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Increased expression of interleukin-6 (IL-6) both in cerebrospinal fluid (CSF) and plasma is closely associated with convulsive epilepsy and symptom severity of depression. By comparison, at present, little is known about the role of this cytokine in childhood (non-convulsive) absence epilepsy. The aim of this work was to investigate the potential effects of acute and chronic treatment with tocilizumab (TCZ, 10 and 30 mg/kg/day), on absence seizures, their development, and related psychiatric comorbidity in WAG/Rij rats. It is known that lipopolysaccharide (LPS)-induced changes in inflammatory processes increase absence epileptic activity. In order to study the central effects of TCZ, we investigated whether administration of this anti-IL-6R antibody could modulate the lipopolysaccharide (LPS) or IL-6-evoked changes in absence epileptic activity in WAG/Rij rats. Our results demonstrate that TCZ, at both doses, significantly reduced the development of absence seizures in adult WAG/Rij rats at 6 months of age (1 month after treatment suspension) compared with untreated controls, thus showing disease-modifying effects. Decreased absence seizure development at 6 months of age was also accompanied by reduced comorbid depressive-like behavior, whereas no effects were observed on anxiety-related behavior. Acute treatment with TCZ, at 30 mg/kg, had anti-absence properties lasting ~25 h. The co-administration TCZ with i.c.v. LPS or IL-6 showed that TCZ inhibited the worsening of absence seizures induced by both proinflammatory agents in the WAG/Rij rats, supporting a central anti-inflammatory-like protective action. These results suggest the possible role of IL-6 and consequent neuroinflammation in the epileptogenic process underlying the development and maintenance of absence seizures in WAG/Rij rats. Accordingly, IL-6 signaling could be a promising pharmacological target in absence epilepsy and depressive-like comorbidity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-020-00893-8) contains supplementary material, which is available to authorized users.

Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. Since we previously demonstrated that LIG3-dependent repair is involved in the genomic instability, drug resistance and survival of MM cells, we here investigated the biological relevance of PARP1, a driver component of Alternative-Non Homologous End Joining (Alt-NHEJ) pathway, in MM. We found a significant correlation between higher PARP1 mRNA expression and poor prognosis of MM patients. PARP1 knockdown or its pharmacological inhibition by Olaparib impaired MM cells viability in vitro and was effective against in vivo xenografts of human MM. Anti-proliferative effects induced by PARP1-inhibition were correlated to increase of DNA double-strand breaks, activation of DNA Damage Response (DDR) and finally apoptosis. Importantly, by comparing a gene expression signature of PARP inhibitors (PARPi) sensitivity to our plasma cell dyscrasia (PC) gene expression profiling (GEP), we identified a subset of MM patients which could benefit from PARP inhibitors. In particular, Gene Set Enrichment Analysis (GSEA) suggested that high MYC expression correlates to PARPi sensitivity in MM. Indeed, we identified MYC as promoter of PARP1-mediated repair in MM and, consistently, we demonstrate that cytotoxic effects induced by PARP inhibition are mostly detectable on MYC-proficient MM cells. Taken together, our findings indicate that MYC-driven MM cells are addicted to PARP1 Alt-NHEJ repair, which represents therefore a druggable target in this still incurable disease.

Simple Summary The oligometastatic prostate cancer state is defined as the presence of a number of lesions ≤ 5 and has been significantly correlated with better survival if compared to a number of metastases > 5. In particular, patients in an oligometastatic setting could benefit from a metastates directed therapy, which could control the disease delaying the start of systemic therapies. For this reason, the selection of true-oligometastatic patients who could benefit from such approach is particularly important in this setting. The aim of the present narrative review is to report the current state of the art on the liquid biopsy-derived analytes and their reliability as biomarkers in the clinics for the identification of true-oligometastatic patients. This kind of molecular profiling could refine current developments in the era of precision oncology allowing patients’ stratification and leading to more refined therapeutic strategies. Abstract In recent years, a growing interest has been directed towards oligometastatic prostate cancer (OMPC), as patients with three to five metastatic lesions have shown a significantly better survival as compared with those harboring a higher number of lesions. The efficacy of local ablative treatments directed on metastatic lesions (metastases-directed treatments) was extensively investigated, with the aim of preventing further disease progression and delaying the start of systemic androgen deprivation therapies. Definitive diagnosis of prostate cancer is traditionally based on histopathological analysis. Nevertheless, a bioptic sample—static in nature—inevitably fails to reflect the dynamics of the tumor and its biological response due to the dynamic selective pressure of cancer therapies, which can profoundly influence spatio-temporal heterogeneity. Furthermore, even with new imaging technologies allowing an increasingly early detection, the diagnosis of oligometastasis is currently based exclusively on radiological investigations. Given these premises, the development of minimally-invasive liquid biopsies was recently promoted and implemented as predictive biomarkers both for clinical decision-making at pre-treatment (baseline assessment) and for monitoring treatment response during the clinical course of the disease. Through liquid biopsy, different biomarkers, commonly extracted from blood, urine or saliva, can be characterized and implemented in clinical routine to select targeted therapies and assess treatment response. Moreover, this approach has the potential to act as a tissue substitute and to accelerate the identification of novel and consistent predictive analytes cost-efficiently. However, the utility of tumor profiling is currently limited in OMPC due to the lack of clinically validated predictive biomarkers. In this scenario, different ongoing trials, such as the RADIOSA trial, might provide additional insights into the biology of the oligometastatic state and on the identification of novel biomarkers for the outlining of true oligometastatic patients, paving the way towards a wider ideal approach of personalized medicine. The aim of the present narrative review is to report the current state of the art on the solidity of liquid biopsy-related analytes such as CTCs, cfDNA, miRNA and epi-miRNA, and to provide a benchmark for their further clinical implementation. Arguably, this kind of molecular profiling could refine current developments in the era of precision oncology and lead to more refined therapeutic strategies in this subset of oligometastatic patients.

Epigenetic mechanisms, such as alterations in histone acetylation based on histone deacetylases (HDACs) activity, have been linked not only to normal brain function but also to several brain disorders including epilepsy and the epileptogenic process. In WAG/Rij rats, a genetic model of absence epilepsy, epileptogenesis and mild-depression comorbidity, we investigated the effects of two HDAC inhibitors (HDACi), namely sodium butyrate (NaB), valproic acid (VPA) and their co-administration, on the development of absence seizures and related psychiatric/neurologic comorbidities following two different experimental paradigms. Treatment effects have been evaluated by EEG recordings (EEG) and behavioural tests at different time points. Prolonged and daily VPA and NaB treatment, started before absence seizure onset (P30), significantly reduced the development of absence epilepsy showing antiepileptogenic effects. These effects were enhanced by NaB/VPA co-administration. Furthermore, early-chronic HDACi treatment improved depressive-like behaviour and cognitive performance 1 month after treatment withdrawal. WAG/Rij rats of 7 months of age showed reduced acetylated levels of histone H3 and H4, analysed by Western Blotting of homogenized brain, in comparison to WAG/Rij before seizure onset (P30). The brain histone acetylation increased significantly during treatment with NaB or VPA alone and more markedly during co-administration. We also observed decreased expression of both HDAC1 and 3 following HDACi treatment compared to control group. Our results suggest that histone modifications may have a crucial role in the development of epilepsy and early treatment with HDACi might be a possible strategy for preventing epileptogenesis also affecting behavioural comorbidities.

Global public health is threatened by new pathogens, antimicrobial resistant microorganisms and a rapid decline of conventional antimicrobials efficacy. Thus, numerous medical procedures become life-threating. Sepsis can lead to tissue damage such as myocardium inflammation, associated with reduction of contractility and diastolic dysfunction, which may cause death. In this perspective, growing interest and attention are paid on host defence peptides considered as new potential antimicrobials. In the present study, we investigated the physiological and biochemical properties of Cateslytin (Ctl), an endogenous antimicrobial chromogranin A-derived peptide, in H9c2 cardiomyocytes exposed to lipopolysaccharide (LPS) infection. We showed that both Ctl (L and D) enantiomers, but not their scrambled counterparts, significantly increased cardiomyocytes viability following LPS, even if L-Ctl was effective at lower concentration (1 nM) compared to D-Ctl (10 nM). L-Ctl mitigated LPS-induced LDH release and oxidative stress, as visible by a reduction of MDA and protein carbonyl groups content, and by an increase of SOD activity. Molecular docking simulations strongly suggested that L-Ctl modulates TLR4 through a direct binding to the partner protein MD-2. Molecular analyses indicated that the protection mediated by L-Ctl against LPS-evoked sepsis targeted the TLR4/ERK/JNK/p38-MAPK pathway, regulating NFkB p65, NFkB p52 and COX2 expression and repressing the mRNA expression levels of the LPS-induced proinflammatory factors IL-1β, IL-6, TNF-α and NOS2. These findings indicate that Ctl could be considered as a possible candidate for the development of new antimicrobials strategies in the treatment of myocarditis. Interestingly, L-enantiomeric Ctl showed remarkable properties in strengthening the anti-inflammatory and anti-oxidant effects on cardiomyocytes. Keywords: Antimicrobial peptides; Cardiomyocytes; Cateslytin; Inflammation; Oxidative stress; Toll-like receptor 4.

The mitochondrial quality control network includes several epigenetically-regulated genes involved in mitochondrial dynamics, mitophagy, and mitochondrial biogenesis under physiologic conditions. Dysregulated expression of such genes has been reported in various disease contexts, including cancer. However, their expression pattern and the possible underlying epigenetic modifications remain to be defined within plasma cell (PC) dyscrasias. Herein, we compared the mRNA expression of mitochondrial quality control genes from multiple myeloma, plasma cell leukemia patients and human myeloma cell lines (HMCLs) with healthy plasma cells; moreover, by applying the Sequenom MassARRAY EpiTYPER technology, we performed a pilot investigation of their CpG methylation status in HMCLs. Overall, the results provided indicate dysregulated expression of several mitochondrial network’s genes, and alteration of the CpG methylation profile, underscoring novel potential myeloma biomarkers deserving in-depth functional investigation in the future.

Simple Summary In the last decade, proteasome inhibitors (PIs) have become a standard for the treatment of multiple myeloma (MM). As a consequence of the pleiotropic effects of PIs on various signaling pathways, synergistic or additive activities with other anti-myeloma therapies have been identified and approved for clinical use. However, the complex biology of the MM disease inevitably triggers resistance also to combined regimens. Complex loops within cellular pathways, crosstalk with the bone marrow microenvironment, and considerable toxicities are accountable for the poor responses of new multidrug treatments. High-throughput functional approaches are allowing the identification of a multitude of previously undescribed synthetic lethal interactions. In the present review, we explore recent investigations on novel combination strategies that could overcome drug resistance and broaden the applicability of PIs to other hematological malignancies and solid tumors. Abstract Multiple myeloma is a malignancy of terminally differentiated plasma cells, characterized by an extreme genetic heterogeneity that poses great challenges for its successful treatment. Due to antibody overproduction, MM cells depend on the precise regulation of the protein degradation systems. Despite the success of PIs in MM treatment, resistance and adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. To this end, the use of rational combinatorial treatments might allow lowering the dose of inhibitors and therefore, minimize their side-effects. Even though the suppression of different cellular pathways in combination with proteasome inhibitors have shown remarkable anti-myeloma activities in preclinical models, many of these promising combinations often failed in clinical trials. Substantial progress has been made by the simultaneous targeting of proteasome and different aspects of MM-associated immune dysfunctions. Moreover, targeting deranged metabolic hubs could represent a new avenue to identify effective therapeutic combinations with PIs. Finally, epigenetic drugs targeting either DNA methylation, histone modifiers/readers, or chromatin remodelers are showing pleiotropic anti-myeloma effects alone and in combination with PIs. We envisage that the positive outcome of patients will probably depend on the availability of more effective drug combinations and treatment of early MM stages. Therefore, the identification of sensitive targets and aberrant signaling pathways is instrumental for the development of new personalized therapies for MM patients.

Multiple myeloma (MM) is an incurable malignancy of plasma cells that grow within a permissive bone marrow microenvironment (BMM). The bone marrow milieu supports the malignant transformation both by promoting uncontrolled proliferation and resistance to cell death in MM cells, and by hampering the immune response against the tumor clone. Hence, it is expected that restoring host anti-MM immunity may provide therapeutic benefit for MM patients. Already several immunotherapeutic approaches have shown promising results in the clinical setting. In this review, we outline recent findings demonstrating the potential advantages of targeting the immunosuppressive bone marrow niche to restore effective anti-MM immunity. We discuss different approaches aiming to boost the effector function of T cells and/or exploit innate or adaptive immunity, and highlight novel therapeutic opportunities to increase the immunogenicity of the MM clone. We also discuss the main challenges that hamper the efficacy of immune-based approaches, including intrinsic resistance of MM cells to activated immune-effectors, as well as the protective role of the immune-suppressive and inflammatory bone marrow milieu. Targeting mechanisms to convert the immunologically “cold” to “hot” MM BMM may induce durable immune responses, which in turn may result in long-lasting clinical benefit, even in patient subgroups with high-risk features and poor survival.

Aim Chromogranin A (CgA), a 439-residue long protein, is an important cardiovascular regulator and a precursor of various bioactive fragments. Under stressful/pathological conditions, CgA cleavage generates the CgA1-373 proangiogenic fragment. The present work investigated the possibility that human CgA1-373 influences the mammalian cardiac performance, evaluating the role of its C-terminal sequence. Methods Haemodynamic assessment was performed on an ex vivo Langendorff rat heart model, while mechanistic studies were performed using perfused hearts, H9c2 cardiomyocytes and in silico. Results On the ex vivo heart, CgA1-373 elicited direct dose-dependent negative inotropism and vasodilation, while CgA1-372 , a fragment lacking the C-terminal R373 residue, was ineffective. Antibodies against the PGPQLR373 C-terminal sequence abrogated the CgA1-373 -dependent cardiac and coronary modulation. Ex vivo studies showed that CgA1-373 -dependent effects were mediated by endothelium, neuropilin-1 (NRP1) receptor, Akt/NO/Erk1,2 pathways, nitric oxide (NO) production and S-nitrosylation. In vitro experiments on H9c2 cardiomyocytes indicated that CgA1-373 also induced eNOS activation directly on the cardiomyocyte component by NRP1 targeting and NO involvement and provided beneficial action against isoproterenol-induced hypertrophy, by reducing the increase in cell surface area and brain natriuretic peptide (BNP) release. Molecular docking and all-atom molecular dynamics simulations strongly supported the hypothesis that the C-terminal R373 residue of CgA1-373 directly interacts with NRP1. Conclusion These results suggest that CgA1-373 is a new cardioregulatory hormone and that the removal of R373 represents a critical switch for turning "off" its cardioregulatory activity.

Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.

The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-coding genes. These molecules can play a pivotal role in the development and progression of human disease, including cancer, and are susceptible to therapeutic intervention. Evidence of biologic function, however, is still missing for the vast majority of them. Both loss-of-function (LOF) and gain-of-function (GOF) studies are therefore necessary to advance our understanding of lncRNA networks and programs driving tumorigenesis. Here, we describe a protocol to perform lncRNA's LOF or GOF studies in multiple myeloma (MM) cells, using CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) technologies, respectively. These approaches have many advantages, including applicability to large-scale genetic screens in mammalian cells and possible reversibility of modulating effects; moreover, CRISPRa offers the unique opportunity to enhance lncRNA expression at the site of transcription, with relevant biologic implications.

Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1&ndash, 2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines&rsquo, ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro, in vivo validation was performed on a zebrafish model and MM patients&rsquo, BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF, and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands.

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression, therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1&alpha, /XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1&alpha, by phosphorylation in S724, accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1&alpha, (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption.

There is a need for continued drug development for nonalcoholic steatohepatitis (NASH). Bergamot is a plant whose fruit juice is enriched with flavonoids and phenolic compounds which improves dyslipidemia and markers of systemic inflammation in patients with Metabolic Syndrome. The aim of this study was to perform a preclinical “proof of concept” study of Bergamot polyphenolic formulation (BPF99) for the treatment of NASH. A disease reversal study was performed in the diet-induced animal model of NAFLD (DIAMOND). Groups of 8 weeks old mice were randomly assigned to receive chow diet, high fat diet with sugar in drinking water (Western diet- WD). Mice on WD were further randomized to continue on WD gavaged with vehicle or continue on WD with additional gavage of BPF99 (50 mg/kg) after 16 weeks of diet. Mice were euthanized after 11 additional weeks. The primary endpoint was resolution of NASH. Secondary endpoints included changes in individual histological features, body weight, liver enzymes, dyslipidemia, markers of oxidative stress and molecular markers of disease activity and fibrosis. The results showed that BPF99 reduced ALT (mean 71.6 vs 44.6 IU/l, p

Increasing evidence indicates that extracellular vesicles (EVs) released from both tumor cells and the cells of the bone marrow microenvironment contribute to the pathobiology of multiple myeloma (MM). Recent studies on the mechanisms by which EVs exert their biological activity have indicated that the non-coding RNA (ncRNA) cargo is key in mediating their effect on MM development and progression. In this review, we will first discuss the role of EV-associated ncRNAs in different aspects of MM pathobiology, including proliferation, angiogenesis, bone disease development, and drug resistance. Finally, since ncRNAs carried by MM vesicles have also emerged as a promising tool for early diagnosis and therapy response prediction, we will report evidence of their potential use as clinical biomarkers.

Despite substantial advancements have been done in the understanding of the pathogenesis of plasma cell (PC) disorders, these malignancies remain hard-to-treat. The discovery and subsequent characterization of non-coding transcripts, which include several members with diverse length and mode of action, has unraveled novel mechanisms of gene expression regulation often malfunctioning in cancer. Increasing evidence indicates that such non-coding molecules also feature in the pathobiology of PC dyscrasias, where they are endowed with strong therapeutic and/or prognostic potential. In this review, we aim to summarize the most relevant findings on the biological and clinical features of the non-coding RNA landscape of malignant PCs, with major focus on multiple myeloma. The most relevant classes of non-coding RNAs will be examined, along with the mechanisms accounting for their dysregulation and the recent strategies used for their targeting in PC dyscrasias. It is hoped these insights may lead to clinical applications of non-coding RNA molecules as biomarkers or therapeutic targets/agents in the near future.

Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability. Here we provide evidence that hyper-activation of DNA ligase III (LIG3) is crucial for genomic instability and survival of MM cells. LIG3 mRNA expression in MM patients correlates with shorter survival and even increases with more advanced stage of disease. Knockdown of LIG3 impairs MM cells viability in vitro and in vivo, suggesting that neoplastic plasmacells are dependent on LIG3-driven repair. To investigate the mechanisms involved in LIG3 expression, we investigated the post-transcriptional regulation. We identified miR-22-3p as effective negative regulator of LIG3 in MM. Enforced expression of miR-22 in MM cells downregulated LIG3 protein, which in turn increased DNA damage inhibiting in vitro and in vivo cell growth. Taken together, our findings demonstrate that myeloma cells are addicted to LIG3, which can be effectively inhibited by miR-22, promoting a novel axis of genome stability regulation.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Arginine methyltransferases critically regulate cellular homeostasis by modulating the functional outcome of their substrates. The protein arginine methyltransferase 5 (PRMT5) is an enzyme involved in growth and survival pathways promoting tumorigenesis. However, little is known about the biologic function of PRMT5 and its therapeutic potential in multiple myeloma (MM). In the present study, we identified and validated PRMT5 as a new therapeutic target in MM. PRMT5 is overexpressed in patient MM cells and associated with decreased progression-free survival and overall survival. Either genetic knockdown or pharmacological inhibition of PRMT5 with the inhibitor EPZ015666 significantly inhibited growth of both cell lines and patient MM cells. Furthermore, PRMT5 inhibition abrogated NF-κB signaling. Interestingly, mass spectrometry identified a tripartite motif-containing protein 21 TRIM21 as a new PRMT5-partner, and we delineated a TRIM21-dependent mechanism of NF-κB inhibition. Importantly, oral administration of EPZ015666 significantly decreased MM growth in a humanized murine model of MM. These data both demonstrate the oncogenic role and prognostic relevance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome.

The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose-limiting cardiotoxicity in approximately 20% of patients. The G-protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G-1 reduced myocardial inflammation, improved immunosuppression, triggered pro-survival signaling cascades, improved myocardial mechanical performance, and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand-activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G-1 (50 μg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF-α, IL-1β, LDH, and ROS levels. Western blotting analysis of cardiac homogenates indicated that G-1 prevents the increase in p-c-jun, BAX, CTGF, iNOS, and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK, and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G-1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G-1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox-induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. J. Cell. Physiol. 232: 1640-1649, 2017. © 2016 Wiley Periodicals, Inc.

Multiple Myeloma (MM) is a clonal late B-cell disorder accounting for about 13% of hematological cancers and 1% of all neoplastic diseases. Recent studies on the molecular pathogenesis and biology of MM have highlighted a complex epigenomic landscape contributing to MM onset, prognosis and high individual variability. Areas covered: We describe here the current knowledge on epigenetic events characterizing MM initiation and progression, focusing on the role of DNA and histone methylation and on the most promising epi-therapeutic approaches targeting the methylation pathway. Expert opinion: Data published so far indicate that alterations of the epigenetic framework, which include aberrant global or gene/non-coding RNA specific methylation profiles, feature prominently in the pathobiology of MM. Indeed, the aberrant expression of components of the epigenetic machinery as well as the reversibility of the epigenetic marks make this pathway druggable, providing the basis for the design of epigenetic therapies against this still fatal malignancy.

Multiple myeloma (MM) is an incurable hematological malignancy driven by several genetic and epigenetic alterations. The hyperactivation of the Polycomb repressive complex 2 (PRC2), a multi-subunit oncogenic histone methyltransferase, has been implicated in the pathogenesis of this malignancy. Upon protein-protein interaction (PPI) between the catalytic subunit EZH2 and EED, PRC2 primarily methylates lysine 27 of histone H3 (H3K27me3), thus modulating the chromatin structure and inducing transcriptional repression. Herein, we highlight a new mechanism of action that can contribute to explain the anti-tumor activity of hydroxychloroquine (HCQ), an anti-malaric agent also known as autophagy inhibitor. By structural studies, we demonstrate that HCQ inhibits the allosteric binding of PRC2 to EED within the H3K27me3-binding pocket, thus antagonizing the PRC2 catalytic activity. In silico results are compatible with the significant reduction of the H3K27me3 levels in MM cells exerted by HCQ. Overall, these findings disclose a novel epigenetic activity of HCQ with potential implications for its clinical repositioning.

Aberrant expression of microRNAs (miRNAs) has been associated to the pathogenesis of multiple myeloma (MM). While miR-155 is considered a therapeutic target in several malignancies, its role in MM is still unclear. The analysis of miR-155 expression indicates its down-regulation in MM patient-derived as compared to healthy plasma cells, thus pointing to a tumor suppressor role in this malignancy. On this finding, we investigated miR-155 replacement as a potential anti-tumor strategy in MM. The miR-155 enforced expression triggered anti-proliferative and pro-apoptotic effects in vitro. Given the lower miR-155 levels in bortezomib-resistant as compared to sensitive MM cells, we analyzed the possible involvement of miR-155 in bortezomib resistance. Importantly, miR-155 replacement enhanced bortezomib anti-tumor activity both in vitro and in vivo in a xenograft model of human MM. In primary MM cells, we observed an inverse correlation between miR-155 and the mRNA encoding the proteasome subunit gene PSM&beta, 5, whose dysregulation has been largely implicated in bortezomib resistance, and we validated PSM&beta, 5 3&prime, UTR mRNA targeting, along with reduced proteasome activity, by miR-155. Collectively, our findings demonstrate that miR-155 elicits anti-MM activity, likely via proteasome inhibition, providing the framework for miR-155-based anti-MM therapeutic strategies.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still open questions. Herein, we investigated the functional significance of the oncogenic lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in MM. Our study demonstrates that NEAT1 expression level is higher in MM than in the majority of hematological malignancies. NEAT1 silencing by novel LNA-gapmeR antisense oligonucleotide inhibits MM cell proliferation and triggers apoptosis in vitro and in vivo murine MM model as well. By transcriptome analyses, we found that NEAT1 targeting downregulates genes involved in DNA repair processes including the Homologous Recombination pathway, which in turn results in massive DNA damage. These findings may explain the synergistic impact on apoptosis observed in MM cell lines co-treated with inhibitors of both NEAT1 and PARP. The translational significance of NEAT1 targeting is further underlined by its synergistic effects with the most common drugs administered for MM treatment, including bortezomib, carfilzomib, and melphalan. Overall, NEAT1 silencing is associated with a chemo-sensitizing effect of both conventional and novel therapies, and its targeting could therefore represent a promising strategy for novel anti-MM therapeutic options.

Simple Summary Genomic instability (GI) plays an important role in the pathobiology of multiple myeloma (MM) by promoting the acquisition of several tumor hallmarks. Molecular determinants of GI in MM are continuously emerging and will be herein discussed, with specific regard to non-coding RNAs. Targeting non-coding RNA molecules known to be involved in GI indeed provides novel routes to dampen such oncogenic mechanisms in MM. Abstract Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal proliferation of malignant plasma cells (PCs) within a permissive bone marrow microenvironment. The pathogenesis of MM is unequivocally linked to the acquisition of genomic instability (GI), which indicates the tendency of tumor cells to accumulate a wide repertoire of genetic alterations. Such alterations can even be detected at the premalignant stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) and, overall, contribute to the acquisition of the malignant traits underlying disease progression. The molecular basis of GI remains unclear, with replication stress and deregulation of DNA damage repair pathways representing the most documented mechanisms. The discovery that non-coding RNA molecules are deeply dysregulated in MM and can target pivotal components of GI pathways has introduced a further layer of complexity to the GI scenario in this disease. In this review, we will summarize available information on the molecular determinants of GI in MM, focusing on the role of non-coding RNAs as novel means to tackle GI for therapeutic intervention.

Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy.

Multiple myeloma (MM) is a hematologic malignancy of differentiated plasma cells that accumulate in the bone marrow, where a complex microenvironment made by different cell types supports proliferation, survival, and drug resistance of tumor cells. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at posttranscriptional level. Emerging evidence indicates that miRNAs are aberrantly expressed or functionally deregulated in MM cells as the result of multiple genetic or epigenetic mechanisms and that also the tumor microenvironment regulates MM cell functions by miRNAs. Consistently, modulation of miRNA levels in MM cells has been demonstrated to impair their functional interaction with the bone marrow microenvironment and to produce significant antitumor activity even able to overcome the protective bone marrowmilieu. This review will describe the most recent findings on miRNA function in the context of MM bone marrow microenvironment, focusing on the therapeutic potential of miRNA-based approaches.

Bone remodeling is uncoupled in the multiple myeloma (MM) bone marrow niche, resulting in enhanced osteoclastogenesis responsible of MM-related bone disease (MMBD). Several studies have disclosed the mechanisms underlying increased osteoclast formation and activity triggered by the various cellular components of the MM bone marrow microenvironment, leading to the identification of novel targets for therapeutic intervention. In this regard, recent attention has been given to non-coding RNA (ncRNA) molecules, that finely tune gene expression programs involved in bone homeostasis both in physiological and pathological settings. In this review, we will analyze major signaling pathways involved in MMBD pathophysiology, and report emerging evidence of their regulation by different classes of ncRNAs.

Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNA) reported to be frequently deregulated in various types of cancers and neurodegenerative processes. NEAT1 is an indispensable structural component of paraspeckles (PSs), which are dynamic and membraneless nuclear bodies that affect different cellular functions, including stress response. Furthermore, increasing evidence supports the crucial role of NEAT1 and essential structural proteins of PSs (PSPs) in the regulation of the DNA damage repair (DDR) system. This review aims to provide an overview of the current knowledge on the involvement of NEAT1 and PSPs in DDR, which might strengthen the rationale underlying future NEAT1-based therapeutic options in tumor and neurodegenerative diseases.

The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an important aspect of investigation, which may contribute to the understanding of the complex pathobiology of the disease whilst also providing novel potential therapeutic targets. Herein, we investigated the expression pattern and the biological significance of the lncRNA ST3 beta-galactoside alpha-2,3 sialyltransferase 6 antisense RNA 1 (ST3GAL6-AS1) in MM. We documented a high ST3GAL6-AS1 expression level in MM compared to normal plasma cells (PCs) or other hematological malignancies. Transcriptome analyses of MM PCs from patients included in the CoMMpass database indicated a potential involvement of ST3GAL6-AS1 in MAPK signaling and ubiquitin-mediated proteolysis pathways. ST3GAL6-AS1 silencing by LNA-gapmeR antisense oligonucleotides inhibits cell proliferation and triggers apoptosis in MM cell line. Notably, ST3GAL6-AS1 silencing in vitro displayed the down-regulation of the MAPK pathway and protein ubiquitination. These data suggest that ST3GAL6-AS1 deregulation may play a pathogenetic role in MM by affecting both proliferation pathways and circuits fundamental for PC survival. However, ST3GAL6-AS1 expression levels seem not to be significantly associated with clinical outcome and its targeting appears to exert antagonistic effects with proteasome inhibitors used in MM. These findings strongly urge the need for further studies investigating the relevance of ST3GAL6-AS1 in MM.

The microRNA (miRNA) cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce ribonuclease H-mediated degradation of MIR17HG primary transcripts and consequently prevent biogenesis of miR-17-92 miRNAs (miR-17-92s). The leading LNA ASO, MIR17PTi, impaired proliferation of several cancer cell lines (n = 48) established from both solid and hematologic tumors by on-target antisense activity, more effectively as compared with miR-17-92 inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells and induces MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo antitumor activity in nonobese diabetic severe combined immunodeficient mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetic profiles in nonhuman primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies.

Preventive therapy can target hormone responsive breast cancer by treatment with selective estrogen receptor modulators (SERMs) and reduce the incidence of breast cancer. Genome wide association studies have identified single nucleotide polymorphisms (SNPs) with relevant predictive values, SNPs in the ZNF423 gene were associated with decreased risk of breast cancer during SERM therapy, and SNPs in the Cathepsin O gene with an increased risk. ZNF423 which was not previously associated with breast cancer is a multifunctional transcription factor known to have a role in development, neurogenesis and adipogenesis, and is implicated in other types of cancer. ZNF423 is transcriptionally controlled by the homolog ZNF521, EBF transcription factor, epigenetic silencing of the promoter by CpG island hyper-methylation, and also by ZNF423 itself in an auto-regulatory loop. In breast cancer cells, ZNF423 expression is found to be induced by estrogen, dependent on the binding of the estrogen receptor and calmodulin-like 3 to SNPs in ZNP423 intronic sites in proximity to consensus estrogen response elements. ZNF423 has also been shown to play a mechanistic role by trans-activating the tumor suppressor BRCA1 and thus modulating the DNA damage response. Even though recent extensive trial studies did not classify these SNPs with the highest predictive values, for inclusion in polygenic SNP analysis, the mechanism unveiled in these studies has introduced ZNF423 as a factor important in the control of the estrogen response. Here, we aim at providing an overview of ZNF423 expression and functional role in human malignancies, with a specific focus on its implication in hormone-responsive breast cancer.

Chemoprevention is based on the use of non-toxic, pharmacologically active agents to prevent tumor progression. In this regard, natural dietary agents have been described by the most recent literature as promising tools for controlling onset and progression of malignancies. Extensive research has been so far performed to shed light on the effects of natural products on tumor growth and survival, disclosing the most relevant signal transduction pathways targeted by such compounds. Overall, anti-inflammatory, anti-oxidant and cytotoxic effects of dietary agents on tumor cells are supported either by results from epidemiological or animal studies and even by clinical trials. : Multiple myeloma is a hematologic malignancy characterized by abnormal proliferation of bone marrow plasma cells and subsequent hypercalcemia, renal dysfunction, anemia, or bone disease, which remains incurable despite novel emerging therapeutic strategies. Notably, increasing evidence supports the capability of dietary natural compounds to antagonize multiple myeloma growth in preclinical models of the disease, underscoring their potential as candidate anti-cancer agents. : In this review, we aim at summarizing findings on the anti-tumor activity of dietary natural products, focusing on their molecular mechanisms, which include inhibition of oncogenic signal transduction pathways and/or epigenetic modulating effects, along with their potential clinical applications against multiple myeloma and its related bone disease.

The deeper understanding of non-coding RNAs has recently changed the dogma of molecular biology assuming protein-coding genes as unique functional biological effectors, while non-coding genes as junk material of doubtful significance. In the last decade, an exciting boom of experimental research has brought to light the pivotal biological functions of long non-coding RNAs (lncRNAs), representing more than the half of the whole non-coding transcriptome, along with their dysregulation in many diseases, including cancer. In this review, we summarize the emerging insights on lncRNA expression and functional role in cancer, focusing on the evolutionary conserved and abundantly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) that currently represents the best characterized lncRNA. Altogether, literature data indicate aberrant expression and dysregulated activity of MALAT1 in human malignancies and envision MALAT1 targeting as a novel treatment strategy against cancer.

Acute myeloid leukemia (AML), the most common acute leukemia in the adult, is believed to arise as a consequence of multiple molecular events that confer on primitive hematopoietic progenitors unlimited self-renewal potential and cause defective differentiation. A number of genetic aberrations, among which a variety of gene fusions, have been implicated in the development of a transformed phenotype through the generation of dysfunctional molecules that disrupt key regulatory mechanisms controlling survival, proliferation, and differentiation in normal stem and progenitor cells. Such genetic aberrations can be recreated experimentally to a large extent, to render normal hematopoietic stem cells “bad”, analogous to the leukemic stem cells. Here, we wish to provide a brief outline of the complementary experimental approaches, largely based on gene delivery and more recently on gene editing, employed over the last two decades to gain insights into the molecular mechanisms underlying AML development and progression and on the prospects that their applications offer for the discovery and validation of innovative therapies.

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κ B, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.

// Maria Angelica Stamato 1, * , Giada Juli 1, * , Enrica Romeo 1 , Domenica Ronchetti 2, 3 , Mariamena Arbitrio 4 , Daniele Caracciolo 1 , Antonino Neri 2, 3 , Pierosandro Tagliaferri 1 , Pierfrancesco Tassone 1, 5 and Nicola Amodio 1 1 Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Catanzaro, Italy 2 Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy 3 Hematology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy 4 ISN-CNR, Roccelletta di Borgia, Catanzaro, Italy 5 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, US * These authors contributed equally to this work Correspondence to: Pierfrancesco Tassone, email: tassone@unicz.it Nicola Amodio, email: amodio@unicz.it Keywords: miR-29b; microRNA; miRNA; multiple myeloma; EZH2 Received: August 31, 2017 Accepted: October 29, 2017 Published: November 20, 2017 ABSTRACT Downregulation of tumor suppressor (TS) microRNAs (miRNAs) commonly occurs in human cancer, including multiple myeloma (MM). We previously demonstrated that miR-29b is a relevant TS miRNA, whose expression in MM cells is inhibited by HDAC4-dependent deacetylation. Here, we provide novel insights into epigenetic mechanisms suppressing miR-29b in MM. In MM patient-derived plasma cells, we found inverse correlation between miR-29b and EZH2 mRNA expression. Both siRNAs and pharmacologic inhibitors of EZH2 led to miR-29b upregulation, and this effect was ascribed to reduced H3K27-trimethylation (H3K27me3) of miR-29a/b-1 promoter regions. Induction of miR-29b upon EZH2 inhibition occurred together with downregulation of major miR-29b pro-survival targets, such as SP1, MCL-1 and CDK6. Knock-down of the EZH2-interacting long non-coding RNA MALAT1 also reduced H3K27me3 of miR-29a/b-1 promoter, along with induction of miR-29b and downregulation of miR-29b targets. Importantly, inhibition of miR-29b by antagomiRs dramatically reduced in vitro anti-MM activity of small molecule EZH2-inhibitors, indicating that functional miR-29b is crucial for the activity of these compounds. Altogether, these results disclose novel epigenetic alterations contributing to the suppression of miR-29b molecular network, which can be instrumental for the development of rationally designed miRNA-based anti-MM therapeutics.

MiR-34a acts as tumor suppressor microRNA (miRNA) in several cancers, including multiple myeloma (MM), by controlling the expression of target proteins involved in cell cycle, differentiation and apoptosis. Here, we have investigated the combination between miR-34a and γ-secretase inhibitor (γSI), Sirtinol or zoledronic acid (ZOL) in order to enhance the inhibitory action of this miRNA on its canonical targets such as Notch1 and SIRT1, and on Ras/MAPK-dependent pathways. Our data demonstrate that miR-34a synthetic mimics significantly enhance the anti-tumor activity of all the above-mentioned anti-cancer agents in RPMI 8226 MM cells. We found that γSI enhanced miR-34a-dependent anti-tumor effects by activating the extrinsic apoptotic pathway which could overcome the cytoprotective autophagic mechanism. Moreover, the combination between miR-34a and γSI increased the cell surface calreticulin (CRT) expression, that is well known for triggering anti-tumor immunological response. The combination between miR-34a and Sirtinol induced the activation of an intrinsic apoptotic pathway along with increased surface expression of CRT. Regarding ZOL, we found a powerful growth inhibition after enforced miR-34a expression, which was not likely attributable to neither apoptosis nor autophagy modulation. Based on our data, the combination of miR-34a with other anti-cancer agents appears a promising anti-MM strategy deserving further investigation.

One of the major challenges in the epilepsy field is identifying disease-modifying drugs in order to prevent or delay spontaneous recurrent seizure onset or to cure already established epilepsy. It has been recently reported that fingolimod, currently approved for the treatment of relapsing-remitting multiple sclerosis, has demonstrated antiepileptogenic effects in 2 different preclinical models of acquired epilepsy. However, to date, no data exist regarding the role of fingolimod against genetic epilepsy. Therefore, we have addressed this issue by studying the effects of fingolimod in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats, a well-established genetic model of absence epilepsy, epileptogenesis, and neuropsychiatric comorbidity. Our results have demonstrated that an early long-term treatment with fingolimod (1 mg/kg/day), started before absence seizure onset, has both antiepileptogenic and antidepressant-like effects in WAG/Rij rats. However, these effects were transitory, as 5 months after treatment discontinuation, both absence seizure and depressive like-behavior returned to control levels. Furthermore, a temporary reduction of mTOR signaling pathway activity, indicated by reduced phosphorylated mammalian target of rapamycin and phosphorylated p70S6k levels, and by increased phosphorylated Akt in WAG/Rij rats of 6 months of age accompanied the transitory antiepileptogenic effects of fingolimod. Surprisingly, fingolimod has demonstrated longer-lasting positive effects on cognitive decline in this strain. This effect was accompanied by an increased acetylation of lysine 8 of histone H4 (at both 6 and 10 months of age). In conclusion, our results support the antiepileptogenic effects of fingolimod. However, the antiepileptogenic effects were transitory. Moreover, fingolimod might also have a positive impact on animal behavior and particularly in protecting the development of memory decline.

Phoenixin-14 (PNX) is a newly identified peptide co-expressed in the hypothalamus with the anorexic and cardioactive Nesfatin-1. Like Nesfatin-1, PNX is able to cross the blood-brain barrier and this suggests a role in peripheral modulation. Preliminary mass spectrography data indicate that, in addition to the hypothalamus, PNX is present in the mammalian heart. This study aimed to quantify PNX expression in the rat heart, and to evaluate whether the peptide influences the myocardial function under basal condition and in the presence of ischemia/reperfusion (I/R). By ELISA the presence of PNX was detected in both hypothalamus and heart. In plasma of normal, but not of obese rats, the peptide concentrations increased after meal. Exposure of the isolated and Langendorff perfused rat heart to exogenous PNX induces a reduction of contractility and relaxation, without effects on coronary pressure and heart rate. As revealed by immunoblotting, these effects were accompanied by an increase of Erk1/2, Akt and eNOS phosphorylation. PNX (EC50 dose), administered after ischemia, induced post-conditioning-like cardioprotection. This was revealed by a smaller infarct size and a better systolic recovery with respect to those detected on hearts exposed to I/R alone. The peptide also activates the cardioprotective RISK and SAFE cascades and inhibits apoptosis. These effects were also observed in the heart of obese rats. Our data provide a first evidence on the peripheral activity of PNX and on its direct cardiomodulatory and cardioprotective role under both normal conditions and in the presence of metabolic disorders.

Osteosarcoma (OS) is a rare primary malignant bone tumour arising from primitive bone-forming mesenchymal cells, with high incidence in children and young adults, accounting for approximately 60% of all malignant bone tumours. Currently, long-term disease-free survival can be achieved by surgical treatment plus chemotherapy in approximately 60% of patients with localized extremity disease, and in 20-30% of patients with metastatic lung or bone disease. Diagnosis of primary lesions and recurrences is achieved by using radiological investigations and standard tissue biopsy, the latter being costly, painful and hardly repeatable for patients. Therefore, despite some recent advances, novel biomarkers for OS diagnosis, prediction of response to therapy, disease progression and chemoresistance, are urgently needed. Biological fluids such as blood represent a rich source of non-invasive cancer biomarkers, which allow to understand what is really happening inside the tumour, either at diagnosis or during disease progression. In this regard, liquid biopsy potentially represents an alternative and non-invasive method to detect tumour onset, progression and response to therapy. In this review, we will summarize the state of the art in this novel area, illustrating recent studies on OS. Although the data reported in literature seem preliminary, liquid biopsy represents a promising tool with the potential to be rapidly translated in the clinical practice.

Epigenetic abnormalities are common in hematologic malignancies, including multiple myeloma, and their effects can be efficiently counteracted by a class of tumor suppressor miRNAs, named epi-miRNAs. Given the oncogenic role of histone deacetylases (HDAC) in multiple myeloma, we investigated whether their activity could be antagonized by miR-29b, a well-established epi-miRNA. We demonstrated here that miR-29b specifically targets HDAC4 and highlighted that both molecules are involved in a functional loop. In fact, silencing of HDAC4 by shRNAs inhibited multiple myeloma cell survival and migration and triggered apoptosis and autophagy, along with the induction of miR-29b expression by promoter hyperacetylation, leading to the downregulation of prosurvival miR-29b targets (SP1, MCL-1). Moreover, treatment with the pan-HDAC inhibitor SAHA upregulated miR-29b, overcoming the negative control exerted by HDAC4. Importantly, overexpression or inhibition of miR-29b, respectively, potentiated or antagonized SAHA activity on multiple myeloma cells, as also shown in vivo by a strong synergism between miR-29b synthetic mimics and SAHA in a murine xenograft model of human multiple myeloma. Altogether, our results shed light on a novel epigenetic circuitry regulating multiple myeloma cell growth and survival and open new avenues for miR-29b–based epi-therapeutic approaches in the treatment of this malignancy. Mol Cancer Ther; 15(6); 1364–75. ©2016 AACR.

Multiple myeloma (MM) is a hematologic cancer characterized by the accumulation of malignant plasma cells in the bone marrow, which causes bone destruction and marrow failure [1]. Despite the fact that clinical introduction of novel drugs has led to a dramatic improvement of disease, MM ultimately relapses and remains an incurable disease [2]. Consistently, research in this field has dramatically grown, as demonstrated by the increased rate of publications during the last decade. Original research and review articles published in this special issue focus on identification of new targets and pathways for prognostic and therapeutic application in myeloma, as well as validation of novel antitumor agents. (1) Diagnosis, Monitoring, and Prognosis of MM. Extensive gene expression profile analysis has provided interesting insight into the disease biology, and its correlation with clinical outcome has opened a new direction for risk stratification as well as novel target and therapy identification. Prognostic models such as the Durie-Salmon staging system and International Staging System (ISS) are available and account for the disease burden [3]; however, the original analysis of ISS system did not include Chinese patients' data and take into account the recent introduction of novel agents. The data provided in the manuscript by J. Lu et al. represent the first multicenter retrospective study analyzing ISS value in a large number of unselected Chinese myeloma patients. The results demonstrate that ISS still has prognostic value in Chinese patients with MM, but not in patients receiving bortezomib-based therapy. Therefore, further studies are needed to develop more suitable and robust stratification systems to predict prognosis and optimize treatment strategy early during the course of the disease. Minimal residual disease (MRD) has emerged as one of the most relevant prognostic factors in MM and should be included in a new definition of complete response (CR) [4]. In the manuscript by M. Fulciniti et al., the authors reviewed current definition of deep response in MM, advantages and limitations of current MRD assessment assays, and clinical evidences for MRD monitoring as a prognostic tool for therapeutic decisions in MM, providing the rationale for the use of MRD assessment in the evolving MM clinical paradigm. (2) Role of the Bone Marrow Milieu. The bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis. MM cells reside in and dynamically interact with various subsets of cells in the BM including mesenchymal cells (MSCs), osteoclasts (OCLs), osteoblasts (OBLs), and vascular endothelial cells, which support growth and survival of the tumor cells and lead to development of drug resistance [5]. These cells not only physically interact with MM cells but also secrete growth and/or antiapoptotic factors. The cross talk between MM cells with bone marrow stromal cells or other cellular components is finely tuned by a plethora of cytokines, growth-factor, and other molecules which can be released either by MM cells or by cells of the BM microenvironment. Several studies have demonstrated deregulation of the cellular and humoral components of the BM in MM including increased OCL activity and inhibited OBL activity, and both are involved in the pathophysiology of the bone lesions in MM [6]. The review by F. Accardi et al. summarizes the preclinical and clinical evidence on the effects of bortezomib and other new Proteasome Inhibitors (PIs) on myeloma bone disease. Osteoclastic formation and activity are inhibited by PIs, mainly through the blockade of RANKL signaling pathway in the osteoclast progenitors. However, the more significant impact of the bone remodeling by this class of drugs is the capacity to stimulate either the osteogenic differentiation of MSC or the osteoblastic function, leading to the consequent bone formation with a considerable anabolic effect. Osteocytes are also possible targets of PIs with a stimulatory effect on their viability. The preclinical evidence, thus, is confirmed in MM patients treated with bortezomib and more recently with carfilzomib. An improvement of the bone remodeling markers was observed in the patients treated with PIs. The histomorphometric data in MM patients treated with bortezomib prominently indicated that PIs can stimulate the bone formation process and induce the bone regeneration process. Bone healing, as well as an increase in the BMD, has also been reported in some of the patients treated with bortezomib. Overall, the literature data support the use of these drugs to restore bone integrity in MM patients. MM cells are cradled within the BM microenvironment by an array of adhesive interactions between the BM extracellular matrix (ECM) components and a variety of adhesion molecules on the surface of MM cells, which results in the “cell adhesion-mediated drug resistance” (CAM-DR) thought to be one of the major mechanisms by which MM cells escape the cytotoxic effects of therapeutic agents [7]. MM is characterized by continuous spread of cancer cells at different sites of the bone marrow, through continuous cell trafficking including adhesion of myeloma cells to vascular wall. This process requires the presence of P-selectin on the endothelium and stroma, as well as PSGL-1 on tumor cells. PSGL-1 was previously suggested as a novel target for immunotherapy in MM using small molecule inhibitor which demonstrated sensitization of MM cells to therapy, controlling tumor growth and dissemination. However, poor pharmacokinetic profile with a very short half-life may hold up further usage of this drug in MM. The study by B. Muz et al. demonstrates that inhibition of P-selectin/PSGL-1 axis using humanized monoclonal antibodies is a promising approach for the treatment of MM with high efficacy in inhibition of P-selectin/PSGL-1 interactions and sensitization of MM cells to therapy, along with favorable pharmacokinetics. Angiogenesis is fundamental to tumor growth and spread in many hematological disorders, particularly MM [8]. The angiogenic potential of MM is regulated by several pro- and antiangiogenesis cytokines produced by myeloma cells and other cell types in the tumor microenvironment. The study by T. Valkovic et al. determines the plasma levels of monocyte chemotactic protein-1 (MCP-1), as well as its possible association with angiogenesis, in 45 newly diagnosed MM patients and 24 healthy controls. The manuscript reveals a positive association between plasma MCP-1 levels, angiogenesis, and clinical features in patients with MM. However, additional prospective studies with a respectable number of patients should be performed to authenticate these results and establish MCP-1 as a possible target of active treatment. (3) Genetic and Epigenetic Abnormalities. MM cells accumulate various genetic and epigenetic abnormalities that drive the malignant phenotype and confer distinct biologic sequelae and disease outcomes [9]. miRNA are important transcriptome modifiers that play important role in myeloma tumor progression, survival, and development of drug resistance [10]. In their review, Raimondi et al. discuss the role of endogenous noncoding RNAs as microRNAs as a novel class of regulators of the intercellular communication between MM cells and other cells of the BM milieu, focusing on the therapeutic potential of experimental strategies aimed at modulating microRNA levels in MM cells and capable of overcoming the tumor-promoting BM microenvironment. Moreover, S.-F. Lin and W.-C. Yang review molecular mechanisms underlying MM development, progression, and resistance to treatment, further highlighting the relevance of microRNAs as well as immune dysfunctions and conventional or novel therapies targeting such vulnerabilities. Overall, authors advocate that understanding the genomic landscape of MM deserves more attention in the prospect of developing personalized and effective targeted therapies that overcome resistance to currently used anti-MM drugs. In addition to microRNAs, dysregulation of transcription factors (TFs) features prominently in the biology of MM and B cell malignancies. TFs are the downstream effectors of signaling pathways within cells, which receive growth and other signals from the microenvironment; they also regulate cellular homeostasis as well as cell survival and proliferation. On this basis, mutations in TF genes or dysregulation of TFs' expression could play a significant role in cancer pathogenesis and drug resistance. The TF EBF1 is the master regulator of the specification, development, and maintenance of the B-lymphoid lineage, and perturbations of EBF1 expression and/or functions have been associated with the development of B cell malignancies [11]. In this regard, M. Mesuraca et al. have reviewed the role of two zinc finger proteins, namely, ZNF423 and ZNF521, as potent inhibitors of EBF1 and as likely contributing to the development of B cell leukaemia diseases. Genome instability, defined by higher rate of genomic changes acquisition per cell division compared to normal cells, represents a prominent feature of MM cells [12]. M. Cea et al. provide a comprehensive overview of the current knowledge of genomic instability in MM both in terms of its contribution to disease development and progression and in terms of possible relevance as therapeutic target. The paper describes mechanisms by which genetic aberrations give rise to multiple pathogenic events required for myelomagenesis and concludes with a discussion of the clinical applications of these findings in MM patients. Altogether, the data presented in this special issue may contribute to increasing our current knowledge of the biology of MM and B cell malignancies. Mariateresa Fulciniti Nicola Amodio Michele Cea Patricia Maiso Abdel Kareem Azab

// Enrica Calura 1,* , Andrea Bisognin 2,* , Martina Manzoni 3 , Katia Todoerti 4 , Elisa Taiana 3 , Gabriele Sales 1 , Gareth J. Morgan 5 , Giovanni Tonon 6 , Nicola Amodio 7 , Pierfrancesco Tassone 7 , Antonino Neri 3 , Luca Agnelli 3,** , Chiara Romualdi 1,** and Stefania Bortoluzzi 2,** 1 Department of Biology, University of Padua, Padua, Italy 2 Department of Molecular Medicine, University of Padua, Padua, Italy 3 Department of Clinical Sciences and Community Health, University of Milan, and Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy 4 Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy 5 Myeloma Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA 6 Functional Genomics of Cancer Unit, Division of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy 7 Department of Experimental and Clinical Medicine, University of Study Magna Graecia, Catanzaro, Italy * The first two authors equally contributed to this work ** A substantial contribution to the conception, design, execution and writing of this work was equally provided by these three groups. LA, CR, SB have therefore to be considered co-last authors Correspondence to: Luca Agnelli, email: // Keywords : multiple myeloma, transciptional regulatory network, t(4;14) translocation, microRNA, expression profiling Received : August 05, 2015 Accepted : September 30, 2015 Published : October 19, 2015 Abstract The identification of overexpressed miRNAs in multiple myeloma (MM) has progressively added a further level of complexity to MM biology. miRNA and gene expression profiles of two large representative MM datasets, available from retrospective and prospective series and encompassing a total of 249 patients at diagnosis, were analyzed by means of i n silico integrative genomics methods, based on MAGIA 2 and Micrographite computational procedures. We first identified relevant miRNA/transcription factors/target gene regulation circuits in the disease and linked them to biological processes. Members of the miR-99b/let-7e/miR-125a cluster, or of its paralog, upregulated in t(4;14), were connected with the specific transcription factors PBX1 and CEBPA and several target genes. These results were validated in two additional independent plasma cell tumor datasets. Then, we reconstructed a non-redundant miRNA-gene regulatory network in MM, linking miRNAs, such as let-7g , miR-19a , mirR-20a , mir-21 , miR-29 family, miR-34 family, miR-125b , miR-155 , miR-221 to pathways associated with MM subtypes, in particular the ErbB, the Hippo, and the Acute myeloid leukemia associated pathways.

Carcinogenesis is a process generally occurring in tumors characterized by a long latency, in which the accumulation of molecular aberrations leads to transformation and uncontrolled cell growth. Given the existence of pre-malignant conditions including monoclonal gammopathy of undetermined significance, indolent multiple myeloma (IMM) and smoldering myeloma, multiple myeloma (MM) represents the prototype of cancers which could benefit from preventive strategies aimed at hampering the malignant transformation of plasma cells. Here, we will briefly discuss the available findings on potential chemopreventive strategies based on the targeting of the most relevant signaling pathways involved in the onset and development of MM.

Background Circulating tumor cells (CTCs) represent one of the most interesting target in improving diagnosis, prognosis and treatment. Herein we evaluate the possibility of using an emo-cytometric approach on the evaluation of the heterogeneous population of CTCs to improve personalized metastatic risk assessment. We benchmarked ex vivo behavior of distinct subsets of circulating colon tumor cells with correspondent clinical behavior of patients from which we isolated CTCs. Methods Isolation and CTC expansion were performed by a gradient protocol. In vitro characterization was determined by flow cytometry, immunofluorescence, western blotting and proteomic profiling. Cell sorter was performed with immunomagnetic beads. Confocal microscopy was used to evaluate tissue sections. Kaplan Mayer curves was cared for through Medcalc program. Results We collected heterogeneous CTCs, derived from the whole blood of seven patients affected by colon cancer, expressing CD133posCD45neg (5 ± 1) and (2 ± 1) and CK20posCD45neg of (29 ± 3) (11 ± 1) cells/ml in Dukes D and A stage respectively. Proliferation rate of 57 ± 16 %, expression for CXCR4pos of 18 ± 7 % and detectable levels of IL-6, IL-8 and SDF-1 cytokines in conditioned culture medium characterized short-time expanded–CTCs (eCTCs). ECTCs organized in tumor sphere were CD45negCD133pos while in adhesion were CXCR4posCK20pos. These two subsets were separately injected in mice. The first group of xenografts developed superficial lesions within 2 weeks. In the second group, in absence of growing tumour, the survival of injected eCTCs was monitored through SDF-1 serum levels detection. The detection of human cancer cells expressing CK20, in mice tissues sections, suggested a different biological behaviour of injected eCTC-subsets: tumorigenic for the first and disseminating for the second. The benchmarking of the experimental data with the clinical course highlights that patients with prevalence of circulating cancer stem cells (CD45negCD133pos) have a lower overall survival. Conversely, patients with prevalence of circulating differentiated cells (CXCR4posCK20pos) have a low disease-free survival. Conclusion On the basis of the heterogeneous composition and despite the low number of CTCs, it was possible to distinguish two subgroups of CTCs, suggesting a different clinical outcome. CTC-subsets detailing is useful to better define the metastatic–risk personalized score thus improving disease management and reducing patient care cost. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0876-y) contains supplementary material, which is available to authorized users.

Purpose: The onset of drug resistance is a major cause of treatment failure in multiple myeloma. Although increasing evidence is defining the role of miRNAs in mediating drug resistance, their potential activity as drug-sensitizing agents has not yet been investigated in multiple myeloma. Experimental Design: Here we studied the potential utility of miR-221/222 inhibition in sensitizing refractory multiple myeloma cells to melphalan. Results: miR-221/222 expression inversely correlated with melphalan sensitivity of multiple myeloma cells. Inhibition of miR-221/222 overcame melphalan resistance and triggered apoptosis of multiple myeloma cells in vitro, in the presence or absence of human bone marrow (BM) stromal cells. Decreased multiple myeloma cell growth induced by inhibition of miR-221/222 plus melphalan was associated with a marked upregulation of pro-apoptotic BBC3/PUMA protein, a miR-221/222 target, as well as with modulation of drug influx–efflux transporters SLC7A5/LAT1 and the ABC transporter ABCC1/MRP1. Finally, in vivo treatment of SCID/NOD mice bearing human melphalan-refractory multiple myeloma xenografts with systemic locked nucleic acid (LNA) inhibitors of miR-221 (LNA-i-miR-221) plus melphalan overcame drug resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and ABCC1 in tumors retrieved from treated mice. Conclusions: Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan resistance in preclinical models of multiple myeloma, providing the framework for clinical trials to overcome drug resistance, and improve patient outcome in multiple myeloma. Clin Cancer Res; 22(5); 1222–33. ©2015 AACR.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom’s macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3′UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells.