Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer.

IMPORTANCE: Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.

PARP1 Inhibition Halts EBV+ Lymphoma Progression by Disrupting the EBNA2/MYC Axis.

PARP1 has been shown to regulate EBV latency. However, the therapeutic effect of PARP1 inhibitors on EBV+ lymphomagenesis has not yet been explored. Here, we show that PARPi BMN-673 has a potent anti-tumor effect on EBV-driven LCL in a mouse xenograft model. We found that PARP1 inhibition induces a dramatic transcriptional reprogramming of LCLs driven largely by the reduction of the MYC oncogene expression and dysregulation of MYC targets, both in vivo and in vitro. PARP1 inhibition also reduced the expression of viral oncoprotein EBNA2, which we previously demonstrated depends on PARP1 for activation of MYC. Further, we show that PARP1 inhibition blocks the chromatin association of MYC, EBNA2, and tumor suppressor p53. Overall, our study strengthens the central role of PARP1 in EBV malignant transformation and identifies the EBNA2/MYC pathway as a target of PARP1 inhibitors and its utility for the treatment of EBNA2-driven EBV-associated cancers.

Three-Dimensional Chromatin Structure of the EBV Genome: A Crucial Factor in Viral Infection.

Epstein-Barr Virus (EBV) is a human gamma-herpesvirus that is widespread worldwide. To this day, about 200,000 cancer cases per year are attributed to EBV infection. EBV is capable of infecting both B cells and epithelial cells. Upon entry, viral DNA reaches the nucleus and undergoes a process of circularization and chromatinization and establishes a latent lifelong infection in host cells. There are different types of latency all characterized by different expressions of latent viral genes correlated with a different three-dimensional architecture of the viral genome. There are multiple factors involved in the regulation and maintenance of this three-dimensional organization, such as CTCF, PARP1, MYC and Nuclear Lamina, emphasizing its central role in latency maintenance.

The nuclear lamina binds the EBV genome during latency and regulates viral gene expression.

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity.

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 and B-Cell Growth Transformation Induce Lipogenesis through Fatty Acid Synthase.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro Several B-cell malignancies are associated with latent LMP1-positive EBV infection, including Hodgkin's and diffuse large B-cell lymphomas. We have previously reported that promotion of B cell proliferation by LMP1 coincided with an induction of aerobic glycolysis. To further examine LMP1-induced metabolic reprogramming in B cells, we ectopically expressed LMP1 in an EBV-negative Burkitt's lymphoma (BL) cell line preceding a targeted metabolic analysis. This analysis revealed that the most significant LMP1-induced metabolic changes were to fatty acids. Significant changes to fatty acid levels were also found in primary B cells following EBV-mediated B-cell growth transformation. Ectopic expression of LMP1- and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. Furthermore, inhibition of lipogenesis caused preferential killing of LMP1-expressing B cells and significantly hindered EBV immortalization of primary B cells. Finally, our investigation also found that USP2a, a ubiquitin-specific protease, is significantly increased in LMP1-positive BL cells and mediates FASN stability. Our findings demonstrate that ectopic expression of LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, fatty acids, and lipid droplet formation, possibly pointing to a reliance on lipogenesis. Therefore, the use of lipogenesis inhibitors could be used in the treatment of LMP1+ EBV-associated malignancies by targeting an LMP1-specific dependency on lipogenesis.IMPORTANCE Despite many attempts to develop novel therapies, EBV-specific therapies currently remain largely investigational, and EBV-associated malignancies are often associated with a worse prognosis. Therefore, there is a clear demand for EBV-specific therapies for both prevention and treatment of virus-associated malignancies. Noncancerous cells preferentially obtain fatty acids from dietary sources, whereas cancer cells will often produce fatty acids themselves by de novo lipogenesis, often becoming dependent on the pathway for cell survival and proliferation. LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, a key enzyme responsible for the catalysis of endogenous fatty acids. Preferential killing of LMP1-expressing B cells following inhibition of FASN suggests that targeting LMP-induced lipogenesis is an effective strategy in treating LMP1-positive EBV-associated malignancies. Importantly, targeting unique metabolic perturbations induced by EBV could be a way to explicitly target EBV-positive malignancies and distinguish their treatment from EBV-negative counterparts.

TGFßR-SMAD3 Signaling Induces Resistance to PARP Inhibitors in the Bone Marrow Microenvironment.

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFßR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-ß1). Genetic and/or pharmacological targeting of the TGF-ß1-TGFßR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFßR inhibitor in patients receiving PARPis.

Poly(ADP-ribosylated) proteins in ß-amyloid peptide-stimulated microglial cells.

Amyloid-treated microglia prime and sustain neuroinflammatory processes in the central nervous system activating different signalling pathways inside the cells. Since a key role for PARP-1 has been demonstrated in inflammation and in neurodegeneration, we investigated PARylated proteins in resting and in ß-amyloid peptide treated BV2 microglial cells. A total of 1158 proteins were identified by mass spectrometry with 117 specifically modified in the amyloid-treated cells. Intervention of PARylation on the proteome of microglia showed to be widespread in different cellular districts and to affect various cellular pathways, highlighting the role of this dynamic post-translational modification in cellular regulation. Ubiquitination is one of the more enriched pathways, encompassing PARylated proteins like NEDD4, an E3 ubiquitine ligase and USP10, a de-ubiquitinase, both associated with intracellular responses induced by ß-amyloid peptide challenge. PARylation of NEDD4 may be involved in the recruiting of this protein to the plasma membrane where it regulates the endocytosis of AMPA receptors, whereas USP10 may be responsible for the increase of p53 levels in amyloid stimulated microglia. Unfolded protein response and Endoplasmic Reticulum Stress pathways, strictly correlated with the Ubiquitination process, also showed enrichment in PARylated proteins. PARylation may thus represent one of the molecular switches responsible for the transition of microglia towards the inflammatory microglia phenotype, a pivotal player in brain diseases including neurodegenerative processes. The establishment of trials with PARP inhibitors to test their efficacy in the containment of neurodegenerative diseases may be envisaged.

Functional heterogeneity as reflected by topological parameters in a classical protein molecular model: t4 phage lysozyme.

A systematic comparison with the Wild-Type (WT) of one-point mutants of bacteriophage T4 lysozyme was carried out using as difference markers the topological parameters of the protein contact networks corresponding to each crystallographic structure. The investigation concerned changes at the resolution level of single residue along the protein sequence. The results were correlated with (reported) changes in functional properties and (observed) changes in the information provided by the energy dissipation algorithm of the "Turbine" software simulation tool. The critical factor leading to significant difference among mutants and WT is in most cases associated to the sensitivity towards mutation of relatively short windows in the amino acidic sequence not necessarily contiguous to the active site.