Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.

It has been four decades since the discovery of p53, the designated ‘Guardian of the Genome’. P53 is primarily known as a master transcription factor and critical tumor suppressor, with countless studies detailing the mechanisms by which it regulates a host of gene targets and their consequent signaling pathways. However, transcription-independent functions of p53 also strongly define its tumor-suppressive capabilities and recent findings shed light on the molecular mechanisms hinted at by earlier efforts. This review highlights the transcription-independent mechanisms by which p53 influences the cellular response to genomic instability (in the form of replication stress, centrosome homeostasis, and transposition) and cell death. We also pinpoint areas for further investigation in order to better understand the context dependency of p53 transcription-independent functions and how these are perturbed when TP53 is mutated in human cancer.

IntroductionNucleophosmin (NPM) is a multifunctional protein that resides predominantly in the nucleoli. It performs a multitude of nuclear functions, such as processing and transporting of ribosomal RNA and proteins, 1 molecular chaperoning, 2 and regulating the stability of tumor suppressors, such as p53, 3 ARF, 4 and c-Myc. 5 However, its cytoplasmic role is largely unknown. In up to one-third of patients with acute myeloid leukemia (AML), a heterogeneous frame-shift mutation in the NPM1 gene results in skewed cytoplasmic accumulation of the NPM mutant protein (NPMc), and this is thought to function in cancer pathogenesis. 6 As NPM1 is the most frequently mutated gene in AML with a normal karyotype, uncovering NPM's diverse cellular functions may hold the key to unraveling specific leukemogenic mechanisms involving the mutant protein.Current AML research centers on the effect of nuclear NPM deprivation on genetic instability, derailment of cell cycle, and cancer progression. NPMc mutants were demonstrated to retain functional interactions with both their nuclear partners and wild-type NPM. Massive dislocation of the mutant protein to the cytoplasm was observed to deplete tumor suppressors and negative controllers of cell proliferation, such as p53, 7 ARF, 8 c-Myc-regulating Fbw7, 5 Miz1, 9 HEXIM1, 10 and NF-B, 11 from the nucleus. The ensuing impairment in cell proliferation control was thus hypothesized to drive carcinogenesis. However, the overexpression of NPMc alone failed to demonstrate any transformation activity or enhancement of cell proliferation 5,7 ; hence, these observations seemed incongruent with the aforementioned hypothesis. Furthermore, loss of p53 from the nucleus, which often results in genetic instability, is also not consistent with the fact that NPMc is exclusively associated with normal karyotypes. 12 More importantly, NPMc mutations are so far identified only in AML, 13 whereas ARF, c-Myc, and p53 defects are found in a multitude of cancers of diverse tissue origins. [14][15][16] Such discrepancies argue for the existence of a hematopoietic-specific mechanism involving the mutant NPM.Here, we identify NPMc as an inhibitor of active caspase-6 and -8, which are pivotal components of cell death signaling. Through the impediment of the caspase signaling cascade, the dislocated NPMc mutant not only raises the threshold for cell death initiation, but more interestingly, hinders the caspase-mediated myeloid differentiation process. Our data thus demonstrate a potential myeloid-specific oncogenic role for the NPMc mutant. Methods ReagentsAntibodies against NPM, caspase-3, -6, and -9 were purchased from Cell Signaling Technology, antibodies against cleaved caspase-3, caspase-8, poly(ADP-ribose) polymerase, and actin from Santa Cruz Biotechnology, antibody against caspase-7 from Neomarker, anti-Fas antibody from Upstate Biotechnology, and antibody against Oct-1 from Chemicon. Recombinant active caspase-3, -6, -7, -8, and -9, recombinant procaspase-3 protein, caspase-6 and -8 inhibitors, and reco...

Mouse double minute (Mdm) genes span an evolutionary timeframe from the ancient eukaryotic placozoa Trichoplax adhaerens to Homo sapiens, implying a significant and possibly conserved cellular role throughout history. Maintenance of DNA integrity and response to DNA damage involve many key regulatory pathways, including precise control over the tumour suppressor protein p53. In most vertebrates, degradation of p53 through proteasomal targeting is primarily mediated by heterodimers of Mdm2 and the Mdm2-related protein Mdm4 (also known as MdmX). Both Mdm2 and Mdm4 have p53-binding regions, acidic domains, zinc fingers, and C-terminal RING domains that are conserved throughout evolution. Vertebrates typically have both Mdm2 and Mdm4 genes, while analyses of sequenced genomes of invertebrate species have identified single Mdm genes, suggesting that a duplication event occurred prior to emergence of jawless vertebrates about 550-440 million years ago. The functional relationship between Mdm and p53 in T. adhaerens, an organism that has existed for 1 billion years, implies that these two proteins have evolved together to maintain a conserved and regulated function.

Although p53 is found mutated in almost 50% of all cancers, p53 mutations in leukaemia are relatively rare. Acute myeloid leukaemia (AML) cells employ other strategies to inactivate their wild type p53 (WTp53), like the overexpression of the p53 negative regulators Mdm2 and Mdm4. As such, AMLs are excellent candidates for therapeutics involving the reactivation of their WTp53 to restrict and destroy cancer cells, and the Mdm2 antagonist nutlin-3 is one such promising agent. Using AML cell lines with WTp53, we identified stable and high levels of p53 in the OCI/AML-2 cell lines. We demonstrate that this nutlin-3 sensitive cell line overexpressed Mdm4 to sequester, stabilise and inhibit p53 in the cytoplasm. We also show that elevated Mdm4 competed with Mdm2-p53 interaction and therefore extended p53 half-life while preventing p53 transcriptional activity. Our results provide biochemical evidence on the dynamics of the p53-Mdm2-Mdm4 interactions in affecting p53 levels and activity, and unlike previously reported findings derived from genetically manipulated systems, AML cells with naturally high levels of Mdm4 remain sensitive to nutlin treatment.Key PointsEndogenously high levels of Mdm4 inhibit and sequester p53 in AML.High levels of Mdm4 do not block function of Mdm2 inhibitors in AML.

Previous publications on stapled peptide inhibitors against Mdm2/Mdm4-p53 interactions have established that this new class of drugs have the potential to be easily optimised to attain high binding affinity and specificity, but the mechanisms controlling their cellular uptake and target engagement remain elusive and controversial. To aid in understanding the rules of peptide and staple design, and to enable rapid optimisation, we employed the newly-developed cellular thermal shift assay (CETSA). CETSA was able to validate stapled peptide binding to Mdm2 and Mdm4, and the method was also used to determine the extent of cellular uptake, cellular availability, and intracellular binding of the endogenous target proteins in its native environment. Our data suggest that while the stapled peptides engage their targets intracellularly, more work is needed to improve their cellular entry and target engagement efficiency in vivo. CETSA now provides a valuable tool to optimize such in vivo properties of stapled peptides.

Peptide stapling is am ethod for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However,o btaining ac ell-active inhibitor can require significant optimization. We report anovel stapling technique based on ad ouble strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cellactive stapled peptides.Asaproof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates,a nd simultaneously evaluated in ap 53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. a-Helicity was confirmed by ac rystal structure of the MDM2-peptide complex. This work introduces in situ stapling as av ersatile biocompatible techniquew ith many other potential high-throughput biological applications.

Vemurafenib is a BRAF kinase inhibitor (BRAFi) that is used to treat melanoma patients harboring the constitutively active BRAF-V600E mutation. However, after a few months of treatment patients often develop resistance to vemurafenib leading to disease progression. Sequence analysis of drug-resistant tumor cells and functional genomic screens has identified several genes that regulate vemurafenib resistance. Reactivation of mitogen-activated protein kinase (MAPK) pathway is a recurrent feature of cells that develop resistance to vemurafenib. We performed a genome-scale CRISPR-based knockout screen to identify modulators of vemurafenib resistance in melanoma cells with a highly improved CRISPR sgRNA library called Brunello. We identified 33 genes that regulate resistance to vemurafenib out of which 14 genes have not been reported before. Gene ontology enrichment analysis showed that the hit genes regulate histone modification, transcription and cell cycle. We discuss how inactivation of hit genes might confer resistance to vemurafenib and provide a framework for follow-up investigations.