The cohesin complex consists of multiple core subunits that play critical roles in mitosis and transcriptional regulation. The cohesin-associated protein Wapal plays a central role in off-loading cohesin to facilitate sister chromatid separation, but its role in regulating mammalian gene expression is not understood. We used embryonic stem cells as a model, given that the well-defined transcriptional regulatory circuits were established through master transcription factors and epigenetic pathways that regulate their ability to maintain a pluripotent state.
RNAi-mediated depletion of Wapal causes a loss of pluripotency, phenocopying loss of core cohesin subunits. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq), we determine that Wapal occupies genomic sites distal to genes in combination with CTCF and core cohesin subunits such as Rad21. Interestingly, genomic sites occupied by Wapal appear enriched for cohesin, implying that Wapal does not off-load cohesin at regions it occupies. Wapal depletion induces derepression of Polycomb group (PcG) target genes without altering total levels of Polycomb-mediated histone modifications, implying that PcG enzymatic activity is preserved. By integrating ChIP-seq and gene expression changes data, we identify that Wapal binding is enriched at the promoters of PcG-silenced genes and is required for proper Polycomb repressive complex 2 (PRC2) recruitment. Lastly, we demonstrate that Wapal is required for the interaction of a distal cis-regulatory element (CRE) with the c-Fos promoter.
Collectively, this work indicates that Wapal plays a critical role in silencing of PcG target genes through the interaction of distal CREs with promoters.

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Ultrasound-induced cavitation has found many applications in the field of cancer therapy. One of its beneficial effects is the enhancement of drug intake by tumor cells. Our group has developed a device that can create and control unseeded cavitation in tissue using ultrasound. We conducted experiments on tumor-bearing mice using our device to assess the impact of sonication on the penetration of fluorescent probes into tumor cells. We studied the influence of pressure level, timing of sonication and sonication duration on treatment efficiency. Our results indicate that fluorescent probes penetrate better into tumors exposed to ultrasound. The best results revealed an increase in penetration of 61% and were obtained when sonicating the tumor in presence of the probes with a peak negative pressure at focus of 19 MPa. At this pressure level, the treatment generated only minor skin damage. Treatments could be significantly accelerated as equivalent enhanced penetration of probes was achieved when multiplying the initial raster scan speed by a factor of four.
Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

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Dolastatin 10 is a powerful antineoplastic agent and microtubule inhibitor that was discovered by Pettit et al. and published in 1987. Since then, many research groups have engaged in SAR studies of synthetic analogues, termed "auristatins". It was eventually discovered that auristatins are of great value as payloads in antibody drug conjugates (ADCs), which led to the FDA-approved ADC brentuximab vedotin (Seattle Genetics). Currently, over 30 ADCs in clinical trials employ auristatins as payloads, and there is a great interest in the research community, both on academic and industrial sides, to further study these analogues. This review will provide an overview of the recent advancements in auristatin development spanning a time frame of about the past ten years. The main focus will be to describe structural changes made to the auristatin peptide and their resulting biological activities in tumor cell proliferation assays. Selected ADC examples will also be described.

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The use of predictive preclinical models in drug discovery is critical for compound selection, optimization, preclinical to clinical translation, and strategic decision-making. Trophoblast glycoprotein (TPBG), also known as 5T4, is the therapeutic target of several anticancer agents currently in clinical development, largely due to its high expression in tumors and low expression in normal adult tissues. In this study, mice were engineered to express human TPBG under endogenous regulatory sequences by replacement of the murine Tpbg coding sequence. The gene replacement was considered functional since the hTPBG knockin (hTPBG-KI) mice did not exhibit clinical observations or histopathological phenotypes that are associated with Tpbg gene deletion, except in rare instances. The expression of hTPBG in certain epithelial cell types and in different microregions of the brain and spinal cord was consistent with previously reported phenotypes and expression patterns. In pharmacokinetic studies, the exposure of a clinical-stage anti-TPBG antibody-drug conjugate (ADC), A1mcMMAF, was lower in hTPBG-KI versus wild-type animals, which was evidence of target-related increased clearance in hTPBG-KI mice. Thus, the hTPBG-KI mice constitute an improved system for pharmacology studies with current and future TPBG-targeted therapies and can generate more precise pharmacokinetic and pharmacodynamic data. In general the strategy of employing gene replacement to improve pharmacokinetic assessments should be broadly applicable to the discovery and development of ADCs and other biotherapeutics.

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There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines. We therefore optimized mitogenicity assay conditions for a panel of five cell lines. All cell lines expressed insulin receptors (IR), IGF-I receptors (IGF-IR) and hybrid receptors, and in all cell lines, insulin as well as the comparator compounds X10 and IGF-I caused phosphorylation of the IR as well as IGF-IR. Insulin exhibited mitogenicity EC(50) values in the single-digit nanomolar to picomolar range. We observed correlations across cell types between (i) mitogenic potency of insulin and IGF-IR/IR ratio, (ii) Akt phosphorylation and mitogenic potency and (iii) Akt phosphorylation and IR phosphorylation. Using siRNA-mediated knockdown of IR and IGF-IR, we observed that in HCT 116 cells the IR appeared dominant in driving the mitogenic response to insulin, whereas in MCF7 cells the IGF-IR appeared dominant in driving the mitogenic response to insulin. Together, our results show that the IR as well as IGF-IR may contribute to the mitogenic potency of insulin. While insulin was a more potent mitogen than IGF-I in cells expressing more IR than IGF-IR, the hyper-mitogenic insulin analogue X10 was a more potent mitogen than insulin across all cell types, supporting that the hyper-mitogenic effect of X10 involves the IR as well as the IGF-IR. These results are relevant for preclinical safety assessment of developmental insulin analogues.
Copyright © 2014 John Wiley & Sons, Ltd.

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