ИНГИБИРОВАНИЕ ЭКСПРЕССИИ ГЕНА REDD1 ДЛЯ СНИЖЕНИЯ ПОБОЧНЫХ ЭФФЕКТОВ ГЛЮКОКОРТИКОИДОВ

Glucocorticoids (GC ) have been an integral component of the treatment of leukemias and lymphomas for several decades. Specific cytotoxic effect of GC on transformed lymphoblasts mediates their use at the stage of the remission induction as well as consolidation of treatment. However, the main problem of the long-term GC use is the development of atrophic and metabolic side effects as well as GC resistance. The biological effects of GC are realized via activation of the glucocorticoid receptor (GR) by two mechanisms: transrepression (TR) associated with the therapeutic effects of GC , and transactivation (TA ), which mediates the development of metabolic and atrophic complications. It was demonstrated that an increase in the expression of the GC - dependent gene REDD1 associated with GC -induced skin, muscle and bone atrophy of the skin, muscle and bone tissue was realized via the induction of transactivation. Therefore, identification of potential inhibitors of REDD1 expression and study of their biological effects in combination with GC in models of leukemia and lymphoma is of particular interest. In our recent study we have selected a number of drugs from the class of PI 3K/Akt/mTO R modulators using bioinformatic screening. These drugs effectively inhibited REDD1 expression, modulated GR activity and shifted it towards transrepression, and prevented the development of GC -induced side effects in mice. Here we aimed to study the effects of potential inhibitors of REDD1 expression from different pharmacological groups, the compounds Emetine and CGP -60474, on leukemia and lymphoma cells in combination with GC . We demonstrated antitumor effect of the compounds in vitro, a decrease in the expression of TA -associated genes and an increase in TR induction. Further studies of the antitumor effects of REDD1 expression inhibitors (Emetine and CGP -60474 is a promising area of research.Глюкокортикоиды (GC ) являются неотъемлемым компонентом терапии лейкозов и лимфом на протяжении нескольких десятков лет. Их специфическое цитотоксическое действие на трансформированные лимфобласты обусловливает применение данных препаратов как при индукции ремиссии, так и в ходе дальнейшего лечения. Однако одной из проблем, осложняющих длительное применение GC , является развитие атрофических и метаболических побочных эффектов, а также резистентности. Биологические эффекты GC реализуются посредством активации глюкокортикоидного рецептора (GR) по двум механизмам: трансрепрессии (TR), обусловливающей терапевтическое действие GC , и трансактивации (TA ), опосредующей развитие побочных эффектов. В частности, с индукцией трансактивации связано увеличение экспрессии GC -зависимого гена REDD1, ассоциированного с GC -индуцированной атрофией кожного покрова, мышечной и костной ткани. В связи с этим актуальным является поиск потенциальных ингибиторов экспрессии REDD1 и изучение их эффектов в комбинации с GC на моделях лейкозов и лимфом. Ранее нами с помощью биоинформатического анализа был отобран ряд препаратов класса модуляторов сигнального пути PI 3K/Akt/mTO R. Данные лекарственные средства оказались эффективными ингибиторами экспрессии гена REDD1, модулировали активность GR, усиливая трансрепрессию, а также предотвращали развитие GC -индуцированных побочных эффектов у мышей. В представленной работе изучены эффекты потенциальных ингибиторов экспрессии REDD1, соединений других фармакологических групп, эметина и CGP -60474 на клетки лейкозов и лимфом совместно с GC . Было отмечено противоопухолевое действие соединений in vitro, снижение экспрессии генов, ассоциированных с TA , и усиление TR. В связи с этим дальнейшее изучение противоопухолевых эффектов ингибиторов экспрессии REDD1 эметина и CGP -60474 является перспективным направлением исследований

P85

Glucocorticoids (GCs) are the most important component of therapy for a number of diseases including combined chemotherapy of hematological malignancies. However, their application is strongly restricted by the development of serious side effects as well as glucocorticoid resistance. Both side effects and resistance affect child cancer patients more intensively. Adverse side-effects of glucocorticoid treatment are the result of glucocorticoid receptor (GR)-mediated gene activation, while the beneficial anti-inflammatory effects result from GR-mediated ‘transrepression’. Difference in mechanisms of therapeutic and side effects of GCs became obvious and selective glucocorticoid agonists (SEGRA) were developed aiming to separate transactivation from transrepression. Recently we have demonstrated that one of the modern SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride or CpdA, selectively induces transrepression in lymphoma cells and reveals GR-dependent anti-lymphoma activity in vitro and in vivo. However, CpdA is a chiral molecule and exists as a racemic mixture of two enantiomers. Enantiomers often possess the same physical and chemical properties but their biological effects may differ drastically. They differently interact with cell receptors and it can lead to the essential diversity in pharmacokinetics and pharmacodynamics. The notorious example of such a molecule is Thalidomide which was originally designed and used as a sedative drug during the pregnancy, but it was withdrawn chiefly because of its severe teratogenicity. As it was demonstrated, only (R)-thalidomide exhibited significant sedative effects, while (S)-thalidomide revealed the teratogenic effects. The application of enantiopure compounds may lead to reduction of metabolism variability in patients and to decrease of drug effective dose. Thereby, the synthesis of enantiopure isomers of CpdA and the study of their anti-cancer activity are of immediate interest in cancer research. We synthesized for the first time enantiomers of the CpdA based on literature precedent Sharpless asymmetric dihydroxylation with AD-mix-alpha or AD-mix-beta. (S)- and (R)-enantiomers with enantiopure excess of 98% were obtained. Then we evaluated the cytotoxic effects on acute lymphoblastic leukemia cells CEM and mantle cell lymphoma cells Granta by direct cell counting and found that cytotoxic activities of both enantiomers were comparable with the effect of racemic mixture on cell growth and survival. Glucocorticoids modulate the expression of genes through transrepression and transactivation mechanisms realized in equal measure. In present work we estimated the potential ability of newly synthesized enantiomers to activate these mechanisms. Transactivation realizes through direct binding of receptor homodimers to specific sequences (glucocorticoid responsive elements (GREs)) in promoter or enhancer regions of GR target genes. Therefore, we studied the level of transactivation as the expression of GR-regulated genes, immunophiline FKBP51 and glucocorticoid-induced leucine zipper GILZ. As shown by Q-RT-PCR, the expression of FKBP51 and GILZ was unaffected after cell treatment by both CpdA enantiomers; hence, these compounds did not induce GR transactivation. GR-transrepression is chiefly mediated by protein–protein interaction between GR and other transcription factors like NF-kB and AP1, followed by inhibition of their transcriptional activity. We evaluated the transrepression by expression of NF-kB-depended genes, cyclines D1 and D2 (CCND and CCND2). (S)-enantiomer of CpdA down-regulated the expression of CCND1 and CCND2 to the level compared with Dex while (R)-enantiomer surprisingly increased level of CCND1 and CCND2 expression. This fact demonstrated that (S)-CpdA is perspective as antiproliferative and cell growth inhibiting agent. Overall, our data provide the potential for further studies of CpdA optical isomers, especially (S)-enantiomer, as GR-dependent anti-cancer agents

T25

Glucocorticoids (GCs) are widely used in treatment of many cancer types due to its ability to induce apoptosis in malignant cells in blood cancer therapy, and to prevent nausea, emesis and chemotherapy-associated hepatotoxicity in case of solid tumors. However, severe dose-limiting side effects occur, including osteoporosis, diabetes and other metabolic complications. Moreover, in therapy of solid tumors GCs strongly affect microenvironment which could be associated with poor prognosis, risk of metastasis and high frequency of relapses. Biological response to GCs is mediated by glucocorticoid receptor (GR), a well-characterized transcription factor. GR controls gene expression via (1) transactivation, which requires binding of GR homodimers to glucocorticoid-responsive elements (GRE) in gene promoters and enhancers, and (2) dimerization-independent transrepression mediated via negative interaction between GR and other transcription factors including major effectors of inflammation and proliferation. Transrepression plays an important role in anti-inflammatory and anti-cancer effects of GR, including normalizing influence on microenvironment, while side effects are associated with GR transactivation. In particular, GCs induce insulin resistance in adipocytes, a major component of the mammary microenvironment, which secrete pro-inflammatory cytokines and growth factors, implicated in tumor progression. Selective GR agonists (SEGRA) that preferentially activate GR transrepression could be a better option for treatment of cancer. Dozens of candidate SEGRAs were identified, synthesized and tested by industry and academia, with some having reached clinical trials. One of the novel GR modulators is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride, or CpdA, synthetic analogue of aziridine precursor isolated from Namibian shrub Salsola tuberculatiformis Botschantzev. It was shown that CpdA acts as “dissociated” GR ligand: it competes with GCs for GR binding and efficiently induces GR transrepression but not transactivation. We and other authors reported recently that CpdA inhibits survival of prostate cancer cells as well as blood cancer cells in GR-dependent fashion. Furthermore, primary leukemia cells from T-ALL patients appeared to be equally sensitive to GCs and CpdA. Our further studies were concentrated on three directions: (1) GC/SEGRA-based chemotherapy. We screened biological effect of CpdA in combination with traditional agents (doxorubicin, vincristine) and newer therapeutics (Bortezomib, Carfilzomib, MLN-4924, Rapamycin). Pretreatment of lymphoma cells with proteasome inhibitor Bortezomib resulted in GR accumulation and enhanced ligand properties of CpdA. We also revealed remarkable GR-dependent cooperation between CpdA and Bortezomib in suppressing survival of lymphoma cells in vitro and in vivo. Also surprising findings were substantial cooperation in anti-cancer effect of immunosuppressant Rapamycin and CpdA in vitro, and unexpected “dissociated” effect of Rapamycin on GR signaling realized through down -regulation of REDD1, mTORC1 inhibitor. These data suggested high clinical potential of Rapamycin/GC combination in cancer treatment. (2) SEGRA list extention We used two approaches to extend SEGRA list: (1) synthesis of CpdA enantiomers and (2) its chemical derivatives. Chemical analogues of CpdA were designed by appending of bulky substituent into benzene ring, alkylation of carbon atom adjacent to chlorine atom or appending of substituents to nitrogen atom. Evaluation of biological properties of enantiomers revealed higher GR-dependent anti-cancer potential of S-CpdA. Cytotoxic and proapoptotic effects of CpdA analogues were comparable with precursor. (3) Selection of tumor types acceptable for SEGRA treatment. CpdA was selected for NCI-60 in Vitro Cell Line Screening Project providing direct support to anticancer drug discovery program. It was shown that CpdA affect viability of some adherent cancer cell lines. We demonstrated that CpdA unlike GCs did not modify microenvironment and disintegrate tight junctions between cells decreasing risk of metastasis in case of solid tumors. It demonstrates reasonability of further investigations. Overall, our data provide the rationale for novel therapy of cancer based on combination of non-steroidal GR modulators with classic and modern chemotherapeutics. Approaches to obtain more SEGRAs were elaborated

N-bromotaurine surrogates for loss of antiproliferative response and enhances cisplatin efficacy in cancer cells with impaired glucocorticoid receptor

Glucocorticoids (GCs) are frequently used in anticancer combination regimens; however, their continuous use adds selective pressure on cancer cells to develop GC-resistance via impairment of the glucocorticoid receptor (GR), therefore creating a need for GC-alternatives. Based on the drug repurposing approach and the commonalities between inflammation and neoplasia, drugs that are either in late-stage clinical trials and/or already marketed for GC-refractory inflammatory diseases could be evaluated as GC-substitutes in the context of cancer. Advantageously, unlike new molecular entities currently being de novo developed to restore GC-responsiveness of cancer cells, such drugs have documented safety and efficacy profile, which overall simplifies their introduction in clinical cancer trials. In this study, we estimated the potential of a well-established, multistage, cell line-based, mouse skin carcinogenesis model to be exploited as an initial screening tool for unveiling covert GC-substitutes. First, we categorized the cell lines of this model to GC-sensitive and GC-resistant, in correlation with their corresponding GR status, localization, and functionality. We found that GC-resistance starts in papilloma stages, due to a dysfunctional GR, which is overexpressed, DNA binding-competent, but transactivation-incompetent in papilloma, squamous, and spindle stages of the model. Then, aided by this tool, we evaluated the ability of N-bromotaurine, a naturally occurring, small-molecule, nonsteroid anti-inflammatory drug which is under consideration for use interchangeably/in replacement to GCs in skin inflammations, to restore antiproliferative response of GC-resistant cancer cells. Unlike GCs, N-bromotaurine inhibited cell-cycle progression in GC-resistant cancer cells and efficiently synergized with cisplatin, thus indicating a potential to be exploited instead of GCs against cancer. © 2016 Elsevier Inc

Nutritional Sensor REDD1 in Cancer and Inflammation: Friend or Foe?

Regulated in Development and DNA Damage Response 1 (REDD1)/DNA Damage-Induced Transcript 4 (DDIT4) is an immediate early response gene activated by different stress conditions, including growth factor depletion, hypoxia, DNA damage, and stress hormones, i.e., glucocorticoids. The most known functions of REDD1 are the inhibition of proliferative signaling and the regulation of metabolism via the repression of the central regulator of these processes, the mammalian target of rapamycin (mTOR). The involvement of REDD1 in cell growth, apoptosis, metabolism, and oxidative stress implies its role in various pathological conditions, including cancer and inflammatory diseases. Recently, REDD1 was identified as one of the central genes mechanistically involved in undesirable atrophic effects induced by chronic topical and systemic glucocorticoids widely used for the treatment of blood cancer and inflammatory diseases. In this review, we discuss the role of REDD1 in the regulation of cell signaling and processes in normal and cancer cells, its involvement in the pathogenesis of different diseases, and the approach to safer glucocorticoid receptor (GR)-targeted therapies via a combination of glucocorticoids and REDD1 inhibitors to decrease the adverse atrophogenic effects of these steroids

A Novel Approach to Safer Glucocorticoid Receptor-Targeted Anti-lymphoma Therapy via RED D1 (Regulated in Development and DNA Damage 1) Inhibition

Glucocorticoids are widely used for therapy of hematologic malignancies. Unfortunately, chronic treatment with glucocorticoids commonly leads to adverse effects including skin and muscle atrophy and osteoporosis. We found recently that REDDI (regulated in development and DNA damage 1) plays central role in steroid atrophy. Here, we tested whether REDD1 suppression makes glucocorticoid-based therapy of blood cancer safer. Unexpectedly, approximately 50% of top putative REDD1 inhibitors selected by bioinforma tics screening of Library of Integrated Network-Based Cellular Signatures database (LINCS) were PI3K/Akt/mTOR inhibitors. We selected Wortmannin, LY294002, and AZD8055 for our studies and showed that they blocked basal and glucocorticoid-induced REDD1 expression. Moreover, all PI3K/mTOR/Akt inhibitors modified glucocorticoid receptor function shifting it toward therapeutically important transrepression. PI3KJAkt/mTOR inhibitors enhanced anti-lymphoma effects of Dexamethasone in vitro and in vivo, in lymphoma xenograft model. The therapeutic effects of PI3K inhibitor+Dexamethasone combinations ranged from cooperative to synergistic, especially in case of LY294002 and Rapamycin, used as a previously characterized reference REDD1 inhibitor. We found that coadministration of LY294002 or Rapamycin with Dexamethasone protected skin against Dexamethasone-induced atrophy, and normalized RANKL/OPG ratio indicating a reduction of Dexamethasone-induced osteoporosis. Together, our results provide foundation for further development of safer and more effective glucocorticoid-based combination therapy of hematologic malignancies using PI3K/Akt/mTOR inhibitors