Cruciform structures are a common DNA feature important for regulating biological processes

DNA cruciforms play an important role in the regulation of natural processes involving DNA. These structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling. Cruciform structures are fundamentally important for a wide range of biological processes, including replication, regulation of gene expression, nucleosome structure and recombination. They also have been implicated in the evolution and development of diseases including cancer, Werner's syndrome and others

Nuclear Export in Non-Hodgkin Lymphoma and Implications for Targeted XPO1 Inhibitors

Exportin-1 (XPO1) is a key player in the nuclear export pathway and is overexpressed in almost all cancers. This is especially relevant for non-Hodgkin lymphoma (NHL), where high XPO1 expression is associated with poor prognosis due to its oncogenic role in exporting proteins and RNA that are involved in cancer progression and treatment resistance. Here, we discuss the proteins and RNA transcripts that have been identified as XPO1 cargo in NHL lymphoma including tumour suppressors, immune modulators, and transcription factors, and their implications for oncogenesis. We then highlight the research to date on XPO1 inhibitors such as selinexor and other selective inhibitors of nuclear export (SINEs), which are used to treat some cases of non-Hodgkin lymphoma. In vitro, in vivo, and clinical studies investigating the anti-cancer effects of SINEs from bench to bedside, both as a single agent and in combination, are also reported. Finally, we discuss the limitations of the current research landscape and future directions to better understand and improve the clinical utility of SINE compounds in NHL

Small Molecule Stat5-Sh2 Domain Inhibitors Exhibit Potent Antileukemia Activity

We investigate the alignment of 4\u27-pentyl-4-cynobiphenyl (5CB) liquid crystal on phase-separated two-component organosilane monolayers that consist of the protruding islands surrounded by a continuous phase. The molecular disordering at the protruding island edge locally perturbs the homeotropic alignment of the 5CB induced the phase-separated monolayers, leading to the formation of ring patterns. The edge effect on the 5CB alignment decreases with the decrease of the height difference between the protruding islands and the surrounding monolayer and increases with the decrease of the size of the protruding islands. © 2011 Elsevier B.V

Eme1 is involved in DNA damage processing and maintenance of genomic stability in mammalian cells

Yeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication. We identified mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3′-flap structures and replication forks rather than Holliday junctions in vitro. We demonstrate that Eme1(–/–) embryonic stem (ES) cells are hypersensitive to the DNA cross-linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment. Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE). Unlike Blm-deficient ES cells, increased SCE was seen only following induced DNA damage in Eme1-deficient cells. Most importantly, Eme1 deficiency led to spontaneous genomic instability. These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity

Metabolic Adaptation to Chronic Inhibition of Mitochondrial Protein Synthesis in Acute Myeloid Leukemia Cells

<div><p>Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.</p> </div

Small Molecule STAT5-SH2 Domain Inhibitors Exhibit Potent Antileukemia Activity

A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an <i>in vitro</i> screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (<i>K</i>_i < 5 μM) and STAT5 selective (>3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, <b>BP-1-107</b>, <b>BP-1-108</b>, <b>SF-1-087</b>, and <b>SF-1-088</b>. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC₅₀’s ∼ 20 μM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes <i>cyclin D1</i>, <i>cyclin D2</i>, <i>C-MYC</i>, and <i>MCL-1</i>. Moreover, lead agent <b>BP-1-108</b> showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date

RTEX+TIG cells have defective oxidative phosphorylation.

<p><b>A</b> Basal oxygen consumption rate of TEX and RTEX+TIG cells was measured with the Seahorse Metabolic Flux Analyzer as described in the Materials and Methods section. Results shown as mean ± SD of three independent experiments. <b>B</b> Basal extracellular acidification rate of TEX and RTEX+TIG cells was measured with the Seahorse Metabolic Flux Analyzer as described in the Materials and Methods section. Results shown as mean ± SD of three independent experiments. <b>C</b> Lactate production of TEX and RTEX+TIG cells (2×10⁷) was measured by NMR as described in the Materials and Methods section. Results shown as mean ± SD of independent experiments. <b>D</b> Intracellular ATP content of TEX and RTEX+TIG cells was measured as described in the Materials and Methods section. Results shown as mean ± SD from a representative experiment. <b>E</b> Resting mitochondrial membrane potential (Δψ, Red/Green ratio) was measured in TEX and RTEX+TIG cells before and after uncoupling the potential with CCCP. Cells were stained with JC-1 dye and analyzed by flow cytometry. Results shown as mean ± SD fluorescence intensity relative to TEX cells. <b>F</b> The number of viable TEX and RTEX+TIG cells was counted using trypan blue staining at 24, 48 and 72 hours. Results shown as mean ± SD viable cells from independent experiments.</p

The defective oxidative phosphorylation in RTEX+TIG is reversible.

<p>Tigecycline was withdrawn from the culture media of RTEX+TIG cells for one week (RTEX-TIG). <b>A</b> Complex III and IV enzyme activity relative to citrate synthase activity was determined in RTEX+TIG cells upon the withdrawal of tigecycline (RTEX-TIG) at various time points as described in the Materials and Methods section. Results shown as mean ± SD of three independent experiments. <b>B</b> Cells were then re-treated with tigecycline (24 µM) for 72 hours (RTEX-TIG (72 hrs TIG)). Complex IV, III, and II activity was measured as described in the Materials and Methods section. Data represent the mean±SD from three independent experiments. <b>C</b> Cells were then re-treated with tigecycline (10 µM) for 72 hours (RTEX-TIG (72 hrs TIG)). Oxygen consumption rate was measured with the Seahorse Metabolic Flux Analyzer. Data represent the mean±SD from a representative experiment. <b>D</b> Cells were then re-treated with tigecycline (10 µM) for 72 hours (RTEX-TIG (72 hrs TIG)). Extracellular acidification rate was measured with the Seahorse Metabolic Flux Analyzer. Data represent the mean±SD from a representative experiments. <b>E</b> Intracellular ATP content was measured in TEX, RTEX+TIG and RTEX-TIG cells. Data represent the mean±SD relative to TEX cells from a representative experiments.</p