Transcriptional Activation Function Research Articles

Sterol Composition Modulates the Response of Saccharomyces cerevisiae to Iron Deficiency.

Iron is a vital micronutrient that functions as an essential cofactor in multiple biological processes, including oxygen transport, cellular respiration, and metabolic pathways, such as sterol biosynthesis. However, its low bioavailability at physiological pH frequently leads to nutritional iron deficiency. The yeast Saccharomyces cerevisiae is extensively used to study iron and lipid metabolisms, as well as in multiple biotechnological applications. Despite iron being indispensable for yeast ergosterol biosynthesis and growth, little is known about their interconnections. Here, we used lipid composition analyses to determine that changes in the pattern of sterols impair the response to iron deprivation of yeast cells. Yeast mutants defective in ergosterol biosynthesis display defects in the transcriptional activation of the iron-acquisition machinery and growth defects in iron-depleted conditions. The transcriptional activation function of the iron-sensing Aft1 factor is interrupted due to its mislocalization to the vacuole. These data uncover novel links between iron and sterol metabolisms that need to be considered when producing yeast-derived foods or when treating fungal infections with drugs that target the ergosterol biosynthesis pathway.

PARROT is a flexible recurrent neural network framework for analysis of large protein datasets.

The rise of high-throughput experiments has transformed how scientists approach biological questions. The ubiquity of large-scale assays that can test thousands of samples in a day has necessitated the development of new computational approaches to interpret this data. Among these tools, machine learning approaches are increasingly being utilized due to their ability to infer complex nonlinear patterns from high-dimensional data. Despite their effectiveness, machine learning (and in particular deep learning) approaches are not always accessible or easy to implement for those with limited computational expertise. Here we present PARROT, a general framework for training and applying deep learning-based predictors on large protein datasets. Using an internal recurrent neural network architecture, PARROT is capable of tackling both classification and regression tasks while only requiring raw protein sequences as input. We showcase the potential uses of PARROT on three diverse machine learning tasks: predicting phosphorylation sites, predicting transcriptional activation function of peptides generated by high-throughput reporter assays, and predicting the fibrillization propensity of amyloid beta with data generated by deep mutational scanning. Through these examples, we demonstrate that PARROT is easy to use, performs comparably to state-of-the-art computational tools, and is applicable for a wide array of biological problems.

Ras/ERK and PI3K/AKT signaling differentially regulate oncogenic ERG mediated transcription in prostate cells

The TMPRSS2/ERG gene rearrangement occurs in 50% of prostate tumors and results in expression of the transcription factor ERG, which is normally silent in prostate cells. ERG expression promotes prostate tumor formation and luminal epithelial cell fates when combined with PI3K/AKT pathway activation, however the mechanism of synergy is not known. In contrast to luminal fates, expression of ERG alone in immortalized normal prostate epithelial cells promotes cell migration and epithelial to mesenchymal transition (EMT). Migration requires ERG serine 96 phosphorylation via endogenous Ras/ERK signaling. We found that a phosphomimetic mutant, S96E ERG, drove tumor formation and clonogenic survival without activated AKT. S96 was only phosphorylated on nuclear ERG, and differential recruitment of ERK to a subset of ERG-bound chromatin associated with ERG-activated, but not ERG-repressed genes. S96E did not alter ERG genomic binding, but caused a loss of ERG-mediated repression, EZH2 binding and H3K27 methylation. In contrast, AKT activation altered the ERG cistrome and promoted expression of luminal cell fate genes. These data suggest that, depending on AKT status, ERG can promote either luminal or EMT transcription programs, but ERG can promote tumorigenesis independent of these cell fates and tumorigenesis requires only the transcriptional activation function.

Mutant p53L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to MortaparibPlus, a Multimodal Small Molecule Inhibitor.

Simple SummaryTumor suppressor protein p53 is a master regulator that inhibits the process of oncogenesis by induction of cell senescence/cell cycle arrest/apoptosis during normal and stressed states of cells. It is functionally inactivated in the majority of cancers. Mortalin, a member of the Hsp70 family of proteins, enriched in cancer cells, is known to cause cytoplasmic sequestration and inactivation of the p53’s transcriptional activation function. Inhibition of mortalin–p53 interaction and reactivation of p53 functions by natural and synthetic drugs has emerged as a possible cancer therapeutic strategy. We recently reported a novel multimodal small molecule, named MortaparibPlus, that inhibited mortalin–p53 interaction and caused reactivation of p53 function in colorectal cancer cells. Here, we report its effect on breast cancer cells with wildtype (MCF-7) or mutant (T47D) p53 status.We previously performed a drug screening to identify a potential inhibitor of mortalin–p53 interaction. In four rounds of screenings based on the shift in mortalin immunostaining pattern from perinuclear to pan-cytoplasmic and nuclear enrichment of p53, we had identified MortaparibPlus (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole) as a novel synthetic small molecule. In order to validate its activity and mechanism of action, we recruited Luminal-A breast cancer cells, MCF-7 (p53wild type) and T47D (p53L194F) and performed extensive biochemical and immunocytochemical analyses. Molecular analyses revealed that MortaparibPlus is capable of abrogating mortalin–p53 interaction in both MCF-7 and T47D cells. Intriguingly, upregulation of transcriptional activation function of p53 (as marked by upregulation of the p53 effector gene—p21WAF1—responsible for cell cycle arrest and apoptosis) was recorded only in MortaparibPlus-treated MCF-7 cells. On the other hand, MortaparibPlus-treated T47D cells exhibited hyperactivation of PARP1 (accumulation of PAR polymer and decrease in ATP levels) as a possible non-p53 tumor suppression program. However, these cells did not show full signs of either apoptosis or PAR-Thanatos. Molecular analyses attributed such a response to the inability of MortaparibPlus to disrupt the AIF–mortalin complexes; hence, AIF did not translocate to the nucleus to induce chromatinolysis and DNA degradation. These data suggested that the cancer cells possessing enriched levels of such complexes may not respond to MortaparibPlus. Taken together, we report the multimodal anticancer potential of MortaparibPlus that warrants further attention in laboratory and clinical studies.

Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes

Steroid hormone receptors activate gene transcription by binding specific DNA sequences and recruiting coactivators to initiate transcription of their target genes. For most nuclear hormone receptors (NRs), the ligand-dependent activation function domain-2 (AF-2), residing in the C-terminal ligand binding domain (LBD), is a primary contributor to the NR transcriptional activity. In contrast to other steroid receptors such as estrogen receptor-α (ERα), the transcriptional activation function of androgen receptor (AR) is thought to be largely dependent on its ligand-independent activation function domain-1 (AF-1) located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different activation function domain from other steroid receptors despite the fact that NRs share similar domain organizations and have a highly conserved DNA binding domain (DBD) and LBD. Here we present cryo-electron microscopic structures of DNA-bound full-length AR and its functional complex structure with its key coactivators, steroid receptor coactivator-3 (SRC-3) and the histone acetyltransferase p300. The structures reveal that androgen-bound full-length AR dimerization follows a unique head-to-head and tail-to-tail manner with all of the domains involved. The DBD and LBD are located at the center of the dimer interface. The NTDs wrap around the LBDs through their unique intra- and inter-molecular N- and C-terminal interactions and connect to each other. We observe that unlike ERα, AR binds a single SRC-3 molecule along with a single p300 molecule. The AR NTD appears to be the primary site for recruitment of both coactivators. The N-terminal region of SRC-3, rather than its receptor interaction domain, is important for this interaction. The structures presented here highlight the importance of the AR NTD for its transcriptional functions and provide a structural basis for understanding the assembly of the AR:coactivator complex and its domain contributions to transcriptional regulation.

Transcriptional and Non-Transcriptional Activation, Posttranslational Modifications, and Antiviral Functions of Interferon Regulatory Factor 3 and Viral Antagonism by the SARS-Coronavirus.

The immune system defends against invading pathogens through the rapid activation of innate immune signaling pathways. Interferon regulatory factor 3 (IRF3) is a key transcription factor activated in response to virus infection and is largely responsible for establishing an antiviral state in the infected host. Studies in Irf3−/− mice have demonstrated the absence of IRF3 imparts a high degree of susceptibility to a wide range of viral infections. Virus infection causes the activation of IRF3 to transcribe type-I interferon (e.g., IFNβ), which is responsible for inducing the interferon-stimulated genes (ISGs), which act at specific stages to limit virus replication. In addition to its transcriptional function, IRF3 is also activated to trigger apoptosis of virus-infected cells, as a mechanism to restrict virus spread within the host, in a pathway called RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA). These dual functions of IRF3 work in concert to mediate protective immunity against virus infection. These two pathways are activated differentially by the posttranslational modifications (PTMs) of IRF3. Moreover, PTMs regulate not only IRF3 activation and function, but also protein stability. Consequently, many viruses utilize viral proteins or hijack cellular enzymes to inhibit IRF3 functions. This review will describe the PTMs that regulate IRF3′s RIPA and transcriptional activities and use coronavirus as a model virus capable of antagonizing IRF3-mediated innate immune responses. A thorough understanding of the cellular control of IRF3 and the mechanisms that viruses use to subvert this system is critical for developing novel therapies for virus-induced pathologies.

Aberrant cytoplasmic localization of ARID1B activates ERK signaling and promotes oncogenesis.

The ARID1B (BAF250b) subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. We employed in silico prediction, intracellular fluorescence and cellular fractionation-based subcellular localization analyses to identify the ARID1B nuclear localization signal (NLS). A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised in its canonical transcription activation and tumor suppressive functions, as expected. Surprisingly however, cytoplasmic localization appeared to induce a gain of oncogenic function for ARID1B, as evidenced from several cell line- and mouse xenograft-based assays. Mechanistically, cytoplasm-localized ARID1B could bind c-RAF (RAF1) and PPP1CA causing stimulation of RAF-ERK signaling and β-catenin (CTNNB1) transcription activity. ARID1B harboring NLS mutations derived from tumor samples also exhibited aberrant cytoplasmic localization and acquired a neo-morphic oncogenic function via activation of RAF-ERK signaling. Furthermore, immunohistochemistry on a tissue microarray revealed significant correlation of ARID1B cytoplasmic localization with increased levels of active forms of ERK1 and ERK2 (also known as MAPK3 and MAPK1) and of β-catenin, as well as with advanced tumor stage and lymph node positivity in human primary pancreatic tumor tissues. ARID1B therefore promotes oncogenesis through cytoplasm-based gain-of-function mechanisms in addition to dysregulation in the nucleus.This article has an associated First Person interview with the first author of the paper.

English

Abiotic stresses like drought affect plant growth and crop yield with climate change worsening. Stress associated proteins (SAPs), as the zinc finger proteins with A20/AN1 domain, play an important role in regulating abiotic stress response. As a typical summer dryland grain crop in the north of China, foxtail millet has the characteristics of drought resistance, making it a valuable resource for anti-stress gene exploitation and utilization. In this study, SiSAP4 gene was cloned from foxtail millet variety Yugu 1. Analysis showed that SiSAP4 gene was expressed in roots, stems and leaves at seedling stage, and the highest expression level was detected in leaves. Expression patterns under different stress conditions showed that expression level of SiSAP4 gene was significantly up-regulated under drought stress, suggesting it may be involved in drought stress response. Subcellular localization indicated that SiSAP4 was present in the nucleus and cytoplasm. It was revealed that SiSAP4 had no function in transcriptional activation in the yeast system. Overexpression of SiSAP4 in transgenic Arabidopsis resulted in enhanced tolerance to drought stress, which was simultaneously demonstrated by increased expression of a broad range of stress response genes. Based on those results, SiSAP4 has the potential to be used in transgenic breeding to improve drought stress tolerance in other crops. © 2021 Friends Science Publishers

Natural variation in the promoter of GsERD15B affects salt tolerance in soybean.

SummarySalt stress has detrimental effects on crop growth and yield, and the area of salt‐affected land is increasing. Soybean is a major source of vegetable protein, oil and feed, but considered as a salt‐sensitive crop. Cultivated soybean (Glycine max) is domesticated from wild soybean (G. soja) but lost considerable amount of genetic diversity during the artificial selection. Therefore, it is important to exploit the gene pool of wild soybean. In this study, we identified 34 salt‐tolerant accessions from wild soybean germplasm and found that a 7‐bp insertion/deletion (InDel) in the promoter of GsERD15B (early responsive to dehydration 15B) significantly affects the salt tolerance of soybean. GsERD15B encodes a protein with transcriptional activation function and contains a PAM2 domain to mediate its interaction with poly(A)‐binding (PAB) proteins. The 7‐bp deletion in GsERD15B promoter enhanced the salt tolerance of soybean, with increased up‐regulation of GsERD15B, two GmPAB genes, the known stress‐related genes including GmABI1, GmABI2, GmbZIP1, GmP5CS, GmCAT4, GmPIP1:6, GmMYB84 and GmSOS1 in response to salt stress. We propose that natural variation in GsERD15B promoter affects soybean salt tolerance, and overexpression of GsERD15B enhanced salt tolerance probably by increasing the expression levels of genes related to ABA‐signalling, proline content, catalase peroxidase, dehydration response and cation transport.

Application of WES Towards Molecular Investigation of Congenital Cataracts: Identification of Novel Alleles and Genes in a Hospital-Based Cohort of South India.

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2–13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2, TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function.

Discovery and functional analysis of a novel ISL1 variant associated with congenital heart defect

To analyze variation of ISL1 gene and explore its functional characteristics in relation with congenital heart defect (CHD). Clinical data and peripheral blood samples of 194 CHD patients and 232 healthy controls were collected for the extraction of genomic DNA. The coding exons and flanking intronic regions of the ISL1 gene were sequenced. Expression plasmid for the wild-type ISL1 gene ISL1-pcDNA3.1 was constructed, and the corresponding variants were obtained by site-specific mutagenesis. The gene expression plasmid was transfected into CHO cells with liposome, and the functional characteristics of ISL1 variant were studied by double luciferase reporter gene analysis. A novel variant of the ISL1 gene c.499C>T (p.Q167X) was detected in a patient with sporadic CHD. Functional study showed that the variant has lost its transcriptional activation function for the MEF2C promoter. A novel variant of the ISL1 gene related to CHD has been identified. The defect of ISL1 gene may underlay the pathogenesis for a proportion of CHD.

Abstract 4376: OP-1250, a complete estrogen receptor antagonist (CERAN) that shrinks estrogen receptor positive tumors and exhibits favorable pharmacokinetics

Abstract Fulvestrant (FASLODEX), a Complete Estrogen Receptor ANtagonist (CERAN) is recognized as the most effective endocrine therapy for estrogen receptor positive (ER+) metastatic breast cancer. Unfortunately, fulvestrant must be injected i.m. and has poor drug exposure. Thus, an oral compound that shares fulvestrant's drug virtues with improved drug exposure might be both more effective and more convenient than fulvestrant. This realization has sparked intensive efforts to develop such a compound, however most efforts have focused on discovering novel selective estrogen receptor degraders (SERDS) most of which have also displayed agonist activities on gene expression and uterus, and are therefore are also selective estrogen receptor modulators (SERMs). We predict that CERANs that fully block the activities of estrogen receptor alpha (ERalpha) will have a clinical advantage over SERM/SERDs in shrinking breast tumors as well as preventing and treating endocrine resistance. We have developed a small molecule, OP-1250 that, like fulvestrant, is a CERAN that completely turns off both transcriptional activation functions of ERalpha, as can be seen in a uterine cell assay, and also prevents uterine weight gain in ovariectomized mice. In addition, it blocks degrades ERalpha and blocks the proliferation of a large variety of ER+ breast cells. Using RNAseq, we found many estrogen-regulated biomarkers that are also regulated by SERM/SERDs, however CERANS such as OP-1250 and fulvestrant have no, or even an inverse effect on many such genes. Unlike fulvestrant, OP-1250 gives high and stable oral drug exposure in multiple species. Consequentially, OP-1250 effectively shrinks ER+ tumors in multiple human tumor xenografts in mice at very low daily doses of 3 mg/kg, even in models containing activating ERalpha mutant receptors. Importantly, the pharmacokinetics and biodistribution of OP-1250 has a favorable profile that results in unusual potency of OP-1250 in vivo. Citation Format: Leslie Hodges-Gallagher, Cyrus L. Harmon, Richard Sun, David C. Myles, Peter Kushner. OP-1250, a complete estrogen receptor antagonist (CERAN) that shrinks estrogen receptor positive tumors and exhibits favorable pharmacokinetics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4376.

The zinc finger transcription factor ATF1 regulates aluminum tolerance in barley.

Aluminum (Al) toxicity is a major abiotic stress that restricts crop production in acid soils. Plants have evolved internal and external mechanisms of tolerance, and among them it is well known that AtSTOP1 and OsART1 are key transcription factors involved in tolerance through regulation of multiple downstream genes. Here, we identified the closest homolog of these two proteins in barley, namely HvATF1, Al-tolerance Transcription Factor 1, and determined its potential function in Al stress. HvATF1 is expressed in the nucleus, and functions in transcriptional activation. The transcription of HvATF1 was found to be constitutive in different tissues, and was little affected by Al stress. Knockdown of HvATF1 by RNAi resulted in increased Al sensitivity. Transcriptomics analysis identified 64 differently expressed genes in the RNAi lines compared to the wild-type, and these were considered as candidate downstream genes regulated by HvATF1. This study provides insights into the different molecular mechanisms of Al tolerance in barley and other plants.

Chromatin remodeler Ino80C acts independently of H2A.Z to evict promoter nucleosomes and stimulate transcription of highly expressed genes in yeast

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.

Genome-wide identification and transcript profiles of walnut heat stress transcription factor involved in abiotic stress

BackgroundWalnut (Juglans regia) is an important tree cultivated worldwide and is exposed to a series of both abiotic and biotic stress during their life-cycles. The heat stress transcription factors (HSFs) play a crucial role in plant response to various stresses by regulating the expression of stress-responsive genes. HSF genes are classified into 3 classes: HSFA, HSFB, and HSFC. HSFA gene has transcriptional activation function and is the main regulator of high temperature-induced gene expression. HSFB gene negatively regulates plant resistance to drought and NaCl. And HSFC gene may be involved in plant response to various stresses. There are some reports about the HSF family in herbaceous plants, however, there are no reports about the HSFs in walnut.ResultIn this study, based on the complete genome sequencing of walnut, the bioinformatics method was used and 29 HSF genes were identified. These HSFs covered 18 HSFA, 9 HSFB, and 2 HSFC genes. Phylogenetic analysis of these HSF proteins along with those from Arabidopsis thaliana showed that the HSFs in the two species are closely related to each other and have different evolutionary processes. The distribution of conserved motifs and the sequence analysis of HSF genes family indicated that the members of the walnut HSFs are highly conserved. Quantitative Real-Time PCR (qRT-PCR) analysis revealed that the most of walnut HSFs were expressed in the walnut varieties of ‘Qingxiang’ and ‘Xiangling’ under high temperature (HT), high salt and drought stress, and some JrHSFs expression pattern are different between the two varieties.ConclusionThe complex HSF genes family from walnut was confirmed by genome-wide identification, evolutionary exploration, sequence characterization and expression analysis. This research provides useful information for future studies on the function of the HSF genes and molecular mechanism in plant stress response.

A Gatekeeping Role of ESR2 to Maintain the Primordial Follicle Reserve.

Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of estrogen receptor β (ESR2) signaling results in increased activation of primordial follicles in Esr2-null (Esr2-/-) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wild-type and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2 signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1, indicating that the regulation of primordial follicle activation is ESR2 specific. Follicle activation was also increased in Esr2 mutants lacking the DNA binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2, suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK, and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles.

Advanced maternal age alters expression of maternal effect genes that are essential for human oocyte quality.

To investigate the effects of maternal age on the quality of oocytes, we used single-cell RNA sequencing to detect global gene transcriptome and identify key genes affected by advanced age in human mature oocytes. We isolated mRNA from mature oocytes obtained from IVF or ICSI patients (three oocytes from younger (≤30 years) and three oocytes from older (≥40 years) patients for scRNA-seq. We identified 357 genes differentially expressed between matured oocytes from older and younger women's. The up-regulated genes were significantly enriched with annotations related to transcriptional activation, oxidative stress and immune function, while down-regulated genes were enriched with catalytic activity. The key candidate gene TOP2B was found by protein interaction network analysis, and knockdown verification on younger mouse matured oocytes showed that TOP2B was a key gene affecting the oocyte quality and early embryo development. These results will contribute new knowledge on the molecular mechanisms of female ovary aging and establish a criterion to evaluate the quality of oocytes in women with advanced maternal age.

Abstract P5-05-02: Preclinical development of OP-1250, an oral complete estrogen receptor antagonist (CERAN) that shrinks ER-positive breast tumors in xenograft models

Abstract Fulvestrant (FASLODEX) is the most effective endocrine therapy for estrogen receptor positive (ER+) metastatic breast cancer (MBC). Despite this superiority, fulvestrant must be delivered by intramuscular injection and its efficacy appears limited by poor drug exposure. Thus, an oral compound that shares fulvestrant’s pharmacodynamic virtues with improved drug exposure might be both more convenient and more effective than fulvestrant. Fulvestrant development was aimed at creating a Complete Estrogen Receptor ANtagonist (CERAN) using the rodent uterine weight gain assay. It is now understood that the ability of fulvestrant to shut off both the AF1 and AF2 transcriptional activation functions of the ER underlies this activity. In contrast to CERANs, selective estrogen receptor modulators (SERMs) such as tamoxifen efficiently shut off AF2 signaling (turned on by estradiol), but incompletely antagonize AF1, a function that is turned on by multiple cell signaling pathways and shown to play a role in the development of endocrine resistance. We have developed a sensitive cell culture assay that distinguish between CERANs and SERMs. Fulvestrant is also a Selective Estrogen Receptor Degrader (SERD), and it has become widely but mistakenly assumed that such ability makes a compound a CERAN. In our assay several SERDs under clinical investigation, including AZD9496 and GDC-0810, were identified as SERMs that exhibited ER-mediated agonism on genes dependent on AF1 activity. Further, these SERM/SERDs stimulate cell proliferation of CAMA-1 and HCC-1500 breast cancer cells even in the absence of estrogen. These proliferation assays may model clinical breast cancer progression in which tumors become less dependent on estrogen and more sensitive to AF1 signaling mediated by SRC family kinases, IGFs, and FGFs. We thus predict that SERM/SERDs will be inferior in preventing and treating resistance compared to CERAN/SERDs. Guided by our cell culture assay, we have developed the CERAN/SERD OP-1250. OP-1250 completely blocks both AF2 and AF1 activity as seen in cell culture and the ovex mouse uterine weight gain assay. Unlike SERM/SERDs, OP-1250 is a potent inhibitor of CAMA1 and HCC-1500 breast cancer cell proliferation. OP-1250 also is a SERD in a large variety of cell lines including those in which some SERM/SERDs have little activity. OP-1250 achieves high and stable drug exposure in multiple animal species unlike fulvestrant and many SERMs. As might be expected for a compound with this spectrum of activities, OP-1250 shrinks tumors in xenograft models in which fulvestrant (because of limited drug exposure) and SERM/SERDs (because of residual estrogen-like action) fail to shrink the tumors. These models include HCC-1500 xenografts with a wild type ER and HCI-013 patient-derived xenografts with ESR1Y537S. These results suggest that OP-1250 has potential to be a superior compound to treat ER+ MBC, especially in patients whose tumors allow high activity of AF1 and including those with activating mutations ins ESR1. We expect to bring OP-1250 into the clinic shortly. Citation Format: Leslie Hodges-Gallagher, Richard Sun, David C Myles, Cyrus L Harmon, Peter J Kushner. Preclinical development of OP-1250, an oral complete estrogen receptor antagonist (CERAN) that shrinks ER-positive breast tumors in xenograft models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-02.

Krüppel homologue 1 interacts directly with Hairy and regulates ecdysis in the brown planthopper.

Juvenile hormone (JH) plays important roles in the growth and development of insects. JH and its receptor methoprene-tolerant (Met) regulate the expression of transcription factors to control the transcription of downstream genes. The expression of Hairy (Hry) and Krüppel homologue 1 (Kr-h1) is regulated by JH and JH receptors. Hry and Kr-h1 are both crucial in mediating JH signalling. However, whether they interact at the gene level in regulating metamorphosis and whether they interact physically at the protein level remain unknown. We used co-immunoprecipitation, glutathione S-transferase pull-down and RNA interference (RNAi) approaches to study the genetic and biochemical interactions of the two proteins Hry and Kr-h1. The results showed that brown planthopper (Nilaparvata lugens) Hry and Kr-h1 interact directly: Hry binds to the N-terminal of Kr-h1, which includes five zinc-finger domains. The RNAi experiment showed that downregulation of Hry reduced the ratio of ecdysis failure caused by knockdown of Kr-h1, indicating that the downregulation of Hry might mitigate ecdysis failure via the downregulation of Kr-h1. The expression of Hry increased significantly when Kr-h1 was downregulated, whereas it did not change significantly when both were downregulated. Our results suggest that the binding of Hry protein with Kr-h1 prevents the N-terminal five zinc-finger domains from binding with DNA, which in turn inactivates the transcription activator or inhibitor function of Kr-h1. Hry could possibly be used as a target for pesticide applications in the future.

Structural Insights into the Ligand Binding Domain of the Glucocorticoid Receptor: A Molecular Dynamics Study.

The glucocorticoid receptor (GR) is a ligand-binding dependent transcription factor that ultimately regulates vital biological processes and inflammation response through specific gene expression control, thus representing a notable drug target to explore. Structurally, its ligand binding domain (LBD) harbors the region for the ligand-dependent transcriptional activation function 2 (AF-2), a majorly hydrophobic groove formed by residues from helices H3, H4, and H12, where the H12 position plays a critical role in AF-2 spatial conformation and GR function as a whole. However, the exact mechanisms underlying how regulatory ligands control the H12 structure and dynamics are yet to be elucidated. In this work, we have explored the correlation between ligand identity and GR LBD H12 behavior through different molecular dynamics (MD) simulations. After building diverse GR LBD systems in agonist and nonagonist states, we studied each system's response in the absence or the presence of an agonist ligand (dexamethasone) or an antagonist ligand (RU486) using classical MD simulations. We complemented them with steered MD for assessing the transition between those states and with the Umbrella Sampling method for free-energy evaluation. On the one hand, successfully obtaining fully folded nonagonist GR LBD states from the partially unfolded crystal GR LBD/RU486 underlines the role of the H1 in the GR LBD folding pathway. On the other hand, our results describe the H12 as a dynamic ensemble of conformations whose relative population is in the end determined by the interacting ligand: while dexamethasone privileges only a few poses (determined by a potential energy surface with a deep minimum), RU486 favors a wider H12 conformational amplitude, as indicated by a flatter potential landscape. By characterizing the H12 conformation in different conditions, we provide novel GR LBD models that represent potential targets for rational glucocorticoid drugs design, with the aim of accurately modulating GR activity.