Anti-tumor Activity of Novel Compounds Targeting BCR-ABL, c-SRC and BCR-ABLT315I in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a hematological stem-cell disorder characterized by the expression of the BCR-ABL fusion protein, a constitutively active tyrosine kinase that causes pathogenesis. The development of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL oncogene has proven an effective approach to treat CML, but a non-negligible proportion of patients develop a resistance to this class of drugs. Of note, the T315I mutant of BCR-ABL is resistant to all known TKIs, with the noticeable exception of ponatinib. To address this unmet medical need, a new series of compounds was designed and tested for anti-tumor effects against BCR-ABLT315I CML. The effects of three OriBase Pharma compounds (OR1001, OR1002 and OR1003) on the kinase activity of wild-type and mutant BCR-ABL proteins, on cell proliferation and on the growth of subcutaneous xenografts of CML cells in athymic mice were investigated. In vitro, the three compounds were potent inhibitors of both ABL and c-SRC kinases and of the main mutants of ABL, including T315I. The three compounds inhibited the proliferation of cell lines expressing the wild-type and several mutated forms of BCR-ABL, including T315I. Finally, in a mouse xenograft model, OR1001, was found to significantly reduce tumor growth. These data support the potential of OR1001 as an effective therapy for the treatment of de novo and TKI-resistant patients

Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements

Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved

Epigenome‐Wide Scan Identifies a Treatment‐Responsive Pattern of Altered DNA Methylation Among Cytoskeletal Remodeling Genes in Monocytes and CD4+ T Cells From Patients With Behçet's Disease

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/107357/1/art38409.pd

NF-kappaB P50/P65 hetero-dimer mediates differential regulation of CD166/ALCAM expression via interaction with micoRNA-9 after serum deprivation, providing evidence for a novel negative auto-regulatory loop

CD166/ALCAM plays an important role in tumor aggression and progression as well as protecting cancer cells against apoptosis and autophagy. However, the mechanism by which pro-cell death signals control CD166 expression remains unclear. Here we show that following serum deprivation (SD), upregulation of CD166 protein is shorter than that of CD166 mRNA. Molecular analysis revealed both CD166 and miR-9-1 as two novel NF-κB target genes in hepatoma cells. In vivo activation and translocation of the NF-κB P50/P65 hetero-dimer into the nucleus following the phosphorylation and accompanied degradation of its inhibitor, IκBα, contributes to efficient transcription of both genes following SD. We show that following serum starvation, delayed up-regulation of miR-9 represses translation of CD166 protein through its target sites in the 3′-UTR of CD166 mRNA. We also propose that miR-9 promotes cell migration largely due to inhibition of CD166. Collectively, the study elucidates a novel negative auto-regulatory loop in which NF-κB mediates differential regulation of CD166 after SD

Dynamic and Polarized Muscle Cell Behaviors Accompany Tail Morphogenesis in the Ascidian Ciona intestinalis

BACKGROUND: Axial elongation is a key morphogenetic process that serves to shape developing organisms. Tail extension in the ascidian larva represents a striking example of this process, wherein paraxially positioned muscle cells undergo elongation and differentiation independent of the segmentation process that characterizes the formation of paraxial mesoderm in vertebrates. Investigating the cell behaviors underlying the morphogenesis of muscle in ascidians may therefore reveal the evolutionarily conserved mechanisms operating during this process. METHODOLOGY/PRINCIPLE FINDINGS: A live cell imaging approach utilizing subcellularly-localized fluorescent proteins was employed to investigate muscle cell behaviors during tail extension in the ascidian Ciona intestinalis. Changes in the position and morphology of individual muscle cells were analyzed in vivo in wild type embryos undergoing tail extension and in embryos in which muscle development was perturbed. Muscle cells were observed to undergo elongation in the absence of positional reorganization. Furthermore, high-speed high-resolution live imaging revealed that the onset and progression of tail extension were characterized by the presence of dynamic and polarized actin-based protrusive activity at the plasma membrane of individual muscle cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that in the Ciona muscle, tissue elongation resulted from gradual and coordinated changes in cell geometry and not from changes in cell topology. Proper formation of muscle cells was found to be necessary not only for muscle tissue elongation, but also more generally for completion of tail extension. Based upon the characterized dynamic changes in cell morphology and plasma membrane protrusive activity, a three-phase model is proposed to describe the cell behavior operating during muscle morphogenesis in the ascidian embryo

Large Isoforms of UNC-89 (Obscurin) Are Required for Muscle Cell Architecture and Optimal Calcium Release in Caenorhabditis elegans

Calcium, a ubiquitous intracellular signaling molecule, controls a diverse array of cellular processes. Consequently, cells have developed strategies to modulate the shape of calcium signals in space and time. The force generating machinery in muscle is regulated by the influx and efflux of calcium ions into the muscle cytoplasm. In order for efficient and effective muscle contraction to occur, calcium needs to be rapidly, accurately and reliably regulated. The mechanisms underlying this highly regulated process are not fully understood. Here, we show that the Caenorhabditis elegans homolog of the giant muscle protein obscurin, UNC-89, is required for normal muscle cell architecture. The large immunoglobulin domain-rich isoforms of UNC-89 are critical for sarcomere and sarcoplasmic reticulum organization. Furthermore, we have found evidence that this structural organization is crucial for excitation-contraction coupling in the body wall muscle, through the coordination of calcium signaling. Thus, our data implicates UNC-89 in maintaining muscle cell architecture and that this precise organization is essential for optimal calcium mobilization and efficient and effective muscle contraction

Rôle de Brm dans le contrôle du cycle cellulaire et Étude de l'équilibre prolifération/différenciation des kératinocytes

The main regulators of the cellular proliferation are the kinases Cdk, whose activity depends on their association with the cyclins. The control of cyclin expression is the first mechanism of cell cycle regulation, that is essential for the balance proliferation/differentiation in the keratinocytes. We show that Brm, main subunit of the chromatin remodeling complexes SWI/SNF, is necessary for cyclin A repression by positioning two nucleosomes on the proximal promoter of the gene. We also demonstrate that the lack of brm leads to genomic instability by accelerating S phase and lengthening mitosis. This may be the consequence of the deregulation of the three oncogenes, c-myc, cyclin E and cyclin A. Finally, we present evidences that the keratinocytes enter differentiation after a p21 dependent cell cycle arrest in G2/M. However, differentiating keratinocytes cannot sustain this state, bypass mitosis and reenter G1 phase with a 4N DNA content.Les principaux régulateurs de la prolifération cellulaire sont les Cdk (cyclin dependent kinase), dont l'activité dépend de leur association avec leurs partenaires, les cyclines. Le contrôle du niveau d'expression des cyclines représente le premier mécanisme par lequel l'activité des Cdk est régulée. Cette régulation est essentielle pour maintenir l'équilibre prolifération/différenciation de la peau. Cependant, les mécanismes mis en jeu restent peu connus.Nous avons montré que Brm, protéine des complexes de remodelage de la chromatine SWI/SNF, est responsable de la répression de la cycline A par la mise en place ou le maintien de deux nucléosomes situés sur les sites d'initiation de la transcription. De plus, nous avons mis en évidence que l'absence de brm conduit à accélérer la progression des cellules dans le cycle cellulaire en jouant sur le déroulement de la phase S. Cependant, les cellules dépourvues de brm présentent également une mitose rallongée et des aberrations chromosomiques. Ceci pourrait être la conséquence de la dérégulation de trois oncogènes : c-myc, cycline A et cycline E et pourrait expliquer pourquoi brm est mutée dans de nombreux cancers.Enfin, nous avons montré que l'entrée en différenciation des kératinocytes s'accompagne d'une forte expression de p21 qui entraîne un arrêt en G2/M en inhibant les complexes Cycline A/Cdk. Cependant, les kératinocytes en différenciation ne peuvent maintenir cet arrêt et entre dans un état G1 à 4N, caractérisé par une forte expression de la Cycline E et l'absence de Cyclines de G2/M

Rôle de Brm dans le contrôle du cycle cellulaire et Étude de l'équilibre prolifération/différenciation des kératinocytes

The main regulators of the cellular proliferation are the kinases Cdk, whose activity depends on their association with the cyclins. The control of cyclin expression is the first mechanism of cell cycle regulation, that is essential for the balance proliferation/differentiation in the keratinocytes. We show that Brm, main subunit of the chromatin remodeling complexes SWI/SNF, is necessary for cyclin A repression by positioning two nucleosomes on the proximal promoter of the gene. We also demonstrate that the lack of brm leads to genomic instability by accelerating S phase and lengthening mitosis. This may be the consequence of the deregulation of the three oncogenes, c-myc, cyclin E and cyclin A. Finally, we present evidences that the keratinocytes enter differentiation after a p21 dependent cell cycle arrest in G2/M. However, differentiating keratinocytes cannot sustain this state, bypass mitosis and reenter G1 phase with a 4N DNA content.Les principaux régulateurs de la prolifération cellulaire sont les Cdk (cyclin dependent kinase), dont l'activité dépend de leur association avec leurs partenaires, les cyclines. Le contrôle du niveau d'expression des cyclines représente le premier mécanisme par lequel l'activité des Cdk est régulée. Cette régulation est essentielle pour maintenir l'équilibre prolifération/différenciation de la peau. Cependant, les mécanismes mis en jeu restent peu connus.Nous avons montré que Brm, protéine des complexes de remodelage de la chromatine SWI/SNF, est responsable de la répression de la cycline A par la mise en place ou le maintien de deux nucléosomes situés sur les sites d'initiation de la transcription. De plus, nous avons mis en évidence que l'absence de brm conduit à accélérer la progression des cellules dans le cycle cellulaire en jouant sur le déroulement de la phase S. Cependant, les cellules dépourvues de brm présentent également une mitose rallongée et des aberrations chromosomiques. Ceci pourrait être la conséquence de la dérégulation de trois oncogènes : c-myc, cycline A et cycline E et pourrait expliquer pourquoi brm est mutée dans de nombreux cancers.Enfin, nous avons montré que l'entrée en différenciation des kératinocytes s'accompagne d'une forte expression de p21 qui entraîne un arrêt en G2/M en inhibant les complexes Cycline A/Cdk. Cependant, les kératinocytes en différenciation ne peuvent maintenir cet arrêt et entre dans un état G1 à 4N, caractérisé par une forte expression de la Cycline E et l'absence de Cyclines de G2/M