De Novo p.Asp3368Gly Variant of Dystrophin Gene Associated with X-Linked Dilated Cardiomyopathy and Skeletal Myopathy: Clinical Features and In Silico Analysis.

Dystrophin (DMD) gene mutations are associated with skeletal muscle diseases such as Duchenne and Becker Muscular Dystrophy (BMD) and X-linked dilated cardiomyopathy (XL-DCM). To investigate the molecular basis of DCM in a 37-year-old woman. Clinical and genetic investigations were performed. Genetic testing was performed with whole exome sequencing (WES) using the Illumina platform. According to the standard protocol, a variant found by WES was confirmed in all available members of the family by bi-directional capillary Sanger resequencing. The effect of the variant was investigated by using an in silico prediction of pathogenicity. The index case was a 37-year-old woman diagnosed with DCM at the age of 33. A germline heterozygous A>G transversion at nucleotide 10103 in the DMD gene, leading to an aspartic acid-glycine substitution at the amino acid 3368 of the DMD protein (c.10103A>G p.Asp3368Gly), was identified and confirmed by PCR-based Sanger sequencing of the exon 70. In silico prediction suggests that this variant could have a deleterious impact on protein structure and functionality (CADD = 30). The genetic analysis was extended to the first-degree relatives of the proband (mother, father, and sister) and because of the absence of the variant in both parents, the p.Asp3368Gly substitution was considered as occurring de novo. Then, the direct sequencing analysis of her 8-year-old son identified as hemizygous for the same variant. The young patient did not present any signs or symptoms attributable to DCM, but reported asthenia and presented with bilateral calf hypertrophy at clinical examination. Laboratory testing revealed increased levels of creatinine kinase (maximum value of 19,000 IU/L). We report an early presentation of dilated cardiomyopathy in a 33-year-old woman due to a de novo pathogenic variant of the dystrophin (DMD) gene (p.Asp3368Gly). Genetic identification of this variant allowed an early diagnosis of a skeletal muscle disease in her son.

Investigation of a Large Kindred Reveals Cardiac Calsequestrin (CASQ2) as a Cause of Brugada Syndrome.

BACKGROUND: Brugada syndrome (BrS) is an inherited primary channelopathy syndrome associated with the risk of ventricular fibrillation (VF) and sudden cardiac death in a structurally normal heart. AIM OF THE STUDY: The aim of this study was to clinically and genetically evaluate a large family with severe autosomal dominant Brugada syndrome. METHODS: Clinical and genetic studies were performed. Genetic analysis was conducted with NGS technologies (WES) using the Illumina instrument. According to the standard procedure, variants found by WES were confirmed in all available families by Sanger sequencing. The effect of the variants was studied by using in silico prediction of pathogenicity. RESULTS: The proband was a 52-year-old man who was admitted to the emergency department for syncope at rest. WES of the index case identified a heterozygous VUS CASQ2, c.532T>C, p.(Tyr178His). We studied the segregation of the variation in all pedigree members. All the patients were heterozygous for the variation CASQ2 p.(Tyr178His), whereas the remaining healthy individuals in the family were homozygous for the normal allele. Structural analysis of CASQ2 p.(Tyr178His) was performed and revealed an important effect of the missense variation on monomer stability. The CASQ2 Tyr180 residue is located inside the sarcoplasmic reticulum (SR) junctional face membrane interaction domain and is predicted to disrupt filamentation. CONCLUSIONS: Our data suggest that the p.Tyr178His substitution is associated with BrS in the family investigated, affecting the stability of the protein, disrupting filamentation at the interdimer interface, and affecting the subsequent formation of tetramers and polymers that contain calcium-binding sites.

Epigenetic Immune Remodeling of Mesothelioma Cells: A New Strategy to Improve the Efficacy of Immunotherapy.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with a severe prognosis, and with a long-standing need for more effective therapeutic approaches. However, treatment with immune checkpoint inhibitors is becoming an increasingly effective strategy for MPM patients. In this scenario, epigenetic modifications may negatively regulate the interplay between immune and malignant cells within the tumor microenvironment, thus contributing to the highly immunosuppressive contexture of MPM that may limit the efficacy of immunotherapy. Aiming to further improve prospectively the clinical efficacy of immunotherapeutic approaches in MPM, we investigated the immunomodulatory potential of different classes of epigenetic drugs (i.e., DNA hypomethylating agent (DHA) guadecitabine, histone deacetylase inhibitors VPA and SAHA, or EZH2 inhibitors EPZ-6438) in epithelioid, biphasic, and sarcomatoid MPM cell lines, by cytofluorimetric and real-time PCR analyses. We also characterized the effects of the DHA, guadecitabine, on the gene expression profiles (GEP) of the investigated MPM cell lines by the nCounter platform. Among investigated drugs, exposure of MPM cells to guadecitabine, either alone or in combination with VPA, SAHA and EPZ-6438 demonstrated to be the main driver of the induction/upregulation of immune molecules functionally crucial in host-tumor interaction (i.e., HLA class I, ICAM-1 and cancer testis antigens) in all three MPM subtypes investigated. Additionally, GEP demonstrated that treatment with guadecitabine led to the activation of genes involved in several immune-related functional classes mainly in the sarcomatoid subtype. Furthermore, among investigated MPM subtypes, DHA-induced CDH1 expression that contributes to restoring the epithelial phenotype was highest in sarcomatoid cells. Altogether, our results contribute to providing the rationale to develop new epigenetically-based immunotherapeutic approaches for MPM patients, potentially tailored to the specific histologic subtypes.

Circulating Levels of PD-L1 in Mesothelioma Patients from the NIBIT-MESO-1 Study: Correlation with Survival.

Targeting of the programmed cell death protein (PD)-1/programmed death-ligand 1 (PD-L1) axis has shown a significant clinical impact in several tumor types. Accordingly, our phase II NIBIT-MESO-1 study demonstrated an improved clinical efficacy in mesothelioma patients treated with the anti-PD-L1 durvalumab combined with the anti-cytotoxic T-lymphocyte antigen (CTLA)-4 tremelimumab, as compared to tremelimumab alone. Due to the promising therapeutic activity of immune check-point inhibitors (ICIs) in mesothelioma patients, the identification of biomarkers predictive of response to treatment is of crucial relevance. The prognostic role of soluble PD-L1 (sPD-L1) proposed in cancer patients prompted us to investigate this protein in sera from mesothelioma patients (n = 40) enrolled in the NIBIT-MESO-1 study. A significant (p < 0.001) increase in sPD-L1 levels was detected in patients after the first cycle and during therapy vs. baseline. A longer overall survival (OS) was observed in patients with sPD-L1 concentrations below (at baseline, d1C2, d1C5 (p < 0.01)) or FC values above (p < 0.05 at d1C2, d1C3, d1C5) their statistically calculated optimal cut-offs. On the basis of these initial results, the specific role of CTLA-4-, PD-L1-, or PD-1-targeting on sPD-L1 release was then investigated in sera from 81 additional ICI-treated solid cancer patients. Results showed a significant (p < 0.001) increase of sPD-L1 levels during therapy compared to baseline only in anti-PD-L1-treated patients, supporting the specific involvement of PD-L1 targeting in the release of its soluble form. Our findings suggest that sPD-L1 represents a predictive biomarker of clinical response to anti-PD-L1 cancer immunotherapy.

Immunomodulatory Properties of DNA Hypomethylating Agents: Selecting the Optimal Epigenetic Partner for Cancer Immunotherapy.

DNA hypomethylating agents (DHAs) play a well-acknowledged role in potentiating the immunogenicity and the immune recognition of neoplastic cells. This immunomodulatory activity of DHAs is linked to their ability to induce or to up-regulate on neoplastic cells the expression of a variety of immune molecules that play a crucial role in host-tumor immune interactions. To further investigate the clinical potential of diverse epigenetic compounds when combined with immunotherapeutic strategies, we have now compared the tumor immunomodulatory properties of the first generation DHAs, azacytidine (AZA) and decitabine (DAC) and of the next generation DHA, guadecitabine. To this end, human melanoma and hematological cancer cells were treated in vitro with 1 µM guadecitabine, DAC or AZA and then studied by molecular and flow cytometry analyses for changes in their baseline expression of selected immune molecules involved in different mechanism(s) of immune recognition. Results demonstrated a stronger DNA hypomethylating activity of guadecitabine and DAC, compared to AZA that associated with stronger immunomodulatory activities. Indeed, the mRNA expression of cancer testis antigens, immune-checkpoint blocking molecules, immunostimulatory cytokines, involved in NK and T cell signaling and recruiting, and of genes involved in interferon pathway was higher after guadecitabine and DAC compared to AZA treatment. Moreover, a stronger up-regulation of the constitutive expression of HLA class I antigens and of Intercellular Adhesion Molecule-1 was observed with guadecitabine and DAC compared to AZA. Guadecitabine and DAC seem to represent the optimal combination partners to improve the therapeutic efficacy of immunotherapeutic agents in combination/sequencing clinical studies.

Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.

Histone macroH2A1.2 promotes metabolic health and leanness by inhibiting adipogenesis.

BACKGROUND: Obesity has tremendous impact on the health systems. Its epigenetic bases are unclear. MacroH2A1 is a variant of histone H2A, present in two alternatively exon-spliced isoforms macroH2A1.1 and macroH2A1.2, regulating cell plasticity and proliferation, during pluripotency and tumorigenesis. Their role in adipose tissue plasticity is unknown. RESULTS: Here, we show evidence that macroH2A1.1 protein levels in the visceral adipose tissue of obese humans positively correlate with BMI, while macroH2A1.2 is nearly absent. We thus introduced a constitutive GFP-tagged transgene for macroH2A1.2 in mice, and we characterized their metabolic health upon being fed a standard chow diet or a high fat diet. Despite unchanged food intake, these mice exhibit lower adipose mass and improved glucose metabolism both under a chow and an obesogenic diet. In the latter regimen, transgenic mice display smaller pancreatic islets and significantly less inflammation. MacroH2A1.2 overexpression in the mouse adipose tissue induced dramatic changes in the transcript levels of key adipogenic genes; genomic analyses comparing pre-adipocytes to mature adipocytes uncovered only minor changes in macroH2A1.2 genomic distribution upon adipogenic differentiation and suggested differential cooperation with transcription factors. MacroH2A1.2 overexpression markedly inhibited adipogenesis, while overexpression of macroH2A1.1 had opposite effects. CONCLUSIONS: MacroH2A1.2 is an unprecedented chromatin component powerfully promoting metabolic health by modulating anti-adipogenic transcriptional networks in the differentiating adipose tissue. Strategies aiming at enhancing macroH2A1.2 expression might counteract excessive adiposity in humans.