Targeting ROS production through inhibition of NADPH oxidases.

NADPH oxidases (NOXs) are transmembrane enzymes that are devoted to the production of reactive oxygen species (ROS). In cancers, dysregulation of NOX enzymes affects ROS production, leading to redox unbalance and tumor progression. Consequently, NOXs are a drug target for cancer therapeutics, although current therapies have off-target effects: there is a need for isoenzyme-selective inhibitors. Here, we describe fully validated human NOX inhibitors, obtained from an in silico screen, targeting the active site of Cylindrospermum stagnale NOX5 (csNOX5). The hits are validated by in vitro and in cellulo enzymatic and binding assays, and their binding modes to the dehydrogenase domain of csNOX5 studied via high-resolution crystal structures. A high-throughput screen in a panel of cancer cells shows activity in selected cancer cell lines and synergistic effects with KRAS modulators. Our work lays the foundation for the development of inhibitor-based methods for controlling the tightly regulated and highly localized ROS sources.

Epigenomic machinery regulating pediatric AML: Clonal expansion mechanisms, therapies, and future perspectives.

Acute myeloid leukemia (AML) is a heterogeneous disease with a genetic, epigenetic, and transcriptional etiology mainly presenting somatic and germline abnormalities. AML incidence rises with age but can also occur during childhood. Pediatric AML (pAML) accounts for 15-20% of all pediatric leukemias and differs considerably from adult AML. Next-generation sequencing technologies have enabled the research community to "paint" the genomic and epigenomic landscape in order to identify pathology-associated mutations and other prognostic biomarkers in pAML. Although current treatments have improved the prognosis for pAML, chemoresistance, recurrence, and refractory disease remain major challenges. In particular, pAML relapse is commonly caused by leukemia stem cells that resist therapy. Marked patient-to-patient heterogeneity is likely the primary reason why the same treatment is successful for some patients but, at best, only partially effective for others. Accumulating evidence indicates that patient-specific clonal composition impinges significantly on cellular processes, such as gene regulation and metabolism. Although our understanding of metabolism in pAML is still in its infancy, greater insights into these processes and their (epigenetic) modulation may pave the way toward novel treatment options. In this review, we summarize current knowledge on the function of genetic and epigenetic (mis)regulation in pAML, including metabolic features observed in the disease. Specifically, we describe how (epi)genetic machinery can affect chromatin status during hematopoiesis, leading to an altered metabolic profile, and focus on the potential value of targeting epigenetic abnormalities in precision and combination therapy for pAML. We also discuss the possibility of using alternative epidrug-based therapeutic approaches that are already in clinical practice, either alone as adjuvant treatments and/or in combination with other drugs.

The m6A-independent role of epitranscriptomic factors in cancer.

Protein function alteration and protein mislocalization are cancer hallmarks that drive oncogenesis. N6-methyladenosine (m6A) deposition mediated by METTL3, METTL16, and METTL5 together with the contribution of additional subunits of the m6A system, has shown a dramatic impact on cancer development. However, the cellular localization of m6A proteins inside tumor cells has been little studied so far. Interestingly, recent evidence indicates that m6A methyltransferases are not always confined to the nucleus, suggesting that epitranscriptomic factors may also have multiple oncogenic roles beyond m6A that still represent an unexplored field. To date novel epigenetic drugs targeting m6A modifiers, such as METTL3 inhibitors, are entering into clinical trials, therefore, the study of the potential onco-properties of m6A effectors beyond m6A is required. Here we will provide an overview of methylation-independent functions of the m6A players in cancer, describing the molecular mechanisms involved and the future implications for therapeutics.

A Combination of Microarray-Based Profiling and Biocomputational Analysis Identified miR331-3p and hsa-let-7d-5p as Potential Biomarkers of Ulcerative Colitis Progression to Colorectal Cancer.

Ulcerative colitis (UC), an inflammatory bowel disease (IBD), may increase the risk of colorectal cancer (CRC) by activating chronic proinflammatory pathways. The goal of this study was to find serum prediction biomarkers in UC to CRC development by combining low-density miRNA microarray and biocomputational approaches. The UC and CRC miRNA expression profiles were compared by low-density miRNA microarray, finding five upregulated miRNAs specific to UC progression to CRC (hsa-let-7d-5p, hsa-miR-16-5p, hsa-miR-145-5p, hsa-miR-223-5p, and hsa-miR-331-3p). The circRNA/miRNA/mRNA competitive endogenous RNA (ceRNA) network analysis showed that the candidate miRNAs were connected to well-known colitis-associated CRC ACVR2A, SOCS1, IGF2BP1, FAM126A, and CCDC85C mRNAs, and circ-SHPRH circRNA. SST and SCARA5 genes regulated by hsa-let-7d-5p, hsa-miR-145-5p, and hsa-miR-331-3p were linked to a poor survival prognosis in a CRC patient dataset from The Cancer Genome Atlas (TCGA). Lastly, our mRNA and miRNA candidates were validated by comparing their expression to differentially expressed mRNAs and miRNAs from colitis-associated CRC tissue databases. A high level of hsa-miR-331-3p and a parallel reduction in SOCS1 mRNA were found in tissue and serum. We propose hsa-miR-331-3p and possibly hsa-let-7d-5p as novel serum biomarkers for predicting UC progression to CRC. More clinical sample analysis is required for further validation.

Interplay between m6 A epitranscriptome and epigenome in cancer: current knowledge and therapeutic perspectives.

Chromatin has an extremely flexible structure that allows the fine regulation of gene expression. To orchestrate this process, small chemical modifications are dynamically added or removed on DNA, RNA and histone substrates. Epigenetic modifications govern a plethora of key cellular functions, whose dysregulation contributes to oncogenesis. The interrelationship between (irreversible) genetic mutations and (reversible) epigenetic alterations and how this crosstalk regulates gene expression has long been a major area of interest. Marks modulating the RNA code (epitranscriptome), such as the well-studied N6 -methyladenosine (m6 A), are known to influence stability, metabolism and life cycle of many mRNAs, including cancer-associated transcripts. Together, epigenetic and epitranscriptomic pathways therefore control the entire cellular expression profile and, eventually, cell fate. Recently, previously undescribed crosstalk between these two pathways has started to be unrevealed. For example, m6 A and its effectors cooperate with histone modifications to localize chromatin-modifying complexes to their target regions. Epigenetic marks governing the expression of m6 A factors can also be found at specific genetic loci. m6 A itself can mark noncoding RNAs (including lncRNAs, circRNAs and miRNAs), influencing their structure, maturation and function. These interactions affect both cell physiology and pathology. Clear evidence that dysregulation of this network plays a role in cancer has emerged, suggesting a new layer of complexity in the landscape of gene expression. Here, we summarize current knowledge on the interplay between m6 A epitranscriptome and epigenome, focusing on cancer processes. We also discuss strategies to target m6 A machinery for future therapeutic intervention.

Epigenetic alterations in glioblastomas: Diagnostic, prognostic and therapeutic relevance.

Glioblastoma, the most common and heterogeneous tumor affecting brain parenchyma, is dismally characterized by a very poor prognosis. Thus, the search of new, more effective treatments is a vital need. Here, we will review the druggable epigenetic features of glioblastomas that are, indeed, currently explored in preclinical studies and in clinical trials for the development of more effective, personalized treatments. In detail, we will review the studies that have led to the identification of epigenetic signatures, IDH mutations, MGMT gene methylation, histone modification alterations, H3K27 mutations and epitranscriptome landscapes of glioblastomas, in each case discussing the corresponding targeted therapies and their potential efficacy. Finally, we will emphasize how recent technological improvements permit to routinely investigate many glioblastoma epigenetic biomarkers in clinical practice, further enforcing the hope that personalized drugs, targeting specific epigenetic features, could be in future a therapeutic option for selected patients.

Panitumumab plus trifluridine/tipiracil as anti-EGFR rechallenge therapy in patients with refractory RAS wild-type metastatic colorectal cancer: Overall survival and subgroup analysis of the randomized phase II VELO trial.

The randomized phase II VELO trial showed that the addition of panitumumab to trifluridine/tipiracil significantly improves progression-free survival (PFS) as compared to trifluridine/tipiracil in third-line therapy in patients with refractory RAS wild-type (WT) metastatic colorectal cancer (mCRC). With longer follow-up, final overall survival results and posttreatment subgroup analysis are presented. Sixty-two patients with refractory RAS WT mCRC were randomly assigned to receive, as third-line therapy, trifluridine/tipiracil alone (arm A) or in combination with panitumumab (arm B). Primary endpoint was PFS; secondary endpoints included overall survival (OS) and overall response rate (ORR). Median OS was 13.1 months (95% CI 9.5-16.7) in arm A compared to 11.6 months (95% CI 6.3-17.0) in arm B (HR: 0.96, 95% CI 0.54-1.71, P = .9). To evaluate the impact of subsequent lines of treatment, subgroup analysis was performed for the 24/30 patients in arm A, that received fourth-line therapy after disease progression. Median PFS was 4.1 months (95% CI 1.44-6.83) for 17 patients treated with anti-EGFR rechallenge as compared to 3.0 months (95% CI 1.61-4.31) for seven patients that received other therapies (HR: 0.29, 95% CI 0.10-0.85, P = .024). Median OS from the start of fourth-line treatment was 13.6 months (95% CI 7.2-20), and 5.1 months (95% CI 1.8-8.3) for patients treated with anti-EGFR rechallenge vs other therapies, respectively (HR: 0.30, 95% CI 0.11-0.81, P = .019). Final results of the VELO trial support the role of anti-EGFR rechallenge in the continuum of care of patients with RAS/BRAF WT mCRC.

FASN multi-omic characterization reveals metabolic heterogeneity in pancreatic and prostate adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) and prostate cancer (PCa) are among the most prevalent malignant tumors worldwide. There is now a comprehensive understanding of metabolic reprogramming as a hallmark of cancer. Fatty acid synthase (FASN) is a key regulator of the lipid metabolic network, providing energy to favor tumor proliferation and development. Whereas the biological role of FASN is known, its response and sensitivity to inhibition have not yet been fully established in these two cancer settings. METHODS: To evaluate the association between FASN expression, methylation, prognosis, and mutational profile in PDAC and PCa, we interrogated public databases and surveyed online platforms using TCGA data. The STRING database was used to investigate FASN interactors, and the Gene Set Enrichment Analysis platform Reactome database was used to perform an enrichment analysis using data from RNA sequencing public databases of PDAC and PCa. In vitro models using PDAC and PCa cell lines were used to corroborate the expression of FASN, as shown by Western blot, and the effects of FASN inhibition on cell proliferation/cell cycle progression and mitochondrial respiration were investigated with MTT, colony formation assay, cell cycle analysis and MitoStress Test. RESULTS: The expression of FASN was not modulated in PDAC compared to normal pancreatic tissues, while it was overexpressed in PCa, which also displayed a different level of promoter methylation. Based on tumor grade, FASN expression decreased in advanced stages of PDAC, but increased in PCa. A low incidence of FASN mutations was found for both tumors. FASN was overexpressed in PCa, despite not reaching statistical significance, and was associated with a worse prognosis than in PDAC. The biological role of FASN interactors correlated with lipid metabolism, and GSEA indicated that lipid-mediated mitochondrial respiration was enriched in PCa. Following validation of FASN overexpression in PCa compared to PDAC in vitro, we tested TVB-2640 as a FASN inhibitor. PCa proliferation arrest was modulated by FASN inhibition in a dose- and time-dependent manner, whereas PDAC proliferation was not altered. In line with this finding, mitochondrial respiration was found to be more affected in PCa than in PDAC. FASN inhibition interfered with metabolic signaling causing lipid accumulation and affecting cell viability with an impact on the replicative processes. CONCLUSIONS: FASN exhibited differential expression patterns in PDAC and PCa, suggesting a different evolution during cancer progression. This was corroborated by the fact that both tumors responded differently to FASN inhibition in terms of proliferative potential and mitochondrial respiration, indicating that its use should reflect context specificity.

SIRT1 activation promotes energy homeostasis and reprograms liver cancer metabolism.

BACKGROUND: Cancer cells are characterized by uncontrolled cell proliferation and impaired bioenergetics. Sirtuins are a family of highly conserved enzymes that play a fundamental role in energy metabolism regulation. SIRT1, in particular, drives many physiological stress responses and metabolic pathways following nutrient deprivation. We previously showed that SIRT1 activation using SCIC2.1 was able to attenuate genotoxic response and senescence. Here, we report that in hepatocellular carcinoma (HCC) cells under glucose-deprived conditions, SCIC2.1 treatment induced overexpression of SIRT1, SIRT3, and SIRT6, modulating metabolic response. METHODS: Flow cytometry was used to analyze the cell cycle. The MTT assay and xCELLigence system were used to measure cell viability and proliferation. In vitro enzymatic assays were carried out as directed by the manufacturer, and the absorbance was measured with an automated Infinite M1000 reader. Western blotting and immunoprecipitation were used to evaluate the expression of various proteins described in this study. The relative expression of genes was studied using real-time PCR. We employed a Seahorse XF24 Analyzer to determine the metabolic state of the cells. Oil Red O staining was used to measure lipid accumulation. RESULTS: SCIC2.1 significantly promoted mitochondrial biogenesis via the AMPK-p53-PGC1α pathway and enhanced mitochondrial ATP production under glucose deprivation. SIRT1 inhibition by Ex-527 further supported our hypothesis that metabolic effects are dependent on SIRT1 activation. Interestingly, SCIC2.1 reprogrammed glucose metabolism and fatty acid oxidation for bioenergetic circuits by repressing de novo lipogenesis. In addition, SCIC2.1-mediated SIRT1 activation strongly modulated antioxidant response through SIRT3 activation, and p53-dependent stress response via indirect recruitment of SIRT6. CONCLUSION: Our results show that SCIC2.1 is able to promote energy homeostasis, attenuating metabolic stress under glucose deprivation via activation of SIRT1. These findings shed light on the metabolic action of SIRT1 in the pathogenesis of HCC and may help determine future therapies for this and, possibly, other metabolic diseases.

Multi-omics analysis reveals attenuation of cellular stress by empagliflozin in high glucose-treated human cardiomyocytes.

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT2) inhibitors constitute the gold standard treatment for type 2 diabetes mellitus (T2DM). Among them, empagliflozin (EMPA) has shown beneficial effects against heart failure. Because cardiovascular diseases (mainly diabetic cardiomyopathy) are the leading cause of death in diabetic patients, the use of EMPA could be, simultaneously, cardioprotective and antidiabetic, reducing the risk of death from cardiovascular causes and decreasing the risk of hospitalization for heart failure in T2DM patients. Interestingly, recent studies have shown that EMPA has positive benefits for people with and without diabetes. This finding broadens the scope of EMPA function beyond glucose regulation alone to include a more intricate metabolic process that is, in part, still unknown. Similarly, this significantly increases the number of people with heart diseases who may be eligible for EMPA treatment. METHODS: This study aimed to clarify the metabolic effect of EMPA on the human myocardial cell model by using orthogonal metabolomics, lipidomics, and proteomics approaches. The untargeted and multivariate analysis mimicked the fasting blood sugar level of T2DM patients (hyperglycemia: HG) and in the average blood sugar range (normal glucose: NG), with and without the addition of EMPA. RESULTS: Results highlighted that EMPA was able to modulate and partially restore the levels of multiple metabolites associated with cellular stress, which were dysregulated in the HG conditions, such as nicotinamide mononucleotide, glucose-6-phosphate, lactic acid, FA 22:6 as well as nucleotide sugars and purine/pyrimidines. Additionally, EMPA regulated the levels of several lipid sub-classes, in particular dihydroceramide and triacylglycerols, which tend to accumulate in HG conditions resulting in lipotoxicity. Finally, EMPA counteracted the dysregulation of endoplasmic reticulum-derived proteins involved in cellular stress management. CONCLUSIONS: These results could suggest an effect of EMPA on different metabolic routes, tending to rescue cardiomyocyte metabolic status towards a healthy phenotype.

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.

Longitudinal single-cell transcriptomics reveals distinct patterns of recurrence in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous and aggressive blood cancer that results from diverse genetic aberrations in the hematopoietic stem or progenitor cells (HSPCs) leading to the expansion of blasts in the hematopoietic system. The heterogeneity and evolution of cancer blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. In this study, we investigated the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs at genetic and transcriptional levels, and unveiled the underlying pathways and genes contributing to recurrence. METHODS: Whole-exome sequencing was used to detect somatic mutations and large copy number variations (CNVs). Single cell RNA-seq was performed to investigate the clonal heterogeneity between Dx-Re pairs and amongst patients. RESULTS: scRNA-seq analysis revealed extensive expression differences between patients and Dx-Re pairs, even for those with the same -presumed- initiating events. Transcriptional differences between and within patients are associated with clonal composition and evolution, with the most striking differences in patients that gained large-scale copy number variations at relapse. These differences appear to have significant molecular implications, exemplified by a DNMT3A/FLT3-ITD patient where the leukemia switched from an AP-1 regulated clone at Dx to a mTOR signaling driven clone at Re. The two distinct AML1-ETO pairs share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells evolved from the Dx cells. CONCLUSIONS: In summary, the single cell RNA data underpinned the tumor heterogeneity not only amongst patient blasts with similar initiating mutations but also between each Dx-Re pair. Our results suggest alternatively and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.

CBX2 shapes chromatin accessibility promoting AML via p38 MAPK signaling pathway.

BACKGROUND: The dynamic epigenome and proteins specialized in the interpretation of epigenetic marks critically contribute to leukemic pathogenesis but also offer alternative therapeutic avenues. Targeting newly discovered chromatin readers involved in leukemogenesis may thus provide new anticancer strategies. Accumulating evidence suggests that the PRC1 complex member CBX2 is overexpressed in solid tumors and promotes cancer cell survival. However, its role in leukemia is still unclear. METHODS: We exploited reverse genetic approaches to investigate the role of CBX2 in human leukemic cell lines and ex vivo samples. We also analyzed phenotypic effects following CBX2 silencing using cellular and molecular assays and related functional mechanisms by ATAC-seq and RNA-seq. We then performed bioinformatic analysis of ChIP-seq data to explore the influence of histone modifications in CBX2-mediated open chromatin sites. Lastly, we used molecular assays to determine the contribution of CBX2-regulated pathways to leukemic phenotype. RESULTS: We found CBX2 overexpressed in leukemia both in vitro and ex vivo samples compared to CD34+ cells. Decreased CBX2 RNA levels prompted a robust reduction in cell proliferation and induction of apoptosis. Similarly, sensitivity to CBX2 silencing was observed in primary acute myeloid leukemia samples. CBX2 suppression increased genome-wide chromatin accessibility followed by alteration of leukemic cell transcriptional programs, resulting in enrichment of cell death pathways and downregulation of survival genes. Intriguingly, CBX2 silencing induced epigenetic reprogramming at p38 MAPK-associated regulatory sites with consequent deregulation of gene expression. CONCLUSIONS: Our results identify CBX2 as a crucial player in leukemia progression and highlight a potential druggable CBX2-p38 MAPK network in AML.

Effects of novel SGLT2 inhibitors on cancer incidence in hyperglycemic patients: a meta-analysis of randomized clinical trials.

Epidemiological evidence shows that diabetic patients have an increased cancer risk and a higher mortality rate. Glucose could play a central role in metabolism and growth of many tumor types, and this possible mechanism is supported by the high rate of glucose demand and uptake in cancer. Thus, growing evidence suggests that hyperglycemia contributes to cancer progression but also to its onset. Many mechanisms underlying this association have been hypothesized, such as insulin resistance, hyperinsulinemia, and increased inflammatory processes. Inflammation is a common pathophysiological feature in both diabetic and oncological patients, and inflammation linked to high glucose levels sensitizes microenvironment to tumorigenesis, promoting the development of malignant lesions by altering and sustaining a pathological condition in tissues. Glycemic control is the first goal of antidiabetic therapy, and glucose level reduction has also been associated with favorable outcomes in cancer. Here, we describe key events in carcinogenesis focusing on hyperglycemia as supporter in tumor progression and in particular, related to the role of a specific hypoglycemic drug class, sodium-glucose linked transporters (SGLTs). We also discuss the use of SGLT2 inhibitors as a novel potential cancer therapy. Our meta-analysis showed that SGLT-2 inhibitors were significantly associated with an overall reduced risk of cancer as compared to placebo (RR = 0.35, CI 0.33-0.37, P = 0. 00) with a particular effectiveness for dapaglifozin and ertuglifozin (RR = 0. 06, CI 0. 06-0. 07 and RR = 0. 22, CI 0. 18-0. 26, respectively). Network Medicine approaches may advance the possible repurposing of these drugs in patients with concomitant diabetes and cancer.

Discovery of a new class of triazole based inhibitors of acetyl transferase KAT2A.

We have recently developed a new synthetic methodology that provided both N-aryl-5-hydroxytriazoles and N-pyridine-4-alkyl triazoles. A selection of these products was carried through virtual screening towards targets that are contemporary and validated for drug discovery and development. This study determined a number of potential structure target dyads of which N-pyridinium-4-carboxylic-5-alkyl triazole displayed the highest score specificity towards KAT2A. Binding affinity tests of abovementioned triazole and related analogs towards KAT2A confirmed the predictions of the in-silico assay. Finally, we have run in vitro inhibition assays of selected triazoles towards KAT2A; the ensemble of binding and inhibition assays delivered pyridyl-triazoles carboxylates as the prototype of a new class of inhibitors of KAT2A.

Heterocycle-containing tranylcypromine derivatives endowed with high anti-LSD1 activity.

As regioisomers/bioisosteres of 1a, a 4-phenylbenzamide tranylcypromine (TCP) derivative previously disclosed by us, we report here the synthesis and biological evaluation of some (hetero)arylbenzoylamino TCP derivatives 1b-6, in which the 4-phenyl moiety of 1a was shifted at the benzamide C3 position or replaced by 2- or 3-furyl, 2- or 3-thienyl, or 4-pyridyl group, all at the benzamide C4 or C3 position. In anti-LSD1-CoREST assay, all the meta derivatives were more effective than the para analogues, with the meta thienyl analogs 4b and 5b being the most potent (IC50 values = 0.015 and 0.005 µM) and the most selective over MAO-B (selectivity indexes: 24.4 and 164). When tested in U937 AML and prostate cancer LNCaP cells, selected compounds 1a,b, 2b, 3b, 4b, and 5a,b displayed cell growth arrest mainly in LNCaP cells. Western blot analyses showed increased levels of H3K4me2 and/or H3K9me2 confirming the involvement of LSD1 inhibition in these assays.

Recent Advancement in Anticancer Compounds from Marine Organisms: Approval, Use and Bioinformatic Approaches to Predict New Targets.

In recent years, the study of anticancer bioactive compounds from marine sources has received wide interest. Contextually, world regulatory authorities have approved several marine molecules, and new synthetic derivatives have also been synthesized and structurally improved for the treatment of numerous forms of cancer. However, the administration of drugs in cancer patients requires careful evaluation since their interaction with individual biological macromolecules, such as proteins or nucleic acids, determines variable downstream effects. This is reflected in a constant search for personalized therapies that lay the foundations of modern medicine. The new knowledge acquired on cancer mechanisms has certainly allowed advancements in tumor prevention, but unfortunately, due to the huge complexity and heterogeneity of cancer, we are still looking for a definitive therapy and clinical approaches. In this review, we discuss the significance of recently approved molecules originating from the marine environment, starting from their organism of origin to their structure and mechanism of action. Subsequently, these bio-compounds are used as models to illustrate possible bioinformatics approaches for the search of new targets that are useful for improving the knowledge on anticancer therapies.

Cytotoxic Potential of the Marine Diatom Thalassiosira rotula: Insights into Bioactivity of 24-Methylene Cholesterol.

Marine microalgae are receiving great interest as sustainable sources of bioactive metabolites for health, nutrition and personal care. In the present study, a bioassay-guided screening allowed identifying an enriched fraction from SPE separation of the methanolic extract of the marine diatom Thalassiosira rotula with a chemically heterogeneous composition of cytotoxic molecules, including PUFAs, the terpene phytol, the carotenoid fucoxanthin and the phytosterol 24-methylene cholesterol (24-MChol). In particular, this latter was the object of deep investigation aimed to gain insight into the mechanisms of action activated in two tumour cell models recognised as resistant to chemical treatments, the breast MCF7 and the lung A549 cell lines. The results of our studies revealed that 24-MChol, in line with the most studied ß-sitosterol (ß-SIT), showed cytotoxic activity in a 3-30 µM range of concentration involving the induction of apoptosis and cell cycle arrest, although differences emerged between the two sterols and the two cancer systems when specific targets were investigated (caspase-3, caspase-9, FAS and TRAIL).

Histone demethylase KDM5C is a SAHA-sensitive central hub at the crossroads of transcriptional axes involved in multiple neurodevelopmental disorders.

A disproportional large number of neurodevelopmental disorders (NDDs) is caused by variants in genes encoding transcription factors and chromatin modifiers. However, the functional interactions between the corresponding proteins are only partly known. Here, we show that KDM5C, encoding a H3K4 demethylase, is at the intersection of transcriptional axes under the control of three regulatory proteins ARX, ZNF711 and PHF8. Interestingly, mutations in all four genes (KDM5C, ARX, ZNF711 and PHF8) are associated with X-linked NDDs comprising intellectual disability as a core feature. in vitro analysis of the KDM5C promoter revealed that ARX and ZNF711 function as antagonist transcription factors that activate KDM5C expression and compete for the recruitment of PHF8. Functional analysis of mutations in these genes showed a correlation between phenotype severity and the reduction in KDM5C transcriptional activity. The KDM5C decrease was associated with a lack of repression of downstream target genes Scn2a, Syn1 and Bdnf in the embryonic brain of Arx-null mice. Aiming to correct the faulty expression of KDM5C, we studied the effect of the FDA-approved histone deacetylase inhibitor suberanilohydroxamic acid (SAHA). In Arx-KO murine ES-derived neurons, SAHA was able to rescue KDM5C depletion, recover H3K4me3 signalling and improve neuronal differentiation. Indeed, in ARX/alr-1-deficient Caenorhabditis elegans animals, SAHA was shown to counteract the defective KDM5C/rbr-2-H3K4me3 signalling, recover abnormal behavioural phenotype and ameliorate neuronal maturation. Overall, our studies indicate that KDM5C is a conserved and druggable effector molecule across a number of NDDs for whom the use of SAHA may be considered a potential therapeutic strategy.

The SGLT-2 inhibitor empagliflozin improves myocardial strain, reduces cardiac fibrosis and pro-inflammatory cytokines in non-diabetic mice treated with doxorubicin.

BACKGROUND: Empagliflozin (EMPA), a selective inhibitor of the sodium glucose co-transporter 2, reduced the risk of hospitalization for heart failure and cardiovascular death in type 2 diabetic patients in the EMPA-REG OUTCOME trial. Recent trials evidenced several cardio-renal benefits of EMPA in non-diabetic patients through the involvement of biochemical pathways that are still to be deeply analysed. We aimed to evaluate the effects of EMPA on myocardial strain of non-diabetic mice treated with doxorubicin (DOXO) through the analysis of NLRP3 inflammasome and MyD88-related pathways resulting in anti-apoptotic and anti-fibrotic effects. METHODS: Preliminary cellular studies were performed on mouse cardiomyocytes (HL-1 cell line) exposed to doxorubicin alone or combined to EMPA. The following analysis were performed: determination of cell viability (through a modified MTT assay), study of intracellular ROS production, lipid peroxidation (quantifying intracellular malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studies were also performed: expression of NLRP3 inflammasome, MyD88 myddosome and p65/NF-κB associated to secretion of cytokines involved in cardiotoxicity (Interleukins 1ß, 8, 6). C57Bl/6 mice were untreated (Sham, n = 6) or treated for 10 days with doxorubicin (DOXO, n = 6), EMPA (EMPA, n = 6) or doxorubicin combined to EMPA (DOXO-EMPA, n = 6). DOXO was injected intraperitoneally. Ferroptosis and xanthine oxidase were studied before and after treatments. Cardiac function studies, including EF, FS and radial/longitudinal strain were analysed through transthoracic echocardiography (Vevo 2100). Cardiac fibrosis and apoptosis were histologically studied through Picrosirius red and TUNEL assay, respectively and quantified through pro-collagen-1α1, MMP-9 and Caspase-3 expression. Tissue NLRP3, MyD88 and cytokines were also quantified before and after treatments through ELISA methods. RESULTS: Cardiomyocytes exposed to doxorubicin increased the intracellular Ca2+ content and expression of several pro-inflammatory markers associated to cell death; co-incubation with EMPA reduced significantly the magnitude of the effects. In preclinical study, EMPA increased EF and FS compared to DOXO groups (p < 0.05), prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin (RS) 30.3% in EMPA-DOXO vs 15.7% in DOXO mice; LS - 17% in EMPA-DOXO vs - 11.7% in DOXO mice (p < 0.001 for both). Significant reductions in ferroptosis, xanthine oxidase expression, cardiac fibrosis and apoptosis in EMPA associated to DOXO were also seen. A reduced expression of pro-inflammatory cytokines, NLRP3, MyD88 and NF-kB in heart, liver and kidneys was also seen in DOXO-EMPA group compared to DOXO (p < 0.001). CONCLUSION: EMPA reduced ferroptosis, fibrosis, apoptosis and inflammation in doxorubicin-treated mice through the involvement of NLRP3 and MyD88-related pathways, resulting in significant improvements in cardiac functions. These findings provides the proof of concept for translational studies designed to reduce adverse cardiovascular outcomes in non-diabetic cancer patients treated with doxorubicin.