RESUMEN
Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Neoplasias Colorrectales/genética , Difenilamina/análogos & derivados , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Evolución Clonal , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Difenilamina/farmacología , Difenilamina/uso terapéutico , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mutación , RetroviridaeRESUMEN
Human papillomavirus (HPV) causes 5% of all cancers and frequently integrates into host chromosomes. The HPV oncoproteins E6 and E7 are necessary but insufficient for cancer formation, indicating that additional secondary genetic events are required. Here, we investigate potential oncogenic impacts of virus integration. Analysis of 105 HPV-positive oropharyngeal cancers by whole-genome sequencing detects virus integration in 77%, revealing five statistically significant sites of recurrent integration near genes that regulate epithelial stem cell maintenance (i.e., SOX2, TP63, FGFR, MYC) and immune evasion (i.e., CD274). Genomic copy number hyperamplification is enriched 16-fold near HPV integrants, and the extent of focal host genomic instability increases with their local density. The frequency of genes expressed at extreme outlier levels is increased 86-fold within ±150 kb of integrants. Across 95% of tumors with integration, host gene transcription is disrupted via intragenic integrants, chimeric transcription, outlier expression, gene breaking, and/or de novo expression of noncoding or imprinted genes. We conclude that virus integration can contribute to carcinogenesis in a large majority of HPV-positive oropharyngeal cancers by inducing extensive disruption of host genome structure and gene expression.
Asunto(s)
Alphapapillomavirus , Proteínas Oncogénicas Virales , Neoplasias Orofaríngeas , Alphapapillomavirus/metabolismo , Carcinogénesis , Humanos , Proteínas Oncogénicas Virales/genética , Neoplasias Orofaríngeas/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Integración Viral/genéticaRESUMEN
Human papillomavirus (HPV) is a necessary but insufficient cause of a subset of oral squamous cell carcinomas (OSCCs) that is increasing markedly in frequency. To identify contributory, secondary genetic alterations in these cancers, we used comprehensive genomics methods to compare 149 HPV-positive and 335 HPV-negative OSCC tumor/normal pairs. Different behavioral risk factors underlying the two OSCC types were reflected in distinctive genomic mutational signatures. In HPV-positive OSCCs, the signatures of APOBEC cytosine deaminase editing, associated with anti-viral immunity, were strongly linked to overall mutational burden. In contrast, in HPV-negative OSCCs, T>C substitutions in the sequence context 5'-ATN-3' correlated with tobacco exposure. Universal expression of HPV E6*1 and E7 oncogenes was a sine qua non of HPV-positive OSCCs. Significant enrichment of somatic mutations was confirmed or newly identified in PIK3CA, KMT2D, FGFR3, FBXW7, DDX3X, PTEN, TRAF3, RB1, CYLD, RIPK4, ZNF750, EP300, CASZ1, TAF5, RBL1, IFNGR1, and NFKBIA Of these, many affect host pathways already targeted by HPV oncoproteins, including the p53 and pRB pathways, or disrupt host defenses against viral infections, including interferon (IFN) and nuclear factor kappa B signaling. Frequent copy number changes were associated with concordant changes in gene expression. Chr 11q (including CCND1) and 14q (including DICER1 and AKT1) were recurrently lost in HPV-positive OSCCs, in contrast to their gains in HPV-negative OSCCs. High-ranking variant allele fractions implicated ZNF750, PIK3CA, and EP300 mutations as candidate driver events in HPV-positive cancers. We conclude that virus-host interactions cooperatively shape the unique genetic features of these cancers, distinguishing them from their HPV-negative counterparts.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de la Boca , Proteínas de Neoplasias , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Femenino , Humanos , Masculino , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Neoplasias de la Boca/virología , Mutación , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Papillomaviridae/metabolismoRESUMEN
Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression.
Asunto(s)
Regulación Viral de la Expresión Génica , Genes Virales , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidad , Factores de Transcripción/metabolismo , Integración Viral/fisiología , Células Cultivadas , Elementos de Facilitación Genéticos , Células HCT116 , Células HeLa , Células Hep G2 , Interacciones Huésped-Patógeno/genética , Células Endoteliales de la Vena Umbilical Humana , Papillomavirus Humano 16/metabolismo , Humanos , Células K562 , Virus Oncogénicos/genética , Virus Oncogénicos/patogenicidad , Papillomaviridae/genética , Papillomaviridae/metabolismo , Papillomaviridae/patogenicidad , Unión Proteica , Multimerización de Proteína , Regulación hacia Arriba/genéticaRESUMEN
Mutations that deregulate Notch1 and Ras/phosphoinositide 3 kinase (PI3K)/Akt signalling are prevalent in T-cell acute lymphoblastic leukaemia (T-ALL), and often coexist. Here we show that the PI3K inhibitor GDC-0941 is active against primary T-ALLs from wild-type and Kras(G12D) mice, and addition of the MEK inhibitor PD0325901 increases its efficacy. Mice invariably relapsed after treatment with drug-resistant clones, most of which unexpectedly had reduced levels of activated Notch1 protein, downregulated many Notch1 target genes, and exhibited cross-resistance to γ-secretase inhibitors. Multiple resistant primary T-ALLs that emerged in vivo did not contain somatic Notch1 mutations present in the parental leukaemia. Importantly, resistant clones upregulated PI3K signalling. Consistent with these data, inhibiting Notch1 activated the PI3K pathway, providing a likely mechanism for selection against oncogenic Notch1 signalling. These studies validate PI3K as a therapeutic target in T-ALL and raise the unexpected possibility that dual inhibition of PI3K and Notch1 signalling could promote drug resistance in T-ALL.
Asunto(s)
Resistencia a Antineoplásicos , Indazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptor Notch1/metabolismo , Sulfonamidas/farmacología , Animales , Benzamidas/farmacología , Benzamidas/uso terapéutico , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Células Clonales/patología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Difenilamina/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Genes ras/genética , Indazoles/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Notch1/química , Receptor Notch1/deficiencia , Receptor Notch1/genética , Transducción de Señal/efectos de los fármacos , Sulfonamidas/uso terapéuticoRESUMEN
RATIONALE: Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr+/- mice after a single systemic delivery of recombinant adeno-associated virus. OBJECTIVE: To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. METHODS AND RESULTS: Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat-associated 9 from Staphylococcus aureus) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr+/- neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. CONCLUSIONS: This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Cardiomiopatías/terapia , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Distrofina/genética , Edición Génica/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Contracción Miocárdica , Músculos Papilares/metabolismo , Animales , Proteínas Asociadas a CRISPR/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Exones , Fibrosis , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Vectores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Mutación , Músculos Papilares/patología , Músculos Papilares/fisiopatología , Fenotipo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Recuperación de la Función , Utrofina/genéticaRESUMEN
Two African apes are the closest living relatives of humans: the chimpanzee (Pan troglodytes) and the bonobo (Pan paniscus). Although they are similar in many respects, bonobos and chimpanzees differ strikingly in key social and sexual behaviours, and for some of these traits they show more similarity with humans than with each other. Here we report the sequencing and assembly of the bonobo genome to study its evolutionary relationship with the chimpanzee and human genomes. We find that more than three per cent of the human genome is more closely related to either the bonobo or the chimpanzee genome than these are to each other. These regions allow various aspects of the ancestry of the two ape species to be reconstructed. In addition, many of the regions that overlap genes may eventually help us understand the genetic basis of phenotypes that humans share with one of the two apes to the exclusion of the other.
Asunto(s)
Evolución Molecular , Variación Genética/genética , Genoma Humano/genética , Genoma/genética , Pan paniscus/genética , Pan troglodytes/genética , Animales , Elementos Transponibles de ADN/genética , Duplicación de Gen/genética , Genotipo , Humanos , Datos de Secuencia Molecular , Fenotipo , Filogenia , Especificidad de la EspecieRESUMEN
Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and in most children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system. The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour. Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies.
Asunto(s)
Evolución Clonal/genética , Meduloblastoma/genética , Meduloblastoma/patología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Animales , Islas de CpG/genética , Metilación de ADN , Elementos Transponibles de ADN/genética , Modelos Animales de Enfermedad , Genes p53/genética , Mutación de Línea Germinal/genética , Humanos , Síndrome de Li-Fraumeni/complicaciones , Síndrome de Li-Fraumeni/genética , Meduloblastoma/complicaciones , Ratones , Mutagénesis Insercional , Tasa de SupervivenciaRESUMEN
The [A] allele of SNP rs965513 in 9q22 has been consistently shown to be highly associated with increased papillary thyroid cancer (PTC) risk with an odds ratio of â¼1.8 as determined by genome-wide association studies, yet the molecular mechanisms remain poorly understood. Previously, we noted that the expression of two genes in the region, forkhead box E1 (FOXE1) and PTC susceptibility candidate 2 (PTCSC2), is regulated by rs965513 in unaffected thyroid tissue, but the underlying mechanisms were not elucidated. Here, we fine-mapped the 9q22 region in PTC and controls and detected an â¼33-kb linkage disequilibrium block (containing the lead SNP rs965513) that significantly associates with PTC risk. Chromatin characteristics and regulatory element signatures in this block disclosed at least three regulatory elements functioning as enhancers. These enhancers harbor at least four SNPs (rs7864322, rs12352658, rs7847449, and rs10759944) that serve as functional variants. The variant genotypes are associated with differential enhancer activities and/or transcription factor binding activities. Using the chromosome conformation capture methodology, long-range looping interactions of these elements with the promoter region shared by FOXE1 and PTCSC2 in a human papillary thyroid carcinoma cell line (KTC-1) and unaffected thyroid tissue were found. Our results suggest that multiple variants coinherited with the lead SNP and located in long-range enhancers are involved in the transcriptional regulation of FOXE1 and PTCSC2 expression. These results explain the mechanism by which the risk allele of rs965513 predisposes to thyroid cancer.
Asunto(s)
Carcinoma/genética , Elementos de Facilitación Genéticos , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , Neoplasias de la Tiroides/genética , Alelos , Carcinoma Papilar , Línea Celular Tumoral , Cromatina/química , Inmunoprecipitación de Cromatina , Factores de Transcripción Forkhead/genética , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos , Histonas/química , Humanos , Oportunidad Relativa , Penetrancia , Cáncer Papilar TiroideoRESUMEN
Genomic instability is a hallmark of human cancers, including the 5% caused by human papillomavirus (HPV). Here we report a striking association between HPV integration and adjacent host genomic structural variation in human cancer cell lines and primary tumors. Whole-genome sequencing revealed HPV integrants flanking and bridging extensive host genomic amplifications and rearrangements, including deletions, inversions, and chromosomal translocations. We present a model of "looping" by which HPV integrant-mediated DNA replication and recombination may result in viral-host DNA concatemers, frequently disrupting genes involved in oncogenesis and amplifying HPV oncogenes E6 and E7. Our high-resolution results shed new light on a catastrophic process, distinct from chromothripsis and other mutational processes, by which HPV directly promotes genomic instability.
Asunto(s)
Replicación del ADN/genética , Inestabilidad Genómica , Papillomavirus Humano 16/genética , Neoplasias/genética , ADN Viral/genética , Femenino , Papillomavirus Humano 16/crecimiento & desarrollo , Humanos , Masculino , Neoplasias/clasificación , Neoplasias/patología , Neoplasias/virología , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Integración Viral/genéticaRESUMEN
Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition.
Asunto(s)
Elementos de Nucleótido Esparcido Largo , Regiones Promotoras Genéticas , ARN sin Sentido/biosíntesis , Proteínas de Unión al ARN/genética , Animales , Secuencia de Bases , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , ARN Polimerasa II/metabolismo , Ribonucleasa III/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Endogenous retrotransposons have caused extensive genomic variation within mammalian species, but the functional implications of such mobilization are mostly unknown. We mapped thousands of endogenous retrovirus (ERV) germline integrants in highly divergent, previously unsequenced mouse lineages, facilitating a comparison of gene expression in the presence or absence of local insertions. Polymorphic ERVs occur relatively infrequently in gene introns and are particularly depleted from genes involved in embryogenesis or that are highly expressed in embryonic stem cells. Their genomic distribution implies ongoing negative selection due to deleterious effects on gene expression and function. A polymorphic, intronic ERV at Slc15a2 triggers up to 49-fold increases in premature transcriptional termination and up to 39-fold reductions in full-length transcripts in adult mouse tissues, thereby disrupting protein expression and functional activity. Prematurely truncated transcripts also occur at Polr1a, Spon1, and up to â¼5% of other genes when intronic ERV polymorphisms are present. Analysis of expression quantitative trait loci (eQTLs) in recombinant BxD mouse strains demonstrated very strong genetic associations between the polymorphic ERV in cis and disrupted transcript levels. Premature polyadenylation is triggered at genomic distances up to >12.5 kb upstream of the ERV, both in cis and between alleles. The parent of origin of the ERV is associated with variable expression of nonterminated transcripts and differential DNA methylation at its 5'-long terminal repeat. This study defines an unexpectedly strong functional impact of ERVs in disrupting gene transcription at a distance and demonstrates that ongoing retrotransposition can contribute significantly to natural phenotypic diversity.
Asunto(s)
Retrovirus Endógenos/genética , Regulación de la Expresión Génica , Transcripción Genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Metilación de ADN , Femenino , Variación Genética , Heterocigoto , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Polimorfismo Genético , Biosíntesis de Proteínas/genética , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ADN , Simportadores/genética , Simportadores/metabolismo , Secuencias Repetidas TerminalesRESUMEN
The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.
Asunto(s)
Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas ras/metabolismo , Animales , Benzamidas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Genes ras , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Ratones , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas ras/genéticaRESUMEN
In this essay, we discuss new insights into the wide-ranging impacts of mammalian transposable elements (TE) on gene expression and function. Nearly half of each mammalian genome is comprised of these mobile, repetitive elements. While most TEs are ancient relics, certain classes can move from one chromosomal location to another even now. Indeed, striking recent data show that extensive transposition occurs not only in the germline over evolutionary time, but also in developing somatic tissues and particular human cancers. While occasional germline TE insertions may contribute to genetic variation, many other, similar TEs appear to have little or no impact on neighboring genes. However, the effects of somatic insertions on gene expression and function remain almost completely unknown. We present a conceptual framework to understand how the ages, allele frequencies, molecular structures, and especially the genomic context of mammalian TEs each can influence their various possible functional consequences.
Asunto(s)
Elementos Transponibles de ADN/genética , Regulación de la Expresión Génica , Variación Genética , Alelos , Animales , Evolución Molecular , Expresión Génica , Frecuencia de los Genes , Humanos , Neoplasias/genéticaRESUMEN
Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.
Asunto(s)
Bancos de Muestras Biológicas , Células Madre Embrionarias/metabolismo , Homocigoto , Mutación/genética , Bancos de Tejidos , Animales , Células Madre Embrionarias/citología , Genómica , Gentamicinas/farmacología , Ratones , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteínas/genética , Puromicina/farmacología , Proteínas de Unión al ARNRESUMEN
Metastatic penile squamous cell carcinoma (PSCC) has only a 50% response rate to first-line combination chemotherapies and there are currently no targeted-therapy approaches. Therefore, we have an urgent need in advanced-PSCC treatment to find novel therapies. Approximately half of all PSCC cases are positive for high-risk human papillomavirus (HR-HPV). Our objective was to generate HPV-positive (HPV+) and HPV-negative (HPV-) patient-derived xenograft (PDX) models and to determine the biological differences between HPV+ and HPV- disease. We generated four HPV+ and three HPV- PSCC PDX animal models by directly implanting resected patient tumor tissue into immunocompromised mice. PDX tumor tissue was found to be similar to patient tumor tissue (donor tissue) by histology and short tandem repeat fingerprinting. DNA mutations were mostly preserved in PDX tissues and similar APOBEC (apolipoprotein B mRNA editing catalytic polypeptide) mutational fractions in donor tissue and PDX tissues were noted. A higher APOBEC mutational fraction was found in HPV+ versus HPV- PDX tissues (p = 0.044), and significant transcriptomic and proteomic expression differences based on HPV status included p16 (CDKN2A), RRM2, and CDC25C. These models will allow for the direct testing of targeted therapies in PSCC and determine their response in correlation to HPV status.
RESUMEN
NRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-Nras(G12D) mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-Nras(G12D) mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-Nras(G12D) mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-Nras(G12D) mice is attenuated compared with Mx1-Cre, LSL-Kras(G12D) mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous Kras(G12D) expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1(+) cells, whereas Mx1-Cre, LSL-Nras(G12D) mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.
Asunto(s)
Genes ras , Hematopoyesis/genética , Leucemia Mieloide Aguda/genética , Mutación , Sustitución de Aminoácidos , Animales , Citocinas/biosíntesis , Eritropoyesis/genética , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Linfopoyesis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Mielopoyesis/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Mice that accurately model the genetic diversity found in human cancer are valuable tools for interrogating disease mechanisms and investigating novel therapeutic strategies. We performed insertional mutagenesis with the MOL4070LTR retrovirus in Mx1-Cre, Kras(G12D) mice and generated a large cohort of T lineage acute lymphoblastic leukemias (T-ALLs). Molecular analysis infers that retroviral integration within Ikzf1 is an early event in leukemogenesis that precedes Kras(G12D) expression and later acquisition of somatic Notch1 mutations. Importantly, biochemical analysis uncovered unexpected heterogeneity, which suggests that Ras signaling networks are remodeled during multistep tumorigenesis. We tested tumor-derived cell lines to identify biomarkers of therapeutic response to targeted inhibitors. Whereas all T-ALLs tested were sensitive to a dual-specificity phosphoinosityl 3-kinase/mammalian target of rapamycin inhibitor, biochemical evidence of Notch1 activation correlated with sensitivity to gamma-secretase inhibition. In addition, Kras(G12D) T-ALLs were more responsive to a MAP/ERK kinase inhibitor in vitro and in vivo. Together, these studies identify a genetic pathway involving Ikzf1, Kras(G12D), and Notch1 in T lineage leukemogenesis, reveal unexpected diversity in Ras-regulated signaling networks, and define biomarkers of drug responses that may inform treatment strategies.
Asunto(s)
Antineoplásicos/farmacología , Linaje de la Célula , Factor de Transcripción Ikaros/metabolismo , Proteínas Mutantes/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptor Notch1/metabolismo , Sustitución de Aminoácidos/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Benzamidas/farmacología , Línea Celular Tumoral , Linaje de la Célula/efectos de los fármacos , Células Clonales , Difenilamina/análogos & derivados , Difenilamina/farmacología , Inhibidores Enzimáticos/farmacología , Sitios Genéticos/genética , Humanos , Factor de Transcripción Ikaros/genética , Integrasas/metabolismo , Ratones , Modelos Inmunológicos , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor Notch1/genética , Retroviridae , Transducción de Señal/efectos de los fármacosRESUMEN
Retroviral insertional mutagenesis in BXH2 and AKXD mice induces a high incidence of myeloid leukemia and B- and T-cell lymphoma, respectively. The retroviral integration sites (RISs) in these tumors thus provide powerful genetic tags for the discovery of genes involved in cancer. Here we report the first large-scale use of retroviral tagging for cancer gene discovery in the post-genome era. Using high throughput inverse PCR, we cloned and analyzed the sequences of 884 RISs from a tumor panel composed primarily of B-cell lymphomas. We then compared these sequences, and another 415 RIS sequences previously cloned from BXH2 myeloid leukemias and from a few AKXD lymphomas, against the recently assembled mouse genome sequence. These studies identified 152 loci that are targets of retroviral integration in more than one tumor (common retroviral integration sites, CISs) and therefore likely to encode a cancer gene. Thirty-six CISs encode genes that are known or predicted to be genes involved in human cancer or their homologs, whereas others encode candidate genes that have not yet been examined for a role in human cancer. Our studies demonstrate the power of retroviral tagging for cancer gene discovery in the post-genome era and indicate a largely unrecognized complexity in mouse and presumably human cancer.
Asunto(s)
Leucemia Mieloide/genética , Linfoma de Células B/genética , Retroviridae/genética , Integración Viral/genética , Animales , Genes Supresores de Tumor , Humanos , Ratones , Ratones Endogámicos , Oncogenes , Reacción en Cadena de la Polimerasa , Provirus/genéticaRESUMEN
The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read sequencing of oropharyngeal cancers and cancer cell lines identified a previously undescribed form of structural variation, "heterocateny," characterized by diverse, interrelated, and repetitive patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoints shared across structural variants facilitated stepwise reconstruction of their evolution from a common molecular ancestor. This analysis revealed that virus and virus-host concatemers are unstable and, upon insertion into and excision from chromosomes, facilitate capture, amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of heterocateny was detected in extrachromosomal and intrachromosomal DNA. These findings indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of an oncogenic DNA virus, thereby extending known consequences of HPV integration to include promotion of intratumoral heterogeneity and clonal evolution. SIGNIFICANCE: Long-read sequencing of HPV-positive cancers revealed "heterocateny," a previously unreported form of genomic structural variation characterized by heterogeneous, interrelated, and repetitive genomic rearrangements within a tumor. Heterocateny is driven by unstable concatemerized HPV genomes, which facilitate capture, rearrangement, and amplification of host DNA, and promotes intratumoral heterogeneity and clonal evolution. See related commentary by McBride and White, p. 814. This article is highlighted in the In This Issue feature, p. 799.