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Neuroblastoma is a peripheral nervous system tumor that almost exclusively occurs in young children. Although intensified treatment modalities have led to increased patient survival, the prognosis for patients with high-risk disease is still around 50%, signifying neuroblastoma as a leading cause of cancer-related deaths in children. Neuroblastoma is an embryonal tumor and is shaped by its origin from cells within the neural crest. Hence, neuroblastoma usually presents with a low mutational burden and is, in the majority of cases, driven by epigenetically deregulated transcription networks. The recent development of Omic techniques has given us detailed knowledge of neuroblastoma evolution, heterogeneity, and plasticity, as well as intra- and intercellular molecular communication networks within the neuroblastoma microenvironment. Here, we discuss the potential of these recent discoveries with emphasis on new treatment modalities, including immunotherapies which hold promise for better future treatment regimens.
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Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the BM. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-Seq to characterize immune and tumor cell alterations in neuroblastoma BM metastases by comparative analysis with patients without metastases. Our results reveal remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the BM metastatic niche, we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of Tregs and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and we identified prognostic markers in malignant cells that are associated with worse clinical outcomes in 3 independent neuroblastoma cohorts. Our results provide insight into the cellular, compositional, and transcriptional shifts underlying neuroblastoma BM metastases that contribute to the development of new therapeutic strategies.
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Médula Ósea , Neuroblastoma , Humanos , Niño , Médula Ósea/patología , Neuroblastoma/genética , Análisis de la Célula Individual , Microambiente TumoralRESUMEN
Neuroblastoma is a neural crest-derived tumor of the peripheral nervous system that is a leading cause of cancer-related deaths in children [...].
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BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.
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Carcinoma de Células Renales , Humanos , Carcinoma de Células Renales/genética , Células del Estroma/patología , Transducción de Señal , Perfilación de la Expresión Génica , Análisis de la Célula Individual , Microambiente TumoralRESUMEN
Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.
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Metiltransferasas , Neuroblastoma , ARN Largo no Codificante , Humanos , Adenina/análogos & derivados , Metiltransferasas/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Estructuras R-Loop , ARN Largo no Codificante/metabolismo , Telómero/genética , Homeostasis del TelómeroRESUMEN
Deregulation of the MYC family of transcription factors c-MYC (encoded by MYC), MYCN, and MYCL is prevalent in most human cancers, with an impact on tumor initiation and progression, as well as response to therapy. In neuroblastoma (NB), amplification of the MYCN oncogene and over-expression of MYC characterize approximately 40% and 10% of all high-risk NB cases, respectively. However, the mechanism and stage of neural crest development in which MYCN and c-MYC contribute to the onset and/or progression of NB are not yet fully understood. Here, we hypothesized that subtle differences in the expression of MYCN and/or c-MYC targets could more accurately stratify NB patients in different risk groups rather than using the expression of either MYC gene alone. We employed an integrative approach using the transcriptome of 498 NB patients from the SEQC cohort and previously defined c-MYC and MYCN target genes to model a multigene transcriptional risk score. Our findings demonstrate that defined sets of c-MYC and MYCN targets with significant prognostic value, effectively stratify NB patients into different groups with varying overall survival probabilities. In particular, patients exhibiting a high-risk signature score present unfavorable clinical parameters, including increased clinical risk, higher INSS stage, MYCN amplification, and disease progression. Notably, target genes with prognostic value differ between c-MYC and MYCN, exhibiting distinct expression patterns in the developing sympathoadrenal system. Genes associated with poor outcomes are mainly found in sympathoblasts rather than in chromaffin cells during the sympathoadrenal development.
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BACKGROUND: p53 mutants contribute to the chronic inflammatory tumour microenvironment (TME). In this study, we address the mechanism of how p53 mutants lead to chronic inflammation in tumours and how to transform it to restore cancer immune surveillance. METHODS: Our analysis of RNA-seq data from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) project revealed that mutant p53 (mtp53) cancers correlated with chronic inflammation. We used cell-based assays and a mouse model to discover a novel gain of function of mtp53 and the effect of the mtp53 reactivating compound APR-246 on the anti-tumour immune response. RESULTS: We found that tumour samples from patients with breast carcinoma carrying mtp53 showed elevated Interferon (IFN) signalling, Tumour Inflammation Signature (TIS) score and infiltration of CD8+ T cells compared to wild type p53 (wtp53) tumours. We showed that the expression of IFN and immune checkpoints were elevated in tumour cells in a mtp53-dependent manner, suggesting a novel gain of function. Restoration of wt function to mtp53 by APR-246 induced the expression of endogenous retroviruses, IFN signalling and repressed immune checkpoints. Moreover, APR-246 promoted CD4+ T cells infiltration and IFN signalling and prevented CD8+ T cells exhaustion within the TME in vivo. CONCLUSIONS: Breast carcinomas with mtp53 displayed enhanced inflammation. APR-246 boosted the interferon response or represses immune checkpoints in p53 mutant tumour cells, and restores cancer immune surveillance in vivo.
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Neoplasias , Proteína p53 Supresora de Tumor , Ratones , Animales , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Mutación con Ganancia de Función , Neoplasias/genética , Interferones/genética , Interferones/metabolismo , Inflamación/genética , Microambiente Tumoral/genéticaRESUMEN
Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.
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Neuroblastoma , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Animales , Dihidroorotato Deshidrogenasa , Humanos , Ratones , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pronóstico , TemozolomidaRESUMEN
Understanding the complete immune cell composition of human neuroblastoma (NB) is crucial for the development of immunotherapeutics. Here, we perform single-cell RNA sequencing (scRNA-seq) on 19 human NB samples coupled with multiplex immunohistochemistry, survival analysis, and comparison with normal fetal adrenal gland data. We provide a comprehensive immune cell landscape and characterize cell-state changes from normal tissue to NB. Our analysis reveals 27 immune cell subtypes, including distinct subpopulations of myeloid, NK, B, and T cells. Several different cell types demonstrate a survival benefit. In contrast to adult cancers and previous NB studies, we show an increase in inflammatory monocyte cell state when contrasting normal and tumor tissue, while no differences in cytotoxicity and exhaustion score for T cells, nor in Treg activity, are observed. Our receptor-ligand interaction analysis reveals a highly complex interactive network of the NB microenvironment from which we highlight several interactions that we suggest for future therapeutic studies.
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Neuroblastoma , Adulto , Humanos , Inmunohistoquímica , Neuroblastoma/genética , Microambiente Tumoral/genéticaRESUMEN
PURPOSE OF REVIEW: The evolving information of the initiation, tumor cell heterogeneity, and plasticity of childhood neuroblastoma has opened up new perspectives for developing therapies based on detailed knowledge of the disease. RECENT FINDINGS: The cellular origin of neuroblastoma has begun to unravel and there have been several reports on tumor cell heterogeneity based on transcriptional core regulatory circuitries that have given us important information on the biology of neuroblastoma as a developmental disease. This together with new insight of the tumor microenvironment which acts as a support for neuroblastoma growth has given us the prospect for designing better treatment approaches for patients with high-risk neuroblastoma. Here, we discuss these new discoveries and highlight some emerging therapeutic options. Neuroblastoma is a disease with multiple facets. Detailed biological and molecular knowledge on neuroblastoma initiation, heterogeneity, and the communications between cells in the tumor microenvironment holds promise for better therapies.
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Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/terapia , Microambiente TumoralRESUMEN
Merkel cell polyomavirus (MCPyV) is a causal factor in Merkel cell carcinoma (MCC). The oncogenic potential is mediated through its viral oncoproteins large T-antigen (LT) and small T-antigen (sT). Cytokines produced by tumor cells play an important role in cancer pathogenesis, and viruses affect their expression. Therefore, we compared human cytokine and receptor transcript levels in virus positive (V+) and virus negative (V-) MCC cell lines. Increased expression of IL-33, a potent modulator of tumor microenvironment, was observed in V+ MCC cell lines when compared to V- MCC-13 cells. Transient transfection studies with luciferase reporter plasmids demonstrated that LT and sT stimulated IL-33, ST2/IL1RL1 and IL1RAcP promoter activity. The induction of IL-33 expression was confirmed by transfecting MCC-13 cells with MCPyV LT. Furthermore, recombinant human cytokine domain IL-33 induced activation of MAP kinase and NF-κB pathways, which could be blocked by a ST2 receptor antibody. Immunohistochemical analysis demonstrated a significantly stronger IL-33, ST2, and IL1RAcP expression in MCC tissues compared to normal skin. Of interest, significantly higher IL-33 and IL1RAcP protein levels were observed in MCC patient plasma compared to plasma from healthy controls. Previous studies have demonstrated the implication of the IL-33/STL2 pathway in cancer. Because our results revealed a T-antigens-dependent induction of the IL-33/ST2 axis, IL-33/ST2 may play a role in the tumorigenesis of MCPyV-positive MCC. Therefore, neutralizing the IL-33/ST2 axis may present a novel therapeutic approach for MCC patients.
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Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Neoplasias Cutáneas , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Carcinogénesis , Carcinoma de Células de Merkel/patología , Citocinas/metabolismo , Humanos , Proteína 1 Similar al Receptor de Interleucina-1/genética , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Poliomavirus de Células de Merkel/fisiología , Neoplasias Cutáneas/patología , Microambiente TumoralRESUMEN
Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.
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Neoplasias de la Mama , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mama/patología , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Femenino , Humanos , Recurrencia Local de Neoplasia , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
AIMS: Medulloblastoma (MB) is one of the most common malignant central nervous system tumors of childhood. Despite intensive treatments that often leads to severe neurological sequelae, the risk for resistant relapses remains significant. In this study we have evaluated the effects of the ω3-long chain polyunsaturated fatty acids (ω3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model. MAIN METHODS: Effects of ω3-LCPUFA treatment of MB cells were assessed using the following: WST-1 assay, cell death probes, clonogenic assay, ELISA and western blot. MB cells were implanted into nude mice and the mice were randomized to DHA, or a combination of DHA and EPA treatment, or to control group. Treatment effects in tumor tissues were evaluated with: LC-MS/MS, RNA-sequencing and immunohistochemistry, and tumors, erythrocytes and brain tissues were analyzed with gas chromatography. KEY FINDINGS: ω3-LCPUFA decreased prostaglandin E2 (PGE2) secretion from MB cells, and impaired MB cell viability and colony forming ability and increased apoptosis in a dose-dependent manner. DHA reduced tumor growth in vivo, and both PGE2 and prostacyclin were significantly decreased in tumor tissue from treated mice compared to control animals. All ω3-LCPUFA and dihomo-γ-linolenic acid increased in tumors from treated mice. RNA-sequencing revealed 10 downregulated genes in common among ω3-LCPUFA treated tumors. CRYAB was the most significantly altered gene and the downregulation was confirmed by immunohistochemistry. SIGNIFICANCE: Our findings suggest that addition of DHA and EPA to the standard MB treatment regimen might be a novel approach to target inflammation in the tumor microenvironment.
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Ácidos Grasos Omega-3/farmacología , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinogénesis , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida/métodos , Dinoprostona/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Ácidos Grasos/metabolismo , Ácidos Grasos Omega-3/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Prostaglandinas/metabolismo , Espectrometría de Masas en Tándem/métodos , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Cadena B de alfa-Cristalina/efectos de los fármacos , Cadena B de alfa-Cristalina/metabolismoRESUMEN
BACKGROUND: Transgenic animal models are crucial for the study of gene function and disease, and are widely utilized in basic biological research, agriculture and pharma industries. Since the current methods for generating transgenic animals result in the random integration of the transgene under study, the phenotype may be compromised due to disruption of known genes or regulatory regions. Unfortunately, most of the tools that predict transgene insertion sites from high-throughput data are not publicly available or not properly maintained. RESULTS: We implemented TC-hunter, Transgene-Construct hunter, an open tool that identifies transgene insertion sites and provides simple reports and visualization aids. It relies on common tools used in the analysis of high-throughput data and makes use of chimeric reads and discordant read pairs to identify and support the transgenic insertion site. To demonstrate its applicability, we applied TC-hunter to four transgenic mice samples harboring the human PPM1D gene, a model used in the study of malignant tumor development. We identified the transgenic insertion site in each sample and experimentally validated them with Touchdown-polymerase chain reaction followed by Sanger sequencing. CONCLUSIONS: TC-hunter is an accessible bioinformatics tool that can automatically identify transgene insertion sites from DNA sequencing data with high sensitivity (98%) and precision (92.45%). TC-hunter is a valuable tool that can aid in evaluating any potential phenotypic complications due to the random integration of the transgene and can be accessed at https://github.com/bcfgothenburg/SSF .
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Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Secuencia de Bases , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ratones , Ratones Transgénicos , TransgenesAsunto(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
Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered "undruggable," and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection of patient-derived primary glioblastoma and acute myeloid leukemia (AML) ex vivo cultures. Importantly, a variety of normal cells become G1 arrested without signs of apoptosis upon MYCMI-7 treatment. Finally, in mouse tumor models of MYC-driven AML, breast cancer, and MYCN-amplified neuroblastoma, treatment with MYCMI-7 downregulates MYC/MYCN, inhibits tumor growth, and prolongs survival through apoptosis with few side effects. In conclusion, MYCMI-7 is a potent and selective MYC inhibitor that is highly relevant for the development into clinically useful drugs for the treatment of MYC-driven cancer. Significance: Our findings demonstrate that the small-molecule MYCMI-7 binds MYC and inhibits interaction between MYC and MAX, thereby hampering MYC-driven tumor cell growth in culture and in vivo while sparing normal cells.
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Neuroblastoma , Animales , Ratones , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Línea Celular Tumoral , Neuroblastoma/tratamiento farmacológico , Proliferación Celular , Ciclo CelularRESUMEN
Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.
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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.
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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.
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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 repression of repetitive elements is an important facet of p53's function as a guardian of the genome. Paradoxically, we found that p53 activated by MDM2 inhibitors induced the expression of endogenous retroviruses (ERV) via increased occupancy on ERV promoters and inhibition of two major ERV repressors, histone demethylase LSD1 and DNA methyltransferase DNMT1. Double-stranded RNA stress caused by ERVs triggered type I/III interferon expression and antigen processing and presentation. Pharmacologic activation of p53 in vivo unleashed the IFN program, promoted T-cell infiltration, and significantly enhanced the efficacy of checkpoint therapy in an allograft tumor model. Furthermore, the MDM2 inhibitor ALRN-6924 induced a viral mimicry pathway and tumor inflammation signature genes in patients with melanoma. Our results identify ERV expression as the central mechanism whereby p53 induction overcomes tumor immune evasion and transforms tumor microenvironment to a favorable phenotype, providing a rationale for the synergy of MDM2 inhibitors and immunotherapy. SIGNIFICANCE: We found that p53 activated by MDM2 inhibitors induced the expression of ERVs, in part via epigenetic factors LSD1 and DNMT1. Induction of IFN response caused by ERV derepression upon p53-targeting therapies provides a possibility to overcome resistance to immune checkpoint blockade and potentially transform "cold" tumors into "hot." This article is highlighted in the In This Issue feature, p. 2945.