Anne Schepers

Bio: Anne Schepers is an academic researcher from University of Antwerp. The author has contributed to research in topics: Epigenome & DNA methylation. The author has an hindex of 4, co-authored 5 publications receiving 55 citations.

The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

Abstract: DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2-6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

Hotspot DAXX, PTCH2 and CYFIP2 mutations in pancreatic neuroendocrine neoplasms.

Abstract: Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs) However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance) Found variants were validated by Sanger sequencing Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476 Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2 This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN

Ferroptosis Induction in Multiple Myeloma Cells Triggers DNA Methylation and Histone Modification Changes Associated with Cellular Senescence

Abstract: Disease relapse and therapy resistance remain key challenges in treating multiple myeloma. Underlying (epi-)mutational events can promote myelomagenesis and contribute to multi-drug and apoptosis resistance. Therefore, compounds inducing ferroptosis, a form of iron and lipid peroxidation-regulated cell death, are appealing alternative treatment strategies for multiple myeloma and other malignancies. Both ferroptosis and the epigenetic machinery are heavily influenced by oxidative stress and iron metabolism changes. Yet, only a limited number of epigenetic enzymes and modifications have been identified as ferroptosis regulators. In this study, we found that MM1 multiple myeloma cells are sensitive to ferroptosis induction and epigenetic reprogramming by RSL3, irrespective of their glucocorticoid-sensitivity status. LC-MS/MS analysis revealed the formation of non-heme iron-histone complexes and altered expression of histone modifications associated with DNA repair and cellular senescence. In line with this observation, EPIC BeadChip measurements of significant DNA methylation changes in ferroptotic myeloma cells demonstrated an enrichment of CpG probes located in genes associated with cell cycle progression and senescence, such as Nuclear Receptor Subfamily 4 Group A member 2 (NR4A2). Overall, our data show that ferroptotic cell death is associated with an epigenomic stress response that might advance the therapeutic applicability of ferroptotic compounds.

Insufficient evidence for a role of SERPINF1 in otosclerosis

Abstract: Otosclerosis is a common form of hearing loss (HL) due to abnormal remodeling of the otic capsule. The genetic causes of otosclerosis remain largely unidentified. Only mutations in a single gene, SERPINF1, were previously published in patients with familial otosclerosis. To unravel the contribution of genetic variation in this gene to otosclerosis, this gene was re-sequenced in a large population of otosclerosis patients and controls. Resequencing of the 5′ and 3′ UTRs, coding regions, and exon–intron boundaries of SERPINF1 was performed in 1604 unrelated otosclerosis patients and 1538 unscreened controls, and in 62 large otosclerosis families. Our study showed no enrichment of rare variants, stratified by type, in SERPINF1 in patients versus controls. Furthermore, the c.392C > A (p.Ala131Asp) variant, previously reported as pathogenic, was identified in three patients and four controls, not replicating its pathogenic nature. We could also not find evidence for a pathogenic role in otosclerosis for 5′ UTR variants in the SERPINF1-012 transcript (ENST00000573763), described as the major transcript in human stapes. Furthermore, no rare variants were identified in the otosclerosis families. This study does not support a pathogenic role for variants in SERPINF1 as a cause of otosclerosis. Therefore, the etiology of the disease remains largely unknown and will undoubtedly be the focus of future studies.

Patterns of Somatic Mutation in Human Cancer Genomes

Abstract: AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore PL02-05 All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death.

Abstract: Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis.

The gasdermins, a protein family executing cell death and inflammation

Abstract: The gasdermins are a family of recently identified pore-forming effector proteins that cause membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. Gasdermins contain a cytotoxic N-terminal domain and a C-terminal repressor domain connected by a flexible linker. Proteolytic cleavage between these two domains releases the intramolecular inhibition on the cytotoxic domain, allowing it to insert into cell membranes and form large oligomeric pores, which disrupts ion homeostasis and induces cell death. Gasdermin-induced pyroptosis plays a prominent role in many hereditary diseases and (auto)inflammatory disorders as well as in cancer. In this Review, we discuss recent developments in gasdermin research with a focus on mechanisms that control gasdermin activation, pore formation and functional consequences of gasdermin-induced membrane permeabilization.

Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation.

Abstract: Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.