BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT

A recent approach for limiting production of pro-inflammatory cytokines has been to target bromodomain and extra-terminal (BET) proteins. These epigenetic readers of histone acetylation regulate transcription of genes involved in inflammation, cardiovascular disease, and cancer. Development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential. Because inflammatory signals and donor T cells promote graft-versus-host disease (GVHD), regulating both pathways could be effective to abrogate this disorder. The objective of the present study was to identify a BETi which did not interfere in vivo with CD4+FoxP3+ regulatory T cell (Treg) expansion and function to utilize together with Tregs following allogeneic hematopoietic stem cell transplantation (aHSCT) to ameliorate GVHD. We have reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively. Here, mice were treated over 7 days (TL1A-Ig + IL-2) together with BETi. We found that the BETi EP11313 did not decrease frequency/numbers or phenotype of expanded Tregs as well as effector molecules, such as IL-10 and TGF-β. However, BETi JQ1 interfered with Treg expansion and altered subset distribution and phenotype. Notably, in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 RNA levels. Remarkably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. MHC-mismatched aHSCT (B6 → BALB/c) was performed using in vivo expanded donor Tregs with or without EP11313 short-term treatment in the recipient. Early post-transplant, improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in aHSCT recipients treated with expanded Tregs + EP11313 was detected. Interestingly, this group exhibited a significant diminution of GVHD clinical score with less skin and ocular involvement. Finally, using low numbers of highly purified expanded Tregs, improved clinical GVHD scores were observed in EP11313 treated recipients. In total, we conclude that use of this novel combinatorial strategy can suppress pre-clinical GVHD and posit, in vivo EP11313 treatment might be useful combined with Treg expansion therapy for treatment of diseases involving inflammatory responses

A Novel RNA Transcript with Antiapoptotic Function Is Silenced in Fragile X Syndrome

Several genome-wide transcriptomics efforts have shown that a large percentage of the mammalian genome is transcribed into RNAs, however, only a small percentage (1–2%) of these RNAs is translated into proteins. Currently there is an intense interest in characterizing the function of the different classes of noncoding RNAs and their relevance to human disease. Using genomic approaches we discovered FMR4, a primate-specific noncoding RNA transcript (2.4 kb) that resides upstream and likely shares a bidirectional promoter with FMR1. FMR4 is a product of RNA polymerase II and has a similar half-life to FMR1. The CGG expansion in the 5′ UTR of FMR1 appears to affect transcription in both directions as we found FMR4, similar to FMR1, to be silenced in fragile X patients and up-regulated in premutation carriers. Knockdown of FMR4 by several siRNAs did not affect FMR1 expression, nor vice versa, suggesting that FMR4 is not a direct regulatory transcript for FMR1. However, FMR4 markedly affected human cell proliferation in vitro; siRNAs knockdown of FMR4 resulted in alterations in the cell cycle and increased apoptosis, while the overexpression of FMR4 caused an increase in cell proliferation. Collectively, our results demonstrate an antiapoptotic function of FMR4 and provide evidence that a well-studied genomic locus can show unexpected functional complexity. It cannot be excluded that altered FMR4 expression might contribute to aspects of the clinical presentation of fragile X syndrome and/or related disorders

Pathogenesis and Treatment of Pancreatic Cancer Related Pain

Pancreatic cancer is often diagnosed due to the patient seeking medical attention for abdominal pain. It is among the most painful cancers, with pain severity strongly correlating with prognosis. Perineural invasion is a prominent feature of pancreatic cancer and often the first route of metastasis resulting in neuropathic pain. While surgical pain is present, it is generally short-lived; chemo- and radio-therapy associated side effect pain is often longer lasting and more difficult to manage. Treatment-induced mucositis in response to chemotherapy occurs throughout the GI tract resulting in infection-prone ulcers on the lip, buccal mucosa, palate or tongue. Cisplatin treatment is associated with axonal neuropathy in the dorsal root ganglion, although other large sensory fibers can be affected. Opioid-induced hyperalgesia can also emerge in patients. Along with traditional means to address pain, neurolytic celiac plexus block of afferent nociceptive fibers has been reported to be effective in 74% of patients. Moreover, as cancer treatments become more effective and result in improved survival, treatment-related side effects become more prevalent. Here, pancreatic cancer and treatment associated pain are reviewed along with current treatment strategies. Potential future therapeutic strategies to target the pathophysiology underlying pancreatic cancer and pain induction are also presented

Functional Rescue of Misfolded Receptor Mutants

Mutant gonadotropin-releasing hormone (GnRH) receptors isolated from patients with GnRH-resistant hypogonadotropic hypogonadism are frequently proteins that are misrouted in the cell. Such mutant receptors are retained in the endoplasmic reticulum and can be rescued by pharmacological chaperones. This understanding contrasts with the view that these mutant receptors lose the ability to bind ligand or effect signal transduction. Pharmacological chaperones, or “pharmacoperones,” bind specifically to GnRH receptors and allow them to escape retention by the cellular quality control systems and route to the plasma membrane, where they function normally. This observation suggests that pharmacoperones have the potential to be used to treat a number of human diseases characterized by misrouted proteins, among these, hypogonadotropic hypogonadism, cystic fibrosis and nephrogenic diabetes insipidus

Naturally occurring compounds as pancreatic cancer therapeutics

Naturally occurring small molecule compounds have long been in the spotlight of pancreatic cancer research as potential therapeutics to prevent cancer progression and sensitize chemoresistant tumors. The hope is that terminal pancreatic cancer patients receiving aggressive chemotherapy can benefit from an increase in treatment efficacy without adding further toxicity by way of utilizing natural compounds. While preclinical studies on a number of natural compounds, such as resveratrol, curcumin, rapalogs and cannabinoids, show promising preclinical results, little has translated into clinical practice, though a number of other compounds hold clinical potential. Nevertheless, recent advances in compound formulation may increase the clinical utility of these compounds

Calnexin regulated gonadotropin-releasing hormone receptor plasma membrane expression

A significant proportion of human gonadotropin-releasing hormone receptors (GnRHRs) are normally retained in the endoplasmic reticulum (ER); however, nearly all rat GnRHRs are routed to the plasma membrane. When mutations are introduced into either receptor, considerably more of the proteins are recognized by the quality control system (QCS) as misfolded and retained compared with wild-type (WT) receptor, resulting in decreased signaling in the presence of agonist. Calnexin, a component of the QCS, decreased plasma membrane expression of the GnRHRs, an effect that was mediated by a physical interaction between the receptor and the calnexin. Only the human receptor showed reduced signaling because it had fewer spare receptors compared with the rat GnRHR, allowing calnexin to affect signaling. Calnexin did not affect receptor signaling when K(191) was deleted from the human WT GnRHR. Removal of this amino acid decreases receptor misfolding and increases plasma membrane expression. K(191) is not present in the rat WT GnRHR. A pharmacological chaperone that corrects GnRHR misfolding, increased expression of the human WT GnRHR in the presence of calnexin. Calnexin apparently retains misfolded GnRHRs but routes correctly folded receptors to the plasma membrane. Mutation of a calnexin protein kinase C consensus phosphorylation site promoted increased retention of the human GnRHR, suggesting that calnexin phosphorylation controls the retention mechanism. We conclude that a proportion of the human and the rat WT GnRHR appears to be retained in the ER by calnexin, an effect that decreases GnRHR signaling capacity

Unexpected effects of epitope and chimeric tags on gonadotropin-releasing hormone receptors: implications for understanding the molecular etiology of hypogonadotropic hypogonadism

In the case of human GnRH receptor (GnRHR) mutants associated with hypogonadotropic hypogonadism, a view emerged that these mutants are correctly routed to the plasma membrane. This view, supported almost entirely by studies using the HA-tag (hemagglutinin influenza virus epitope tag) and other epitope and chimeric tags, obscured recognition that GnRHR mutants frequently become misrouted proteins. The underlying assumption in epitope and chimeric tagging studies is that the cell does not distinguish tagged from unmodified proteins. It should not have been surprising, in retrospect, to find that even a single amino acid mutation dramatically alters protein function or routing because increased plasma membrane expression is associated with deletion of a single amino acid in the human GnRHR (K191), and point mutations have been shown to block plasma membrane routing of many receptors, including most of those responsible for the hypogonadotropic hypogonadism phenotype. Our present observations suggest that epitope and chimeric tags do have a significant effect on protein localization and function. Although rarely provided, control experiments addressing the effects of epitope or chimeric tagging are an essential part of any study relying on these proteomic tools

A Gα s mutation (D 229S) differentially effects gonadotropin-releasing hormone receptor regulation by RGS10, RGS3 and RGS3T

Regulators of G protein signaling (RGS) act as GTPase-activating proteins for Gα i and for Gα q/11. There is recent evidence for interaction of RGS proteins with Gα s, and substitution of Ser for Asp 229 in RGS proteins enhances interactions with G proteins. Site-directed mutagenesis of Asp 229 was used to assess the effect of this site on the gonadotropin-releasing hormone receptor (GnRHR)-Gα s mediated signaling in the absence or presence of over-expressed RGS3, RGS10 or a truncated form of RGS3 (RGS3T). We observed increased cAMP release with the mutant Gα s(D 229S) compared to wt Gα s when GGH 3 cells (GH 3 cells stably expressing the GnRH receptor) were stimulated with a GnRH agonist. In the presence of RGS3, we did not observe any difference in cAMP release with wt Gα s or with Gα s(D 229S) compared to control values; in the presence of RGS3T there was an increase of cAMP release with wt Gα s compared to the control but there was no difference between the Gα s(D 229S) and the control values. When cells co-expressed wt Gα s and RGS10, there was a significant increase of cAMP release compared with cells co-expressing wt Gα s and Lac Z. Cells co-expressing Gα s(D 229S) and RGS10 showed a significant increase of cAMP release compared to control cells. These results indicate differential regulation of the GnRHR-Gα s mediated signaling by a single mutation in Gα s in the presence of RGS10 and RGS3T, but not with RGS3. This is the first report of an effect of the Gα s(D 229S) mutation on G protein-coupled receptor-mediated activation

Emerging treatment strategies for glioblastoma multiforme

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor with a more than 90% 5-year mortality. GBM has a paltry median survival of 12.6 months attributed to the unique treatment limitations such as the high average age of onset, tumor location, and poor current understandings of the tumor pathophysiology. The resection techniques, chemotherapic strategies, and radiation therapy currently used to treat GBM have slowly evolved, but the improvements have not translated to marked increases in patient survival. Here, we will discuss the recent progress in our understanding of GBM pathophysiology, and the diagnostic techniques and treatment options. The discussion will include biomarkers, tumor imaging, novel therapies such as monoclonal antibodies and small-molecule inhibitors, and the heterogeneity resulting from the GBM cancer stem cell population

mRNA-to-protein translation in hypoxia

Abstract Cells respond to hypoxia by shifting cellular processes from general housekeeping functions to activating specialized hypoxia-response pathways. Oxygen plays an important role in generating ATP to maintain a productive rate of protein synthesis in normoxia. In hypoxia, the rate of the canonical protein synthesis pathway is significantly slowed and impaired due to limited ATP availability, necessitating an alternative mechanism to mediate protein synthesis and facilitate adaptation. Hypoxia adaptation is largely mediated by hypoxia-inducible factors (HIFs). While HIFs are well known for their transcriptional functions, they also play imperative roles in translation to mediate hypoxic protein synthesis. Such adaptations to hypoxia are often hyperactive in solid tumors, contributing to the expression of cancer hallmarks, including treatment resistance. The current literature on protein synthesis in hypoxia is reviewed here, inclusive of hypoxia-specific mRNA selection to translation termination. Current HIF targeting therapies are also discussed as are the opportunities involved with targeting hypoxia specific protein synthesis pathways