A network-based computational and experimental framework for repurposing compounds toward the treatment of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is among the most common liver pathologies, however, none approved condition-specific therapy yet exists. The present study introduces a drug repositioning (DR) approach that combines in vitro steatosis models with a network-based computational platform, constructed upon genomic data from diseased liver biopsies and compound-treated cell lines, to propose effectively repositioned therapeutic compounds. The introduced in silico approach screened 20'000 compounds, while complementary in vitro and proteomic assays were developed to test the efficacy of the 46 in silico predictions. This approach successfully identified six compounds, including the known anti-steatogenic drugs resveratrol and sirolimus. In short, gallamine triethiotide, diflorasone, fenoterol, and pralidoxime ameliorate steatosis similarly to resveratrol/sirolimus. The implementation holds great potential in reducing screening time in the early drug discovery stages and in delivering promising compounds for in vivo testing.

Tumor Protein 53 Gene Mutations Without 17p13 Deletion Have No Significant Clinical Implications in Chronic Lymphocytic Leukemia. Detection of a New Mutation.

BACKGROUND: The tumor protein p53 (TP53) gene may be inactivated through 17p13 deletion, somatic mutations, or both. In chronic lymphocytic leukemia (CLL) although 17p13 deletion is correlated with poor prognosis, the role of sole TP53 mutations remains controversial. MATERIALS AND METHODS: We carried out a mutation analysis of TP53 gene in 72 patients with CLL. RESULTS: Seventy-one (98.6%) patients carried the polymorphic site c.215C>G, p.Pro72Arg, but its presence was not correlated with overall survival (OS). Moreover, 19 (26.4%) patients carried a mutation of TP53. Among the eight detected mutations, to our knowledge, one (c.587G>A) has never been reported in the past. There was a correlation of the mutation burden with the stage of the disease (p=0.022), but not with OS. None of the detected mutations was individually correlated with OS. CONCLUSION: The clinical significance of TP53 mutations is still a matter of debate and larger studies and meta-analyses are required to reach an unequivocal conclusion.

Prognostic significance of signal transducer and activator of transcription 5 and 5b expression in Epstein-Barr virus-positive patients with chronic lymphocytic leukemia.

Signal transducer and activator of transcription (STAT) proteins have been intensively studied in hematologic malignancies, and the efficacy of agents against STATs in lymphomas is already under research. We investigated the expression of total STAT5 and STAT5b in peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) in correlation with the presence of Epstein-Barr Virus (EBV) and its major oncoprotein (latent membrane protein 1, LMP1). The EBV load was measured in the peripheral blood by real-time PCR for the BXLF1 gene and the levels of LMP1 by PCR and ELISA. Western blotting was performed for total STAT5 and STAT5b in protein extracts. STAT5b was only expressed in patients (not in healthy subjects) and STAT5 but particularly STAT5b expression was correlated with the presence of the virus (77.3% vs. 51.2%, P = 0.006 for STAT5b) and to the expression of LMP1 (58.3% vs. 21.6%, P = 0.011 for STAT5b). Moreover, the expression of STAT5b and the presence of EBV and LMP1 were strongly negatively correlated with the overall survival of the patients (log-rank test P = 0.011, 0.015, 0.006, respectively). Double positive (for EBV and STAT5b) patients had the lowest overall survival (log-rank test P = 0.013). This is the first report of a survival disadvantage of EBV+ patients with CLL, and the first time that STAT5b expression is correlated with survival. The correlation of STAT5 expression with the presence of the virus, along with our survival correlations defines a subgroup of patients with CLL that may benefit from anti-STAT agents.

PARP1-driven apoptosis in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is considered a malignancy resulting from defects in apoptosis. For this reason, targeting apoptotic pathways in CLL may be valuable for its management. Poly [ADP-ribose] polymerase 1 (PARP1) is the main member of a family of nuclear enzymes that act as DNA damage sensors. Through binding on DNA damaged structures, PARP1 recruits repair enzymes and serves as a survival factor, but if the damage is severe enough, its action may lead the cell to apoptosis through caspase activation, or necrosis. We measured the PARP1 mRNA and protein pretreatment levels in 26 patients with CLL and the corresponding posttreatment levels in 15 patients after 3 cycles of immunochemotherapy, as well as in 15 healthy blood donors. No difference was found between the pre- and posttreatment levels of PARP1, but we found a statistically significant relative increase of the 89 kDa fragment of PARP1 that is cleaved by caspases in the posttreatment samples, indicating PARP1-related apoptosis in CLL patients after treatment. Our findings constitute an important step in the field, especially in the era of PARP1 inhibitors, and may serve as a base for future clinical trials with these agents in CLL.

Survivin messenger RNA levels in Epstein-Barr virus-positive patients with leukemic low-grade B-cell lymphomas expressing the latent membrane protein 1: evidence of apoptotic function?

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous pathogen that chronically infects B lymphocytes and is implicated in the pathogenesis of lymphoproliferative diseases. Latent membrane protein 1 (LMP1), the major oncoprotein of the virus, has been shown to inhibit apoptosis and trigger survivin expression in malignant cell lines. LMP1 expression has been detected in patients with chronic lymphocytic leukemia, but its properties have not been studied in patients with low-grade B-cell lymphomas. Recent data show that LMP1 can simultaneously induce and inhibit apoptosis in B cells. We detected LMP1 messenger RNA (mRNA) in patients with leukemic low-grade B-cell lymphoma and correlated the expression of the antiapoptotic molecule survivin to that of LMP1 in this group of patients. PATIENTS AND METHODS: Peripheral whole blood from 64 patients with low-grade B-cell lymphoma was tested by quantitative reverse transcriptase-polymerase chain reaction (PCR) for the presence of the BXLF-1 gene of EBV, and positive samples were tested by conventional PCR for LMP1 expression. Accordingly, survivin mRNA levels were measured by quantitative reverse transcriptase PCR in all samples and compared between LMP1-positive (LMP1(+)) and LMP1(-) patients. RESULTS: The BXLF-1 gene was detected in 27 of 64 patients (42%). LMP1 was expressed in 22 of 27 (81%) EBV(+) patients. Survivin expression was found to be 6.36 times higher in LMP1(-) patients than in LMP1(+) patients (P = .008). CONCLUSION: Our results imply that in patients with non-EBV-related leukemic low-grade B-cell lymphoma, LMP1 expression is possibly correlated to apoptosis, as indicated by the lower survivin mRNA levels in LMP1(+) patients.

Rituximab in the treatment of EBV-positive low grade B-cell lymphoma.

BACKGROUND: Following infection of B lymphocytes by Epstein Barr virus (EBV), the viral genome remains in the nucleus, and a latency phase is established, during which only a small proportion of the viral genes are expressed. Among them, LMP1 is essential for transformation. Rituximab is a potent agent used in the treatment of low grade B-cell lymphomas and is also widely used for the treatment of post-transplant lymphoproliferative disorders caused by EBV. The effect of rituximab treatment on the latent EBV infection in non-transplant patients with lymphoproliferative disorders has never been studied to our knowledge. PATIENTS AND METHODS: We studied, the effect of rituximab-based immunochemotherapy on the EBV status of 44 patients with leukemic low grade B-cell lymphoma. RESULTS: After three cycles of rituximab-based treatment, only 1/17 patients was still positive for EBV. DISCUSSION: Our results suggest that rituximab used in the treatment of EBV-positive low-grade lymphoma is efficient in eradicating the virus from the peripheral blood, a fact with potential implications in the course and prognosis of the disease.

Correlation of Fc-γ RIIA polymorphisms with latent Epstein-Barr virus infection and latent membrane protein 1 expression in patients with low grade B-cell lymphomas.

Fc-γ RIIA (CD32), a member of the family of Fc-γ receptors, participates in the phagocytosis of bound to antibody antigens. The effectiveness of this function varies for its several haplotypes, and it participates in the pathogenesis of viral infections, according to recent studies. The genetic locus of Fc-γ RIIA consists of two allelic genes: 131-Arg (R131) and 131-His (H131). Our aim was to correlate Fc-γ RIIA polymorphisms, by studying the prevalence of each allele using PCR-RFLPs (polymerase chain reaction-restriction fragment length polymorphisms), with latent Epstein-Barr virus (EBV) infection and the expression of latent membrane protein 1 (LMP1) in 40 patients with leukemic low grade B-cell lymphomas. R131 was found in 84.2% of EBV-positive patients, but only in 28.5% of EBV-negative patients (p = 0.001). A similar correlation was found for R131 and LMP1 expression (84.6% vs. 28.5%) (p = 0.002). Our results support the hypothesis that Fc-γ RIIA polymorphisms are a genetic risk factor for latent EBV infection and the expression of its oncogenic latency proteins.