Cmpd10357 to treat B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of cancer found in children. Although the overall survival rates are now >80%, 15%-20% of pediatric patients relapse, with survival rates subsequently dropping to 5%-10%. Cmpd10357, 3-amino-5-arylamino-6-chloro-N- (diaminomethylene) pyrazine-2-carboximide, is a highly potent, cell-permeant compound recently shown to have cytotoxic effects on solid tumors, including human breast cancer and high-grade gliomas, independent of their proliferative status. Cmpd10357 demonstrated concentration-dependent cytotoxicity in two human B-ALL cell lines, JM1 and Reh, at half-maximal inhibitory concentrations (IC50) of 3.2 and 3.3 μM, respectively. Cmpd10357, at a dose of 5 mg/kg, significantly prolonged survival in our B-ALL xenograft mouse model, with a median survival time of 49.0 days compared with 45.5 days in the control group (p < 0.05). The cytotoxicity of Cmpd10357 demonstrated caspase-independent, nonapoptotic cancer cell demise associated with the nuclear translocation of apoptosis-inducing factor (AIF). The cytotoxicity of Cmpd10357 in B-ALL cells was inhibited by Necrostatin-1 but not by Necrosulfonamide. These studies suggest that an AIF-mediated, caspase-independent necrosis mechanism of Cmpd10357 in B-ALL could be used in combination with traditional apoptotic chemotherapeutic agents

A Genome-Wide Screen Identifies Yeast Genes Required for Tolerance to Technical Toxaphene, an Organochlorinated Pesticide Mixture

<div><p>Exposure to toxaphene, an environmentally persistent mixture of chlorinated terpenes previously utilized as an insecticide, has been associated with various cancers and diseases such as amyotrophic lateral sclerosis. Nevertheless, the cellular and molecular mechanisms responsible for these toxic effects have not been established. In this study, we used a functional approach in the model eukaryote <i>Saccharomyces cerevisiae</i> to demonstrate that toxaphene affects yeast mutants defective in (1) processes associated with transcription elongation and (2) nutrient utilization. Synergistic growth defects are observed upon exposure to both toxaphene and the known transcription elongation inhibitor mycophenolic acid (MPA). However, unlike MPA, toxaphene does not deplete nucleotides and additionally has no detectable effect on transcription elongation. Many of the yeast genes identified in this study have human homologs, warranting further investigations into the potentially conserved mechanisms of toxaphene toxicity. </p> </div

Determining the toxaphene IC20 for functional profiling.

<p>(<b>A</b>) The structure of two chlorinated congeners present in the toxaphene technical mixture. (<b>B</b>) Representative growth curves in YPD media for the BY4743 wild-type strain treated with toxaphene. For clarity, only the 250, 550, and 1000μM toxaphene doses are shown, although additional curves were performed at 400, 700, and 850μM. (<b>C</b>) The area under the curve (AUC) was calculated at each dose for three independent experiments, expressed as the mean and SE, and plotted as a percentage of the untreated control. The toxaphene IC20 was determined to be 640μM, as indicated by the dashed lines.</p

Toxaphene exhibits synergy with MPA and 4-NQO.

<p>Growth curve assays were performed for three independent cultures of the BY4743 wild-type strain with the indicated compounds. Both MPA (a transcription elongation inhibitor) and 4-NQO (a genotoxicant) displayed synergy with toxaphene, while tunicamycin (an endoplasmic reticulum stressor) did not. Statistical significance was determined with one-way ANOVA with a Tukey post-test. *** represents significance observed at <i>p</i><0.001 between both toxaphene/toxaphene+synergist and synergist/toxaphene+synergist comparisons.</p

Toxaphene does not inhibit transcription elongation.

<p>(<b>A</b>) GLAM-ratios are not altered upon toxaphene treatment. The acid phosphatase activity of long (<i>PHO5-lacZ</i> or <i>PHO5-LAC4</i>) versus short (PHO5) transcriptional units was measured and the means and SE are shown for three independent experiments. The <i>mft1</i>Δ strain was used as a positive control. (<b>B</b>) Toxaphene does not affect RNA processivity. Levels of RNA polymerase II bound to different regions of a long gene were measured by chromatin immunoprecipitation.</p

Transcription elongation mutants are sensitive to toxaphene.

<p>Growth curves for three independent cultures were obtained for the indicated strains and toxaphene concentrations. Mutants with known defects in transcription elongation are sensitive to toxaphene, including members of the THO, Paf1p, SAGA, and TREX-2 complexes. Additional mutants lacking genes implicated in transcription elongation exhibit sensitivity as well. The AUC was calculated for each curve and is shown as a percentage of the untreated strain. Statistical significance between the wild-type and mutant strains was calculated with Student's <i>t</i>-test, where ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. </p

Neither guanine nor uracil rescues toxaphene sensitivity of transcription elongation mutants.

<p>Relative growth ratios (treatment vs. control) to a GFP-expressing wild-type strain were obtained for three independent cultures and the means and SEs are shown. One-way ANOVA followed by a Bonferroni post-test determined statistical significance. ***<i>p</i><0.001 for wild-type/mutant comparisons. (<b>A</b>) The toxaphene sensitivity of the <i>thp2</i>Δ strain cannot be rescued by guanine. YPD media was supplemented with the indicated concentrations of guanine and toxaphene. (<b>B</b>) Uracil cannot reverse the toxaphene sensitivity of the <i>mft1</i>Δ or <i>thp2</i>Δ strains. Uracil and toxaphene were added to YPD media at the indicated concentrations. The impact of toxaphene on <i>mft1</i>Δ and <i>thp2</i>Δ was stronger in these relative growth experiments versus growth curve assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081253#pone-0081253-g003" target="_blank">Figure 3</a>). Most likely, this is due to the competitive nature of the relative growth assays, where even in control experiments, deletions with intrinsic growth defects are outcompeted by wild-type. Exposure to toxaphene likely magnified the relative growth defects between <i>mft1</i>Δ or <i>thp2</i>Δ and the wild-type strain in this assay.</p

Nitrogen utilization and aromatic amino acid synthesis genes are required for toxaphene tolerance.

<p>The AUC was calculated for strains treated with 640μM or 960μM toxaphene and expressed as a percentage of the AUC for the untreated strain. Bars signify the mean and SE for three independent cultures.</p

Biological attributes required for toxaphene tolerance are identified by network mapping.

<p>Cytoscape was used to map fitness scores (the ratio of the log₂ hybridization signals between DMSO and toxaphene exposures) for toxaphene-sensitive strains onto the <i>Saccharomyces cerevisiae</i> BioGRID interaction dataset. A subnetwork (<i>n</i> = 104) containing genetic and physical interactions between the sensitive, non-sensitive, and essential genes was created and significantly overrepresented (<i>p</i> value cutoff of 0.03) GO categories were identified. The green node color corresponds to the GO <i>p</i>-value while the node size correlates to the number of genes in the category. Edge arrows indicate hierarchy of GO terms. Networks for various GO categories are shown, where node color corresponds to deletion strain fitness score and edge defines the type of interaction between the genes.</p