The Nitro Group Reshapes the Effects of Pyrido[3,4-g]quinazoline Derivatives on DYRK/CLK Activity and RNA Splicing in Glioblastoma Cells.

Serine-threonine protein kinases of the DYRK and CLK families regulate a variety of vital cellular functions. In particular, these enzymes phosphorylate proteins involved in pre-mRNA splicing. Targeting splicing with pharmacological DYRK/CLK inhibitors emerged as a promising anticancer strategy. Investigation of the pyrido[3,4-g]quinazoline scaffold led to the discovery of DYRK/CLK binders with differential potency against individual enzyme isoforms. Exploring the structure-activity relationship within this chemotype, we demonstrated that two structurally close compounds, pyrido[3,4-g]quinazoline-2,10-diamine 1 and 10-nitro pyrido[3,4-g]quinazoline-2-amine 2, differentially inhibited DYRK1-4 and CLK1-3 protein kinases in vitro. Unlike compound 1, compound 2 efficiently inhibited DYRK3 and CLK4 isoenzymes at nanomolar concentrations. Quantum chemical calculations, docking and molecular dynamic simulations of complexes of 1 and 2 with DYRK3 and CLK4 identified a dramatic difference in electron donor-acceptor properties critical for preferential interaction of 2 with these targets. Subsequent transcriptome and proteome analyses of patient-derived glioblastoma (GBM) neurospheres treated with 2 revealed that this compound impaired CLK4 interactions with spliceosomal proteins, thereby altering RNA splicing. Importantly, 2 affected the genes that perform critical functions for cancer cells including DNA damage response, p53 signaling and transcription. Altogether, these results provide a mechanistic basis for the therapeutic efficacy of 2 previously demonstrated in in vivo GBM models.

Genetically modified mice as a tool for the study of human diseases.

Modeling a human disease is an essential part of biomedical research. The recent advances in the field of molecular genetics made it possible to obtain genetically modified animals for the study of various diseases. Not only monogenic disorders but also chromosomal and multifactorial disorders can be mimicked in lab animals due to genetic modification. Even human infectious diseases can be studied in genetically modified animals. An animal model of a disease enables the tracking of its pathogenesis and, more importantly, to test new therapies. In the first part of this paper, we review the most common DNA modification technologies and provide key ideas on specific technology choices according to the task at hand. In the second part, we focus on the application of genetically modified mice in studying human diseases.

Key Proteins of Replication Stress Response and Cell Cycle Control as Cancer Therapy Targets.

Replication stress (RS) is a characteristic state of cancer cells as they tend to exchange precision of replication for fast proliferation and increased genomic instability. To overcome the consequences of improper replication control, malignant cells frequently inactivate parts of their DNA damage response (DDR) pathways (the ATM-CHK2-p53 pathway), while relying on other pathways which help to maintain replication fork stability (ATR-CHK1). This creates a dependency on the remaining DDR pathways, vulnerability to further destabilization of replication and synthetic lethality of DDR inhibitors with common oncogenic alterations such as mutations of TP53, RB1, ATM, amplifications of MYC, CCNE1 and others. The response to RS is normally limited by coordination of cell cycle, transcription and replication. Inhibition of WEE1 and PKMYT1 kinases, which prevent unscheduled mitosis entry, leads to fragility of under-replicated sites. Recent evidence also shows that inhibition of Cyclin-dependent kinases (CDKs), such as CDK4/6, CDK2, CDK8/19 and CDK12/13 can contribute to RS through disruption of DNA repair and replication control. Here, we review the main causes of RS in cancers as well as main therapeutic targets-ATR, CHK1, PARP and their inhibitors.

A novel chromatin-remodeling complex variant, dcPBAF, is involved in maintaining transcription in differentiated neurons.

The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains (called PHF10D). In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes.

New Approach for Studying of Isoforms and High-Homology Proteins in Mammalian Cells.

In mammals, a large number of proteins are expressed as more than one isoform, resulting in the increased diversity of their proteome. Understanding the functions of isoforms is very important, since individual isoforms of the same protein can have oncogenic or pathogenic properties, or serve as disease markers. The high homology of isoforms with ubiquitous expression makes it difficult to study them. In this work, we propose a new approach for the study of protein isoforms in mammalian cells, which makes it possible to individually detect and investigate the functions of an individual isoform. The approach was developed to study the functions of isoforms of the PHF10 protein, a chromatin subunit of the PBAF remodeling complex. We demonstrated the possibility of induced simultaneous suppression of all endogenous PHF10 isoforms and the expression of a single recombinant FLAG-tagged isoform. For this purpose, we created constructs based on the pSLIK plasmid with a cloned cassette containing the recombinant gene of interest and miR30 with the corresponding shRNAs. The doxycycline-induced activation of the cassette allows on and off switching. Using this construct, we achieved the preferential expression of only one recombinant PHF10 isoform with a simultaneously reduced number of all endogenous isoforms. Our approach can be used to study the role of point mutations, the functions of individual domains and important sites, or to individually detect untagged isoforms with knockdown of all endogenous isoforms.

Genetically Engineered Mice Unveil In Vivo Roles of the Mediator Complex.

The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.

Inhibition of Cyclin-Dependent Kinases 8/19 Restricts Bacterial and Virus-Induced Inflammatory Responses in Monocytes.

Hyperactivation of the immune system remains a dramatic, life-threatening complication of viral and bacterial infections, particularly during pneumonia. Therapeutic approaches to counteract local and systemic outbreaks of cytokine storm and to prevent tissue damage remain limited. Cyclin-dependent kinases 8 and 19 (CDK8/19) potentiate transcriptional responses to the altered microenvironment, but CDK8/19 potential in immunoregulation is not fully understood. In the present study, we investigated how a selective CDK8/19 inhibitor, Senexin B, impacts the immunogenic profiles of monocytic cells stimulated using influenza virus H1N1 or bacterial lipopolysaccharides. Senexin B was able to prevent the induction of gene expression of proinflammatory cytokines in THP1 and U937 cell lines and in human peripheral blood-derived mononuclear cells. Moreover, Senexin B substantially reduced functional manifestations of inflammation, including clustering and chemokine-dependent migration of THP1 monocytes and human pulmonary fibroblasts (HPF).

Therapy-Induced Tumor Cell Senescence: Mechanisms and Circumvention.

Plasticity of tumor cells (multitude of molecular regulation pathways) allows them to evade cytocidal effects of chemo- and/or radiation therapy. Metabolic adaptation of the surviving cells is based on transcriptional reprogramming. Similarly to the process of natural cell aging, specific features of the survived tumor cells comprise the therapy-induced senescence phenotype. Tumor cells with this phenotype differ from the parental cells since they become less responsive to drugs and form aggressive progeny. Importance of the problem is explained by the general biological significance of transcriptional reprogramming as a mechanism of adaptation to stress, and by the emerging potential of its pharmacological targeting. In this review we analyze the mechanisms of regulation of the therapy-induced tumor cell senescence, as well as new drug combinations aimed to prevent this clinically unfavorable phenomenon.

Graphene Oxide Nanosurface Reduces Apoptotic Death of HCT116 Colon Carcinoma Cells Induced by Zirconium Trisulfide Nanoribbons.

Due to their chemical, mechanical, and optical properties, 2D ultrathin nanomaterials have significant potential in biomedicine. However, the cytotoxicity of such materials, including their mutual increase or decrease, is still not well understood. We studied the effects that graphene oxide (GO) nanolayers (with dimensions 0.1-3 µm and average individual flake thickness less than 1 nm) and ZrS3 nanoribbons (length more than 10 µm, width 0.4-3 µm, and thickness 50-120 nm) have on the viability, cell cycle, and cell death of HCT116 colon carcinoma cells. We found that ZrS3 exhibited strong cytotoxicity by causing apoptotic cell death, which was in contrast to GO. When adding GO to ZrS3, ZrS3 was significantly less toxic, which may be because GO inhibits the effects of cytotoxic hydrogen sulfide produced by ZrS3. Thus, using zirconium trisulfide nanoribbons as an example, we have demonstrated the ability of graphene oxide to reduce the cytotoxicity of another nanomaterial, which may be of practical importance in biomedicine, including the development of biocompatible nanocoatings for scaffolds, theranostic nanostructures, and others.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

Alternative RNA splicing modulates ribosomal composition and determines the spatial phenotype of glioblastoma cells.

Glioblastoma (GBM) is characterized by exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here, we found that the compositions of ribosomes of GBM cells in the tumour core and edge differ due to alternative RNA splicing. The acidic pH in the core switches before messenger RNA splicing of the ribosomal gene RPL22L1 towards the RPL22L1b isoform. This allows cells to survive acidosis, increases stemness and correlates with worse patient outcome. Mechanistically, RPL22L1b promotes RNA splicing by interacting with lncMALAT1 in the nucleus and inducing its degradation. Contrarily, in the tumour edge region, RPL22L1a interacts with ribosomes in the cytoplasm and upregulates the translation of multiple messenger RNAs including TP53. We found that the RPL22L1 isoform switch is regulated by SRSF4 and identified a compound that inhibits this process and decreases tumour growth. These findings demonstrate how distinct GBM cell populations arise during tumour growth. Targeting this mechanism may decrease GBM heterogeneity and facilitate therapy.

Evaluation of New Antimicrobial Agents Based on tris(1H-Indol-3-yl)methylium Salts: Activity, Toxicity, Suppression of Experimental Sepsis in Mice.

The antimicrobial activity and toxicity of three novel synthetic antibacterial agents containing tris(1H-indol-3-yl)methylium fragment were studied in vitro and in vivo. All compounds in vitro revealed high activity (minimal inhibitory concentration (MIC) 0.13-1.0 µg/mL) against bacteria that were either sensitive or resistant to antibiotics, including multidrug-resistant clinical isolates. The derivatives combining high antimicrobial activity with relatively low cytotoxicity against human donor fibroblasts HPF-hTERT were subjected to further testing on mice. In vivo they revealed fairly good tolerance and relatively low toxicity. Acute toxicity was evaluated, and the main indicators of toxicity, including LD50 and LD10, were determined. A study of compounds in vivo showed their efficiency in the model of staphylococcal sepsis in mice. The efficiency of compounds may be due to the ability of indolylmethylium salts to form pores in the cytoplasmic membrane of microbial cells and thereby facilitate the penetration of molecules into the pathogen.

Novel O-acylated (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-one oximes targeting HSP90-HER2 axis in breast cancer cells.

Novel O-acylated (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-one oximes were designed as potential HSP90 inhibitors. A series of the compounds was synthesized by oximation of (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-ones followed by O-acylation with acylamidobenzoic acids. The obtained compounds showed an antiproliferative effect on three breast cancer cell lines (MCF7, MDA-MB-231 and HCC1954). Compound 16s exhibited high antiproliferative potency against HCC1954 breast cancer cells with the IC50 value of 6 µM was selected for in-depth evaluation. Compound 16s did not inhibit the growth of normal epithelial cells. We have demonstrated that the compound 16s can induce apoptosis in cancer cells via inhibition of HSP90 "client" proteins including a key oncogenic receptor, HER2/neu. Described here compounds can be considered for further basic and preclinical investigation as a part of HSP90/HER2-targeted therapies.

Copper-Containing Nanoparticles and Organic Complexes: Metal Reduction Triggers Rapid Cell Death via Oxidative Burst.

Copper-containing agents are promising antitumor pharmaceuticals due to the ability of the metal ion to react with biomolecules. In the current study, we demonstrate that inorganic Cu2+ in the form of oxide nanoparticles (NPs) or salts, as well as Cu ions in the context of organic complexes (oxidation states +1, +1.5 and +2), acquire significant cytotoxic potency (2-3 orders of magnitude determined by IC50 values) in combinations with N-acetylcysteine (NAC), cysteine, or ascorbate. In contrast, other divalent cations (Zn, Fe, Mo, and Co) evoked no cytotoxicity with these combinations. CuO NPs (0.1-1 µg/mL) together with 1 mM NAC triggered the formation of reactive oxygen species (ROS) within 2-6 h concomitantly with perturbation of the plasma membrane and caspase-independent cell death. Furthermore, NAC potently sensitized HCT116 colon carcinoma cells to Cu-organic complexes in which the metal ion coordinated with 5-(2-pyridylmethylene)-2-methylthio-imidazol-4-one or was present in the coordination sphere of the porphyrin macrocycle. The sensitization effect was detectable in a panel of mammalian tumor cell lines including the sublines with the determinants of chemotherapeutic drug resistance. The components of the combination were non-toxic if added separately. Electrochemical studies revealed that Cu cations underwent a stepwise reduction in the presence of NAC or ascorbate. This mechanism explains differential efficacy of individual Cu-organic compounds in cell sensitization depending on the availability of Cu ions for reduction. In the presence of oxygen, Cu+1 complexes can generate a superoxide anion in a Fenton-like reaction Cu+1L + O2 → O2-. + Cu+2L, where L is the organic ligand. Studies on artificial lipid membranes showed that NAC interacted with negatively charged phospholipids, an effect that can facilitate the penetration of CuO NPs across the membranes. Thus, electrochemical modification of Cu ions and subsequent ROS generation, as well as direct interaction with membranes, represent the mechanisms of irreversible membrane damage and cell death in response to metal reduction in inorganic and organic Cu-containing compounds.

Conserved Structure and Evolution of DPF Domain of PHF10-The Specific Subunit of PBAF Chromatin Remodeling Complex.

Transcription activation factors and multisubunit coactivator complexes get recruited at specific chromatin sites via protein domains that recognize histone modifications. Single PHDs (plant homeodomains) interact with differentially modified H3 histone tails. Double PHD finger (DPF) domains possess a unique structure different from PHD and are found in six proteins: histone acetyltransferases MOZ and MORF; chromatin remodeling complex BAF (DPF1-3); and chromatin remodeling complex PBAF (PHF10). Among them, PHF10 stands out due to the DPF sequence, structure, and functions. PHF10 is ubiquitously expressed in developing and adult organisms as four isoforms differing in structure (the presence or absence of DPF) and transcription regulation functions. Despite the importance of the DPF domain of PHF10 for transcription activation, its structure remains undetermined. We performed homology modeling of the human PHF10 DPF domain and determined common and distinct features in structure and histone modifications recognition capabilities, which can affect PBAF complex chromatin recruitment. We also traced the evolution of DPF1-3 and PHF10 genes from unicellular to vertebrate organisms. The data reviewed suggest that the DPF domain of PHF10 plays an important role in SWI/SNF-dependent chromatin remodeling during transcription activation.

Clinical Correlations of Polycomb Repressive Complex 2 in Different Tumor Types.

PRC2 (Polycomb repressive complex 2) is an evolutionarily conserved protein complex required to maintain transcriptional repression. The core PRC2 complex includes EZH2, SUZ12, and EED proteins and methylates histone H3K27. PRC2 is known to contribute to carcinogenesis and several small molecule inhibitors targeting PRC2 have been developed. The present study aimed to identify the cancer types in which PRC2 targeting drugs could be beneficial. We queried genomic and transcriptomic (cBioPortal, KMplot) database portals of clinical tumor samples to evaluate clinical correlations of PRC2 subunit genes. EZH2, SUZ12, and EED gene amplification was most frequently found in prostate cancer, whereas lymphoid malignancies (DLBCL) frequently showed EZH2 mutations. In both cases, PRC2 alterations were associated with poor prognosis. Moreover, higher expression of PRC2 subunits was correlated with poor survival in renal and liver cancers as well as gliomas. Finally, we generated a Python application to analyze the correlation of EZH2/SUZ12/EED gene knockouts by CRISPR with the alterations detected in the cancer cell lines using DepMap data. As a result, we were able to identify mutations that correlated significantly with tumor cell sensitivity to PRC2 knockout, including SWI/SNF, COMPASS/COMPASS-like subunits and BCL2, warranting the investigation of these genes as potential markers of sensitivity to PRC2-targeting drugs.

The Inhibition of CDK8/19 Mediator Kinases Prevents the Development of Resistance to EGFR-Targeting Drugs.

Drug resistance is the main obstacle to achieving cures with both conventional and targeted anticancer drugs. The emergence of acquired drug resistance is initially mediated by non-genetic transcriptional changes, which occur at a much higher frequency than mutations and may involve population-scale transcriptomic adaptation. CDK8/19 kinases, through association with transcriptional Mediator complex, regulate transcriptional reprogramming by co-operating with different signal-responsive transcription factors. Here we tested if CDK8/19 inhibition could prevent adaptation to drugs acting on epidermal growth factor receptor (EGFR/ERBB1/HER1). The development of resistance was analyzed following long-term exposure of BT474 and SKBR3 breast cancer cells to EGFR-targeting small molecules (gefitinib, erlotinib) and of SW48 colon cancer cells to an anti-EGFR monoclonal antibody cetuximab. In all cases, treatment of small cell populations (~105 cells) with a single dose of the drug initially led to growth inhibition that was followed by the resumption of proliferation and development of drug resistance in the adapted populations. However, this adaptation was always prevented by the addition of selective CDK8/19 inhibitors, even though such inhibitors alone had only moderate or no effect on cell growth. These results indicate that combining EGFR-targeting drugs with CDK8/19 inhibitors may delay or prevent the development of tumor resistance to therapy.

RNA Splicing: Basic Aspects Underlie Antitumor Targeting.

BACKGROUND: RNA splicing, a fundamental step in gene expression, is aimed at intron removal and ordering of exons to form the protein's reading frame. OBJECTIVE: This review is focused on the role of RNA splicing in cancer biology; the splicing abnormalities that lead to tumor progression emerge as targets for therapeutic intervention. METHODS: We discuss the role of aberrant mRNA splicing in carcinogenesis and drug response. RESULTS AND CONCLUSION: Pharmacological modulation of RNA splicing sets the stage for treatment approaches in situations where mRNA splicing is a clinically meaningful mechanism of the disease.

Novel curcumin derivatives as P-glycoprotein inhibitors: Molecular modeling, synthesis and sensitization of multidrug resistant cells to doxorubicin.

The MDR1/P-glycoprotein (Pgp)/ABCB1 multidrug transporter is being investigated as a druggable target for antitumor therapy for decades. The natural product curcumin is known to provide an efficient scaffold for compounds capable of blocking Pgp mediated efflux and sensitization of multidrug resistant (MDR) cells to the Pgp transported drug doxorubicin (Dox). We performed molecular dynamics simulations and docking of curcumin derivatives into the Pgp model. Based on these calculations, a series of pyrazolocurcumin derivatives with predicted metabolic stability and/or improved binding affinity were proposed for synthesis and evaluation of MDR reversal potency against Dox selected K562/4 subline, a derivative of K562 human chronic myelogenous leukemia cell line. Compounds 16 and 19 which are both dimethylcurcumin pyrazole derivatives bearing an N-p-phenylcarboxylic amide substitution, were the most potent Pgp blockers as determined by intracellular Dox accumulation. Furthermore, at non-toxic submicromolar concentrations 16 and 19 dramatically sensitized K562/4 cells to Dox. Together with good water solubility of 16 and 19, these results indicate that the new pyrazolo derivatives of curcumin are a promising scaffold for development of clinically applicable Pgp antagonists.

The sequential phosphorylation of PHF10 subunit of the PBAF chromatin-remodeling complex determines different properties of the PHF10 isoforms.

The mammalian PBAF subfamily of SWI/SNF chromatin remodeling complexes plays a wide role in the regulation of gene expression. PHF10 is a subunit of the signature module of PBAF, responsible for its interaction with chromatin. PHF10 is represented by four different isoforms, which are alternatively incorporated in the complex. Two of PHF10 isoforms lacking C-terminal PHD domains contain a cluster of phosphorylated serine residues, designated as X-cluster. In the present study, we explore the phosphorylation of the X-cluster in detail. We identified additional phosphorylated serine residues and designated them as either frequently or rarely phosphorylated. The X-cluster consists of two independently phosphorylated subclusters. Phosphorylation of the second subcluster depends on phosphorylation of a primary serine 327. These two subclusters surround a sequence, which is predicted to be a nuclear localization sequence (NLS3). The NLS3 does not affect localization of PHF10 isoforms. However, it is essential for X-cluster phosphorylation and increased stability of isoforms that lack PHD. Conversely, the presence of NLS3 signal in isoforms that contain C-terminal PHD domains reduces their stability. Thus, phosphorylation of PHF10 isoforms regulates their cell level, determining the rate of incorporation in PBAF. This may alter the pattern of PBAF regulated genes.