VID22 counteracts G-quadruplex-induced genome instability

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism

Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5 ' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs

EPIGENETIC ALTERATIONS INDUCED BY THE PML-RAR ONCOGENE DURING THE TRANSFORMATION PROCESS OF ITS TARGET CELLS

Acute promyelocytic leukaemia (APL) is a subtype of acute myeloid leukaemia, caused by the t(15;17) translocation that generates an aberrant transcription factor PML-RAR\u3b1. Owing to its ability to affect gene expression through chromatin modifications, PML-RAR\u3b1 is potentially capable of a multitude of alterations in the chromatin landscape, most of which are still poorly characterized. To investigate PML-RAR\u3b1 mechanistical activity at early stages of tumor initiation, first we started elucidating the target cell(s) in which the leukemogenesis takes place. Our results showed that phenotypically defined common myeloid progenitor hematopoietic (CMPs), are capable of initiating leukemogenesis. We found that PML-RAR\u3b1 can induce an adult stem cell signature in normal myeloid progenitors, distinct from that observed in frankly established leukemic stem cells. Among the transcriptional targets identified, the cell cycle inhibitor p21, required for self-renewal of normal and leukemic stem cells, was shown to be indispensable for this phenomenon. To mechanistically dissect the pre-leukemic epigenome of the identified hematopoietic subpopulation, we developed ad hoc technological approaches to study small population of cells (miniChIP-sequencing, Nuclease Accessible Site Sequencing and DNA methylation sequencing). The analysis of the changes between chromatin states show that the oncogene in the pre-leukemic state does not impose a global alteration of chromatin but, rather, it functions as a local modulator of chromatin accessibility involving genomic regions enriched in important regulators of normal and aberrant hematopoiesis

TMBleR: A bioinformatic tool to optimize TMB estimation and predictive power

Motivation: Tumor mutational burden (TMB) has been proposed as a predictive biomarker for immunotherapy response in cancer patients, as it is thought to enrich for tumors with high neoantigen load. TMB assessed by whole-exome sequencing is considered the gold standard but remains confined to research settings. In the clinical setting, targeted gene panels sampling various genomic sizes along with diverse strategies to estimate TMB were proposed and no real standard has emerged yet. Results: We provide the community with TMBleR, a tool to measure the clinical impact of various strategies of panel-based TMB measurement

NA-Seq: a discovery tool for the analysis of chromatin structure and dynamics during differentiation

It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements

Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer

Tumor evolution is shaped by many variables, potentially involving external selective pressures induced by therapies1. After surgery, patients with estrogen receptor (ERα)-positive breast cancer are treated with adjuvant endocrine therapy2, including selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs)3. However, more than 20% of patients relapse within 10 years and eventually progress to incurable metastatic disease4. Here we demonstrate that the choice of therapy has a fundamental influence on the genetic landscape of relapsed diseases. We found that 21.5% of AI-treated, relapsed patients had acquired CYP19A1 (encoding aromatase) amplification (CYP19A1amp). Relapsed patients also developed numerous mutations targeting key breast cancer–associated genes, including ESR1 and CYP19A1. Notably, CYP19A1amp cells also emerged in vitro, but only in AI-resistant models. CYP19A1 amplification caused increased aromatase activity and estrogen-independent ERα binding to target genes, resulting in CYP19A1amp cells showing decreased sensitivity to AI treatment. These data suggest that AI treatment itself selects for acquired CYP19A1amp and promotes local autocrine estrogen signaling in AI-resistant metastatic patients

Prospective Validation of the Italian Alliance Against Cancer Lung Panel in Patients With Advanced Non-Small-Cell Lung Cancer

none26Background: The deeper knowledge of non-small-cell lung cancer (NSCLC) biology and the discovery of driver molecular alterations have opened the era of precision medicine in lung oncology, thus significantly revolutionizing the diagnostic and therapeutic approach to NSCLC. In Italy, however, molecular assessment remains heterogeneous across the country, and numbers of patients accessing personalized treatments remain relatively low. Nationwide programs have demonstrated that the creation of consortia represent a successful strategy to increase the number of patients with a molecular classification. Patients and methods: The Alliance Against Cancer (ACC), a network of 25 Italian Research Institutes, has developed a targeted sequencing panel for the detection of genomic alterations in 182 genes in patients with a diagnosis of NSCLC (ACC lung panel). One thousand metastatic NSCLC patients will be enrolled onto a prospective trial designed to measure the sensitivity and specificity of the ACC lung panel as a tool for molecular screening compared to standard methods. Results and conclusion: The ongoing trial is part of a nationwide strategy of ACC to develop infrastructures and improve competences to make the Italian research institutes independent for genomic profiling of cancer patients.mixedGregorc, Vanesa; Mazzarella, Luca; Lazzari, Chiara; Graziano, Paolo; Vigneri, Paolo; Genova, Carlo; Toschi, Luca; Ciliberto, Gennaro; Bonanno, Laura; Delmonte, Angelo; Bucci, Gabriele; Rossi, Antonio; Motta, Gianmarco; Coco, Simona; Marinello, Arianna; Buglioni, Simonetta; Cangi, Maria Giulia; Di Micco, Concetta; Bandiera, Alessandro; Bonfiglio, Silvia; Pecciarini, Lorenza; Guida, Alessandro; Ceol, Arnaud; Frige', Gianmaria; De Maria, Ruggero; Pelicci, Pier GiuseppeGregorc, Vanesa; Mazzarella, Luca; Lazzari, Chiara; Graziano, Paolo; Vigneri, Paolo; Genova, Carlo; Toschi, Luca; Ciliberto, Gennaro; Bonanno, Laura; Delmonte, Angelo; Bucci, Gabriele; Rossi, Antonio; Motta, Gianmarco; Coco, Simona; Marinello, Arianna; Buglioni, Simonetta; Cangi, Maria Giulia; Di Micco, Concetta; Bandiera, Alessandro; Bonfiglio, Silvia; Pecciarini, Lorenza; Guida, Alessandro; Ceol, Arnaud; Frige', Gianmaria; De Maria, Ruggero; Pelicci, Pier Giusepp

GLP-1 receptor agonists-SGLT-2 inhibitors combination therapy and cardiovascular events after acute myocardial infarction: an observational study in patients with type 2 diabetes

Background: Few studies explored the effect of the combination of glucose sodium-cotransporter-2 inhibitors (SGLT-2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) on the incidence of cardiovascular events in patients with type 2 diabetes (T2D) and acute myocardial infarction (AMI). Methods: We recruited patients with T2D and AMI undergoing percutaneous coronary intervention, treated with either SGLT-2i or GLP-1RA for at least 3 months before hospitalization. Subjects with HbA1c < 7% at admission were considered in good glycemic control and maintained the same glucose-lowering regimen, while those with poor glycemic control (HbA1c ≥ 7%), at admission or during follow-up, were prescribed either a SGLT-2i or a GLP-1RA to obtain a SGLT-2i/GLP-1RA combination therapy. The primary outcome was the incidence of major adverse cardiovascular events (MACE) defined as cardiovascular death, re-acute coronary syndrome, and heart failure related to AMI during a 2-year follow-up. After 3 months, the myocardial salvage index (MSI) was assessed by single-photon emission computed tomography. Findings: Of the 537 subjects screened, 443 completed the follow-up. Of these, 99 were treated with SGLT-2i, 130 with GLP-1RA, and 214 with their combination. The incidence of MACE was lower in the combination therapy group compared with both SGLT-2i and GLP-1RA treated patients, as assessed by multivariable Cox regression analysis adjusted for cardiovascular risk factors (HR = 0.154, 95% CI 0.038-0.622, P = 0.009 vs GLP-1RA and HR = 0.170, 95% CI 0.046-0.633, P = 0.008 vs SGLT-2i). The MSI and the proportion of patients with MSI > 50% was higher in the SGLT-2i/GLP-1RA group compared with both SGLT-2i and GLP-1RA groups. Interpretation: The combination of SGLT-2i and GLP-1RA is associated with a reduced incidence of cardiovascular events in patients with T2D and AMI compared with either drug used alone, with a significant effect also on peri-infarcted myocardial rescue in patients without a second event. Trial registraition ClinicalTrials.gov ID: NCT06017544