Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Chromatin epigenetics and nuclear lamina keep the nucleus in shape: Examples from natural and accelerated aging.

As the repository of genetic information, the cell nucleus must protect DNA integrity from mechanical stresses. The nuclear lamina, which resides within the nuclear envelope (NE), is made up of lamins, intermediate filaments bound to DNA. The nuclear lamina provides the nucleus with the ability to deal with inward as well as outward mechanical stimuli. Chromatin, in turn, through its degrees of compaction, shares this role with the nuclear lamina, thus, ensuring the plasticity of the nucleus. Perturbation of chromatin condensation or the nuclear lamina has been linked to a plethora of biological conditions, that range from cancer and genetic diseases (laminopathies) to aging, both natural and accelerated, such as the case of Hutchinson-Gilford Progeria Syndrome (HGPS). From the experimental results accumulated so far on the topic, a direct link between variations of the epigenetic pattern and nuclear lamina structure would be suggested, however, it has never been clarified thoroughly. This relationship, instead, has a downstream important implication on nucleus shape, genome preservation, force sensing, and, ultimately, aging-related disease onset. With this review, we aim to collect recent studies on the importance of both nuclear lamina components and chromatin status in nuclear mechanics. We also aim to bring to light evidence of the link between DNA methylation and nuclear lamina in natural and accelerated aging.

Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells.

It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations.

Aneuploid IMR90 cells induced by depletion of pRB, DNMT1 and MAD2 show a common gene expression signature.

Chromosome segregation defects lead to aneuploidy which is a major feature of solid tumors. How diploid cells face chromosome mis-segregation and how aneuploidy is tolerated in tumor cells are not completely defined yet. Thus, an important goal of cancer genetics is to identify gene networks that underlie aneuploidy and are involved in its tolerance. To this aim, we induced aneuploidy in IMR90 human primary cells by depleting pRB, DNMT1 and MAD2 and analyzed their gene expression profiles by microarray analysis. Bioinformatic analysis revealed a common gene expression profile of IMR90 cells that became aneuploid. Gene Set Enrichment Analysis (GSEA) also revealed gene-sets/pathways that are shared by aneuploid IMR90 cells that may be exploited for novel therapeutic approaches in cancer. Furthermore, Protein-Protein Interaction (PPI) network analysis identified TOP2A and KIF4A as hub genes that may be important for aneuploidy establishment.

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool "REPAIRv2" (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in different cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFPopal and CFTRW1282X, respectively.

Proliferation of aneuploid cells induced by CENP-E depletion is counteracted by the p14ARF tumor suppressor.

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that ensures the fidelity of chromosomes segregation. Reduced expression of some of its components weakens the SAC and induces chromosome instability and aneuploidy, which are both well-known hallmarks of cancer cells. Centromere protein-E (CENP-E) is a crucial component of the SAC and its function is to facilitate kinetochore microtubule attachment required to achieve and maintain chromosome alignment. The present study investigates the possible role of p14ARF as a controller of aneuploid cells proliferation. We used RNA interference to induce aneuploidy by partial depletion of CENP-E in human primary fibroblasts (IMR90) and in near diploid tumor cells (HCT116). In contrast to IMR90 aneuploid cell number, which was drastically reduced and leaned towards the WT condition, HCT116 aneuploid cell numbers were slightly decreased at later time points. This euploidy restoration was accompanied by increased p14ARF expression in IMR90 cells and followed ectopic p14ARF re-expression in p14ARF-null HCT116 cells. Collectively, our results suggest that hampering proliferation of aneuploid cells could be an additional role of the p14ARF tumor suppressor.

p14(ARF) Prevents Proliferation of Aneuploid Cells by Inducing p53-Dependent Apoptosis.

Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14(ARF) is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14(ARF) is involved in the maintenance of aneuploid cells in primary human cells, suggesting that it could be part of a pathway controlling their proliferation. To investigate this aspect further, p14(ARF) was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14(ARF) Re-expression reduced the number of aneuploid cells in MAD2 post-transcriptionally silenced cells. Also aberrant mitoses, frequently displayed in MAD2-depleted cells, were decreased when p14(ARF) was expressed at the same time. In addition, p14(ARF) ectopic expression in MAD2-depleted cells induced apoptosis associated with increased p53 protein levels. Conversely, p14(ARF) ectopic expression did not induce apoptosis in HCT116 p53KO cells. Collectively, our results suggest that the tumor suppressor p14(ARF) may have an important role in counteracting proliferation of aneuploid cells by activating p53-dependent apoptosis.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.

A Glimpse into Chromatin Organization and Nuclear Lamina Contribution in Neuronal Differentiation.

During embryonic development, stem cells undergo the differentiation process so that they can specialize for different functions within the organism. Complex programs of gene transcription are crucial for this process to happen. Epigenetic modifications and the architecture of chromatin in the nucleus, through the formation of specific regions of active as well as inactive chromatin, allow the coordinated regulation of the genes for each cell fate. In this mini-review, we discuss the current knowledge regarding the regulation of three-dimensional chromatin structure during neuronal differentiation. We also focus on the role the nuclear lamina plays in neurogenesis to ensure the tethering of the chromatin to the nuclear envelope.

MAD2 depletion triggers premature cellular senescence in human primary fibroblasts by activating a p53 pathway preventing aneuploid cells propagation.

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that ensures faithful chromosome segregation during mitosis and its failure can result in aneuploidy. Previously, it was suggested that reduction of the MAD2 gene, encoding a major component of the SAC, induced aneuploidy in human tumor cells. However, tumor cell lines contain multiple mutations that might affect or exacerbate the cellular response to Mad2 depletion. Thus, the scenario resulting by Mad2 depletion in primary human cells could be different and more complex that the one depicted so far. We used primary human fibroblasts (IMR90) and epithelial breast cells (MCF10A) to gain further insight on the effects of genomic instability caused by transient Mad2 depletion. To this aim we depleted Mad2 by RNAi to a level shown by Mad2 haplo-insufficient cells and found that induced aneuploidy caused premature cellular senescence in IMR90 cells. IMR90 cells showed typical features of senescent cells, like senescence-associated (SA) beta galactosidase expression, including up-regulation of p53 and p14ARF proteins and of p21(waf1) as well, but not of p16(INK4A) cyclin-dependent kinase (Cdk) inhibitor. In contrast, after MAD2 post-transcriptional silencing MCF10A cells in which the INK4A/ARF locus is deleted, showed both aneuploidy and a small increase of p53 and p21(waf1) proteins, but not premature cellular senescence. Finally, our results provides an explanation of how a p53 controlled pathway, involving initially p21(waf1) and then p14ARF, could minimize the occurrence of genomic alterations derived from chromosome instability induced by low amounts of MAD2 protein.

Specific Irreversible Cell-Cycle Arrest and Depletion of Cancer Cells Obtained by Combining Curcumin and the Flavonoids Quercetin and Fisetin.

Background: Induced senescence could be exploited to selectively counteract the proliferation of cancer cells and target them for senolysis. We examined the cellular senescence induced by curcumin and whether it could be targeted by fisetin and quercetin, flavonoids with senolytic activity. Methods: Cell-cycle profiles, chromosome number and structure, and heterochromatin markers were evaluated via flow cytometry, metaphase spreads, and immunofluorescence, respectively. The activation of p21waf1/cip1 was assessed via RT-qPCR and immunoblotting. Senescent cells were detected via SA-ß-Galactosidase staining. Results: We report that curcumin treatment specifically triggers senescence in cancer cells by inducing mitotic slippage and DNA damage. We show that curcumin-induced senescence is p21waf1/cip1-dependent and characterized by heterochromatin loss. Finally, we found that flavonoids clear curcumin-induced senescent cancer cells. Conclusions: Our findings expand the characterization of curcumin-induced cellular senescence in cancer cells and lay the foundation for the combination of curcumin and flavonoids as a possible anti-cancer therapy.

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells.

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

Losing DNA methylation at repetitive elements and breaking bad.

BACKGROUND: DNA methylation is an epigenetic chromatin mark that allows heterochromatin formation and gene silencing. It has a fundamental role in preserving genome stability (including chromosome stability) by controlling both gene expression and chromatin structure. Therefore, the onset of an incorrect pattern of DNA methylation is potentially dangerous for the cells. This is particularly important with respect to repetitive elements, which constitute the third of the human genome. MAIN BODY: Repetitive sequences are involved in several cell processes, however, due to their intrinsic nature, they can be a source of genome instability. Thus, most repetitive elements are usually methylated to maintain a heterochromatic, repressed state. Notably, there is increasing evidence showing that repetitive elements (satellites, long interspersed nuclear elements (LINEs), Alus) are frequently hypomethylated in various of human pathologies, from cancer to psychiatric disorders. Repetitive sequences' hypomethylation correlates with chromatin relaxation and unscheduled transcription. If these alterations are directly involved in human diseases aetiology and how, is still under investigation. CONCLUSIONS: Hypomethylation of different families of repetitive sequences is recurrent in many different human diseases, suggesting that the methylation status of these elements can be involved in preservation of human health. This provides a promising point of view towards the research of therapeutic strategies focused on specifically tuning DNA methylation of DNA repeats.

Transcriptomic Changes Following Partial Depletion of CENP-E in Normal Human Fibroblasts.

The centromere is a fundamental chromosome structure in which the macro-molecular kinetochore assembles and is bound by spindle microtubules, allowing the segregation of sister chromatids during mitosis. Any alterations in kinetochore assembly or functioning or kinetochore-microtubule attachments jeopardize chromosome stability, leading to aneuploidy, a common feature of cancer cells. The spindle assembly checkpoint (SAC) supervises this process, ensuring a faithful segregation of chromosomes. CENP-E is both a protein of the kinetochore and a crucial component of the SAC required for kinetochore-microtubule capture and stable attachment, as well as congression of chromosomes to the metaphase plate. As the function of CENP-E is restricted to mitosis, its haploinsufficiency has been used to study the induced cell aneuploidy; however, the gene expression profile triggered by CENP-E reduction in normal cells has never been explored. To fill this gap, here we investigated whether a gene network exists that is associated with an siRNA-induced 50% reduction in CENP-E and consequent aneuploidy. Gene expression microarray analyses were performed at early and late timepoints after transfection. Initially, cell cycle regulation and stress response pathways were downregulated, while afterwards pathways involved in epithelial-mesenchymal transition, hypoxia and xenobiotic metabolism were altered. Collectively, our results suggest that CENP-E reduction triggers a gene expression program that recapitulates some features of tumor cells.

FANCD2 modulates the mitochondrial stress response to prevent common fragile site instability.

Common fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability involves transcription- and replication-dependent mechanisms. Here, we uncover a role for the mitochondrial stress response pathway in the regulation of CFS stability in human cells. We show that FANCD2, a master regulator of CFS stability, dampens the activation of the mitochondrial stress response and prevents mitochondrial dysfunction. Genetic or pharmacological activation of mitochondrial stress signaling induces CFS gene expression and concomitant relocalization to CFSs of FANCD2. FANCD2 attenuates CFS gene transcription and promotes CFS gene stability. Mechanistically, we demonstrate that the mitochondrial stress-dependent induction of CFS genes is mediated by ubiquitin-like protein 5 (UBL5), and that a UBL5-FANCD2 dependent axis regulates the mitochondrial UPR in human cells. We propose that FANCD2 coordinates nuclear and mitochondrial activities to prevent genome instability.

Pyrazole[3,4-d]pyrimidine derivatives loaded into halloysite as potential CDK inhibitors.

Uncontrolled cell proliferation is a hallmark of cancer as a result of rapid and deregulated progression through the cell cycle. The inhibition of cyclin-dependent kinases (CDKs) activities is a promising therapeutic strategy to block cell cycle of tumor cells. In this work we reported a new example of nanocomposites based on halloysite nanotubes (HNTs)/pyrazolo[3,4-d]pyrimidine derivatives (Si306 and Si113) as anticancer agents and CDK inhibitors. HNTs/Si306 and HNTs/Si113 nanocomposites were synthesized and characterized. The release kinetics were also investigated. Antitumoral activity was evaluated on three cancer cell lines (HeLa, MDA-MB-231 and HCT116) and the effects on cell cycle arrest in HCT116 cells were evaluated. Finally, molecular dynamics simulations were performed of the complexes between Si113 or Si306 and the active site of both CDK 1 and 2.

P14ARF: The Absence that Makes the Difference.

P14ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation.

Dicer prevents genome instability in response to replication stress.

Dicer, an endoribonuclease best-known for its role in microRNA biogenesis and RNA interference pathway, has been shown to play a role in the DNA damage response and repair of double-stranded DNA breaks (DSBs) in mammalian cells. However, it remains unknown whether Dicer is also important to preserve genome integrity upon replication stress. To address this question, we focused our study on common fragile sites (CFSs), which are susceptible to breakage after replication stress. We show that inhibition of the Dicer pathway leads to an increase in CFS expression upon induction of replication stress and to an accumulation of 53BP1 nuclear bodies, indicating transmission of replication-associated damage. We also show that in absence of a functional Dicer or Drosha, the assembly into nuclear foci of the Fanconi anemia (FA) protein FANCD2 and of the replication and checkpoint factor TopBP1 in response to replication stress is impaired, and the activation of the S-phase checkpoint is defective. Based on these results, we propose that Dicer pre-vents genomic instability after replication stress, by allowing the proper recruitment to stalled forks of proteins that are necessary to maintain replication fork stability and activate the S-phase checkpoint, thus limiting cells from proceeding into mitosis with under-replicated DNA.

Phosphorylation of CENP-A on serine 7 does not control centromere function.

CENP-A is the histone H3 variant necessary to specify the location of all eukaryotic centromeres via its CENP-A targeting domain and either one of its terminal regions. In humans, several post-translational modifications occur on CENP-A, but their role in centromere function remains controversial. One of these modifications of CENP-A, phosphorylation on serine 7, has been proposed to control centromere assembly and function. Here, using gene targeting at both endogenous CENP-A alleles and gene replacement in human cells, we demonstrate that a CENP-A variant that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability. Thus, we conclude that phosphorylation of CENP-A on serine 7 is dispensable to maintain correct centromere dynamics and function.

DNA demethylation caused by 5-Aza-2'-deoxycytidine induces mitotic alterations and aneuploidy.

Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation.Here, we report that the DNA hypomethylating drug 5-aza-2'-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduction of 5-Methyl Cytosine at the pericentromeric region of chromosomes correlates with aneuploidy and mitotic defects.Our results suggest that DNA hypomethylation leads to aneuploidy by altering the DNA methylation landscape at the centromere that is necessary to ensure proper chromosomes segregation by recruiting the proteins necessary to build up a functional kinetochore.