Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers

There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) + SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and CRC

Additional file 7: Figure S7. of Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing

Multiplex captures have comparable uniformity to individual captures. Uniform coverage enables accurate variant calling with minimal sequencing cost. (PDF 115 kb

Reuse-based Online Models for Caches

We develop a reuse distance/stack distance based analytical modeling framework for efficient, online prediction of cache performance for a range of cache configurations and replacement policies LRU, PLRU, RANDOM, NMRU. Our framework unifies existing cache miss rate prediction techniques such as Smith’s associativity model, Poisson variants, and hardware way-counter based schemes. We also show how to adapt LRU way-counters to work when the number of sets in the cache changes. As an example application, we demonstrate how results from our models can be used to select, based on workload access characteristics, last-level cache configurations that aim to minimize energy-delay product. Categories andSubjectDescriptor

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells

Abstract 3135: <i>In vitro</i> and <i>in vivo</i> synthetic lethal screens to identify novel targets in the context of PTEN deficiency.

Abstract Functional genomic screens have been employed by many groups to identify novel targets for cancer therapeutics. However, translation of these data sets into new drug discovery programs has proved challenging, in part due to difficulties in obtaining strong on-target knockdown, coupled with misleading off-target effects. Most commercially available pooled shRNA screening libraries provide 5-6 shRNAs per gene, with little knowledge of knock-down efficiency. In an effort to overcome these issues, we designed a custom shRNA library targeting 580 human genes, including key nodes in signal transduction pathways and genes of interest for oncology drug development. This pathway-centric design allows us to determine critical pathways for cancer cell growth in addition to individual gene phenotypes. For each gene, we have total of 11 shRNAs, a subset of which have been previously shown high efficiency knock-down. In particular, we emphasized genes where tool or lead compounds were available for rapid follow-up. Both in size and scope, this library was designed to quickly identify important signaling pathways that are essential on their own or when perturbed in the presence of small molecule drugs in our pipeline. With this focused library, we performed functional genomic shRNA screens in PTEN mutant or null lines across cancer types in vitro, including four cell lines that are PTEN deficient (UACC62, MDA-MB-468, U87-MG and PC3), and one cell line, (HCT-116) that is PTEN wild type. Meanwhile, we performed in vivo screens with PC3 cells. To identify hits from our screens, we used an internally developed algorithm, c-FOLD, to calculate p-value and fold changes for each shRNA. As confirmation that our library and methods can identify essential genes in a context specific manner, we observe BRAF as the top essential gene hit in the cell line which contains an activated BRAF allele, UACC62, but not in other lines tested. We then examined the concordance of hits in our in vitro and in vivo experiments with PC3 cells. In general, individual shRNAs were highly correlated between in vitro and in vivo assays, but a subset is specific to one setting, and may present particularly interesting targets. To discover synthetic lethal partners to PTEN deficiency, we required that a gene is essential in all four PTEN deficient lines in vitro, as well as in PC3 in vivo, but not essential in HCT-116. Among the top hits are multiple shRNAs against ENTPD5, an ER enzyme hydrolyzing UDP to UMP. In addition, using our pathway approach, we identified components in JNK pathways as synthetic lethal partners to PTEN deficiency. The results obtained from these studies established a strong platform for in vivo and in vitro identification of novel targets and novel combination partners. These approaches can also be integrated with other Omics data, such as mutation and expression, to ultimately find promising targets to develop novel cancer therapeutics. Citation Format: Jing-xin Zhang, Joshua Murtie, Oleg Iartchouk, Hui Cao, Gary Shapiro, Zhifang Li, Hongyun Wang, Zhihu Ding, Yu-an Zhang, Madelyn Light, Dietmar Hoffman, May Cindhuchao, Saurin Jani, Richard Newcombe, Eva Bric-Furlong, Bin Wu, Angela Virone-Oddos, Stephan Reiling, Joachim Theihaber, Christoph Lengauer, Jack Pollard, James Watters, Serena Silver, Venkat Reddy. In vitro and in vivo synthetic lethal screens to identify novel targets in the context of PTEN deficiency. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3135. doi:10.1158/1538-7445.AM2013-3135</jats:p