Iatrogenic endometriosis harbors somatic cancer-driver mutations.

STUDY QUESTION: Does incisional endometriosis (IE) harbor somatic cancer-driver mutations? SUMMARY ANSWER: We found that approximately one-quarter of IE cases harbor somatic-cancer mutations, which commonly affect components of the MAPK/RAS or PI3K-Akt-mTor signaling pathways. WHAT IS KNOWN ALREADY: Despite the classification of endometriosis as a benign gynecological disease, it shares key features with cancers such as resistance to apoptosis and stimulation of angiogenesis and is well-established as the precursor of clear cell and endometrioid ovarian carcinomas. Our group has recently shown that deep infiltrating endometriosis (DE), a form of endometriosis that rarely undergoes malignant transformation, harbors recurrent somatic mutations. STUDY DESIGN, SIZE, DURATION: In a retrospective study comparing iatrogenically induced and endogenously occurring forms of endometriosis unlikely to progress to cancer, we examined endometriosis specimens from 40 women with IE and 36 women with DE. Specimens were collected between 2004 and 2017 from five hospital sites in either Canada, Germany or the Netherlands. IE and DE cohorts were age-matched and all women presented with histologically typical endometriosis without known history of malignancy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Archival tissue specimens containing endometriotic lesions were macrodissected and/or laser-capture microdissected to enrich endometriotic stroma and epithelium and a hypersensitive cancer hotspot sequencing panel was used to assess for presence of somatic mutations. Mutations were subsequently validated using droplet digital PCR. PTEN and ARID1A immunohistochemistry (IHC) were performed as surrogates for somatic events resulting in functional loss of respective proteins. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, we detected somatic cancer-driver events in 11 of 40 (27.5%) IE cases and 13 of 36 (36.1%) DE cases, including hotspot mutations in KRAS, ERBB2, PIK3CA and CTNNB1. Heterogeneous PTEN loss occurred at similar rates in IE and DE (7/40 vs 5/36, respectively), whereas ARID1A loss only occurred in a single case of DE. While rates of detectable somatic cancer-driver events between IE and DE are not statistically significant (P > 0.05), KRAS activating mutations were more prevalent in DE. LIMITATIONS, REASONS FOR CAUTION: Detection of somatic cancer-driver events were limited to hotspots analyzed in our panel-based sequencing assay and loss of protein expression by IHC from archival tissue. Whole genome or exome sequencing, or epigenetic analysis may uncover additional somatic alterations. Moreover, because of the descriptive nature of this study, the functional roles of identified mutations within the context of endometriosis remain unclear and causality cannot be established. WIDER IMPLICATIONS OF THE FINDINGS: The alterations we report may be important in driving the growth and survival of endometriosis in ectopic regions of the body. Given the frequency of mutation in surgically displaced endometrium (IE), examination of similar somatic events in eutopic endometrium, as well as clinically annotated cases of other forms of endometriosis, in particular endometriomas that are most commonly linked to malignancy, is warranted. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by a Canadian Cancer Society Impact Grant [701603, PI Huntsman], Canadian Institutes of Health Research Transitional Open Operating Grant [MOP-142273, PI Yong], the Canadian Institutes of Health Research Foundation Grant [FDN-154290, PI Huntsman], the Canadian Institutes of Health Research Project Grant [PJT-156084, PIs Yong and Anglesio], and the Janet D. Cottrelle Foundation through the BC Cancer Foundation [PI Huntsman]. D.G. Huntsman is a co-founder and shareholder of Contextual Genomics Inc., a for profit company that provides clinical reporting to assist in cancer patient treatment. R. Aguirre-Hernandez, J. Khattra and L.M. Prentice have a patent MOLECULAR QUALITY ASSURANCE METHODS FOR USE IN SEQUENCING pending and are current (or former) employees of Contextual Genomics Inc. The remaining authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Biophysical investigation of the mode of inhibition of tetramic acids, the allosteric inhibitors of undecaprenyl pyrophosphate synthase.

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C(55) undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS.TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS.TA complex, but the quaternary complex, UPPS.TA.FPP.IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.

Rapid discovery of potent sulfotransferase inhibitors by diversity-oriented reaction in microplates followed by in situ screening.

Rapid diversity-oriented microplate library synthesis and in situ screening with a high-throughput fluorescence-based assay were used to develop potent inhibitors of beta-arylsulfotransferase IV (beta-AST-IV). This strategy leads to facile inhibitor synthesis and study as it allows protecting-group manipulation and product isolation from other library components to be avoided. Through repeated library formation, three aspects of inhibitor makeup, the identities of the two binding groups and the length of the linker between them, were independently optimized. Several potent inhibitors were obtained, one of which was determined to have an inhibition constant K(i) of 5 nM. This compound is the most potent beta-AST-IV inhibitor developed to date, with a K(i) value more than five orders of magnitude lower than the Michaelis constant K(m) for the substrate whose binding it inhibits.

High-throughput identification of fucosyltransferase inhibitors using carbohydrate microarrays.

A noncovalent carbohydrate microarray was used to screen for possible inhibitors to fucosyltransferases, which are critical to the synthesis of inflammation mediators like sialyl Lewis x (SLe(x)). Inhibition was followed by observation of the transferred fucose on the carbohydrate array with the lectin Tetragonolobus purpureas. Of these compounds, four inhibitors with nanomolar Ki(s) were discovered, with three of the top five inhibitors exhibiting a common architecture.

Inhibition of the proteolytic activity of anthrax lethal factor by aminoglycosides.

The anthrax lethal factor (LF), a Zn-dependent endopeptidase, is considered the dominant virulence factor of anthrax. Because pharmacological inhibition of the catalytic activity of LF is considered a plausible mechanism for preventing the lethality of anthrax, a high-throughput screening experiment based on LF-catalyzed cleavage of a fluorescent substrate was performed to identify novel inhibitors of LF. The RNA-targeting antibiotics, neomycin B and some synthetic dimeric aminoglycosides, were found to be nanomolar active-site inhibitors of LF.

Synthesis of N-acetyllactosamine derivatives with variation in the aglycon moiety for the study of inhibition of sialyl Lewis x expression.

Herein we describe an inhibition study of the sialyl Lewis x (sLe(x)) expression on a human monocytic cell line (U937), using a series of peracetylated N-Acetyllactosamine (LacNAc) analogues with variation at the aglycon moiety. It was found that the extent of inhibition was related to the hydrophobicity and structure of the aglycon. In general, peracetylated LacNAc analogues with a naphthyl or biphenyl aglycon (3, 4, 6, and 7) were better in suppression of sLe(x) expression than a benzyl derivative (2). Steady-state kinetic experiments with human alpha-1,3-fucosyltransferases IV and VI (FucT IV and VI, EC 2.4.1.65) revealed that the deacetylated LacNAc-aglycons with naphthyl (18, 19, and 20) or biphenyl (17) moieties exhibited higher affinity to the fucosyltransferases than aglycon moieties with smaller hydrophobic groups (14, 15, and 16). These results are in agreement with the findings of the U937 cell-based experiments, and suggest that the higher enzyme affinity LacNAc-aglycons make better acceptor decoys and, hence, the observed differences in LacNAc-aglycon inhihitory effects on sLe(x) expression.

A potent and highly selective inhibitor of human alpha-1,3-fucosyltransferase via click chemistry.

Potent inhibitors of fucosyltransferases, and glycosyltransferases in general, have been elusive due to the inherent barriers surrounding the family of glycosyltransfer reactions. The problems of weak substrate affinity and low catalytic proficiency of fucosyltransferase was offset by recruiting additional binding features, in this case hydrophobic interactions, to produce a high affinity inhibitor, 24, with Ki = 62 nM. The molecule was identified from a GDP-triazole library of 85 compounds, which was produced by the Cu(I)-catalyzed [2 + 3] cycloaddition reaction between azide and acetylene reactants, followed by in situ screening without product isolation.