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Identifying the mechanisms of action of anticancer drugs is an important step in the development of new drugs. In this study, we established a comprehensive screening platform consisting of 68 oncogenes (MANO panel), encompassing 243 genetic variants, to identify predictive markers for drug efficacy. Validation was performed using drugs that targeted EGFR, BRAF, and MAP2K1, which confirmed the utility of this functional screening panel. Screening of a BRCA2-knockout DLD1 cell line (DLD1-KO) revealed that cells expressing SMO and GLI1 were resistant to olaparib. Gene set enrichment analysis identified genes associated with DNA damage repair that were enriched in cells overexpressing SMO and GLI1. The expression of genes associated with homologous recombination repair (HR), such as the FANC family and BRCA1/2, was significantly upregulated by GLI1 expression, which is indicative of PARP inhibitor resistance. Although not all representative genes of the nucleotide excision repair (NER) pathway were upregulated, NER activity was enhanced by GLI1. The GLI1 inhibitor was effective against DLD1-KO cells overexpressing GLI1 both in vitro and in vivo. Furthermore, the combination therapy of olaparib and GLI1 inhibitor exhibited a synergistic effect on DLD1-KO, suggesting the possible clinical application of GLI1 inhibitor targeting cancer with defective DNA damage repair. This platform enables the identification of biomarkers associated with drug sensitivity, and is a useful tool for drug development.
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E7820 and Indisulam (E7070) are sulfonamide molecular glues that modulate RNA splicing by degrading the splicing factor RBM39 via ternary complex formation with the E3 ligase adaptor DCAF15. To identify biomarkers of the antitumor efficacy of E7820, we treated patient-derived xenograft (PDX) mouse models established from 42 patients with solid tumors. The overall response rate was 38.1% (16 PDXs), and tumor regression was observed across various tumor types. Exome sequencing of the PDX genome revealed that loss-of-function mutations in genes of the homologous recombination repair (HRR) system, such as ATM, were significantly enriched in tumors that responded to E7820 (p = 4.5 × 103). Interestingly, E7820-mediated double-strand breaks in DNA were increased in tumors with BRCA2 dysfunction, and knockdown of BRCA1/2 transcripts or knockout of ATM, ATR, or BAP1 sensitized cancer cells to E7820. Transcriptomic analyses revealed that E7820 treatment resulted in the intron retention of mRNAs and decreased transcription, especially for HRR genes. This induced HRR malfunction probably leads to the synthetic lethality of tumor cells with homologous recombination deficiency (HRD). Furthermore, E7820, in combination with olaparib, exerted a synergistic effect, and E7820 was even effective in an olaparib-resistant cell line. In conclusion, HRD is a promising predictive biomarker of E7820 efficacy and has a high potential to improve the prognosis of patients with HRD-positive cancers.
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PURPOSE: Various molecular profiles are needed to classify malignant brain tumors, including gliomas, based on the latest classification criteria of the World Health Organization, and their poor prognosis necessitates new therapeutic targets. The Todai OncoPanel 2 RNA Panel (TOP2-RNA) is a custom-target RNA-sequencing (RNA-seq) using the junction capture method to maximize the sensitivity of detecting 455 fusion gene transcripts and analyze the expression profiles of 1,390 genes. This study aimed to classify gliomas and identify their molecular targets using TOP2-RNA. METHODS: A total of 124 frozen samples of malignant gliomas were subjected to TOP2-RNA for classification based on their molecular profiles and the identification of molecular targets. RESULTS: Among 55 glioblastoma cases, gene fusions were detected in 11 cases (20%), including novel MET fusions. Seven tyrosine kinase genes were found to be overexpressed in 15 cases (27.3%). In contrast to isocitrate dehydrogenase (IDH) wild-type glioblastoma, IDH-mutant tumors, including astrocytomas and oligodendrogliomas, barely harbor fusion genes or gene overexpression. Of the 34 overexpressed tyrosine kinase genes, MDM2 and CDK4 in glioblastoma, 22 copy number amplifications (64.7%) were observed. When comparing astrocytomas and oligodendrogliomas in gene set enrichment analysis, the gene sets related to 1p36 and 19q were highly enriched in astrocytomas, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis. CONCLUSIONS: TOP2-RNA is a highly sensitive assay for detecting fusion genes, exon skipping, and aberrant gene expression. Alterations in targetable driver genes were identified in more than 50% of glioblastoma. Molecular profiling by TOP2-RNA provides ample predictive, prognostic, and diagnostic biomarkers that may not be identified by conventional assays and, therefore, is expected to increase treatment options for individual patients with glioma.
Assuntos
Astrocitoma , Neoplasias Encefálicas , Glioblastoma , Glioma , Oligodendroglioma , Humanos , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioblastoma/patologia , Oligodendroglioma/patologia , Mutação , Glioma/diagnóstico , Glioma/genética , Glioma/patologia , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Astrocitoma/patologia , Proteínas Tirosina Quinases/genética , Biomarcadores , Isocitrato Desidrogenase/genéticaRESUMO
Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.
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Células-Tronco Pluripotentes Induzidas/citologia , Fígado/enzimologia , Organoides/citologia , Organoides/embriologia , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Fígado/citologiaRESUMO
Intrahepatic cholangiocarcinoma and combined hepatocellular cholangiocarcinoma show varying degrees of biliary epithelial differentiation, which can be defined as liver cancer displaying biliary phenotype (LCB). LCB is second in the incidence for liver cancers with and without chronic hepatitis background and more aggressive than hepatocellular carcinoma (HCC). To gain insight into its molecular alterations, we performed whole-genome sequencing analysis on 30 LCBs. Here we show, the genome-wide substitution patterns of LCBs developed in chronic hepatitis livers overlapped with those of 60 HCCs, whereas those of hepatitis-negative LCBs diverged. The subsequent validation study on 68 LCBs identified recurrent mutations in TERT promoter, chromatin regulators (BAP1, PBRM1 and ARID2), a synapse organization gene (PCLO), IDH genes and KRAS. The frequencies of KRAS and IDHs mutations, which are associated with poor disease-free survival, were significantly higher in hepatitis-negative LCBs. This study reveals the strong impact of chronic hepatitis on the mutational landscape in liver cancer and the genetic diversity among LCBs.
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Neoplasias Hepáticas/genética , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA , Feminino , Hepatite/genética , Hepatite/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Neuropeptídeos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Telomerase/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Proteínas ras/genéticaRESUMO
Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV)-related hepatocellular carcinomas (HCCs) and their matched controls. Comparison of whole genome sequence (WGS) and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3), and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome.
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Carcinoma Hepatocelular/genética , Evolução Clonal , Genoma Humano , Neoplasias Hepáticas/genética , Mutação , Transcriptoma , Carcinoma Hepatocelular/metabolismo , Estudos de Casos e Controles , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Oncogenes/genéticaRESUMO
In mammals, female development has traditionally been considered a default process in the absence of the testis-determining gene, Sry. Recently, it has been documented that the gene for R-spondin1 (RSPO1), a novel class of soluble activator for Wnt/beta-catenin signaling, is mutated in two Italian families with female-to-male (XX) sex reversal. To elucidate the role of Rspo1 as a candidate female-determining gene in a mouse model, we generated Rspo1-null (Rspo1(-/-)) mice and found that Rspo1(-/-) XX mice displayed masculinized features including pseudohermaphroditism in genital ducts, depletion of fetal oocytes, male-specific coelomic vessel formation and ectopic testosterone production in the ovaries. Thus, although Rspo1 is required to fully suppress the male differentiation program and to maintain germ cell survival during the development of XX gonads, the loss of its activity has proved to be insufficient to cause complete XX sex reversal in mice. Interestingly, these partial sex-reversed phenotypes of Rspo1(-/-) XX mice recapitulated those of previously described Wnt-4(-/-) XX mice. In accordance with this finding, the expression of Wnt-4 and its downstream genes was deregulated in early Rspo1(-/-) XX gonads, suggesting that Rspo1 may participate in suppressing the male pathway in the absence of Sry and maintaining oocyte survival through positively regulating Wnt-4 signaling.
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Transtornos do Desenvolvimento Sexual/fisiopatologia , Ovário/crescimento & desenvolvimento , Diferenciação Sexual , Transdução de Sinais , Trombospondinas/genética , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Transtornos do Desenvolvimento Sexual/patologia , Feminino , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Hormônios Ectópicos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Oócitos/citologia , Ovário/patologia , Ovário/fisiopatologia , Especificidade da Espécie , Testosterona/metabolismo , Proteínas Wnt/genética , Proteína Wnt4RESUMO
A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.