Addition of OP-1250 to CDK4/6 inhibitors enhances tumor suppression and prolongs animal survival in ER+ breast cancer xenograft models. A, B, Percent change in tumor volume of individual animals in listed treatment groups at day 29 of the MCF7 xenograft study, normalized to day 1 tumor volume. C, D, Percent change in tumor volume of individual animals in listed treatment groups at day 29 of the ST941 xenograft study, normalized to day 1 tumor volume. E, F, Kaplan–Meier graphs of animal survival over time in listed treatment groups of mice implanted with the ESR1Y537S ST941 PDX model. Dosing ended on day 29 of the study, and animals were sacrificed when tumor volume reached 2000 mm3. G, H, Mean and SEM tumor volume over the full study interval with monotherapy and combination treatments in the ESR1Y537S ST941 PDX breast cancer model, with the dotted line representing tumor stasis. Dosing ended on day 29, as indicated by the dashed line.

OP-1250 acts as a selective ER degrader (SERD) in wild-type and ESR1 mutant cell lines. A, B, Representative blot images from the simple western assay shown in Figure 3 (see main article) after 24-h treatment with 300 nM compound or 1 nM E2 with indicated compound treatments and cell lines. C, D, Representative blot images from simple western assay of MCF7 cells treated for 2 h, 4 h, or 8 h with the listed concentrations of compounds in the presence or absence of MG-132 and simple western analysis of ERα protein levels after treatment with listed compounds. Samples were normalized to untreated control, with mean and SD across at least three independent experiments shown. E, F, ERα degradation in CAMA-1 cells with the indicated homozygous mutant as assessed by simple western assay after 24-h treatment with 300 nM compound or 1 nM E2. Samples were normalized to untreated control, represented by a dotted line, with mean and SD across at least three independent experiments shown. G, H, Representative blot images from simple western data shown in panels E and F with indicated compound treatments and cell lines.

Effects of E2 and antiestrogens on transcriptional activity and cellular proliferation in E2-depleted media. A, Volcano plot of genes differentially expressed by 15-min E2 treatment relative to vehicle in PRO-seq experiment. Each dot represents a gene in the annotation set. Adjusted p-value is plotted on the y-axis and fold-change relative to time-matched vehicle on the x-axis. The number in the top right-hand corner represents the number of genes changed, and red dots indicate genes with observed change in expression. B, PRO-seq read distribution at estrogen target genes CCND1 and MYC after 15 min of E2 treatment (red) compared to vehicle control (blue). The x-axis refers to the position along the genome, and the y-axis refers to the number of reads aligning to that position normalized for the depth of each sample. Replicates were averaged. Bars that are less than zero are reverse-strand reads; bars above zero are forward-strand reads. C, PRO-seq read distribution at estrogen target gene GREB1 after 6 h of OP-1250 treatment following E2 pretreatment (red) compared to 24-h E2 treatment (blue). The x-axis refers to the position along the genome, and the y-axis refers to the number of reads aligning to that position normalized for the depth of each sample. Replicates were averaged. Bars that are less than zero are reverse-strand reads; bars above zero are forward-strand reads. D, Heatmap of annotated G2/M checkpoint genes in CAMA-1 and MCF7 cells after 24 h of compound treatment in PRO-seq experiment. Red indicates high expression and blue low expression. Samples and genes with similar expression patterns are grouped together using hierarchical clustering, with sample relationships depicted by dendrograms at the top and left of graph. E, Chart showing number of differentially expressed genes compared to vehicle for each treatment group in CAMA-1 mRNA-seq experiment. Cells were treated with 100 pM E2 or 316 nM antiestrogen in E2-depleted media for 24 h.

OP-1250 specifically inhibits the ER without cross-reactivity with other hormone receptors. A, LanthaScreen co-activator recruitment assay using wild-type ERα LBD and fluorescein tagged PGC1a comparing OP-1250 and other approved and in-trial comparator molecules. Data are normalized to DMSO vehicle and 10 µM E2-treated wells (100% recruitment) and presented as mean and SD of at least two separate experiments. B, Reporter gene activity assay in CAMA-1 and MCF7 cells transiently transfected with a luciferase reporter gene construct containing an estrogen response element and minimal tk promoter. Cells were cultured in estrogen-depleted media and reporter gene activity was normalized to E2 vehicle. The dotted line represents activity in the absence of estrogen, the hashed line represents activity with E2 supplementation. Data shown represent mean and SD from a representative of at least three independent experiments. C, Reporter gene assay of ER-negative SK-BR-3 cells transiently transfected with wild-type or mutant ESR1 vectors along with a luciferase reporter gene cloned to 3× estrogen response element and TATA minimal promoter. Transcriptional activity was measured as luciferase activity. Cells were cultured in estrogen-depleted media and reporter gene activity was normalized to wild-type E2 vehicle and empty vector + E2. The dotted line represents activity with empty vector control, the hashed line represents activity following supplementation with 31.6 nM E2. Data shown represent mean and SD from a representative of at least three independent experiments. D, Reporter gene activity assay of HeLa cells transiently transfected with glucocorticoid, androgen, or progesterone receptor and a luciferase reporter cloned to 3× PRE and TATA minimal promoter, measuring the impact of OP-1250 on the glucocorticoid receptor in the presence and absence of 31.6 nM dexamethasone, progesterone receptor in the presence and absence of 500 nM MPA (medroxyprogesterone acetate), and androgen receptor in the presence and absence of 50 nM R1881. Data are normalized to the indicated amount of agonist (either MPA, dexamethasone, or R1881). Figures show data as the mean and SD from a representative of at least three independent experiments. E, Reporter gene assay of ER-negative SK-BR-3 cells transiently transfected with ESR1 or ESR2 along with a luciferase reporter gene cloned to 3× estrogen response element and TATA minimal promoter. Transcriptional activity was measured as luciferase activity. Cells were cultured in estrogen-depleted media and reporter gene activity was normalized to E2 vehicle. Data shown represent mean and SD from a representative of at least three independent experiments. F, Uterine wet weight assay following treatment with a dose response of OP-1250 or fulvestrant in the presence of E2 supplementation. Mean and SEM from six mice per treatment group are plotted with asterisks indicating significance relative to vehicle by one-way analysis of variance. * represents adjusted p-value <0.05, *** represents adjusted p-value <0.001, **** represents adjusted p-value <0.0001. G, Simple western analysis of ERα protein levels in Ishikawa cells after 72-h treatment with listed compounds. Samples are normalized to untreated control. Data shown represent mean and SD from a representative of at least three independent experiments. H, Representative blot image from simple western assay shown in panel G after 72-h treatment with 300 nM compound or 1 nM E2 with indicated compound treatments and cell line. AR=androgen receptor; GR=glucocorticoid receptor; PR= progesterone receptor; WT=wild-type.

OP-1250 demonstrates potent activity in an ER+ breast cancer intracranial xenograft and outperforms ribociclib. A, Schematic of intracranial xenograft experiment with data shown in B–D. B, Mean and SEM intracranial tumor volume over time with listed treatments as assessed by MRI in an ESR1Y537S ST941 PDX intracranial xenograft model. C, Percent change in intracranial tumor volume of individual animals in listed treatment groups at day 101 or at the last recorded timepoint in the ST941 intracranial xenograft study, normalized to day 1 tumor volume. D, Kaplan–Meier graphs of animal survival over time in listed treatment groups of mice implanted intracranially with the ST941 PDX xenograft. Dosing ended on day 100 of the study in the OP-1250 10 mg/kg group, as indicated by the shaded area. E, Representative intracranial MRIs from ST941-implanted mice with data shown in Figure 6 (see main article) at indicated timepoints and treatment groups. PO=orally.

RNASeq dataset of gene pathway changes following treatment with OP-1250, palbociclib and ribociclib in an MCF7 xenograft model.

Supplementary text including supplementary materials and methods and references.

PROSeq analysis of gene expression changes following treatment of MCF7 and CAMA-1 cells with ER-alpha ligands.

Abstract The estrogen receptor (ER) is a well-established target for the treatment of breast cancer, with the majority of patients presenting as ER-positive (ER+). Endocrine therapy is a mainstay of breast cancer treatment but the development of resistance mutations in response to aromatase inhibitors, poor pharmacokinetic properties of fulvestrant, agonist activity of tamoxifen, and limited benefit for elacestrant leave unmet needs for patients with or without resistance mutations in ESR1, the gene that encodes the ER protein. Here we describe palazestrant (OP-1250), a novel, orally bioavailable complete ER antagonist and selective ER degrader. OP-1250, like fulvestrant, has no agonist activity on the ER and completely blocks estrogen-induced transcriptional activity. In addition, OP-1250 demonstrates favorable biochemical binding affinity, ER degradation, and antiproliferative activity in ER+ breast cancer models that is comparable or superior to other agents of interest. OP-1250 has superior pharmacokinetic properties relative to fulvestrant, including oral bioavailability and brain penetrance, as well as superior performance in wild-type and ESR1-mutant breast cancer xenograft studies. OP-1250 combines well with cyclin-dependent kinase 4 and 6 inhibitors in xenograft studies of ER+ breast cancer models and effectively shrinks intracranially implanted tumors, resulting in prolonged animal survival. With demonstrated preclinical efficacy exceeding fulvestrant in wild-type models, elacestrant in ESR1-mutant models, and tamoxifen in intracranial xenografts, OP-1250 has the potential to benefit patients with ER+ breast cancer.

Abstract OP-1250, a complete estrogen receptor antagonist (CERAN) and selective estrogen receptor degrader (SERD), is currently in Phase I/II clinical trials for the treatment of estrogen receptor positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. OP-1250 has previously demonstrated efficacy in both estrogen receptor 1 (ESR1) wild-type and Y537S mutant breast cancer xenograft models. Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are a standard first-line treatment for ER+ advanced or metastatic breast cancer in combination with endocrine therapy. To characterize OP-1250 efficacy in this setting, we explored the combination of OP-1250 with CDK4/6 inhibitors palbociclib and ribociclib in ER+/HER2- breast cancer preclinical models, and profiled the transcriptional changes associated with single agent and combination therapy. OP-1250 and CDK4/6 inhibitors were tested alone and in combination in ESR1 wild-type MCF7 and ESR1 Y537S mutant ST941 xenograft models. Higher doses of OP-1250 and either CDK4/6 inhibitor, when given as a monotherapy, suppress tumor growth and increase animal survival, while lower doses of the compounds display a modest or minimal effect on tumor growth or survival. In combination, lower doses of OP-1250 and either CDK4/6 inhibitor result in significantly greater tumor growth suppression (both xenograft models), tumor regression (MCF7) and animal survival (ST491) than each compound individually. RNA sequencing revealed substantially more gene expression changes with combination treatment than monotherapy. In particular, pathways associated with cell cycle progression displayed greater changes with combination treatment, similar to what was observed with a much higher dose of monotherapy. This study supports the clinical evaluation of OP-1250 in combination with CDK4/6 inhibitors (palbociclib or ribociclib) and provides additional context to mechanisms of combination efficacy. OP-1250 is currently being evaluated in a Phase Ib clinical trial in combination with palbociclib and a study including evaluation of the combination of OP-1250 with ribociclib is planned to initiate in Q3 2022. Citation Format: Alison D. Parisian, Gopinath S. Palanisamy, Fabian Ortega, Judevin L. Sapugay, William J. Bodell, David Kulp, Peter Kushner, Cyrus Harmon. Combination of complete estrogen receptor antagonist, OP-1250, and CDK4/6 inhibitors enhances tumor suppression and inhibition of cell cycle-related gene expression [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-07.

OP-1250 is an orally-bioavailable complete estrogen receptor antagonist (CERAN) previously shown to shrink wild-type and Y537S mutant estrogen receptor-positive (ER+) tumors in multiple preclinical xenograft models. To better understand the effects of OP-1250 treatment on ER+ breast cancer cells, we used precision run-on sequencing (PRO-seq) to profile the direct transcriptional effects of OP-1250 in two commonly used breast cancer cell lines, MCF7 and CAMA-1. PRO-seq detects nascent RNA, and thus can detect changes at shorter timescales than traditional RNA-seq methods. We conducted PRO-seq 15 minutes, 1 hour, 6 hours and 24 hours after administration of OP-1250, estradiol, fulvestrant and tamoxifen to compare the effects of these compounds across the two cell lines. OP-1250 was administered in the absence of estradiol and after estradiol pretreatment to generate a detailed profile of the time course of its activity. We identified genes that display early transcriptional changes in response to the compounds tested, many of which have been previously identified as estrogen response genes. At later time points we observed transcriptional changes consistent with modulation of pathways involved in cell cycle progression. We show that OP-1250 treatment reverses the estradiol-induced transcriptional changes associated with estrogen receptor activation and chronicle the time course of these changes. Finally, we show greater similarity in the transcriptional changes induced by CERANs OP-1250 and fulvestrant compared to tamoxifen, as well as cell line-specific differences in estrogen response and response to anti-estrogen treatment. These data provide additional insights into the mechanisms of OP-1250 action on ER+ cell lines and patient tumors. OP-1250 is currently in a Phase I/II clinical trial for the treatment of ER+ breast cancer. Citation Format: Alison D. Parisian, Caitlin Miller, Fabian Ortega, Leslie Hodges-Gallagher, Susanna Barratt, Joey Azofeifa, Peter J. Kushner, David Kulp, Cyrus L. Harmon. Precision run-on sequencing (PRO-seq) analysis of a treatment time course in ER+ breast cancer cell lines reveals the transcriptional changes underlying response to complete estrogen receptor antagonist (CERAN) OP-1250 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5375.

Western culture discourages discussion of death and dying, especially with healthy emerging adults. Yet, research shows that engaging this population in conversations about death and dying is empowering and important for young people’s decision-making around and understanding of the end of life. We show that students are indeed ill-informed on such issues but that they desire to learn more. We describe and assess a pilot undergraduate course in palliative care addressing this need, and we demonstrate its success in engaging and educating students using pedagogical approaches built to develop a social and intellectual community of trust.

Introduction: Counseling families at risk for extremely preterm birth presents significant challenges to providers due to the uncertainty of infant outcomes. Aim: To determine lay-public parental preferences for approaches to prenatal counseling and preferred descriptive terminology usage by providers when discussing an anticipated extremely preterm delivery. Methods: This exploratory pilot study recruited parents through social media to participate in a survey centered around a hypothetical delivery at 23 weeks. Questions assessed participants’ understanding of medical terminology and preferences for terminology, discussion topics, and approaches to decision-making related to anticipated extremely preterm birth. Results: A total of 142 (72%) parents participated. Understanding of medical compared with nonmedical terminology was limited (P < .01). Parents had stronger preferences for the use of certain words and terminology over others. They preferred discussions focused on the possibility of infant survival and information about intensive care, over general statistics, and outcome percentages. Most would choose attempted stabilization and neonatal intensive care unit care in this hypothetical case. Discussion/Conclusions: Parents have preferences regarding physician communication and approaches to decision-making around extremely preterm delivery, which may offer guidance to providers during counseling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Primates and rodents, which descended from a common ancestor around 90 million years ago1, exhibit profound differences in behaviour and cognitive capacity; the cellular basis for these differences is unknown. Here we use single-nucleus RNA sequencing to profile RNA expression in 188,776 individual interneurons across homologous brain regions from three primates (human, macaque and marmoset), a rodent (mouse) and a weasel (ferret). Homologous interneuron types—which were readily identified by their RNA-expression patterns—varied in abundance and RNA expression among ferrets, mice and primates, but varied less among primates. Only a modest fraction of the genes identified as ‘markers’ of specific interneuron subtypes in any one species had this property in another species. In the primate neocortex, dozens of genes showed spatial expression gradients among interneurons of the same type, which suggests that regional variation in cortical contexts shapes the RNA expression patterns of adult neocortical interneurons. We found that an interneuron type that was previously associated with the mouse hippocampus—the ‘ivy cell’, which has neurogliaform characteristics—has become abundant across the neocortex of humans, macaques and marmosets but not mice or ferrets. We also found a notable subcortical innovation: an abundant striatal interneuron type in primates that had no molecularly homologous counterpart in mice or ferrets. These interneurons expressed a unique combination of genes that encode transcription factors, receptors and neuropeptides and constituted around 30% of striatal interneurons in marmosets and humans. Single-nucleus RNA-sequencing analyses of brain from humans, macaques, marmosets, mice and ferrets reveal diverse ways that interneuron populations have changed during evolution.

Primates and rodents, which descended from a common ancestor more than 90 million years ago, exhibit profound differences in behavior and cognitive capacity. Modifications, specializations, and innovations to brain cell types may have occurred along each lineage. We used Drop-seq to profile RNA expression in more than 184,000 individual telencephalic interneurons from humans, macaques, marmosets, and mice. Conserved interneuron types varied significantly in abundance and RNA expression between mice and primates, but varied much more modestly among primates. In adult primates, the expression patterns of dozens of genes exhibited spatial expression gradients among neocortical interneurons, suggesting that adult neocortical interneurons are imprinted by their local cortical context. In addition, we found that an interneuron type previously associated with the mouse hippocampus—the “ivy cell”, which has neurogliaform characteristics—has become abundant across the neocortex of humans, macaques, and marmosets. The most striking innovation was subcortical: we identified an abundant striatal interneuron type in primates that had no molecularly homologous cell population in mouse striatum, cortex, thalamus, or hippocampus. These interneurons, which expressed a unique combination of transcription factors, receptors, and neuropeptides, including the neuropeptide TAC3 , constituted almost 30% of striatal interneurons in marmosets and humans. Understanding how gene and cell-type attributes changed or persisted over the evolutionary divergence of primates and rodents will guide the choice of models for human brain disorders and mutations and help to identify the cellular substrates of expanded cognition in humans and other primates.

The mammalian brain is composed of diverse, specialized cell populations, few of which we fully understand. To more systematically ascertain and learn from cellular specializations in the brain, we used Drop-seq to perform single-cell RNA sequencing of 690,000 cells sampled from nine regions of the adult mouse brain: frontal and posterior cortex (156,000 and 99,000 cells, respectively), hippocampus (113,000), thalamus (89,000), cerebellum (26,000), and all of the basal ganglia – the striatum (77,000), globus pallidus externus/nucleus basalis (66,000), entopeduncular/subthalamic nuclei (19,000), and the substantia nigra/ventral tegmental area (44,000). We developed computational approaches to distinguish biological from technical signals in single-cell data, then identified 565 transcriptionally distinct groups of cells, which we annotate and present through interactive online software we developed for visualizing and re-analyzing these data ( DropViz ). Comparison of cell classes and types across regions revealed features of brain organization. These included a neuronal gene-expression module for synthesizing axonal and presynaptic components; widely shared patterns in the combinatorial co-deployment of voltage-gated ion channels by diverse neuronal populations; functional distinctions among cells of the brain vasculature; and specialization of glutamatergic neurons across cortical regions to a degree not observed in other neuronal or non-neuronal populations. We describe systematic neuronal classifications for two complex, understudied regions of the basal ganglia, the globus pallidus externus and substantia nigra reticulata. In the striatum, where neuron types have been intensely researched, our data reveal a previously undescribed population of striatal spiny projection neurons (SPNs) comprising 4% of SPNs. The adult mouse brain cell atlas can serve as a reference for analyses of development, disease, and evolution.

The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in 690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.

This tutorial is a learning resource that outlines the basic process and provides specific software tools for implementing a complete genome‐wide association analysis. Approaches to post‐analytic visualization and interrogation of potentially novel findings are also presented. Applications are illustrated using the free and open‐source R statistical computing and graphics software environment, Bioconductor software for bioinformatics and the UCSC Genome Browser. Complete genome‐wide association data on 1401 individuals across 861,473 typed single nucleotide polymorphisms from the PennCATH study of coronary artery disease are used for illustration. All data and code, as well as additional instructional resources, are publicly available through the Open Resources in Statistical Genomics project: http://www.stat-gen.org. © 2015 The Authors. Statistics in Medicine Published by John Wiley & Sons Ltd.