The potential of the nutraceutical berberine in the treatment of hepatocellular carcinoma and other liver diseases such as NAFLD and NASH.

Hepatocellular carcinoma (HCC) is a common cancer which unfortunately has poor outcomes. Common anti-cancer treatments such as chemotherapy and targeted therapy have not increased patient survival significantly. A common treatment for HCC patients is transplantation, however, it has limitations and complications. Novel approaches are necessary to more effectively treat HCC patients. Berberine (BBR) is a nutraceutical derived from various fruits and trees, which has been used for centuries in traditional medicine to treat various diseases such as diabetes and inflammation. More recently, the anti-proliferation effects of BBR have been investigated in the treatment of patients with various cancers, especially colorectal cancer, and in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In this review, we will focus on studies with BBR in liver diseases.

Differential activation of basal and IL-7-induced PI3K/Akt/mTOR and JAK/STAT5 signaling distinguishes pediatric from adult acute lymphoblastic leukemia.

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Nuclear Phospholipids and Signaling: An Update of the Story.

In the last three decades, the presence of phospholipids in the nucleus has been shown and thoroughly investigated. A considerable amount of interest has been raised about nuclear inositol lipids, mainly because of their role in signaling acting. Here, we review the main issues of nuclear phospholipid localization and the role of nuclear inositol lipids and their related enzymes in cellular signaling, both in physiological and pathological conditions.

Molecular and clinical presentation of UBA1-mutated myelodysplastic syndromes.

Mutations in UBA1, which are disease-defining for VEXAS syndrome, have been reported in patients diagnosed with myelodysplastic syndromes (MDS). Here, we define the prevalence and clinical associations of UBA1 mutations in a representative cohort of patients with MDS. Digital droplet PCR profiling of a selected cohort of 375 male patients lacking MDS disease-defining mutations or established WHO disease classification identified 28 patients (7%) with UBA1 p.M41T/V/L mutations. Using targeted sequencing of UBA1 in a representative MDS cohort (n=2,027), we identified an additional 27 variants in 26 patients (1%), which we classified as likely/pathogenic (n=12) and unknown significance (n=15). Among the total 40 patients with likely/pathogenic variants (2%), all were male and 63% were classified by WHO2016 as MDS-MLD/SLD. Patients had a median of one additional myeloid gene mutation, often in TET2 (n=12), DNMT3A (n=10), ASXL1 (n=3), or SF3B1 (n=3). Retrospective clinical review where possible showed that 83% (28/34) UBA1-mutant cases had VEXAS-associated diagnoses or inflammatory clinical presentation. The prevalence of UBA1-mutations in MDS patients argues for systematic screening for UBA1 in the management of MDS.

DNA methylation profiling of myelodysplastic syndromes and clinical response to azacitidine: A multicentre retrospective study.

Real-world data have revealed that a substantial portion of patients with myelodysplastic syndromes (MDS) does not respond to epigenetic therapy with hypomethylating agents (HMAs). The cellular and molecular reasons for this resistance to the demethylating agent and biomarkers that would be able to predict the treatment refractoriness are largely unknown. In this study, we shed light on this enigma by characterizing the epigenomic profiles of patients with MDS treated with azacitidine. Our approach provides a comprehensive view of the evolving DNA methylation architecture of the disease and holds great potential for advancing our understanding of MDS treatment responses to HMAs.

Detection of Cancer Stem Cells in Normal and Dysplastic/Leukemic Human Blood.

The hierarchical organization of the leukemic stem cells (LSCs) is identical to that of healthy counterpart cells. It may be split into roughly three stages: a small number of pluripotent stem cells at the top, few lineage-restricted cells in the middle, and several terminally differentiated blood cells at the bottom. Although LSCs can differentiate into the hematopoietic lineage, they can also accumulate as immature progenitor cells, also known as blast cells. Since blast cells are uncommon in healthy bloodstreams, their presence might be a sign of cancer. For instance, a 20% blast cutoff in peripheral blood or bone marrow is formally used to distinguish acute myeloid leukemia from myelodysplastic neoplasms, which is essential to plan the patients' management. Many techniques may be useful for blast enumeration: one of them is flow cytometry, which can perform analyses on many cells by detecting the expression of cell surface markers. Leukemic and non-leukemic blast cells might indeed be characterized by the same surface markers, but these markers are usually differently expressed. Here we propose to use CD45, in combination with CD34 and other cell surface markers, to identify and immunophenotype blast cells in patient-derived samples.

Deciphering signaling pathways in hematopoietic stem cells: the molecular complexity of Myelodysplastic Syndromes (MDS) and leukemic progression.

Myelodysplastic Syndromes, a heterogeneous group of hematological disorders, are characterized by abnormalities in phosphoinositide-dependent signaling, epigenetic regulators, apoptosis, and cytokine interactions within the bone marrow microenvironment, contributing to disease pathogenesis and neoplastic growth. Comprehensive knowledge of these pathways is crucial for the development of innovative therapies that aim to restore normal apoptosis and improve patient outcomes.

Nuclear Phosphoinositides as Key Determinants of Nuclear Functions.

Polyphosphoinositides (PPIns) are signalling messengers representing less than five per cent of the total phospholipid concentration within the cell. Despite their low concentration, these lipids are critical regulators of various cellular processes, including cell cycle, differentiation, gene transcription, apoptosis and motility. PPIns are generated by the phosphorylation of the inositol head group of phosphatidylinositol (PtdIns). Different pools of PPIns are found at distinct subcellular compartments, which are regulated by an array of kinases, phosphatases and phospholipases. Six of the seven PPIns species have been found in the nucleus, including the nuclear envelope, the nucleoplasm and the nucleolus. The identification and characterisation of PPIns interactor and effector proteins in the nucleus have led to increasing interest in the role of PPIns in nuclear signalling. However, the regulation and functions of PPIns in the nucleus are complex and are still being elucidated. This review summarises our current understanding of the localisation, biogenesis and physiological functions of the different PPIns species in the nucleus.

A miRNA screening identifies miR-192-5p as associated with response to azacitidine and lenalidomide therapy in myelodysplastic syndromes.

BACKGROUND: miRNAs are small non-coding RNAs that regulate gene expression and are linked to cancer development and progression. miRNA profiles are currently studied as new prognostic factors or therapeutic perspectives. Among hematological cancers, myelodysplastic syndromes at higher risk of evolution into acute myeloid leukemia are treated with hypomethylating agents, like azacitidine, alone or in combination with other drugs, such as lenalidomide. Recent data showed that, during azacitidine and lenalidomide therapy, the concurrent acquisition of specific point mutations affecting inositide signalling pathways is associated with lack or loss of response to therapy. As these molecules are implicated in epigenetic processes, possibly involving miRNA regulation, and in leukemic progression, through the regulation of proliferation, differentiation and apoptosis, here we performed a new miRNA expression analysis of 26 high-risk patients with myelodysplastic syndromes treated with azacitidine and lenalidomide at baseline and during therapy. miRNA array data were processed, and bioinformatic results were correlated with clinical outcome to investigate the translational relevance of selected miRNAs, while the relationship between selected miRNAs and specific molecules was experimentally tested and proven. RESULTS: Patients' overall response rate was 76.9% (20/26 cases): complete remission (5/26, 19.2%), partial remission (1/26, 3.8%), marrow complete remission (2/26, 7.7%), hematologic improvement (6/26, 23.1%), hematologic improvement with marrow complete remission (6/26, 23.1%), whereas 6/26 patients (23.1%) had a stable disease. miRNA paired analysis showed a statistically significant up-regulation of miR-192-5p after 4 cycles of therapy (vs baseline), that was confirmed by real-time PCR analyses, along with an involvement of BCL2, that was proven to be a miR-192-5p target in hematopoietic cells by luciferase assays. Furthermore, Kaplan-Meier analyses showed a significant correlation between high levels of miR-192-5p after 4 cycles of therapy and overall survival or leukemia-free survival, that was stronger in responders, as compared with patients early losing response and non-responders. CONCLUSIONS: This study shows that high levels of miR-192-5p are associated with higher overall survival and leukemia-free survival in myelodysplastic syndromes responding to azacitidine and lenalidomide. Moreover, miR-192-5p specifically targets and inhibits BCL2, possibly regulating proliferation and apoptosis and leading to the identification of new therapeutic targets.

Advances in MDS/AML and inositide signalling.

Aberrant signaling pathways regulating proliferation and differentiation of hematopoietic stem cells (HSCs) can contribute to disease pathogenesis and neoplastic growth. Phosphoinositides (PIs) are inositol phospholipids that are implicated in the regulation of critical signaling pathways: aberrant regulation of Phospholipase C (PLC) beta1, PLCgamma1 and the PI3K/Akt/mTOR pathway play essential roles in the pathogenesis of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML).

Effects of chloroquine and hydroxychloroquine on the sensitivity of pancreatic cancer cells to targeted therapies.

Approaches to improve pancreatic cancer therapy are essential as this disease has a very bleak outcome. Approximately 80% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is a cancer which is difficult to effectively treat as it is often detected late in the disease process. Almost all PDACs (over 90%) have activating mutations in the GTPase gene KRAS. These mutations result in constitutive KRas activation and the mobilization of downstream pathways such as the Raf/MEK/ERK pathway. Small molecule inhibitors of key components of the KRas/Raf/MEK/ERK pathways as well as monoclonal antibodies (MoAbs) specific for upstream growth factor receptors such insulin like growth factor-1 receptor (IGF1-R) and epidermal growth factor receptors (EGFRs) have been developed and have been evaluated in clinical trials. An additional key regulatory gene frequently mutated (∼75%) in PDAC is the TP53 tumor suppressor gene which controls the transcription of multiple genes involved in cell cycle progression, apoptosis, metabolism, cancer progression and other growth regulatory processes. Small molecule mutant TP53 reactivators have been developed which alter the structure of mutant TP53 protein and restore some of its antiproliferative activities. Some mutant TP53 reactivators have been examined in clinical trials with patients with mutant TP53 genes. Inhibitors to the TP53 negative regulator Mouse Double Minute 2 (MDM2) have been developed and analyzed in clinical trials. Chloroquine and hydroxychloroquine are established anti-malarial and anti-inflammatory drugs that also prevent the induction of autophagy which can have effects on cancer survival. Chloroquine and hydroxychloroquine have also been examined in various clinical trials. Recent studies are suggesting effective treatment of PDAC patients may require chemotherapy as well as targeting multiple pathways and biochemical processes.

Effects of TP53 Mutations and miRs on Immune Responses in the Tumor Microenvironment Important in Pancreatic Cancer Progression.

Approximately 90% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is the fourth leading cause of cancer death world-wide. Therapies for PDAC are largely ineffective due to the dense desmoplastic tumor microenvironment which prevents chemotherapeutic drugs and small molecule inhibitors from exerting effective anti-cancer effects. In this review, we will discuss the roles of TP53 and miRs on the PDAC tumor microenvironment and how loss of the normal functions of TP53 promote tumor progression. The TP53 gene is mutated in approximately 50% of pancreatic cancers. Often, these TP53 mutations are point mutations which confer additional functions for the TP53 proteins. These are called gain of function (GOF) mutations (mut). Another class of TP53 mutations are deletions which result in loss of the TP53 protein; these are referred to TP53-null mutations. We have organized this review into various components/properties of the PDAC microenvironment and how they may be altered in the presence of mutant TP53 and loss of certain miR expression.

Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy.

BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Role of PLCγ1 in the modulation of cell migration and cell invasion in glioblastoma.

Phosphoinositide-specific phospholipases C (PLCs) are a class of enzymes involved in several cell activities, such as cell cycle regulation, proliferation, differentiation and cytoskeletal dynamics. Among these enzymes, PLCγ1 is one of the most expressed PLCs in the brain, contributing to a complex network in the developing nervous system. Several studies have shown that PLCγ1 signaling imbalance is linked to several brain disorders, including glioblastoma, the most aggressive brain tumor in adults. Indeed, it has been demonstrated a link between PLCγ1 inhibition and the arrest of glioma cell motility of fetal rat brain aggregates and the impairment of cell invasion abilities following its down-regulation. This study aims to determine the pathological influence of PLCγ1 in glioblastoma, through a translational study which combines in silico data, data from glioblastoma patients' samples and data on engineered cell lines. We found out that PLCγ1 gene expression correlates with the pathological grade of gliomas, and it is higher in fifty patients' glioblastoma tissue samples compared to twenty healthy controls. Moreover, it was demonstrated that PLCγ1 silencing in U87-MG leads to a reduction in cell migration and invasion abilities. The opposite trend was observed following PLCγ1 overexpression, suggesting an interesting possible involvement of PLCγ1 in gliomas' aggressiveness.

Molecular International Prognostic Scoring System for Myelodysplastic Syndromes.

MDS Molecular International Prognostic Scoring SystemSamples from over 2500 patients with MDS were profiled for gene mutations and used to develop the International Prognostic Scoring System-Molecular (IPSS-M). TP53multihit, FLT3 mutations, and MLLPTD were identified as top genetic predictors of adverse outcomes. IPSS-M improves prognostic discrimination across all clinical end points versus prior versions.

"Modulating Phosphoinositide Profiles as a Roadmap for Treatment in Acute Myeloid Leukemia".

Polyphosphoinositides (PPIns) and their modulating enzymes are involved in regulating many important cellular functions including proliferation, differentiation or gene expression, and their deregulation is involved in human diseases such as metabolic syndromes, neurodegenerative disorders and cancer, including Acute Myeloid Leukemia (AML). Given that PPIns regulating enzymes are highly druggable targets, several studies have recently highlighted the potential of targeting them in AML. For instance many inhibitors targeting the PI3K pathway are in various stages of clinical development and more recently other novel enzymes such as PIP4K2A have been implicated as AML targets. PPIns have distinct subcellular organelle profiles, in part driven by the specific localisation of enzymes that metabolise them. In particular, in the nucleus, PPIns are regulated in response to various extracellular and intracellular pathways and interact with specific nuclear proteins to control epigenetic cell state. While AML does not normally manifest with as many mutations as other cancers, it does appear in large part to be a disease of dysregulation of epigenetic signalling and many novel therapeutics are aimed at reprogramming AML cells toward a differentiated cell state or to one that is responsive to alternative successful but limited AML therapies such as ATRA. Here, we propose that by combining bioinformatic analysis with inhibition of PPIns pathways, especially within the nucleus, we might discover new combination therapies aimed at reprogramming transcriptional output to attenuate uncontrolled AML cell growth. Furthermore, we outline how different part of a PPIns signalling unit might be targeted to control selective outputs that might engender more specific and therefore less toxic inhibitory outcomes.

Clinical and Molecular Insights in Erythropoiesis Regulation of Signal Transduction Pathways in Myelodysplastic Syndromes and ß-Thalassemia.

Erythropoiesis regulation is essential in normal physiology and pathology, particularly in myelodysplastic syndromes (MDS) and ß-thalassemia. Several signaling transduction processes, including those regulated by inositides, are implicated in erythropoiesis, and the latest MDS or ß-thalassemia preclinical and clinical studies are now based on their regulation. Among others, the main pathways involved are those regulated by transforming growth factor (TGF)-ß, which negatively regulates erythrocyte differentiation and maturation, and erythropoietin (EPO), which acts on the early-stage erythropoiesis. Also small mother against decapentaplegic (SMAD) signaling molecules play a role in pathology, and activin receptor ligand traps are being investigated for future clinical applications. Even inositide-dependent signaling, which is important in the regulation of cell proliferation and differentiation, is specifically associated with erythropoiesis, with phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K) as key players that are becoming increasingly important as new promising therapeutic targets. Additionally, Roxadustat, a new erythropoiesis stimulating agent targeting hypoxia inducible factor (HIF), is under clinical development. Here, we review the role and function of the above-mentioned signaling pathways, and we describe the state of the art and new perspectives of erythropoiesis regulation in MDS and ß-thalassemia.

Location-dependent role of phospholipase C signaling in the brain: Physiology and pathology.

Phosphoinositide-specific phospholipases C (PI-PLCs) are a class of enzymes involved in the phosphatidylinositol metabolism, which is implicated in the activation of several signaling pathways and which controls several cellular processes. The scientific community has long accepted the existence of a nuclear phosphoinositide (PI) metabolism, independent from the cytoplasmic one, critical in nuclear function control. Indeed, nuclear PIs are involved in many activities, such as cell cycle regulation, cell proliferation, cell differentiation, membrane transport, gene expression and cytoskeletal dynamics. There are several types of PIs and enzymes implicated in brain activities and among these enzymes, PI-PLCs contribute to a specific and complex network in the developing nervous system. Moreover, considering the abundant presence of PI-PLCß1, PI-PLCγ1 and PI-PLCß4 in the brain, a specific role for each PLC subtype has been suggested in the control of neuronal activity, which is important for synapse function, development and other mechanisms. The focus of this review is to describe the latest research about the involvement of PI-PLC signaling in the nervous system, both physiologically and in pathological conditions. Indeed, PI-PLC signaling imbalance seems to be also linked to several brain disorders including epilepsy, movement and behavior disorders, neurodegenerative diseases and, in addition, some PI-PLC subtypes could become potential novel signature genes for high-grade gliomas.