Identification of therapeutic targets in osteoarthritis by combining heterogeneous transcriptional datasets, drug-induced expression profiles, and known drug-target interactions.

BACKGROUND: Osteoarthritis (OA) is a multifactorial, hypertrophic, and degenerative condition involving the whole joint and affecting a high percentage of middle-aged people. It is due to a combination of factors, although the pivotal mechanisms underlying the disease are still obscure. Moreover, current treatments are still poorly effective, and patients experience a painful and degenerative disease course. METHODS: We used an integrative approach that led us to extract a consensus signature from a meta-analysis of three different OA cohorts. We performed a network-based drug prioritization to detect the most relevant drugs targeting these genes and validated in vitro the most promising candidates. We also proposed a risk score based on a minimal set of genes to predict the OA clinical stage from RNA-Seq data. RESULTS: We derived a consensus signature of 44 genes that we validated on an independent dataset. Using network analysis, we identified Resveratrol, Tenoxicam, Benzbromarone, Pirinixic Acid, and Mesalazine as putative drugs of interest for therapeutics in OA for anti-inflammatory properties. We also derived a list of seven gene-targets validated with functional RT-qPCR assays, confirming the in silico predictions. Finally, we identified a predictive subset of genes composed of DNER, TNFSF11, THBS3, LOXL3, TSPAN2, DYSF, ASPN and HTRA1 to compute the patient's risk score. We validated this risk score on an independent dataset with a high AUC (0.875) and compared it with the same approach computed using the entire consensus signature (AUC 0.922). CONCLUSIONS: The consensus signature highlights crucial mechanisms for disease progression. Moreover, these genes were associated with several candidate drugs that could represent potential innovative therapeutics. Furthermore, the patient's risk scores can be used in clinical settings.

Stratification of Patients with Coronary Artery Disease by Circulating Cytokines Profile: A Pilot Study.

Coronary artery disease (CAD) is a long-term inflammatory process, with atherosclerosis as its underlying pathophysiological mechanism. Endothelial dysfunction is the first step towards atherosclerosis, where damaged endothelial cells release large amounts of pro-inflammatory cytokines and mediators, thus promoting vascular inflammation and disease progression. However, the correlation between serum cytokines and CAD severity remains to be defined. Serum samples from patients performing cardiac computed tomography for suspected CAD (n = 75) were analyzed with a multiplex bead-based immunoassay panel for simultaneous assessment of the concentration of 11 cytokines using flow cytometric technology. The analysis showed statistically significant increases in sRAGE, CCL2_MCP1, FLT1, and IL6 levels in CAD patients compared with healthy subjects and a gradual increase trend towards a more severe form of the disease for most cytokines (e.g., sCD40L, FLT1, sRAGE, CCL2-MCP1, TNFα). Lastly, we explored the performance of cytokines in predicting the diagnosis of CAD and found that an increase in IL6 levels will increase the odds of being non-obstructive CAD-positive. In contrast, an increase in CCL2-MCP1 or FLT1 levels will increase the probability of being obstructive CAD-positive. These results suggest that the combination of serum cytokines may contribute to the not-invasive stratification risk for patients with suspected CAD.

A Comprehensive Analysis of the Expression Profiles of KCTD Proteins in Acute Lymphoblastic Leukemia: Evidence of Selective Expression of KCTD1 in T-ALL.

Acute leukemia is the most common pediatric cancer. In most cases, this disease results from the malignant transformation of either the B-cell (B-ALL) or, less frequently, T-cell progenitors (T-ALL). Recently, a marked overexpression of KCTD15, a member of the emerging class of the potassium (K) channel tetramerization domain-containing proteins (KCTDs) has been detected in both patients and continuous cell lines as in vitro model systems. Because there is growing evidence of the key, yet diversified, roles played by KCTDs in cancers, we here report an exhaustive analysis of their expression profiles in both B-ALL and T-ALL patients. Although for most KCTDs, no significant alterations were found in these pathological states, for some members of the family, significant up- and down-regulations were detected in comparison with the values found in healthy subjects in the transcriptome analysis. Among these, particularly relevant is the upregulation of the closely related KCTD1 and KCTD15 in T-ALL patients. Interestingly, KCTD1 is barely expressed in both unaffected controls and B-ALL patients. Therefore, not only does this analysis represent the first study in which the dysregulation of all KCTDs is simultaneously evaluated in specific pathological contexts, but it also provides a promising T-ALL biomarker that could be suitable for clinical applications.

Specific lncRNA signatures discriminate childhood acute leukaemias: a pilot study.

BACKGROUND: Long non-coding RNAs are RNAs longer than 200 bps that do not encode any proteins and are able to alter gene expression by acting on different steps of regulation, including DNA methylation and chromatin structure. They represent a class of biomarkers of crescent interest in the hematologic and oncologic fields. Recent studies showed that the expression levels of specific lncRNAs correlate with the prognosis of paediatric patients with Acute Lymphoblastic Leukaemia. METHODS: We used NGS approaches to analyse the transcriptome of 9 childhood B-ALL patients and 6 childhood T-ALL patients, in comparison with B and T healthy lymphocytes from cord blood. We validate our findings both ex vivo, in a different cohort of 10 B-ALL and 10 T-ALL patients, and in silico using public datasets. RESULTS: We characterised the lncRNA landscape for B-ALL, T-ALL, healthy B, and T cell progenitors. From the characterised signature, we selected candidate lncRNAs able to discriminate not only B-ALL and T-ALL from healthy subjects but also between the two types of leukaemia, and subsequently validated their potential as a diagnostic tool in an additional cohort of paediatric patients. We confirmed our finding with open access transcriptomic data, comparing ALL lncRNAs with AML lncRNA landscape as well. Finally, expression correlation analyses of T-ALL selected lncRNA biomarkers suggested a possible role in lymphocyte activation and the ß-catenin signalling pathway for AC247036.1 and involvement in hedgehog signalling for HHIP-AS1. CONCLUSIONS: Our work identified a lncRNA signature discriminating paediatric B-ALL and T-ALL from healthy subjects, between them and from AML. This study provides the keystone to future clinical studies determining the theragnostic value of the characterised long non coding transcriptome panorama in a clinical setting for childhood patient management.

SIRT1 pharmacological activation rescues vascular dysfunction and prevents thrombosis in MTHFR deficiency.

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

SIRT1 Activation by Natural Phytochemicals: An Overview.

Sirtuins are class III histone deacetylases, whose enzymatic activity is dependent on NAD+ as a cofactor. Sirtuins are reported to modulate numerous activities by controlling gene expression, DNA repair, metabolism, oxidative stress response, mitochondrial function, and biogenesis. Deregulation of their expression and/or action may lead to tissue-specific degenerative events involved in the development of several human pathologies, including cancer, neurodegeneration, and cardiovascular disease. The most studied member of this class of enzymes is sirtuin 1 (SIRT1), whose expression is associated with increasing insulin sensitivity. SIRT1 has been implicated in both tumorigenic and anticancer processes, and is reported to regulate essential metabolic pathways, suggesting that its activation might be beneficial against disorders of the metabolism. Via regulation of p53 deacetylation and modulation of autophagy, SIRT1 is implicated in cellular response to caloric restriction and lifespan extension. In recent years, scientific interest focusing on the identification of SIRT1 modulators has led to the discovery of novel small molecules targeting SIRT1 activity. This review will examine compounds of natural origin recently found to upregulate SIRT1 activity, such as polyphenolic products in fruits, vegetables, and plants including resveratrol, fisetin, quercetin, and curcumin. We will also discuss the potential therapeutic effects of these natural compounds in the prevention and treatment of human disorders, with particular emphasis on their metabolic impact.

Oxidative nucleophilic substitution selectively produces cambinol derivatives with antiproliferative activity on bladder cancer cell lines.

Methyltrioxorhenium mediated oxidative addition/elimination nucleophilic substitution yielded alkylamino and arylamino cambinol derivatives characterized by anti-proliferative activity against wild-type and p53 mutated MGH-U1 and RT112 bladder cancer cell lines. Some of the novel compounds showed an activity higher than that of the lead compound. The reaction was highly regioselective, affording for the first time a panel of C-2 cambinol substitution products. Aliphatic primary and secondary amines, and primary aromatic amines, were used as nitrogen centered nucleophiles. Surprisingly, the antiproliferative activity of C-2 substituted cambinol derivatives was not correlated to the induction of p53 protein, as evaluated by the analysis of the cell viability on wild-type and p53 mutated cancer cell lines, and further confirmed by western blot analyses. These data suggest that they exert their antiproliferative activity by a mechanism completely different from cambinol.

Novel spiroindoline HDAC inhibitors: Synthesis, molecular modelling and biological studies.

This paper describes the rational development of a series of novel spiroindoline derivatives endowed with selective inhibitory activity on the HDAC6 isoform. A convenient multicomponent one-pot protocol was applied for the assembly of the desired N1-substituted spiroindoline core which allowed a straightforward analoging. Computational studies and in vitro determination of inhibitory potency for the developed compounds against HDAC6 and HDAC1 isoforms were flanked by cell-based studies on histone H3 and α-tubulin acetylation. The effects on cancer cell cycle and apoptosis of the best performing derivatives were assessed on cancer cell lines highlighting a promising antitumor potential. In view of cell-based data and calculated drug-like properties, the selective HDAC6 inhibitor 5b, with a spiroindoline-based hydroxamate bearing a tert-butyl carbamate functionality, was selected to be further investigated for its potential in inhibiting tumor cells migration. It was able to potently inhibit cell migration in SH-SY5Y neuroblastoma cells and did not display toxicity in NIH3T3 mouse fibroblasts. Taken together, these data foster further investigation and optimization for this class of compounds as novel anticancer agents.

Phenylpyrrole-based HDAC inhibitors: synthesis, molecular modeling and biological studies.

AIM: Histone deacetylases (HDACs) regulate the expression and activity of numerous proteins involved in the initiation and progression of cancer. Currently, three hydroxamate-containing HDAC pan-inhibitors have been approved as antitumor agents. RESULTS: We herein present the development of a series of novel phenylpyrrole-based derivatives stemmed from combined computational and medicinal chemistry efforts to rationally modulate HDAC1/6 isoform selectivity. In vitro activity on HDAC1 and HDAC6 isoforms and the effects of selected analogs on histone H3 and α-tubulin acetylation levels were determined. Cell-based data evidenced, for selected compounds, a promising antitumor potential and low toxicity on normal cells. CONCLUSION: The newly developed compounds represent a valuable starting point for the development of novel anticancer agents.

Epigenetic-based therapy: From single- to multi-target approaches.

The treatment of cancer has traditionally been based on the identification of a single molecule and/or enzymatic function (target) responsible for a particular phenotype, and therefore on the ability to stimulate, attenuate or inhibit its activity through the use of selective compounds. However, cancer is no longer considered a disease caused by a single factor, but is now recognized as a multi-factorial disorder. Genetic, epigenetic and metabolic factors all contribute to neoplasia, causing significant changes in molecular networks that govern cell growth, development, death and specialization. Consequently, many antitumor therapies are no longer directed against a single target but the biological system as a whole, in which functions determining the onset and maintenance of a physio-pathological state are modulated. The field of epi-drug discovery is currently in a transitional phase where the search for putative anticancer drugs is shifting from single-target-oriented molecules to network-active compounds and to epi-drugs used in combination with other epi-agents and with traditional chemotherapeutics. This review illustrates the pros and cons of each therapeutic option, providing examples in support of single-target and multi (network)-target epi-drug approaches.

Genetic mutations in epigenetic modifiers as therapeutic targets in acute myeloid leukemia.

INTRODUCTION: Despite enormous insights into the molecular mechanisms of acute myeloid leukemia (AML) pathophysiology, this disease is still fatal in the majority of patients, highlighting the urgent need for novel biomarkers useful in AML prognosis and therapy. AREAS COVERED: The advent of modern sequencing technologies has allowed the identification of genetic mutations in genes encoding for specific enzymes involved in the epigenetic regulation of gene expression. The authors review recent data demonstrating the involvement of mutations in genes encoding for epigenetic players and their complex combination with somatic genetic mutations in the pathogenesis of AML. They also discuss the prognostic and therapeutic implications of these findings. EXPERT OPINION: Current clinical and preclinical studies are underscoring the importance of targeting epigenetic modifiers as new biomarkers for a better prognostic risk stratification and therapeutic evaluation of intermediate-risk patients. Combining data from traditional and modern methodologies will allow a definition of the complex networks of epigenetic changes and molecular interactions between candidate epitargets and key regulators of hematopoiesis. It will thus be possible to achieve an overview of potential aberrant mechanisms driving leukemogenesis in different classes of AML patients. Such an improved approach could pave the way towards 'personalized' therapies.