RESUMO
Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.
Assuntos
Histona-Lisina N-Metiltransferase/imunologia , NF-kappa B/imunologia , Infecções por Orthomyxoviridae/imunologia , Orthomyxoviridae/imunologia , Pneumonia/imunologia , Superinfecção/imunologia , Animais , Quimiocina CXCL1/imunologia , Suscetibilidade a Doenças , Feminino , Interferon Tipo I/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/enzimologia , Pneumonia/virologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Superinfecção/enzimologia , Superinfecção/microbiologiaRESUMO
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.
Assuntos
Alphapapillomavirus , Proteínas Oncogênicas Virais , Neoplasias Orofaríngeas , Alphapapillomavirus/metabolismo , Carcinogênese , Humanos , Proteínas Oncogênicas Virais/genética , Neoplasias Orofaríngeas/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Integração Viral/genéticaRESUMO
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.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Bucais , Proteínas de Neoplasias , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Feminino , Humanos , Masculino , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/virologia , Mutação , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Papillomaviridae/metabolismoRESUMO
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.
Assuntos
Regulação Viral da Expressão Gênica , Genes Virais , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Fatores de Transcrição/metabolismo , Integração Viral/fisiologia , Células Cultivadas , Elementos Facilitadores Genéticos , Células HCT116 , Células HeLa , Células Hep G2 , Interações Hospedeiro-Patógeno/genética , Células Endoteliais da Veia Umbilical Humana , Papillomavirus Humano 16/metabolismo , Humanos , Células K562 , Vírus Oncogênicos/genética , Vírus Oncogênicos/patogenicidade , Papillomaviridae/genética , Papillomaviridae/metabolismo , Papillomaviridae/patogenicidade , Ligação Proteica , Multimerização Proteica , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Cardiomiopatias/terapia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Distrofina/genética , Edição de Genes/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Contração Miocárdica , Músculos Papilares/metabolismo , Animais , Proteínas Associadas a CRISPR/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Dependovirus/genética , Modelos Animais de Doenças , Distrofina/metabolismo , Éxons , Fibrose , Regulação da Expressão Gênica , Predisposição Genética para Doença , Vetores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Mutação , Músculos Papilares/patologia , Músculos Papilares/fisiopatologia , Fenótipo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Recuperação de Função Fisiológica , Utrofina/genéticaRESUMO
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.
Assuntos
Evolução Molecular , Variação Genética/genética , Genoma Humano/genética , Genoma/genética , Pan paniscus/genética , Pan troglodytes/genética , Animais , Elementos de DNA Transponíveis/genética , Duplicação Gênica/genética , Genótipo , Humanos , Dados de Sequência Molecular , Fenótipo , Filogenia , Especificidade da EspécieRESUMO
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.
Assuntos
Carcinoma/genética , Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Neoplasias da Glândula Tireoide/genética , Alelos , Carcinoma Papilar , Linhagem Celular Tumoral , Cromatina/química , Imunoprecipitação da Cromatina , Fatores de Transcrição Forkhead/genética , Predisposição Genética para Doença , Genótipo , Haplótipos , Histonas/química , Humanos , Razão de Chances , Penetrância , Câncer Papilífero da TireoideRESUMO
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.
Assuntos
Replicação do DNA/genética , Instabilidade Genômica , Papillomavirus Humano 16/genética , Neoplasias/genética , DNA Viral/genética , Feminino , Papillomavirus Humano 16/crescimento & desenvolvimento , Humanos , Masculino , Neoplasias/classificação , Neoplasias/patologia , Neoplasias/virologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Integração Viral/genéticaRESUMO
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.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Regiões Promotoras Genéticas , RNA Antissenso/biossíntese , Proteínas de Ligação a RNA/genética , Animais , Sequência de Bases , Linhagem Celular , Humanos , Camundongos , Dados de Sequência Molecular , RNA Polimerase II/metabolismo , Ribonuclease III/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
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.
Assuntos
Retrovirus Endógenos/genética , Regulação da Expressão Gênica , Transcrição Gênica , Animais , Sequência de Bases , Mapeamento Cromossômico , Metilação de DNA , Feminino , Variação Genética , Heterozigoto , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Polimorfismo Genético , Biossíntese de Proteínas/genética , Locos de Características Quantitativas , Análise de Sequência de DNA , Simportadores/genética , Simportadores/metabolismo , Sequências Repetidas TerminaisRESUMO
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.
Assuntos
Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Variação Genética , Alelos , Animais , Evolução Molecular , Expressão Gênica , Frequência do Gene , Humanos , Neoplasias/genéticaRESUMO
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.
Assuntos
Neoplasias Orofaríngeas , Infecções por Papillomavirus , Humanos , Papillomavirus Humano , Rearranjo Gênico , Evolução Clonal/genética , Integração Viral/genética , Papillomaviridae/genéticaRESUMO
Multiple myeloma (MM) is accompanied by alterations to the normal plasma cell (PC) proteome, leading to changes to the tumor microenvironment and disease progression. There is a great need for understanding the consequences that lead to MM progression and for the discovery of new biomarkers that can aid clinical diagnostics and serve as targets for therapeutics. This study demonstrates the applicability of utilizing the single-cell high-definition liquid biopsy assay (HDSCA) and imaging mass cytometry to characterize the proteomic profile of myeloma. In our study, we analyzed ~87,000 cells from seven patient samples (bone marrow and peripheral blood) across the myeloma disease spectrum and utilized our multiplexed panel to characterize the expression of clinical markers for PC classification, additional potential therapeutic targets, and the tumor microenvironment cells. Our analysis showed BCMA, ICAM3 (CD50), CD221, and CS1 (SLAMF7) as the most abundantly expressed markers on PCs across all myeloma stages, with BCMA, ICAM3, and CD221 having significantly higher expression levels on disease versus precursor PCs. Additionally, we identify significantly elevated levels of expression for CD74, MUM1, CD229, CD44, IGLL5, Cyclin D1, UBA52, and CD317 on PCs from overt disease conditions compared to those from precursor states.
RESUMO
OBJECTIVES: Somatic mutations may predict prognosis, therapeutic response, or cancer progression. We evaluated targeted sequencing of oral rinse samples (ORS) for non-invasive mutational profiling of oral squamous cell carcinomas (OSCC). MATERIALS AND METHODS: A custom hybrid capture panel targeting 42 frequently mutated genes in OSCC was used to identify DNA sequence variants in matched ORS and fresh-frozen tumors from 120 newly-diagnosed patients. Receiver operating characteristic (ROC) curves determined the optimal variant allele fraction (VAF) cutoff for variant discrimination in ORS. Behavioral, clinical, and analytical factors were evaluated for impacts on assay performance. RESULTS: Half of tumors involved oral tongue (50 %), and a majority were T1-T2 tumor stage (55 %). Median depth of sequencing coverage was 260X for OSCC and 1,563X for ORS. Frequencies of single nucleotide variants (SNVs) at highly mutated genes (including TP53, FAT1, HRAS, NOTCH1, CDKN2A, CASP8, NFE2L2, and PIK3CA) in OSCC were highly correlated with TCGA data (R = 0.96, p = 2.5E-22). An ROC curve with area-under-the-curve (AUC) of 0.80 showed that, at an optimal VAF cutoff of 0.10 %, ORS provided 76 % sensitivity, 96 % specificity, but precision of only 2.6E-4. At this VAF cutoff, 206 of 270 SNVs in OSCC were detected in matched ORS. Sensitivity varied by patient, T stage and target gene. Neither downsampled ORS as matched control nor a naïve Bayesian classifier adjusting for sequencing bias appreciably improved assay performance. CONCLUSION: Targeted sequencing of ORS provides moderate assay performance for noninvasive detection of SNVs in OSCC. Our findings strongly rationalize further clinical and laboratory optimization of this assay, including strategies to improve precision.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Teorema de Bayes , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carcinoma de Células Escamosas/patologia , Mutação , GenômicaRESUMO
Multiple myeloma remains an incurable disease, and the cellular and molecular evolution from precursor conditions, including monoclonal gammopathy of undetermined significance and smoldering multiple myeloma, is incompletely understood. Here, we combine single-cell RNA and B cell receptor sequencing from fifty-two patients with myeloma precursors in comparison with myeloma and normal donors. Our comprehensive analysis reveals early genomic drivers of malignant transformation, distinct transcriptional features, and divergent clonal expansion in hyperdiploid versus non-hyperdiploid samples. Additionally, we observe intra-patient heterogeneity with potential therapeutic implications and identify distinct patterns of evolution from myeloma precursor disease to myeloma. We also demonstrate distinctive characteristics of the microenvironment associated with specific genomic changes in myeloma cells. These findings add to our knowledge about myeloma precursor disease progression, providing valuable insights into patient risk stratification, biomarker discovery, and possible clinical applications.
Assuntos
Pesquisa Biomédica , Mieloma Múltiplo , Mieloma Múltiplo Latente , Humanos , Mieloma Múltiplo/genética , Aneuploidia , Progressão da Doença , Microambiente Tumoral/genéticaRESUMO
The severity of numerous developmental abnormalities can vary widely despite shared genetic causes. Mice deficient in Twisted gastrulation (Twsg1(-/-)) display such phenotypic variation, developing a wide range of craniofacial malformations on an isogenic C57BL/6 strain background. To examine the molecular basis for this reduced penetrance and variable expressivity, we used exon microarrays to analyze gene expression in mandibular arches from several distinct, morphologically defined classes of Twsg1(-/-) and wild type (WT) embryos. Hierarchical clustering analysis of transcript levels identified numerous differentially expressed genes, clearly distinguishing severely affected and unaffected Twsg1(-/-) mutants from WT embryos. Several genes that play well-known roles in craniofacial development were upregulated in unaffected Twsg1(-/-) mutant embryos, suggesting that they may compensate for the loss of TWSG1. Imprinted genes were overrepresented among genes that were differentially expressed particularly between affected and unaffected mutants. The most severely affected embryos demonstrated increased p53 signaling and increased expression of its target, Trp53inp1. The frequency of craniofacial defects significantly decreased with a reduction of p53 gene dosage from 44% in Twsg1(-/-)p53(+/+) pups (N=675) to 30% in Twsg1(-/-)p53(+/-) (N=47, p=0.04) and 15% in Twsg1(-/-)p53(-/-) littermates (N=39, p=0.001). In summary, these results demonstrate that phenotypic variability in Twsg1(-/-) mice is associated with differential expression of certain developmentally regulated genes, and that craniofacial defects can be partially rescued by reduced p53 levels. We postulate that variable responses to stress may contribute to variable craniofacial phenotypes by triggering differential expression of genes and variable cellular apoptosis.
Assuntos
Anormalidades Craniofaciais/genética , Proteínas/genética , Animais , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
MouseIndelDB is an integrated database resource containing thousands of previously unreported mouse genomic indel (insertion and deletion) polymorphisms ranging from approximately 100 nt to 10 Kb in size. The database currently includes polymorphisms identified from our alignment of 26 million whole-genome shotgun sequence traces from four laboratory mouse strains mapped against the reference C57BL/6J genome using GMAP. They can be queried on a local level by chromosomal coordinates, nearby gene names or other genomic feature identifiers, or in bulk format using categories including mouse strain(s), class of polymorphism(s) and chromosome number. The results of such queries are presented either as a custom track on the UCSC mouse genome browser or in tabular format. We anticipate that the MouseIndelDB database will be widely useful for research in mammalian genetics, genomics, and evolutionary biology. Access to the MouseIndelDB database is freely available at: http://variation.osu.edu/.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Polimorfismo Genético , Animais , Biologia Computacional/tendências , Bases de Dados de Proteínas , Genoma , Armazenamento e Recuperação da Informação/métodos , Internet , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Software , Especificidade da Espécie , Interface Usuário-ComputadorRESUMO
Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.
Assuntos
Mieloma Múltiplo , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Progressão da Doença , Humanos , Mieloma Múltiplo/genética , Plasmócitos/metabolismo , Plasmócitos/patologiaRESUMO
Human papillomavirus (HPV) insertions in cancer genomes have been linked to various forms of focal genomic instability and altered expression of neighboring genes. Here we tested the hypothesis that investigation of HPV insertions in a head and neck cancer squamous cell carcinoma (HNSCC) cell line would identify targetable driver genes contributing to oncogenesis of other HNSCC. In the cell line UPCI:SCC090 HPV16 integration amplified the PIM1 serine/threonine kinase gene ~16-fold, thereby increasing transcript and protein levels. We used genetic and pharmacological approaches to inhibit PIM kinases in this and other HNSCC cell lines. Knockdown of PIM1 transcripts by transfected short hairpin RNAs reduced UPCI:SCC090 viability. CRISPR/Cas9-mediated mutagenesis of PIM1 caused cell cycle arrest and apoptosis. Pharmacological inhibition of PIM family kinases decreased growth of UPCI:SCC090 and additional HNSCC cell lines in vitro and a xenograft UPCI:SCC090 model in vivo. Based on established interactions between intracellular signaling pathways and relatively high levels of gene expression in almost all HNSCC, we also evaluated combinations of PIM kinase and epidermal growth factor receptor (EGFR) inhibitors. Dual inhibition of these pathways resulted in supra-additive cell death. These data support clinical testing of PIM inhibitors alone or in combination in HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço/genética , Papillomavirus Humano 16/genética , Infecções por Papillomavirus/complicações , Proteínas Proto-Oncogênicas c-pim-1/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Integração Viral/genética , Animais , Apoptose , Proliferação de Células , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Camundongos , Camundongos Nus , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Prognóstico , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Oral human papillomavirus (HPV) infection is a risk factor for head and neck squamous cell carcinoma (HNSCC), and is a concern for patients with HPV-positive HNSCC and their partners. The prevalence of oral HPV infection before and after cancer therapy was investigated among patients with HPV16-positive and HPV16-negative HNSCC. EXPERIMENTAL DESIGN: Serial oral rinse samples (ORS) were collected from a cohort of 135 HNSCC cases as frequently as every 3 months for up to 3 years. Tumor HPV status was determined by HPV16 in situ hybridization. HPV was detected in ORS by consensus PCR and line blot hybridization. The HPV16 variants in positive oral rinse-tumor pairs were determined by sequencing. The odds of oral HPV infection among HPV16-positive and HPV16-negative cases were compared by use of generalized estimating equations. RESULTS: Patients were followed for a median of 21 months and provided a median of 4 samples. Forty-four of 135 patients had HPV16-positive tumors. HPV16-positive cases were more likely than HPV16-negative cases to have an oral HPV infection detected before (odds ratio, 8.6; 95% confidence interval, 3.5-21) and after therapy (OR, 2.9; 95% confidence interval, 1.1-7.4). Oral infections by HPV16 and other high-risk, but not low-risk, types were more common among HPV16-positive cases both before and after therapy. Most HPV16 variants in ORS were European, unique, and identical to that in the tumor. Persistence of a type-specific oral infection was demonstrable for as long as 5 years. CONCLUSION: Oral high-risk HPV infections are more frequent among patients with HPV16-positive than HPV16-negative HNSCC, consistent with a behavioral and/or biological disposition to infection.