Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.United States. Dept. of Defense (Grant W81-XWH-13-1-0323)National Cancer Institute (U.S.) (Cancer Center Support Grant P30-CA14051

Effect of Chromosome Tethering on Nuclear Organization in Yeast

<div><p>Interphase chromosomes in <i>Saccharomyces cerevisiae</i> are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB) at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the silent mating locus (<i>HML</i>) on chromosome III in wild–type and mutant yeast strains that contain altered chromosome-tethering interactions. In wild-type yeast cells we find that disruption of the telomere tether does not dramatically change the position of <i>HML</i> with respect to the SPB, in agreement with theoretical predictions. Alternatively, using a mutant strain with a synthetic tether that localizes an <i>HML</i>-proximal site to the nuclear envelope, we find a significant change in the SPB-<i>HML</i> distance, again as predicted by theory. Our study quantifies the importance of tethering at telomeres on the organization of interphase chromosomes in yeast, which has been shown to play a significant role in determining chromosome function such as gene expression and recombination.</p></div

Random walk model of yeast chromosomes.

<p>A single arm of the yeast interphase chromosome is modeled as a random-walk polymer confined to a sphere of radius <i>R</i> and tethered at its ends to the surface of the sphere. The spindle pole body (SPB) tether (gray circle) is positioned at the north pole while the telomere tether (gray circle) is allowed to take any position on the surface of the sphere. The random walk polymer is made up of rigid segments of equal length (Kuhn length) connected by flexible links. In addition to spherical confinement, an impenetrable sub volume (red spherical cap) representing the nucleolar region limits the space available for the chromosome.</p

The effect of telomere tethering on gene positioning.

<p>A) Heat maps of the probability distributions for the position of genetic loci within the nucleus. The genes are located along a 100 kb chromosome arm at distances 0 kb, 10 kb, 20 kb, 40 kb and 60 kb away from the telomere. The probability distribution is projected to a plane that contains the north-south direction defined by the SPB and the nucleolus position, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102474#pone-0102474-g001" target="_blank">Figure 1</a>). The relative probability density (normalized by the maximum) is shown for one half the nuclear sphere while the other half is equivalent by symmetry. For each gene, we show its spatial distribution when the telomere is attached to the nuclear envelope, and when the telomere is not attached. The “difference” heat maps were calculated by subtracting the “no tether” heat map from the “with tether” heat map – i.e. they show the change in the spatial distribution of the gene upon attachment of the telomere to the nuclear envelope. B) The root-mean-square of the probability difference (RMSDs) map quickly decays as the gene is moved away from the telomere. For all genetic loci, except the ones at 0 and 3 kb away from the telomere, the decay of the RMSD with increasing distance from the telomere is roughly exponential with a characteristic length of 20 kb. (The best fitting line shown in the figure is fit to all points except the point at 0 and 3 kb.) When calculating RMSDs, we do not apply the normalization mentioned above in which the maximum probability density of each “no tether” heat map is assigned a value of 1. Rather, we use the absolute probabilities for each pixel when subtracting the “no tether” heat maps from the “with tether” heat maps to obtain the “difference” heat maps.</p

Quantitative fluorescent microscopy of the spindle pole body (SPB) and an <i>HML</i> proximal locus.

<p>A) Schematic view of budding yeast chromosome III (top line indicates the distance of each locus from the left telomere end in kb). 256 tandem repeats of Lac operators are inserted at a site 1.5 kb proximal to HML. Expression of GFP-fused to LacI or LacI-FFAT marks the locus in the proximity of HML. SPB component SPC29 is fused with RFP. B) Representative wide field microscopy images of yeast strain YDB271 are shown; top left: bright field, top right: green channel, bottom left: red channel and bottom right: merged and pseudo colored view of fluorescence channels red and green (scale bar 1 micrometer). Unbudded and G1 (cells with no duplicated SPB) – marked with boxes 1 to 4 – were selected to be analyzed for distance measurements. C) Experimental distributions of SPB-<i>HML</i> distances of 1,266 wild type (red bars) and 1,049 <i>yku80/esc1</i> double mutant (blue bars) cells. Error bars represent counting errors, which we estimated as twice the standard deviation of the number of measurements of distance that falls into each bin, calculated from the binomial distribution. The Kolmogorov-Smirnov test was used to check if these two data sets are indeed from a different distribution and it returned a p-value of 0.011. D) Experimental distributions of SPB-<i>HML</i> distances in case of 657 cells with <i>HML</i> tethering via LacI-FFAT-GFP bound to the <i>HML</i> proximal <i>LacO</i> array in addition to the wild type tethering of telomeres (green), and for 1049 <i>yku80</i>Δ <i>esc1</i>Δ double mutant cells (blue; same as in Figure 4C). Error bars are calculated as explained in C. The Kolmogorov-Smirnov test for these two data sets returns a p-value of 3.5×10⁻⁹, much lower than obtained by comparing the tethered and untethered distributions in Figure 4C.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Comparison of theoretical and experimental distributions.

<p>Column (A): Telomere tethered – wild type; column (B): untethered - <i>yku80/esc1</i> double mutant; column (C): Telomere and <i>LacO</i> tethered – mutant carrying LacI-FFAT-GFP. Top row: a schematic diagram of the polymer models used for each strain. Bottom row: comparison of the experimental PDFs for wild type (red), <i>yku80/esc1</i> double mutant (blue), and mutant carrying LacI-FFAT-GFP (green) cells, and the corresponding theoretical PDFs (black curves in each graph). The parameters of the model are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102474#pone-0102474-t001" target="_blank">Table 1</a>. The p-values for the one-sample Kolmogorov-Smirnov test that compares the experimental and corresponding theoretical distributions, are 5.4×10⁻⁷ for the wild type, 4.9×10⁻³ for untethered telomere mutant, and 3.3×10⁻⁸ for the <i>HML</i>-bound mutant (these p-values are also shown in the plots).</p

Model Parameters.

<p>Model Parameters.</p