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Summary: Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERβ loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERβ and act as ERβ antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives. [ABSTRACT FROM AUTHOR]

Summary: To increase the chemical diversity of bioactive molecules by incorporating unusual elements, we have examined the replacement of a C=C double bond with the isoelectronic, isostructural B-N bond in the context of nonsteroidal estrogen receptor (ER) ligands. While the B-N bond was hydrolytically labile in the unhindered cyclofenil system, the more hindered anilino dimesitylboranes, analogs of triarylethylene estrogens, were easily prepared, hydrolytically stable, and demonstrated substantial affinity for ERs. X-ray analysis of one ERα-ligand complex revealed steric clashes with the para methyl groups distorting the receptor; removal of these groups resulted in an increase in affinity, potency, and transcriptional efficacy. These studies define the structural determinants of stability and cellular bioactivity of a B-N for C=C substitution in nonsteroidal estrogens and provide a framework for further exploration of “elemental isomerism” for diversification of drug-like molecules. [Copyright &y& Elsevier]

The initial step in estrogen-regulated transcription is the binding of a ligand to its cognate receptors, named estrogen receptors (ERα and ERβ). Phytochemicals present in foods and environment can compete with endogenous hormones to alter physiological responses. We screened 224 flavonoids in our engineered biosensor ERα and ERβ PRL-array cell lines to characterize their activity on several steps of the estrogen signaling pathway. We identified 83 and 96 flavonoids that can activate ERα or ERβ, respectively. While most act on both receptors, many appear to be subtype-selective, including potent flavonoids that activate ER at sub-micromolar concentrations. We employed an orthogonal assay using a transgenic zebrafish in vivo model that validated the estrogenic potential of these compounds. To our knowledge, this is the largest study thus far on flavonoids and the ER pathway, facilitating the identification of a new set of potential endocrine disruptors acting on both ERα and ERβ., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells., Competing Interests: The authors declare no competing interests. Patent No: US 11,428,693 B2; Data of Patent: Aug. 30, 2022 “Method of high throughput screening of chemical compounds suppressing nucleolar hypertrophy” Applicant: Baylor College of Medicine, Houston, TX (US) Inventor: Tattym E. Shaiken, Houston, TX (US) Patent materials were incorporated, in part, in the Result section “Assessment of protein synthesis in the cytosol and the cytomatrix”, (© 2023 The Author(s).)

The United States Environmental Protection Agency (EPA) has been pursuing new high throughput in vitro assays to characterize endocrine disrupting chemicals (EDCs) that interact with estrogen receptor signaling. We characterize two new PRL-HeLa cell models expressing either inducible C-terminal (iGFP-ER) or N-terminal (iER-GFP) tagged estrogen receptor-α (ERα) that allows direct visualization of chromatin binding. These models are an order of magnitude more sensitive, detecting 87 - 93% of very weak estrogens tested compared to only 27% by a previous PRL-HeLa variant and compares favorably to the 73% detected by an EPA-developed computational model using in vitro data. Importantly, the chromatin binding assays distinguished agonist- and antagonist-like phenotypes without activity specific assays. Finally, analysis of complex environmentally relevant chemical mixtures demonstrated how chromatin binding data can be used in risk assessment models to predict activity. These new assays should be a useful in vitro tool to screen for estrogenic activity., Competing Interests: The authors declare no competing interests., (© 2022.)

Transcription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop., Competing Interests: C.Y.L. is an inventor of intellectual property licensed to Syros Pharmaceuticals, and is the Vice President of Biology and a shareholder of Kronos Bio, Inc. S.L.S. is a shareholder and serves on the Board of Directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics and Decibel Therapeutics; is a shareholder and advises Kojin Therapeutics, Kisbee Therapeutics, Exo Therapeutics, and Eikonizo Therapeutics; serves on the Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma Foundation, Biogen, Inc., and F-Prime Capital Partners and the Board of Advisers of the Genomics Institute of the Novartis Research Foundation; and is a Novartis Faculty Scholar. M.J.W. is an employee and equity holder of Kojin Therapeutics. P.A.C. is an advisor to Pfizer, Inc. and nference, Inc. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. P.W. is a current employee of The Institute of Cancer Research, which has a Rewards to Inventors scheme and has a commercial interest in development of inhibitors of the WNT pathway, CDK8/19, and other CDKs, with intellectual property licensed to Merck and Cyclacel Pharmaceuticals. P.W. is also a consultant for Astex Pharmaceuticals, CV6 Therapeutics, Nextechinvest, Nuevolution, Black Diamond, and STORM Therapeutics and holds equity in Chroma Therapeutics, Nextech, Black Diamond, and STORM. B.N. is an inventor on patent applications related to the dTAG system (WO/2017/024318, WO/2017/024319, WO/2018/148443, and WO/2018/148440). N.S.G. is a Scientific Founder, member of the Scientific Advisory Board (SAB), and equity holder in C4 Therapeutics, Syros, Soltego (board member), B2S, Allorion, and Inception. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voroni, Arbella, Deerfield, and Sanofi., (© 2020 The Authors.)

Telomere shortening is associated with stem cell decline, fibrotic disorders, and premature aging through mechanisms that are incompletely understood. Here, we show that telomere shortening in livers of telomerase knockout mice leads to a p53-dependent repression of all seven sirtuins. P53 regulates non-mitochondrial sirtuins (Sirt1, 2, 6, and 7) post-transcriptionally through microRNAs (miR-34a, 26a, and 145), while the mitochondrial sirtuins (Sirt3, 4, and 5) are regulated in a peroxisome proliferator-activated receptor gamma co-activator 1 alpha-/beta-dependent manner at the transcriptional level. Administration of the NAD(+) precursor nicotinamide mononucleotide maintains telomere length, dampens the DNA damage response and p53, improves mitochondrial function, and, functionally, rescues liver fibrosis in a partially Sirt1-dependent manner. These studies establish sirtuins as downstream targets of dysfunctional telomeres and suggest that increasing Sirt1 activity alone or in combination with other sirtuins stabilizes telomeres and mitigates telomere-dependent disorders., (Copyright © 2019 Elsevier Inc. All rights reserved.)

By integrating growth pathways on which cancer cells rely, steroid receptor coactivators (SRC-1, SRC-2, and SRC-3) represent emerging targets in cancer therapeutics. High-throughput screening for SRC small molecule inhibitors (SMIs) uncovered MCB-613 as a potent SRC small molecule "stimulator" (SMS). We demonstrate that MCB-613 can super-stimulate SRCs' transcriptional activity. Further investigation revealed that MCB-613 increases SRCs' interactions with other coactivators and markedly induces ER stress coupled to the generation of reactive oxygen species (ROS). Because cancer cells overexpress SRCs and rely on them for growth, we show that we can exploit MCB-613 to selectively induce excessive stress in cancer cells. This suggests that over-stimulating the SRC oncogenic program can be an effective strategy to kill cancer cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)