determination of effect-level cutoffs allows for robust interpretation of biomarkers with significant interpatient and intratumor heterogeneity; simultaneous assessment of biomarkers induced at different phases of the DDR guards against the risk of false negatives due to an ill-timed biopsy.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Daño del ADN , Animales , Proteínas de Ciclo Celular/metabolismo , Clofarabina/farmacología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Reparación del ADN , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Células HCT116 , Células HT29 , Histonas/metabolismo , Humanos , Ratones , Ratones Desnudos , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Topotecan/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Two new cassaine-type diterpenoids, namely erythrofordins D (1) and E (2), sourced from a Cameroon collection of Erythrophleum suaveolens were isolated and assessed for anti-tumor activity. In the NCI-60 cancer cell assay, erythrofordins D (1) and E (2) were found to be cytotoxic in the low micro molar ranges with a mean GI50 value of 2.45 and 0.71⯵M, mean TGI value of 9.77 and 2.29⯵M, and a mean LC50 of 26.92 and 11.48⯵M for 1 and 2 respectively. Using the COMPARE algorithm, the new compounds were found to have similar NCI-60 response profiles to the known cardiac glycosides hyrcanoside and strophanthin. In addition, in an assay examining the viability and contractile function in human cardiomyocytes derived from induced pluripotent stem-cells, erythrofordins showed cardiotoxicity effects at concentrations as low as 0.03⯵g/mL.