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PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.
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Proteínas de la Ataxia Telangiectasia Mutada , Neoplasias , Animales , Humanos , Ratones , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Mutación con Pérdida de Función , Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.
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Neoplasias , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/radioterapia , Línea Celular TumoralRESUMEN
Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.
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Reparación del ADN por Unión de Extremidades , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Ligandos , ADN/metabolismo , ADN Polimerasa thetaRESUMEN
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Reparación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Mutaciones Letales Sintéticas/efectos de los fármacos , Regulación Alostérica , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleasas/antagonistas & inhibidores , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Recombinación Homóloga/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Organoides/efectos de los fármacos , Neoplasias Ováricas/genética , Ratas , Mutaciones Letales Sintéticas/genética , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , ADN Polimerasa thetaRESUMEN
Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.
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Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma , Proteínas/genética , Animales , Apoptosis , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Unión al ARN/genética , TranscriptomaAsunto(s)
Resistencia a Antineoplásicos , Pruebas Genéticas , Vectores Genéticos/metabolismo , Lentivirus/genética , Melanoma/enzimología , Melanoma/genética , Mutagénesis Insercional/genética , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismoRESUMEN
5-Nitrofurans are antibiotic pro-drugs that have potential as cancer therapeutics. Here, we show that 5-nitrofurans can be bio-activated by aldehyde dehydrogenase (ALDH) 1A1/1A3 enzymes that are highly expressed in a subpopulation of cancer-initiating (stem) cells. We discover that the 5-nitrofuran, nifuroxazide, is selective for bio-activation by ALDH1 isoforms over ALDH2, whereby it both oxidizes ALDH1 and is converted to cytotoxic metabolites in a two-hit pro-drug mechanism. We show that ALDH1High melanoma cells are sensitive to nifuroxazide, while ALDH1A3 loss-of-function mutations confer drug resistance. In tumors, nifuroxazide targets ALDH1High melanoma subpopulations with the subsequent loss of melanoma-initiating cell potential. BRAF and MEK inhibitor therapy increases ALDH1 expression in patient melanomas, and effectively combines with nifuroxazide in melanoma cell models. The selective eradication of ALDH1High cells by nifuroxazide-ALDH1 activation goes beyond current strategies based on inhibiting ALDH1 and provides a rational basis for the nifuroxazide mechanism of action in cancer.
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Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Hidroxibenzoatos/metabolismo , Hidroxibenzoatos/farmacología , Isoenzimas/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/patología , Células Madre Neoplásicas/efectos de los fármacos , Nitrofuranos/metabolismo , Nitrofuranos/farmacología , Retinal-Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Antineoplásicos/química , Línea Celular Tumoral , Hidroxibenzoatos/química , Isoenzimas/antagonistas & inhibidores , Melanoma/genética , Melanoma/metabolismo , Ratones , Estructura Molecular , Células Madre Neoplásicas/patología , Nitrofuranos/química , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Retinal-Deshidrogenasa/antagonistas & inhibidoresRESUMEN
Background: Olfactory receptors (ORs) recognize odorant molecules and activate a signal transduction pathway that ultimately leads to the perception of smell. This process also modulates the apoptotic cycle of olfactory sensory neurons in an olfactory receptor-specific manner. Recent reports indicate that some olfactory receptors are expressed in tissues other than the olfactory epithelium suggesting that they may have pleiotropic roles. Methods: We investigated the expression of 301 olfactory receptor genes in a comprehensive panel of 968 cancer cell lines. Results: Forty-nine per cent of cell lines show expression of at least one olfactory receptor gene. Some receptors display a broad pattern of expression across tumour types, while others were expressed in cell lines from a particular tissue. Additionally, most of the cancer cell lines expressing olfactory receptors express the effectors necessary for OR-mediated signal transduction. Remarkably, among cancer cell lines, OR2C3 is exclusively expressed in melanoma lines. We also confirmed the expression of OR2C3 in human melanomas, but not in normal melanocytes. Conclusions: The pattern of OR2C3 expression is suggestive of a functional role in the development and/or progression of melanoma. Some olfactory receptors may contribute to tumorigenesis.
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Great progress has been made in the treatment of melanoma through use of targeted therapies and immunotherapy. One approach that has not been fully explored is synthetic lethality, which exploits somatically acquired changes, usually driver mutations, to specifically kill tumour cells. We outline the various approaches that may be applied to identify synthetic lethal interactions and define how these interactions may drive drug discovery efforts.
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Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Melanoma/tratamiento farmacológico , Melanoma/genética , Terapia Molecular Dirigida , Mutaciones Letales Sintéticas/genética , HumanosRESUMEN
Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting 'off-target' sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools.
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Variaciones en el Número de Copia de ADN/genética , Exoma/genética , Genoma Humano , Algoritmos , Exones/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ADNRESUMEN
We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia.
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Terapia Genética , Hemofilia B/terapia , Lentivirus/genética , Hígado/patología , Animales , Coagulación Sanguínea , Modelos Animales de Enfermedad , Perros , Femenino , Vectores Genéticos/metabolismo , Ratones Endogámicos C57BL , Mutágenos/toxicidad , Factores de Tiempo , Transducción Genética , TransgenesAsunto(s)
GTP Fosfohidrolasas/metabolismo , Melanoma/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Mutación/genética , Neurofibromina 1/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Línea Celular Tumoral , Análisis Mutacional de ADN , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Melanoma/genética , Piridonas/farmacología , Pirimidinonas/farmacologíaRESUMEN
Deep-sequencing of a primary tumor and metastasis from a single patient, and functional validation in culture, reveals that TGFBR2 and FGFR2 act as drivers of gastric cancer.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Análisis de Secuencia de ADN/métodos , Neoplasias Gástricas/genética , Progresión de la Enfermedad , Amplificación de Genes , Humanos , Metástasis de la Neoplasia , Receptor Tipo II de Factor de Crecimiento Transformador beta , Eliminación de Secuencia , Neoplasias Gástricas/patologíaRESUMEN
The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2(+) breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors.
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Neoplasias de la Mama/genética , Resistencia a Antineoplásicos , Mutagénesis Insercional/métodos , Neoplasias Pancreáticas/genética , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Clorhidrato de Erlotinib , Femenino , Vectores Genéticos/genética , Humanos , Lapatinib , Lentivirus/genética , Terapia Molecular Dirigida , Fosfatidilinositol 3-Quinasas/genética , PronósticoRESUMEN
A new study describes a tool, Lentihop, for somatic insertional mutagenesis in human cells and uses this system in combination with cancer genome data to define new genes and pathways involved in sarcoma development. Gene discovery in this way suggests that we are far from a complete catalog of cancer drivers.
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Mutagénesis Insercional , Sarcoma/genética , HumanosRESUMEN
Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.
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Carcinogénesis/genética , Terapia Genética , Vectores Genéticos/efectos adversos , Lentivirus/genética , Vectores Genéticos/uso terapéutico , Humanos , Lentivirus/patogenicidad , Mutagénesis Insercional/genética , Regiones Promotoras GenéticasRESUMEN
Insertional mutagenesis has been used as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. Retroviruses have been used for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using retrovirus-based approaches. Most recently, lentiviral vectors have appeared on the scene for use in cancer gene screens. Lentiviral vectors are replication-defective integrating vectors that have the advantage of being able to infect nondividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations, and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future.
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Genes Relacionados con las Neoplasias , Vectores Genéticos , Mutagénesis Insercional/métodos , Neoplasias/genética , Animales , Elementos Transponibles de ADN , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Ratones , Especificidad de Órganos , Proto-Oncogenes , Retroviridae/genéticaRESUMEN
Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.
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Carcinoma Hepatocelular/genética , Lentivirus/genética , Neoplasias Hepáticas/genética , Mutagénesis Insercional , Oncogenes , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Vectores Genéticos , Humanos , Ratones , Fosfohidrolasa PTEN/deficiencia , Prealbúmina/genética , Receptor de Interferón alfa y beta/deficienciaRESUMEN
Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.