Asunto(s)
Benzamidas/uso terapéutico , Indazoles/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasa de Linfoma Anaplásico/fisiología , Cromosomas Humanos Par 2/genética , Citocinas/genética , Variación Genética , Células Germinativas , Humanos , Lactante , Masculino , Neuroblastoma/genética , Neuroblastoma/secundario , Receptor trkA/fisiologíaRESUMEN
PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.
RESUMEN
Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.
Asunto(s)
NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/antagonistas & inhibidores , Animales , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Enzimas Desubicuitinizantes/metabolismo , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones Desnudos , Fenotipo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Especificidad por Sustrato/efectos de los fármacos , Ubiquitina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.
Asunto(s)
Antineoplásicos/farmacología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Ratones , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neuroblastoma/metabolismo , Prostaglandina-E Sintasas/metabolismoRESUMEN
Despite recent progress in diagnosis and treatment, survival for children with high-risk metastatic neuroblastoma is still poor. Prostaglandin E2 (PGE2)-driven inflammation promotes tumor growth, immune suppression, angiogenesis and resistance to established cancer therapies. In neuroblastoma, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment are the primary source of PGE2. However, clinical targeting of PGE2 with current non-steroidal anti-inflammatory drugs or cyclooxygenase inhibitors has been limited due to risk of adverse side effects. By specifically targeting microsomal prostaglandin E synthase-1 (mPGES-1) activity with a small molecule inhibitor we could block CAF-derived PGE2 production leading to reduced tumor growth, impaired angiogenesis, inhibited CAF migration and infiltration, reduced tumor cell proliferation and a favorable shift in the M1/M2 macrophage ratio. In this study, we provide proof-of-principle of the benefits of targeting mPGES-1 in neuroblastoma, applicable to a wide variety of tumors. This non-toxic single drug treatment targeting infiltrating stromal cells opens up for combination treatment options with established cancer therapies.
Asunto(s)
Inflamación/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neuroblastoma/tratamiento farmacológico , Prostaglandina-E Sintasas/genética , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/administración & dosificación , Dinoprostona/genética , Dinoprostona/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Inflamación/genética , Inflamación/patología , Microsomas/efectos de los fármacos , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neuroblastoma/genética , Neuroblastoma/patología , Prostaglandina-E Sintasas/antagonistas & inhibidores , Microambiente Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: The TH-MYCN transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies. METHODS: We followed tumor development in 395 TH-MYCN (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of MYCN in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations. RESULTS: DNA sequencing confirmed presence of human MYCN cDNA in genomic TH-MYCN DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6-19 weeks (median 9.1) and homozygous mice at 4.0-6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17. CONCLUSION: Hemizygous and homozygous TH-MYCN mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-MYCN tumors have few genetic aberrations.