Publications

<p>(A) GOT2 western blot of HeLa cells stably silenced for GOT2 expression by shRNA transfection compared to non-silenced scrambled (Scr) control cells. β-actin was used as a loading control. (B) Number of GOT2 KD HeLa cells after transfection with empty vector (EV), GOT2- WT cDNA, and GOT2- c.357A>T. Cells were seeded into 12-well plates and incubated for various times, as indicated. The counts are reported as means (n=3). A t-test was applied to test for differences. n.s.: not significant.</p>

Abstract Anaplastic thyroid cancer (ATC) is a clinically aggressive malignancy with a dismal prognosis. Combined BRAF/MEK inhibition offers significant therapeutic benefit in patients with BRAF V600E -mutant ATCs. However, relapses are common and overall survival remains poor. Compared with differentiated thyroid cancer, a hallmark of ATCs is significant infiltration with myeloid cells, particularly macrophages. ATCs are most common in the aging population, which also has an increased incidence of TET2 -mutant clonal hematopoiesis (CH). CH-mutant macrophages have been shown to accelerate CH-associated pathophysiology including atherosclerosis. However, the clinical and mechanistic contribution of CH-mutant clones to solid tumour biology, prognosis and therapeutic response has not been elucidated. Here we show that TET2 -mutant CH is enriched in the tumour microenvironment of patients with solid tumours and associated with adverse prognosis in ATC patients. We find that Tet2 -mutant macrophages selectively infiltrate mouse Braf V600E -mutant ATC and that their overexpression of Tgfβ-family ligands mediates resistance to BRAF/MEK inhibition. Importantly, inhibition of Tgfβ signaling restores sensitivity to MAPK pathway inhibition, opening a path for synergistic strategies to improve outcomes of patients with ATCs and concurrent CH.

Abstract Sirtuins are key players in the response to oxidative, metabolic and genotoxic stress, and are involved in genome stability, metabolic homeostasis and aging. Originally described as NAD + -dependent deacetylases, some sirtuins are also characterized by a poorly understood mono-ADP-ribosyltransferase (mADPRT) activity. Here we report that the deacetylase SirT7 is a dual sirtuin as it also features auto-mADPRT activity. Molecular and structural evidence suggests that this novel activity occurs at a second previously undefined active site that is physically separated in another domain. Specific abrogation of this activity alters SirT7 chromatin distribution, suggesting a role for this modification in SirT7 chromatin binding specificity. We uncover an epigenetic pathway by which ADP-ribosyl-SirT7 is recognized by the ADP-ribose reader macroH2A1.1, a histone variant involved in chromatin organization, metabolism and differentiation. Glucose starvation (GS) boosts this interaction and promotes SirT7 relocalization to intergenic regions in a macroH2A1-dependent manner. Both SirT7 activities are in turn required to promote GS-dependent enrichment of macroH2A1 in a subset of nearby genes, which results in their specific up- or downregulation. Consistently, the expression changes of these genes associated to calorie restriction (CR) or aging are abrogated in SirT7 -/- mice, reinforcing the link between Sirtuins, CR and aging. Our work provides a novel perspective about sirtuin duality and suggests a key role for SirT7/macroH2A1.1 axis in mammalian glucose homeostasis, calorie restriction signaling and aging.

<p>Histologic expression of basal markers in HR-positive breast tumors at different tumor stages. <b>A,</b> Representative sections of HR-positive samples at different tumor stages (LGDCIS, DCIS, and IDC) stained with the indicated basal markers. Scale bar, 50 μm. <b>B,</b> Bar plots representing the percentage of cells expressing each marker in in HR-positive samples across the different tumor stages. Statistics calculated two-way ANOVA. *, <i>P</i> < 0.05; **, <i>P</i> < 0.005; ns, not significant.</p>

<p>Primary antibodies used for immunohistochemistry</p>

<p>Linear regression analysis of 15,798 genes with with transcript expression, DNA methylation, and DNA copy number data available.</p>

Mitochondrial DNA (mtDNA) mutations scattered through coding and noncoding regions have been reported in cancer. The mechanisms that generate such mutations and the importance of mtDNA mutations in tumor development are still not clear. Here we present the identification of a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, as a mutational hotspot in primary tumors. Twenty-two % of the 247 primary tumors analyzed harbored somatic deletions/insertions at this mononucleotide repeat. Moreover, these alterations were also present in head and neck preneoplastic lesions. We further characterized the D310 variants that appeared in the lung and head and neck tumors. Most of the somatic alterations found in tumors showed deletion/insertions of 1- or 2-bp generating D310 variants identical to constitutive polymorphisms described previously. Sequencing analysis of individual clones from lymphocytes revealed that patients with D310 mutations in the tumors had statistically significant higher levels of D310 heteroplasmy (more than one length variant) in the lymphocyte mtDNA as compared with the patients without D310 mutations in the tumor mtDNA. On the basis of our observations, we propose a model in which D310 alterations are already present in normal cells and achieve homoplasmy in the tumor through a restriction/amplification event attributable to random genetic drift and clonal expansion.

Intrinsic disorder plays an important functional role in proteins. Disordered regions are linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and disordered regions displaying reciprocal, interdependent features. Although lacking well-defined conformation, disordered regions may affect the intrinsic stability and functional properties of ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl-binding domain (MBD) is one of the key domains in MeCP2, responsible for DNA recognition. It has been reported previously that the two disordered domains flanking MBD, the N-terminal domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single tryptophan in MBD) or from driving the protein unfolding by temperature, we have studied the MBD stability by differential scanning calorimetry (reporting on the global unfolding process) and chemical denaturation (altering intramolecular interactions by a different mechanism compared to thermal denaturation).

<p>Expression of let-7c (A) and miR-29c (B) miRNAs was evaluated in Karpas1718 parental and resistant lines by real time PCR upon previous exposure to DMSO (control, grey), decitabine (100nM, yellow) or tazemetostat (5µM, red). RQ values calculated by the DDCt method, RNU6 was used as housekeeping miRNA. Data derived from three independent experiments.</p>

The longevity-promoting NAD + –dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.

This Report has been prepared for the European Commission, DG TAXUD under contract TAXUD/2017/DE/329, “Study and Reports on the VAT Gap in the EU-28 Member States” and serves as a follow-up to the six reports published between 2013 and 2018. This Study contains new estimates of the Value Added Tax (VAT) Gap for 2017, as well as updated estimates for 2013-2016. As a novelty in this series of reports, so called “fast VAT Gap estimates” are also presented the year immediately preceding the analysis, namely for 2018. In addition, the study reports the results of the econometric analysis of VAT Gap determinants initiated and initially reported in the 2018 Report (Poniatowski et al., 2018). It also scrutinises the Policy Gap in 2017 as well as the contribution that reduced rates and exemptions made to the theoretical VAT revenue losses.

Supplementary Materials and Tables 1-3 from Chromosomal Instability Correlates with Genome-wide DNA Demethylation in Human Primary Colorectal Cancers

Abstract Early stages of type 1 diabetes (T1D) are characterized by local autoimmune inflammation and progressive loss of insulin-producing pancreatic β cells. We show here that exposure to pro-inflammatory cytokines unmasks a marked plasticity of the β-cell regulatory landscape. We expand the repertoire of human islet regulatory elements by mapping stimulus-responsive enhancers linked to changes in the β-cell transcriptome, proteome and 3D chromatin structure. Our data indicates that the β cell response to cytokines is mediated by the induction of novel regulatory regions as well as the activation of primed regulatory elements pre-bound by islet-specific transcription factors. We found that T1D-associated loci are enriched of the newly mapped cis-regulatory regions and identify T1D-associated variants disrupting cytokine-responsive enhancer activity in human β cells. Our study illustrates how β cells respond to a pro-inflammatory environment and implicate a role for stimulus-response islet enhancers in T1D.