RESUMO
Pathways that direct the selection of the telomerase-dependent or recombination-based, alternative lengthening of telomere (ALT) maintenance pathway in cancer cells are poorly understood. Using human lung cancer cells and tumor organoids we show that formation of the 2,2,7-trimethylguanosine (TMG) cap structure at the human telomerase RNA 5' end by the Trimethylguanosine Synthase 1 (TGS1) is central for recruiting telomerase to telomeres and engaging Cajal bodies in telomere maintenance. TGS1 depletion or inhibition by the natural nucleoside sinefungin impairs telomerase recruitment to telomeres leading to Exonuclease 1 mediated generation of telomere 3' end protrusions that engage in RAD51-dependent, homology directed recombination and the activation of key features of the ALT pathway. This indicates a critical role for 2,2,7-TMG capping of the RNA component of human telomerase (hTR) in enforcing telomerase-dependent telomere maintenance to restrict the formation of telomeric substrates conductive to ALT. Our work introduces a targetable pathway of telomere maintenance that holds relevance for telomere-related diseases such as cancer and aging.
Assuntos
Telomerase , Guanosina , Humanos , RNA/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
PURPOSE: Total proctocolectomy with ileal J-pouch-anorectal anastomosis (IPAA) remains the preferred surgical treatment for ulcerative colitis (UC) in children. Considering the well-known advantages of minimally invasive approach, and its main application for the deep pelvis, robotic surgery may be used in UC reconstructive procedures. The aim of the study is to report our experience with Robotic IPAA in children. METHODS: Single surgeon experience on Robotic IPAA were prospectively included. Data on patient demographics, surgical details, complications, and length of stay (LOS), were collected. RESULTS: Fifteen patients were included. Median age was 13.2 years, median body weight 45 kg. Median operative time was 240 min. Median LOS was 7 days and mean follow-up time 1 year. No intraoperative complication occurred. Five postoperative complications happened: 3 minors treated conservatively (CD I-II), 2 majors needing reintervention under anesthesia (CD IIIb). No mortality was observed. CONCLUSION: Our preliminary experience reveals that Robotic IPAA is a safe and feasible option for the surgical treatment of UC in children. A bigger patient sample and a long-term follow-up are needed to confirm our findings.
Assuntos
Colite Ulcerativa , Bolsas Cólicas , Laparoscopia , Proctocolectomia Restauradora , Procedimentos Cirúrgicos Robóticos , Adolescente , Canal Anal/cirurgia , Anastomose Cirúrgica , Criança , Colite Ulcerativa/cirurgia , Humanos , Complicações Pós-Operatórias/epidemiologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.
Assuntos
Mutação , Neoplasias , Biomarcadores Tumorais , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Reprodutibilidade dos Testes , Carga TumoralRESUMO
Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are rare oncogenic drivers in solid tumours. This study aimed to interrogate a large real-world database of comprehensive genomic profiling data to describe the genomic landscape and prevalence of NTRK gene fusions. NTRK fusion-positive tumours were identified from the FoundationCORE® database of >295,000 cancer patients. We investigated the prevalence and concomitant genomic landscape of NTRK fusions, predicted patient ancestry and compared the FoundationCORE cohort with entrectinib clinical trial cohorts (ALKA-372-001 [EudraCT 2012-000148-88]; STARTRK-1 [NCT02097810]; STARTRK-2 [NCT02568267]). Overall NTRK fusion-positive tumour prevalence was 0.30% among 45 cancers with 88 unique fusion partner pairs, of which 66% were previously unreported. Across all cases, prevalence was 0.28% and 1.34% in patients aged ≥18 and <18 years, respectively; prevalence was highest in patients <5 years (2.28%). The highest prevalence of NTRK fusions was observed in salivary gland tumours (2.62%). Presence of NTRK gene fusions did not correlate with other clinically actionable biomarkers; there was no co-occurrence with known oncogenic drivers in breast, or colorectal cancer (CRC). However, in CRC, NTRK fusion-positivity was associated with spontaneous microsatellite instability (MSI); in this MSI CRC subset, mutual exclusivity with BRAF mutations was observed. NTRK fusion-positive tumour types had similar frequencies in FoundationCORE and entrectinib clinical trials. NTRK gene fusion prevalence varied greatly by age, cancer type and histology. Interrogating large datasets drives better understanding of the characteristics of very rare molecular subgroups of cancer and allows identification of genomic patterns and previously unreported fusion partners not evident in smaller datasets.
RESUMO
Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.
Assuntos
Aminopeptidases/metabolismo , DNA Polimerase beta/metabolismo , Quadruplex G , Serina Proteases/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Linhagem Celular Tumoral , Humanos , Complexos Multiproteicos , Proteína de Replicação A/metabolismo , Complexo Shelterina , Telômero/química , Telômero/metabolismoRESUMO
DNA damage response (DDR) is essential for the maintenance of genomic integrity. We have recently discovered the generation of noncoding RNA from a DNA double-strand break (DSB) in an MRE11-RAD50-NBS1 complex-dependent manner, which are necessary for full DDR activation. The low abundance of these noncoding RNA makes them difficult to identify and study. In this chapter, we describe an in vitro biochemical assay to study the generation of damage-induced long noncoding RNA (dilncRNA) from a DNA DSB. In this assay, transcriptionally competent cell-free extracts upon incubation with a linear DNA support RNA synthesis from DNA ends, as monitored by incorporation of 32P[UTP] in discrete products resolved on a denaturing polyacrylamide gel. This approach can be used to identify the role of different DDR proteins in generating dilncRNA.
Assuntos
Dano ao DNA/genética , DNA/genética , RNA Longo não Codificante/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sistema Livre de Células/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Células K562 , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Transcrição Gênica/genéticaRESUMO
The synthesis of middle-to-late-replicating DNA can be affected independently of the rest of the genome by down-regulating the tumor suppressor PREP1 (PKNOX1). Indeed, DNA combing shows that PREP1 down-regulation affects DNA replication rate, increases the number of simultaneously firing origins and the asymmetry of DNA replication, leading to DNA damage. Genome-wide analysis of replication timing by Repli-seq shows that, upon PREP1 down-regulation, 25% of the genome is replicated earlier in the S-phase. The targeted DNA sequences correspond to Lamin-Associated Domains (LADs), and include late-replicating (LRRs) and temporal transition regions (TTRs). Notably, the distribution of PREP1 DNA binding sites and of its target genes indicates that DNA replication defects are independent of the overall PREP1 transcriptional activity. Finally, PREP1 down-regulation causes a substantial decrease in Lamin B1 levels. This suggests that DNA is released from the nuclear lamina earlier than in the control cells and is available for replication, thus explaining timing defects and DNA damage.This is the first evidence that the replication timing of a specific fraction of the human genome is affected by PREP1 tumor suppressor. This previously unknown function might significantly contribute to the genomic instability observed in human tumors.
Assuntos
Período de Replicação do DNA/fisiologia , Genes Supressores de Tumor/fisiologia , Instabilidade Genômica , Proteínas de Homeodomínio/fisiologia , Sítios de Ligação , Dano ao DNA , Período de Replicação do DNA/genética , Regulação da Expressão Gênica , Genoma Humano , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Lamina Tipo B/metabolismoRESUMO
BACKGROUND: Relapsed/metastatic salivary gland carcinomas (SGCs) have a wide diversity of histologic subtypes associated with variable clinical aggressiveness and response to local and systemic therapies. We queried whether comprehensive genomic profiling could define the tumor subtypes and uncover clinically relevant genomic alterations, revealing new routes to targeted therapies for patients with relapsed and metastatic disease. PATIENTS AND METHODS: From a series of 85 686 clinical cases, DNA was extracted from 40 µm of formalin-fixed paraffin embedded (FFPE) sections for 623 consecutive SGC. CGP was carried out on hybridization-captured, adaptor ligation-based libraries (mean coverage depth, >500×) for up to 315 cancer-related genes. Tumor mutational burden was determined on 1.1 Mb of sequenced DNA. All classes of alterations, base substitutions, short insertions/deletions, copy number changes, and rearrangements/fusions were determined simultaneously. RESULTS: The clinically more indolent SGC including adenoid cystic carcinoma, acinic cell carcinoma, polymorphous low-grade adenocarcinoma, mammary analog secretory carcinoma, and epithelial-myoepithelial carcinomas have significantly fewer genomic alterations, TP53 mutations, and lower tumor mutational burden than the typically more aggressive SGCs including mucoepidermoid carcinoma, salivary duct carcinoma, adenocarcinoma, not otherwise specified, carcinoma NOS, and carcinoma ex pleomorphic adenoma. The more aggressive SGCs are commonly driven by ERBB2 PI3K pathway genomic alterations. Additional targetable GAs are frequently seen. CONCLUSIONS: Genomic profiling of SGCs demonstrates important differences between traditionally indolent and aggressive cancers. These differences may provide therapeutic options in the future.
Assuntos
Carcinoma/genética , Recidiva Local de Neoplasia/genética , Neoplasias das Glândulas Salivares/genética , Idoso , Carcinoma/patologia , DNA de Neoplasias/genética , Feminino , Formaldeído , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Inclusão em Parafina , Neoplasias das Glândulas Salivares/patologia , Fixação de TecidosRESUMO
In response to ionizing radiation (IR), cells activate a DNA damage response (DDR) pathway to re-program gene expression. Previous studies using total cellular RNA analyses have shown that the stress kinase ATM and the transcription factor p53 are integral components required for induction of IR-induced gene expression. These studies did not distinguish between changes in RNA synthesis and RNA turnover and did not address the role of enhancer elements in DDR-mediated transcriptional regulation. To determine the contribution of synthesis and degradation of RNA and monitor the activity of enhancer elements following exposure to IR, we used the recently developed Bru-seq, BruChase-seq and BruUV-seq techniques. Our results show that ATM and p53 regulate both RNA synthesis and stability as well as enhancer element activity following exposure to IR. Importantly, many genes in the p53-signaling pathway were coordinately up-regulated by both increased synthesis and RNA stability while down-regulated genes were suppressed either by reduced synthesis or stability. Our study is the first of its kind that independently assessed the effects of ionizing radiation on transcription and post-transcriptional regulation in normal human cells.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Processamento Pós-Transcricional do RNA , Radiação Ionizante , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Dano ao DNA/efeitos da radiação , Elementos Facilitadores Genéticos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Estabilidade de RNA/efeitos da radiação , Ativação TranscricionalRESUMO
Primary mouse embryonic fibroblasts (MEFs) are a popular tool for molecular and cell biology studies. However, when MEFs are grown in vitro under standard tissue culture conditions, they proliferate only for a limited number of population doublings (PD) and eventually undergo cellular senescence. Presently, the molecular mechanisms halting cell cycle progression and establishing cellular senescence under these conditions are unclear. Here, we show that a robust DNA damage response (DDR) is activated when MEFs undergo replicative cellular senescence. Senescent cells accumulate senescence-associated DDR foci (SDFs) containing the activated form of ATM, its phosphorylated substrates and gammaH2AX. In senescent MEFs, DDR markers do not preferentially accumulate at telomeres, the end of linear chromosomes. It has been observed that proliferation of MEFs is extended if they are cultured at low oxygen tension (3% O(2)). We observed that under these conditions, DDR is not observed and senescence is not established. Importantly, inactivation of ATM in senescent MEFs allows escape from senescence and progression through the S-phase. Therefore, MEFs undergoing cellular senescence arrest their proliferation due to the activation of a DNA damage checkpoint mediated by ATM kinase. Finally, we observed that spontaneously immortalized proliferating MEFs display markers of an activated DDR, indicating the presence of chromosomal DNA damage in these established cell lines.