p97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation.

The ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5'-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.

Highly Conserved Homotrimer Cavity Formed by the SARS-CoV-2 Spike Glycoprotein: A Novel Binding Site.

An important stage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) life cycle is the binding of the spike (S) protein to the angiotensin converting enzyme-2 (ACE2) host cell receptor. Therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (MD) simulation. The findings indicated that S2 subunit (heptad-repeat 1 (HR1), central helix (CH), and connector domain (CD) domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the receptor binding domain (RBD) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. Interpretations from MD simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an "up" and "down" state. In addition, the trimer cavity may function as a "bouncing spring" that may facilitate the homotrimer spike protein interactions with the ACE2 receptor. The feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. Several hits were identified that have already been validated or suggested to inhibit the SARS-CoV-2 virus in published cell models. In particular, the data suggest an action mechanism for molecules including Chitosan and macrolides such as the mTOR (mammalian target of Rapamycin) pathway inhibitor Rapamycin. These findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (CoV) family of viruses.

An Integrative "Omics" Approach, for Identification of Bona Fides PLK1 Associated Biomarker in Esophageal Adenocarcinoma.

BACKGROUND: The rapid expansion of genome-wide profiling techniques offers the opportunity to utilize various types of information collected in the study of human health and disease. Overexpression of Polo like kinase 1 (PLK1) is associated with esophageal adenocarcinoma (OAC), however biological functions and molecular targets of PLK1 in OAC are still unknown. OBJECTIVES: Here we performed integrative analysis of two "omics" data sources to reveal high-level interactions of PLK1 associated with OAC. METHODS: Initially, quantitative gene expression (RPKM) was measured from transcriptomics data set of four OAC patients. In parallel, alteration in phosphorylation levels was evaluated in the proteomics data set (mass spectrometry) in OAC cell line (PLK1 inhibited). Next, two "omics" data sets were integrated and through comprehensive analysis possible true PLK1 targets that may serve as OAC biomarkers were assembled. RESULTS: Through experimental validation, small ubiquitin-related modifier 1 (SUMO1) and heat shock protein beta-1 (HSPB1) were identified as novel phosphorylation targets of PLK1. Consequently in vivo, in situ and in silico experiments clearly demonstrated the interaction of PLK1 with putative novel targets (SUMO1 and HSPB1). CONCLUSION: Identification of a PLK1 dependent biosignature in OAC with high confidence in two omics levels proven the robustness and efficacy of our integrative approach.

No additional prognostic value for MRE11 in squamous cell carcinomas of the anus treated with chemo-radiotherapy.

This corrects the article DOI: 10.1038/bjc.2017.188.

Radiosensitization In Vivo by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity.

As the population ages, more elderly patients require radiotherapy-based treatment for their pelvic malignancies, including muscle-invasive bladder cancer, as they are unfit for major surgery. Therefore, there is an urgent need to find radiosensitizing agents minimally toxic to normal tissues, including bowel and bladder, for such patients. We developed methods to determine normal tissue toxicity severity in intestine and bladder in vivo, using novel radiotherapy techniques on a small animal radiation research platform (SARRP). The effects of panobinostat on in vivo tumor growth delay were evaluated using subcutaneous xenografts in athymic nude mice. Panobinostat concentration levels in xenografts, plasma, and normal tissues were measured in CD1-nude mice. CD1-nude mice were treated with drug/irradiation combinations to assess acute normal tissue effects in small intestine using the intestinal crypt assay, and later effects in small and large intestine at 11 weeks by stool assessment and at 12 weeks by histologic examination. In vitro effects of panobinostat were assessed by qPCR and of panobinostat, TMP195, and mocetinostat by clonogenic assay, and Western blot analysis. Panobinostat resulted in growth delay in RT112 bladder cancer xenografts but did not significantly increase acute (3.75 days) or 12 weeks' normal tissue radiation toxicity. Radiosensitization by panobinostat was effective in hypoxic bladder cancer cells and associated with class I HDAC inhibition, and protein downregulation of HDAC2 and MRE11. Pan-HDAC inhibition is a promising strategy for radiosensitization, but more selective agents may be more useful radiosensitizers clinically, resulting in fewer systemic side effects. Mol Cancer Ther; 17(2); 381-92. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

No additional prognostic value for MRE11 in squamous cell carcinomas of the anus treated with chemo-radiotherapy.

BACKGROUND: The majority of anal cancers (84-95%) are driven by infection with human papillomavirus (HPV). HPV-positive tumours show significantly better responses to chemo-radiotherapy when compared with HPV-negative tumours. HPV infection is linked to alterations in DNA damage response proteins, including MRE11. MRE11 is a potential predictive biomarker for response to radiotherapy in muscle-invasive bladder cancer and may hold predictive power in other cancers. METHODS: Using a previously reported cohort, we evaluated the levels of MRE11 in anal cancer and assessed its predictive value in this disease. RESULTS: We found no association between the level of MRE11 and relapse-free survival following chemo-radiotherapy. CONCLUSIONS: MRE11 has no predictive value in the analysis of relapse-free survival after chemo-radiotherapy in anal cancer and does not add to the prognostic value of p16 and tumour-infiltrating lymphocyte scores. Further investigation into the role of DNA repair proteins in anal cancer is required.

E3 Ligase cIAP2 Mediates Downregulation of MRE11 and Radiosensitization in Response to HDAC Inhibition in Bladder Cancer.

The MRE11/RAD50/NBS1 (MRN) complex mediates DNA repair pathways, including double-strand breaks induced by radiotherapy. Meiotic recombination 11 homolog (MRE11) is downregulated by histone deacetylase inhibition (HDACi), resulting in reduced levels of DNA repair in bladder cancer cells and radiosensitization. In this study, we show that the mechanism of this downregulation is posttranslational and identify a C-terminally truncated MRE11, which is formed after HDAC inhibition as full-length MRE11 is downregulated. Truncated MRE11 was stabilized by proteasome inhibition, exhibited a decreased half-life after treatment with panobinostat, and therefore represents a newly identified intermediate induced and degraded in response to HDAC inhibition. The E3 ligase cellular inhibitor of apoptosis protein 2 (cIAP2) was upregulated in response to HDAC inhibition and was validated as a new MRE11 binding partner whose upregulation had similar effects to HDAC inhibition. cIAP2 overexpression resulted in downregulation and altered ubiquitination patterns of MRE11 and mediated radiosensitization in response to HDAC inhibition. These results highlight cIAP2 as a player in the DNA damage response as a posttranscriptional regulator of MRE11 and identify cIAP2 as a potential target for biomarker discovery or chemoradiation strategies in bladder cancer. Cancer Res; 77(11); 3027-39. ©2017 AACR.

Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein-protein interactions by the MDM2 ligand nutlin-3.

Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein-protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein-protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein-protein complexes in drug-treated cells.

The use of ion mobility mass spectrometry to probe modulation of the structure of p53 and of MDM2 by small molecule inhibitors.

Developing drug-like molecules to inhibit the interactions formed by disordered proteins is desirable due to the high correlation of disorder with protein implicated in disease, but is challenging due in part to the lack of atomistically resolved and resolvable structures from conformationally dynamic systems. Ion mobility mass spectrometry (IM-MS) is well-positioned to assess protein ligand interactions along with the effect of a given inhibitor on conformation. Here we demonstrate the use of IM-MS to characterize the effect of two inhibitors RITA and Nutlin-3 on their respective binding partners: p53 and MDM2. RITA binds N-terminal transactivation domain of p53 (Np53) weakly, preventing direct observation of the complex in the gas phase. Nonetheless, upon incubation with RITA, we observe an alteration in the charge state distribution and in the conformational distributions adopted by Np53 in the gas phase. This finding supports the hypothesis that RITAs mode of action proceeds via a conformational change in p53. Circular dichroism corroborates our gas phase findings, showing a slight increase in secondary structure content on ligand incubation, and HDX-MS experiments also highlight the dynamic properties of this protein. Using the same approach we present data to show the effect of Nutlin-3 binding to the N-terminal domain of MDM2 (N-MDM2), N-MDM2 presents as at least two conformational families in the absence of Nutlin-3. Upon Nutlin-3 binding, the protein undergoes a compaction event similar to that exhibited by RITA on Np53. This multi-technique approach highlights the inherent disorder in these systems; and in particular exemplifies the power of IM-MS as a technique to study transient interactions between small molecule inhibitors and intrinsically disordered proteins.

A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2.

Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors.

Quantitative mass spectrometry for colorectal cancer proteomics.

This review documents the uses of quantitative MS applied to colorectal cancer (CRC) proteomics for biomarker discovery and molecular pathway profiling. Investigators are adopting various labeling and label-free MS approaches to quantitate differential protein levels in cells, tumors, and plasma/serum. We comprehensively review recent uses of this technology to examine mouse models of CRC, CRC cell lines, their secretomes and subcellular fractions, CRC tumors, CRC patient plasma/serum, and stool samples. For biomarker discovery these approaches are uncovering proteins with potential diagnostic and prognostic utility, while in vitro cell culture experiments are characterizing proteomic and phosphoproteomic responses to disrupted signaling pathways due to mutations or to inhibition of drugable enzymes.

An iTRAQ proteomics screen reveals the effects of the MDM2 binding ligand Nutlin-3 on cellular proteostasis.

Mouse double minute 2 (MDM2) participates in protein synthesis, folding, and ubiquitin-mediated degradation and is therefore a proteostasic hub protein. The MDM2 interactome contains over 100 proteins, yet stratification of dominant MDM2-interacting proteins has not been achieved. 8-plex iTRAQ (nanoLC-MS/MS) of MCF7 cells treated with the MDM2-binding ligand Nutlin-3 identified the most abundant cellular protein changes over early time points; 1,323 unique proteins were identified including 35 with altered steady-state levels within 2 h of Nutlin-3 treatment, identifying a core group of MDM2 related proteins. Six of these proteins were previously identified MDM2 interactors, and the effects of Nutlin-3 on the MDM2-nucleophosmin interaction (NPM) was further validated. This revealed that Nutlin-3 mediates the in vivo conversion of NPM from an oligomer to a monomer as an MDM2-dependent phenomenon, with Nutlin-3 stimulating MDM2 binding to a peptide motif derived from the oligomerization interface of NPM. These data form the first proteomic screen of Nutlin-3 in cells whereby we (i) identify the most abundant MDM2-interacting proteins whose steady-state levels change early after Nutlin-3 treatment; (ii) identify the first protein apart from p53, nucleophosmin (NPM), whose interaction with MDM2 can be stimulated allosterically by Nutlin-3; and (iii) raise the possibility that Nutlin-3 might act as a general agonist of other MDM2 protein-protein interactions.

Chemical states of the N-terminal "lid" of MDM2 regulate p53 binding: simulations reveal complexities of modulation.

Phosphorylation of S17 in the N-terminal "lid" of MDM2 (residues 1-24) is proposed to regulate the binding of p53. The lid is composed of an intrinsically disordered peptide motif that is not resolved in the crystal structure of the MDM2 N-terminal domain. Molecular dynamics simulations of MDM2 provide novel insight into how the lid undergoes complex dynamics depending on its phosphorylation state that have not been revealed by NMR analyses. The difference in charges between the phosphate and the phosphomimetic 'Asp' and the change in shape from tetrahedral to planar are manifested in differences in strengths and durations of interactions that appear to modulate access of the binding site to ligands and peptides differentially. These findings unveil the complexities that underlie protein-protein interactions and reconcile some differences between the biochemical and NMR data suggesting that lid mutation or deletion can change the specific activity of MDM2 and provide concepts for future approaches to evaluate the effects of S17 modification on p53 binding.

The molecular dynamics of MDM2.

The pro-oncogenic signals of a vast number of anti-cancer drug targets are mediated by protein-protein interactions. This has made such targets less attractive to classic drug discovery programmes. New paradigms in the protein science field have revealed, however, that many protein-protein complexes are stabilized by an interaction between an intrinsically disordered peptide motif and a highly structured globular domain. This type of protein-protein interaction embodied by the MDM2-p53 complex can form a drugable interface. Extensive research has already uncovered the structure of the MDM2-bound p53 peptide to create p53 mimetics like Nutlin, but there has been less emphasis on understanding the dynamic nature of MDM2 itself. The work summarized by Joseph et al. forms a comprehensive and innovative roadmap using molecular dynamics simulations that provide solutions for understanding the flexible nature of a peptide-protein interface. This includes concepts on the plasticity of the peptide-binding groove and induced-fit mechanisms that explain the diversity of linear peptide motifs accommodated by globular domains. The success of molecular dynamics should inspire us to build further the structural biology of full-length MDM2 and other challenging oncoproteins for developing rules on how to develop small molecules that allosterically regulate multi-protein complexes.

Five plus three: legislating for the Five Freedoms and the Three Rs--Animal Welfare Act 1999 (New Zealand).

The Animal Welfare Act 1999 (New Zealand) commenced on 1 January 2000. Rather than focusing on punishing cruelty, the Act establishes a positive duty of care that every owner or person in charge of an animal must provide for its physical, health and behavioural needs. The Five Freedoms, which were initiated by the Farm Animal Welfare Council (UK), were modified as the five basic needs of animals, relating to proper and sufficient food and water, adequate shelter, the ability to display normal patterns of behaviour, physical handling that minimises distress and protection from and rapid diagnosis of injury or disease. Minimum standards are provided in a series of codes of welfare, which is tertiary legislation under the Act. Promotion of the Three Rs--reduction, refinement and replacement--first championed by Russell & Burch, have been incorporated as a purpose of Part 6 of the Act, which restricts projects that use animals, and establishes codes of ethical conduct and animal ethics committees. The legislative process that enabled this to be realised is examined and analysed, and the process by which other Commonwealth countries have emulated this legislation is considered.