RESUMO
Differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic lineages offers great therapeutic potential. During embryogenesis, hemogenic endothelium (HE) gives rise to hematopoietic stem and progenitor cells through the endothelial-to-hematopoietic transition (EHT). Understanding this process using iPSCs is key to generating functional hematopoietic stem cells (HSCs), a currently unmet challenge. In this study, we examined the role of the transcriptional factor GFI1B and its co-factor LSD1/KDM1A in EHT. To this end, we employed patient-derived iPSC lines with a dominant negative dysfunctional GFI1BQ287* and irreversible pharmacological LSD1/KDM1A inhibition in healthy iPSC lines. The formation of HE remained unaffected; however, hematopoietic output was severely reduced in both conditions. Single-cell RNA sequencing (scRNAseq) performed on the CD144+/CD31+ population derived from healthy iPSCs revealed similar expression dynamics of genes associated with in vivo EHT. Interestingly, LSD1/KDM1A inhibition in healthy lines before EHT resulted in a complete absence of hematopoietic output. However, uncommitted HE cells did not display GFI1B expression, suggesting a timed transcriptional program. To test this hypothesis, we ectopically expressed GFI1B in uncommitted HE cells, leading to downregulation of endothelial genes and upregulation of hematopoietic genes, including GATA2, KIT, RUNX1, and SPI1. Thus, we demonstrate that LSD1/KDM1A and GFI1B can function at distinct temporal points in different cellular subsets during EHT. Although GFI1B is not detected in uncommitted HE cells, its ectopic expression allows for partial hematopoietic specification. These data indicate that precisely timed expression of specific transcriptional regulators during EHT is crucial to the eventual outcome of EHT.
RESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 mutations contribute to HCC development remains unclear. METHODS: In this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines. RESULTS: For each cell line we successfully obtained 2-4 correctly repaired clones, which all show reduced ß-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored ß-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive ß-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling. CONCLUSIONS: Our study provides insights into the effects of repairing AXIN1 mutations on ß-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations.
Assuntos
Proteína Axina , Sistemas CRISPR-Cas , Carcinoma Hepatocelular , Neoplasias Hepáticas , Mutação , beta Catenina , Proteína Axina/genética , Proteína Axina/metabolismo , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Linhagem Celular Tumoral , beta Catenina/metabolismo , beta Catenina/genética , Regulação Neoplásica da Expressão Gênica , Edição de Genes , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after surgery and chemotherapy. Unfortunately, many patients experience disease progression because of acquired therapy resistance. This study aims to characterize epigenetic and genomic changes in cell-free DNA (cfDNA) associated with PARPi resistance. MATERIALS AND METHODS: Blood was taken from 31 EOC patients receiving PARPi therapy before treatment and at disease progression during/after treatment. Resistance was defined as disease progression within 6 months after starting PARPi and was seen in fifteen patients, while sixteen patients responded for 6 to 42 months. Blood cfDNA was evaluated via Modified Fast Aneuploidy Screening Test-Sequencing System (mFast-SeqS to detect aneuploidy, via Methylated DNA Sequencing (MeD-seq) to find differentially methylated regions (DMRs), and via shallow whole-genome and -exome sequencing (shWGS, exome-seq) to define tumor fractions and mutational signatures. RESULTS: Aneuploid cfDNA was undetectable pre-treatment but observed in six patients post-treatment, in five resistant and one responding patient. Post-treatment ichorCNA analyses demonstrated in shWGS and exome-seq higher median tumor fractions in resistant (7% and 9%) than in sensitive patients (7% and 5%). SigMiner analyses detected predominantly mutational signatures linked to mismatch repair and chemotherapy. DeSeq2 analyses of MeD-seq data revealed three methylation signatures and more tumor-specific DMRs in resistant than in responding patients in both pre- and post-treatment samples (274 vs. 30 DMRs, 190 vs. 57 DMRs, Χ2-test p < 0.001). CONCLUSION: Our genome-wide Next-Generation Sequencing (NGS) analyses in PARPi-resistant patients identified epigenetic differences in blood before treatment, whereas genomic alterations were more frequently observed after progression. The epigenetic differences at baseline are especially interesting for further exploration as putative predictive biomarkers for PARPi resistance.
Assuntos
Carcinoma Epitelial do Ovário , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Feminino , Resistencia a Medicamentos Antineoplásicos/genética , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Idoso , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/patologia , Adulto , Aneuploidia , Genômica/métodosRESUMO
Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway, suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however, their role in the hematopoietic development in vivo remains unknown. Here, we identify a subpopulation of NG2+Runx1+ perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2+ cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs, endothelial cells and hematopoietic cells in the murine AGM. Importantly, this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental, as NG2+Runx1+ cells were not detected in the adult bone marrow, demonstrating the existence of a specialised pericyte population in the HSC-generating niche, unique to the embryo.
Assuntos
Células Endoteliais , Músculo Liso Vascular , Camundongos , Animais , Músculo Liso Vascular/metabolismo , Células Endoteliais/metabolismo , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Hematopoese/genética , Mesonefro , Gônadas/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismoRESUMO
Colorectal cancer (CRC) colonoscopic surveillance is effective but burdensome. Circulating tumor DNA (ctDNA) analysis has emerged as a promising, minimally invasive tool for disease detection and management. Here, we assessed which ctDNA assay might be most suitable for a ctDNA-based CRC screening/surveillance blood test. In this prospective, proof-of-concept study, patients with colonoscopies for Lynch surveillance or the National Colorectal Cancer screening program were included between 7 July 2019 and 3 June 2022. Blood was drawn, and if advanced neoplasia (adenoma with villous component, high-grade dysplasia, ≥10 mm, or CRC) was detected, it was analyzed for chromosomal copy number variations, single nucleotide variants, and genome-wide methylation (MeD-seq). Outcomes were compared with corresponding patients' tissues and the MeD-seq results of healthy blood donors. Two Lynch carriers and eight screening program patients were included: five with CRC and five with advanced adenomas. cfDNA showed copy number variations and single nucleotide variants in one patient with CRC and liver metastases. Eight patients analyzed with MeD-seq showed clustering of Lynch-associated and sporadic microsatellite instable lesions separate from microsatellite stable lesions, as did healthy blood donors. In conclusion, whereas copy number changes and single nucleotide variants were only detected in one patient, cfDNA methylation profiles could discriminate all microsatellite instable advanced neoplasia, rendering this tool particularly promising for LS surveillance. Larger studies are warranted to validate these findings.
RESUMO
Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants. We used cfDNA of 70 MBC patients profiled with both the small targeted Oncomine breast panel (Thermofisher) and the much larger Qiaseq Human Breast Cancer Panel (Qiagen). The model was trained on the panels' common regions using Oncomine hotspot mutations as ground truth. Applied to Qiaseq data, it achieved 35% sensitivity and 36% precision, outperforming basic filtering. For 20 patients we used germline DNA to filter for somatic variants and obtained 245 variants in total, while our model found seven variants, of which six were also detected using the germline strategy. In ten tumor-free individuals, our method detected in total one (potentially germline) variant, in contrast to 521 variants detected without our model. These results indicate that our model largely detects somatic variants.
Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , Humanos , Feminino , Neoplasias da Mama/genética , Ácidos Nucleicos Livres/genética , Mutação , Mama , Sequenciamento de Nucleotídeos em Larga Escala , Aprendizado de MáquinaRESUMO
Myelodysplastic syndromes (MDS) consist of a group of hematological malignancies characterized by ineffective hematopoiesis, cytogenetic abnormalities, and often a high risk of transformation to acute myeloid leukemia (AML). So far, there have been only a very limited number of studies assessing the epigenetics component contributing to the pathophysiology of these disorders, but not a single study assessing this at a genome-wide level. Here, we implemented a generic high throughput epigenomics approach, using methylated DNA sequencing (MeD-seq) of LpnPI digested fragments to identify potential epigenomic targets associated with MDS subtypes. Our results highlighted that PCDHG and ZNF gene families harbor potential epigenomic targets, which have been shown to be differentially methylated in a variety of comparisons between different MDS subtypes. Specifically, CpG islands, transcription start sites and post-transcriptional start sites within ZNF124, ZNF497 and PCDHG family are differentially methylated with fold change above 3,5. Overall, these findings highlight important aspects of the epigenomic component of MDS syndromes pathogenesis and the pharmacoepigenomic basis to the hypomethylating agents drug treatment response, while this generic high throughput whole epigenome sequencing approach could be readily implemented to other genetic diseases with a strong epigenetic component.
Assuntos
Metilação de DNA , Síndromes Mielodisplásicas , Humanos , Metilação de DNA/genética , Epigenômica , Epigênese Genética , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Progressão da Doença , Ilhas de CpG/genética , Proteínas de Ligação a DNA/genéticaRESUMO
Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.
Assuntos
Vírus da Raiva , Raiva , Humanos , Vírus da Raiva/genética , Transcriptoma , Macrófagos/metabolismo , Antivirais/farmacologiaRESUMO
BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) patients with positive AR-V7 expression in their circulating tumour cells (CTCs) rarely derive benefit from abiraterone and enzalutamide. DESIGN: We performed a prospective, multicenter, single arm phase II clinical trial (CABA-V7) in mCRPC patients previously treated with docetaxel and androgen deprivation therapy. OBJECTIVE: In this trial, we investigated whether cabazitaxel treatment resulted in clinically meaningful PSA response rates in patients with positive CTC-based AR-V7 expression and collected liquid biopsies for genomic profiling. RESULTS: Cabazitaxel was found to be modestly effective, with only 12% of these patients obtaining a PSA response. Genomic profiling revealed that CTC-based AR-V7 expression was not associated with other known mCRPC-associated alterations. CTC-based AR-V7 status and dichotomised CTC counts were observed as independent prognostic markers at baseline. CONCLUSIONS: AR-V7 positivity predicted poor overall survival (OS). However, cabazitaxel-treated AR-V7 positive patients and those lacking AR-V7 positivity, who received cabazitaxel as standard of care, appeared to have similar OS. Therefore, despite the low response rate, cabazitaxel may still be an effective treatment in this poor prognosis, AR-V7 positive patient population.
Assuntos
Células Neoplásicas Circulantes , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/patologia , Antígeno Prostático Específico , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/uso terapêutico , Isoformas de Proteínas/genética , Células Neoplásicas Circulantes/patologia , Nitrilas/uso terapêuticoRESUMO
Ubiquitin-specific protease 7 (USP7) has been implicated in cancer progression and neurodevelopment. However, its molecular targets remain poorly characterized. We combined quantitative proteomics, transcriptomics, and epigenomics to define the core USP7 network. Our multi-omics analysis reveals USP7 as a control hub that links genome regulation, tumor suppression, and histone H2A ubiquitylation (H2AK119ub1) by noncanonical Polycomb-repressive complexes (ncPRC1s). USP7 strongly stabilizes ncPRC1.6 and, to a lesser extent, ncPRC1.1. Moreover, USP7 represses expression of AUTS2, which suppresses H2A ubiquitylation by ncPRC1.3/5. Collectively, these USP7 activities promote the genomic deposition of H2AK119ub1 by ncPRC1, especially at transcriptionally repressed loci. Notably, USP7-dependent changes in H2AK119ub1 levels are uncoupled from H3K27me3. Even complete loss of the PRC1 catalytic core and H2AK119ub1 has only a limited effect on H3K27me3. Besides defining the USP7 regulome, our results reveal that H2AK119ub1 dosage is largely disconnected from H3K27me3.
RESUMO
Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRß signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification in vivo and whether PDGF-B/PDGFRß is involved. Here, we show that PDGFRß is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRß+ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRß+ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of HSPCs in vitro.
Assuntos
Mesonefro , Peixe-Zebra , Animais , Hematopoese , Células-Tronco Hematopoéticas , Camundongos , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Células EstromaisRESUMO
Tissue-resident macrophages of the brain, including microglia, are implicated in the pathogenesis of various CNS disorders and are possible therapeutic targets by their chemical depletion or replenishment by hematopoietic stem cell therapy. Nevertheless, a comprehensive understanding of microglial function and the consequences of microglial depletion in the human brain is lacking. In human disease, heterozygous variants in CSF1R, encoding the Colony-stimulating factor 1 receptor, can lead to adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) possibly caused by microglial depletion. Here, we investigate the effects of ALSP-causing CSF1R variants on microglia and explore the consequences of microglial depletion in the brain. In intermediate- and late-stage ALSP post-mortem brain, we establish that there is an overall loss of homeostatic microglia and that this is predominantly seen in the white matter. By introducing ALSP-causing missense variants into the zebrafish genomic csf1ra locus, we show that these variants act dominant negatively on the number of microglia in vertebrate brain development. Transcriptomics and proteomics on relatively spared ALSP brain tissue validated a downregulation of microglia-associated genes and revealed elevated astrocytic proteins, possibly suggesting involvement of astrocytes in early pathogenesis. Indeed, neuropathological analysis and in vivo imaging of csf1r zebrafish models showed an astrocytic phenotype associated with enhanced, possibly compensatory, endocytosis. Together, our findings indicate that microglial depletion in zebrafish and human disease, likely as a consequence of dominant-acting pathogenic CSF1R variants, correlates with altered astrocytes. These findings underscore the unique opportunity CSF1R variants provide to gain insight into the roles of microglia in the human brain, and the need to further investigate how microglia, astrocytes, and their interactions contribute to white matter homeostasis.
Assuntos
Doenças Desmielinizantes , Leucoencefalopatias , Doenças por Armazenamento dos Lisossomos , Doenças Neurodegenerativas , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Adulto , Animais , Astrócitos/patologia , Doenças Desmielinizantes/patologia , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Microglia/patologia , Doenças Neurodegenerativas/patologia , Fenótipo , Receptores Proteína Tirosina Quinases/genética , Peixe-ZebraRESUMO
BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor. Its cellular composition is very heterogeneous, with cells exhibiting stem-cell characteristics (GSCs) that co-determine therapy resistance and tumor recurrence. Bone Morphogenetic Protein (BMP)-4 promotes astroglial and suppresses oligodendrocyte differentiation in GSCs, processes associated with superior patient prognosis. We characterized variability in cell viability of patient-derived GBM cultures in response to BMP4 and, based on single-cell transcriptome profiling, propose predictive positive and early-response markers for sensitivity to BMP4. METHODS: Cell viability was assessed in 17 BMP4-treated patient-derived GBM cultures. In two cultures, one highly-sensitive to BMP4 (high therapeutic efficacy) and one with low-sensitivity, response to treatment with BMP4 was characterized. We applied single-cell RNA-sequencing, analyzed the relative abundance of cell clusters, searched for and identified the aforementioned two marker types, and validated these results in all 17 cultures. RESULTS: High variation in cell viability was observed after treatment with BMP4. In three cultures with highest sensitivity for BMP4, a substantial new cell subpopulation formed. These cells displayed decreased cell proliferation and increased apoptosis. Neuronal differentiation was reduced most in cultures with little sensitivity for BMP4. OLIG1/2 levels were found predictive for high sensitivity to BMP4. Activation of ribosomal translation (RPL27A, RPS27) was up-regulated within one day in cultures that were very sensitive to BMP4. CONCLUSION: The changes in composition of patient-derived GBM cultures obtained after treatment with BMP4 correlate with treatment efficacy. OLIG1/2 expression can predict this efficacy, and upregulation of RPL27A and RPS27 are useful early-response markers.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Proliferação de Células , Perfilação da Expressão Gênica , Biomarcadores/metabolismo , RNA/metabolismo , Células-Tronco Neoplásicas/metabolismo , Diferenciação Celular , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/farmacologia , Proteína Morfogenética Óssea 4/metabolismoRESUMO
Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry-/-RAG2-/- mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.
Assuntos
Células-Tronco Pluripotentes Induzidas , Teratoma , Animais , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/patologia , Camundongos , Organogênese , Organoides/metabolismo , Teratoma/patologiaRESUMO
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) have been identified in bacteria, archaea and mitochondria of plants, but not in eukaryotes. Here, we report the discovery of 12,572 putative CRISPRs randomly distributed across the human chromosomes, which we termed hCRISPRs. By using available transcriptome datasets, we demonstrate that hCRISPRs are distinctively expressed as small non-coding RNAs (sncRNAs) in cell lines and human tissues. Moreover, expression patterns thereof enabled us to distinguish normal from malignant tissues. In prostate cancer, we confirmed the differential hCRISPR expression between normal adjacent and malignant primary prostate tissue by RT-qPCR and demonstrate that the SHERLOCK and DETECTR dipstick tools are suitable to detect these sncRNAs. We anticipate that the discovery of CRISPRs in the human genome can be further exploited for diagnostic purposes in cancer and other medical conditions, which certainly will lead to the development of point-of-care tests based on the differential expression of the hCRISPRs.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Pequeno RNA não Traduzido , Archaea/genética , Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma Humano , Humanos , MasculinoRESUMO
PURPOSE: In a post hoc analysis of the CATNON trial (NCT00626990), we explored whether adding temozolomide to radiotherapy improves outcome in patients with IDH1/2 wildtype (wt) anaplastic astrocytomas with molecular features of glioblastoma [redesignated as glioblastoma, isocitrate dehydrogenase-wildtype (IDH-wt) in the 2021 World Health Organization (WHO) classification of central nervous system tumors]. PATIENTS AND METHODS: From the randomized phase III CATNON study examining the addition of adjuvant and concurrent temozolomide to radiotherapy in anaplastic astrocytomas, we selected a subgroup of IDH1/2wt and H3F3Awt tumors with presence of TERT promoter mutations and/or EGFR amplifications and/or combined gain of chromosome 7 and loss of chromosome 10. Molecular abnormalities including MGMT promoter methylation status were determined by next-generation sequencing, DNA methylation profiling, and SNaPshot analysis. RESULTS: Of the 751 patients entered in the CATNON study, 670 had fully molecularly characterized tumors. A total of 159 of these tumors met the WHO 2021 molecular criteria for glioblastoma, IDH-wt. Of these patients, 47 received radiotherapy only and 112 received a combination of radiotherapy and temozolomide. There was no added effect of temozolomide on either overall survival [HR, 1.19; 95% confidence interval (CI), 0.82-1.71] or progression-free survival (HR, 0.87; 95% CI, 0.61-1.24). MGMT promoter methylation was prognostic for overall survival, but was not predictive for outcome to temozolomide treatment either with respect to overall survival or progression-free survival. CONCLUSIONS: In this cohort of patients with glioblastoma, IDH-wt temozolomide treatment did not add benefit beyond that observed from radiotherapy, regardless of MGMT promoter status. These findings require a new well-powered prospective clinical study to explore the efficacy of temozolomide treatment in this patient population.
Assuntos
Astrocitoma , Neoplasias Encefálicas , Glioblastoma , Antineoplásicos Alquilantes , Astrocitoma/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/radioterapia , Humanos , Isocitrato Desidrogenase/genética , Estudos Prospectivos , Temozolomida/uso terapêuticoRESUMO
Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFß/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.
Assuntos
Apoptose/genética , Carcinoma Ductal Pancreático/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Expressão Gênica , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Mutações Sintéticas Letais , Carcinoma Ductal Pancreático/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Humanos , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG's. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM.
Assuntos
Melanoma/genética , Melanoma/metabolismo , Metástase Neoplásica/genética , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Análise Mutacional de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
MIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.
Assuntos
Leucemia Mieloide Aguda/genética , MicroRNAs/genética , RNA Polimerase II/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
BACKGROUND: DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. RESULTS: The MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR. CONCLUSIONS: The MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.