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Demyelination is a common pathologic feature in many neurodegenerative diseases including infection with leprosy-causing Mycobacterium leprae. Because of the long incubation time and highly complex disease pathogenesis, the management of nerve damage in leprosy, as in other demyelinating diseases, is extremely difficult. Therefore, an important challenge in therapeutic interventions is to identify the molecular events that occur in the early phase before the progression of the disease. Here we provide evidence that M. leprae–induced demyelination is a result of direct bacterial ligation to and activation of ErbB2 receptor tyrosine kinase (RTK) signaling without ErbB2-ErbB3 heterodimerization, a previously unknown mechanism that bypasses the neuregulin-ErbB3–mediated ErbB2 phosphorylation. MEK-dependent Erk1 and Erk2 (hereafter referred to as Erk1/2) signaling is identified as a downstream target of M. leprae–induced ErbB2 activation that mediates demyelination. Herceptin (trastuzumab), a therapeutic humanized ErbB2-specific antibody, inhibits M. leprae binding to and activation of ErbB2 and Erk1/2 in human primary Schwann cells, and the blockade of ErbB2 activity by the small molecule dual ErbB1-ErbB2 kinase inhibitor PKI-166 (ref. 11) effectively abrogates M. leprae–induced myelin damage in in vitro and in vivo models. These results may have implications for the design of ErbB2 RTK–based therapies for both leprosy nerve damage and other demyelinating neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Activation of extracellular signal-regulated kinase (Erk) 1/2, which plays a critical role in diverse cellular processes, including cell proliferation, is known to be mediated by the canonical Raf- mitogen-activated protein kinase kinase (MEK) kinase cascade. Alternative MEK-independent signaling pathways for Erk 1/2 activation in mammalian cells are not known. During our studies of human primary Schwann cell response to long-term infection of Mycobacterium leprae, the causative organism of leprosy, we identified that intracellular M. Leprae activated Erk 1/2 directly by lymphoid cell kinase (p56Lck), a Src family member, by means of a PKCe-dependent and MEK-independent signaling pathway. Activation of this signaling induced nuclear accumulation of cyclin D1, G1/S-phase progression, and continuous proliferation, but without transformation. Thus, our data reveal a previously unknown signaling mechanism of glial cell proliferation, which might play a role in dedifferentiation as well as nerve regeneration and degeneration. Our findings may also provide a potential mechanism by which an obligate intracellular bacterial pathogen like M. Leprae subverts nervous system signaling to propagate its cellular niche for colonization and long-term bacterial survival. [ABSTRACT FROM AUTHOR]

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DEAD-box helicase 4 (DDX4), a germline factor and RNA helicase conserved in all multicellular organisms, contributes to increased cell motility and cisplatin-mediated drug resistance in small cell lung cancer (SCLC) cells. Proteomic analysis suggests that DDX4 expression upregulates proteins related to DNA repair and immune/inflammatory response. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Further, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of immune/inflammatory response markers. Taken together, we propose that DDX4 increases SCLC cell survival, by increasing the DNA damage and immune response pathways, especially under challenging conditions such as cisplatin treatment. DDX4, a conserved germline factor and RNA helicase, increases small cell lung cancer cell survival by regulating DNA damage and immune response pathways and contributes to cisplatin-mediated drug resistance. [ABSTRACT FROM AUTHOR]

R3259 --> 686.24 --> Background: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. GBM has an ominous prognosis with a survival rate of 14‐15 months after diagnosis and despite worldwide initiatives to optimize therapeutic approaches, GBM is still among the most challenging diseases to treat and the fastest to relapse in clinical oncology. The inevitable treatment resistance and relapse are mainly attributed to the presence of glioma stem cells (GSCs), which have the ability to self‐renew, indefinitely proliferate and to differentiate into different cell types. We hypothesize that HDAC7, as an epigenetic regulator, is playing a crucial mechanistic role in GBM and constitutes a novel druggable target for this universally fatal disease. By uncovering the HDAC7 interactome in the GSC nucleus, we provide mechanistic insights into the role of HDAC7 in regulating cancer stemness and possible unprecedented targeting strategies. Methodology: We quantified the HDAC7 expression in tissue samples from 80 GBM patients admitted at Rhode Island Hospital using Custom Nanostring Panels and correlated the HDAC7 expression to overall patient survival. Also, we compared HDAC7 mRNA expression in GBM to the non‐tumor samples from TCGA data using GlioVis webtool. Next, we performed siRNA knock‐down (KD) of HDAC7 in GSCs followed by RNA‐seq and transcriptomic, gene enrichment and stemness enrichment analysis. We used mass spectrometry (Mod Spec) in si‐HDAC7 KD versus Control to quantify the potential enzymatic de‐acetylation properties of HDAC7 on the global post‐translational modifications of histones. Finally, to unravel the protein partners for HDAC7 in the GSC nucleus, we performed Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME). Results: Survival analysis of GBM patients showed significant decrease in survival for patients with high HDAC7 mRNA expression. In addition, GlioVis webtool revealed that HDAC7 expression is significantly higher in GBM tumors relative to non‐tumor samples. Differential gene expression analysis of RNASeq performed on HDAC7 siRNA KD GSCs versus control, revealed 4963 differentially expressed genes with the Gene Ontology enrichment showing significant enrichment for cell cycle and cell division suppression in HDAC7 KD GSCs. Additionally, 653 genes that were downregulated in HDAC7 KD GSCS were found to play significant role in stemness processes of embryonic stem cells and embryonal carcinoma stem cells. Mass spectrometry on GSCs following HDAC7 siRNA KD, revealed non‐significant de‐acetylation changes on global histone marks suggesting that HDAC7 has minimal or no histone deacetylation activity. Finally, RIME revealed novel protein partners in the proximity of 2.5 A° to HDAC7 in the GSCs nucleus. The newly discovered protein complex of HDAC7 suggests that HDAC7 regulates heterochromatin formation and spreading, which mechanistically control transcriptional programs of GSCs. Conclusion: Our data suggest that HDAC7 plays a pivotal role in GBM. It regulates expression of transcripts that define cancer stemness, while inhibition of HDAC7 results in global increase in heterochromatin and transcriptional repression in GSCs. This study supports HDAC7 as a novel druggable target for GBM in a preclinical setting. [ABSTRACT FROM AUTHOR]

Single-Cell Analysis is a growing field that endeavors to obtain genetic profiles of individual cells. Disruption of cell–cell junctions and digestion of extracellular matrix in tissues requires tissue-specific mechanical and chemical dissociation protocols. Here, a new approach for dissociating tissues into constituent cells is described. Placing a tissue biopsy core within a liquid-filled cavity and applying an electric field between two parallel plate electrodes facilitates rapid dissociation of complex tissues into single cells. Different solution compositions, electric field strengths, and oscillation frequencies are investigated experimentally and with COMSOL Multiphysics. The method is compared with standard chemical and mechanical approaches for tissue dissociation. Treatment of tissue samples at 100 V/cm 1 kHz facilitated dissociation of 95 ± 4% of biopsy tissue sections in as little as 5 min, threefold faster than conventional chemical–mechanical techniques. The approach affords good dissociation of tissues into single cells while preserving cell viability, morphology, and cell cycle progression, suggesting utility for sample preparation of tissue specimens for direct Single-Cell Analysis. [ABSTRACT FROM AUTHOR]

Injury response in the peripheral nervous system (PNS) is characterized by rapid alterations in the genetic program of Schwann cells. However, the epigenetic mechanisms modulating these changes remain elusive. Here we show that sciatic nerve injury in mice induces a cohort of 22 miRNAs, which coordinate Schwann cell differentiation and dedifferentiation through a combinatorial modulation of their positive and negative gene regulators. These miRNAs and their targeted mRNAs form functional complexes with the Argonaute-2 protein to mediate post-transcriptional gene silencing. MiR-138 and miR-709 show the highest affinity amongst the cohort, for binding and regulation of Egr2, Sox-2 and c-Jun expression following injury. Moreover, miR-709 participates in the formation of epigenetic silencing complexes with H3K27me3 and Argonaute-1 to induce transcriptional gene silencing of the Egr2 promoter. Collectively, we identified a discrete cohort of miRNAs as the central epigenetic regulators of the transition between differentiation and dedifferentiation during the acute phase of PNS injury. [ABSTRACT FROM AUTHOR]

ONC201/TIC10 activates TRAIL signaling through ATF4 and the integrated stress response (ISR). ONC201 demonstrated tumor regressions and disease stability in patients with histone H3K27M-mutated midline-glioma. H3K27M-mutation prevents H3K27-methylation on the mutated allele. EZH2 inhibitors (EZH2i) reduce H3K27 methylation and have anti-tumor effects. We hypothesized ONC201 sensitivity and tumor apoptosis may increase by reducing H3K27-methylation with EZH2i or HDACi as mimics of H3K27M-mutation. EZH2i EPZ-6438 (tazemetostat) or PF-06821497 and HDACi vorinostat were combined with ONC201 to treat multiple cancer cell lines and cell viability and histone modifications were analyzed. We observed synergistic effects towards cell viability in multiple cancers by EPZ-6438 or PF-06821497 plus ONC201 or triple therapy with vorinostat, EPZ-6438, and ONC201. EPZ-6438 and vorinostat synergized with ONC201 to enhance apoptosis. Activation of the ISR and TRAIL-DR5 were observed in cells treated with ONC201 -/+ epigenetic modulators. Knockdown of ATF4 reduced DR5 induction and apoptosis following EZH2i and ONC201 treatment of U251 glioma cells. mRNA expression of dopamine-receptors did not correlate with ONC201 sensitivity in the tumor cell lines tested (N = 12), including changes after epigenetic drugs. Dopamine did not rescue apoptosis by ONC201 in different tumor cell lines (N = 10) including 2 GBM, 3 DIPG and did not prevent DR5 activation or apoptosis. DRD2 agonist sumanirole did not protect brain tumor cells (N = 6 including 4 DIPG cell lines) from ONC201 reduction in viability. Although synergy was observed with ONC201 and vorinostat, there was no significant increase in H3K27 acetylation in cell lines including DIPG as compared to vorinostat alone, and in some cases the acetylation was less than vorinostat alone at 72 H. H3K27 methylation reduction correlated with synergy from combinations of either EPZ-6438 or vorinostat with ONC201 or triple combination. Our findings provide a rationale for combination of ONC201 and epigenetic modulators including triple therapy for in vivo and clinical testing in treatment of human malignancies including brain tumors and DIPG. [ABSTRACT FROM AUTHOR]

Demyelination results in severe disability in many neurodegenerative diseases and nervous system infections, and it is typically mediated by inflammatory responses. Mycobacterium leprae, the causative organism of leprosy, induced rapid demyelination by a contact-dependent mechanism in the absence of immune cells in an in vitro nerve tissue culture model and in Rag1-knockout (Rag1[sup -/-]) mice, which lack mature B and T lymphocytes. Myelinated Schwann cells were resistant to M. leprae invasion but undergo demyelination upon bacterial attachment, whereas nonmyelinated Schwann cells harbor intracellular M. leprae in large numbers. During M. leprae-induced demyelination, Schwann cells proliferate significantly both in vitro and in vivo and generate a more nonmyelinated phenotype, thereby securing the intracellular niche for M. leprae. [ABSTRACT FROM AUTHOR]

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition. Author summary: Cellular plasticity and epigenetic adaptation of human glioblastoma stem cells to the tumor microenvironment is a hallmark of this devastating disease. With our present work, we discover the relationship between miRNAs and the RNA methylation machinery in human glioblastoma and show how miRNA-induced loss of m6A results in increase in protein translation of clinically important transcripts during glioblastoma stem cell differentiation. Leveraging the dynamic functions of these miRNAs can be important in the design of optimal therapeutics targeted at cancer cell plasticity. [ABSTRACT FROM AUTHOR]

Study Design: A preclinical animal model of chronic ligation of the sciatic nerve was used to compare the effectiveness of a slow-release hydrogel carrying methylprednisolone to methylprednisolone injection alone, which simulates the current standard of care for chronic compressive radiculopathy (CR).Objective: To extend the short-term benefits of steroid injections by using a nonswelling, biodegradable hydrogel as carrier to locally release methylprednisolone in a regulated and sustained way at the site of nerve compression.Summary Of Background Data: CR affects millions worldwide annually, and is a cause of costly disability with significant societal impact. Currently, a leading nonsurgical therapy involves epidural injection of steroids to temporarily alleviate the pain associated with CR. However, an effective way to extend the short-term effect of steroid treatment to address the chronic component of CR does not exist.Methods: We induced chronic compression injury of the sciatic nerves of rats by permanent ligation. Forty-eight hours later we injected our methylprednisolone infused hydrogel and assessed the effectiveness of our treatment for 4 weeks. We quantified mechanical hyperalgesia using a Dynamic Plantar Aesthesiometer (Ugo Basile, Stoelting Co., IL, USA), whereas gait analysis was conducted using the Catwalk automated gait analysis platform (Noldus, Leesburg, VA, USA). Macrophage staining was performed with immunohistochemistry and quantification of monocyte chemoattractant protein-1 in sciatic nerve lysates was performed with multiplex immunoassay using a SECTOR Imager 2400A (Meso Scale Discovery, Rockville, MA, USA).Results: We demonstrate that using the hydrogel to deliver methylprednisolone results in significant (P < 0.05) reduction of hyperalgesia and improvement in the gait pattern of animals with chronic lesions as compared with animals treated with steroid alone. In addition, animals treated with hydrogel plus steroid showed significant reduction in the number of infiltrating macrophages at the sciatic nerve and reduced expression of the neuroinflammatory chemokine monocyte chemoattractant protein-1 (P < 0.05).Conclusion: Use of hydrogels as carriers for sustained local release of steroids provides significantly better control of pain in an animal model of chronic CR. Our steroid-infused hydrogel could be an effective extender of the short-term benefits of epidural steroid injections for patients with chronic compression-induced radicular pain.Level Of Evidence: N/A. [ABSTRACT FROM AUTHOR]

Short interfering ribonucleic acids (siRNAs) are important agents for RNA interference (RNAi) that have proven useful in gene function studies and therapeutic applications. However, the efficacy of exogenous siRNAs for gene knockdown remains hampered by their susceptibility to cellular nucleases and impermeability to cell membranes. We report here new covalent polymer-escort siRNA constructs that address both of these constraints simultaneously. By simple postsynthetic click conjugation of polymers to the passenger strand of an siRNA duplex followed by annealing with the complementary guide strand, we obtained siRNA in which one strand includes terminal polymer escorts. The polymer escorts both confer protection against nucleases and facilitate cellular internalization of the siRNA. These autotransfecting polymer-escort siRNAs are viable in RNAi and effective in knocking down reporter and endogenous genes. [ABSTRACT FROM AUTHOR]

The article presents a study on key regulators of Glioblastoma aggressiveness and progression. Topics include information on the importance of lipid signaling and phospholipases (PLCs) in the regulation of different mechanisms in the central nervous system; information on pathogenesis of human glioblastoma; and how Glioblastoma is the most aggressive and lethal brain tumor in adults.

Reciprocal interactions between glia and neurons are essential for the proper organization and function of the nervous system. Recently, the interaction between ErbB receptors (ErbB2 and ErbB3) on the surface of Schwann cells and neuronal Neuregulin-1 (NRG1) has emerged as the pivotal signal that controls Schwann cell development, association with axons, and myelination. To understand the function of NRG1-ErbB2/3 signaling axis in adult Schwann cell biology, we are studying the specific role of ErbB3 receptor tyrosine kinase (RTK) since it is the receptor for NRG1 on the surface of Schwann cells. Here, we show that alternative transcription initiation results in the formation of a nuclear variant of ErbB3 (nuc-ErbB3) in rat primary Schwann cells. nuc-ErbB3 possesses a functional nuclear localization signal sequence and binds to chromatin. Using chromatin immunoprecipitation (ChIP)- chip arrays, we identified the promoters that associate with nuc-ErbB3 and clustered the active promoters in Schwann cell gene expression. nuc-ErbB3 regulates the transcriptional activity of ezrin and HMGB1 promoters, whereas inhibition of nuc-ErbB3 expression results in reduced myelination and altered distribution of ezrin in the nodes of Ranvier. Finally, we reveal that NRG1 regulates the translation of nuc-ErbB3 in rat Schwann cells. For the first time, to our knowledge, we show that alternative transcription initiation from a gene that encodes a RTK is capable to generate a protein variant of the receptor with a distinct role in molecular and cellular regulation. We propose a new concept for the molecular regulation of myelination through the expression and distinct role of nuc-ErbB3. [ABSTRACT FROM AUTHOR]

R4856 --> The H3K27M oncohistone mutation has been identified in approximately 80% of diffuse intrinsic pontine gliomas (DIPG), and now represents a potential target for urgently needed new therapies. ONC201, a first‐in‐class imipridone therapy, is known to selectively induce apoptosis of cancer cells independent of p53, and has shown efficacy against H3K27M mutant DIPG. We sought to identify synergy between ONC201 or second generation imipridones (ONC206 and ONC212), and other chemotherapeutics with known preclinical activity against DIPG. We hypothesized that second generation imipridones would demonstrate superior activity to ONC201, and that the combination of imipridones and other chemotherapeutics may be superior to single agent treatment in H3K27M mutant DIPG. Seven patient derived DIPG cell lines (SU‐DIPG‐IV, SU‐DIPG‐13, SU‐DIPG‐25, SUDIPG‐27, SU‐DIPG‐29, SU‐DIPG‐36, SF8628) were grown in culture and exposed to imipridones as a monotherapy and in combination with other FDA approved chemotherapeutics (histone de‐acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide). Cell viability was measured by CellTiter‐Glo. Dose dependent response to imipridone therapy was noted in all cell lines with half maximal inhibitory concentration (IC50) of 1.46 µM, 0.11 µM, and 0.03 µM, in ONC201, ONC206, and ONC212 respectively. Synergy was identified via combination analyses, with combination indices of <1 indicating some degree of synergy, and <0.5 indicating very strong synergy. Strong synergy was identified between ONC201 and HDACi Panobinostat (CI 0.01), Romidepsin (CI 0.08) and protease inhibitor Marizomib (CI 0.19). Lesser synergy was demonstrated between ONC201 and Entinostat (CI 0.71) or Etoposide (CI 0.54). Importantly, temozolomide and ONC201 showed a lack of combinational efficacy against H3K27M mutant DIPG, consistent with the literature to date. Second generation imipridones showed similar synergistic effects with Panobinostat, Romidepsin, and Marizomib. Additionally, induction of apoptosis as measured by the induction of poly (ADP‐ribose) polymerase (PARP) cleavage via immunoblotting, was demonstrated with a combination of imipridones and Panobinostat. These results suggest increased sensitivity of H3K27M mutant DIPG cell lines to second generation imipridone therapies, as compared to ONC201. Additionally, there is evidence of promising synergy between imipridones and Panobinostat, Romidepsin, or Marizomib, which should be taken into consideration for future clinical trials. [ABSTRACT FROM AUTHOR]

Nanomedicines have shown great potential in cancer therapy; in particular, the combination of chemotherapy and immunotherapy (namely chemoimmunotherapy) that is revolutionizing cancer treatment. Currently, most nanomedicines for chemoimmunotherapy are still in preclinical and clinical trials. Lipid-based nanoparticles, the most widely used nanomedicine platform in cancer therapy, is a promising delivery platform for chemoimmunotherapy. In this review, we introduce the commonly used immunotherapy agents and discuss the opportunities for chemoimmunotherapy mediated by lipid-based nanoparticles. We summarize the clinical trials involving lipid-based nanoparticles for chemoimmunotherapy. We also highlight different chemoimmunotherapy strategies based on lipid-based nanoparticles such as liposomes, nanodiscs, and lipid-based hybrid nanoparticles in preclinical research. Finally, we discuss the challenges that have hindered the clinical translation of lipid-based nanoparticles for chemoimmunotherapy, and their future perspectives. [ABSTRACT FROM AUTHOR]