The oncogenic transcription factor FOXM1 and the cyclin-dependent kinases 4 and 6 all promote cancer progression and aggressiveness that can be suppressed initially by targeted inhibitors, but resistance almost always develops. We show that ER-positive breast cancer cells that acquire resistance to FOXM1 inhibitors (FOXM1i) or CDK4/6 inhibitors (CDK4/6i) exhibit some key similarities including an increased JAK/STAT-interferon-ISGylation signaling network with elevated ISG15 and ISG15 protein conjugates, but with differences in magnitudes and patterns of ISGylated proteins. All the resistant cell lines also express higher levels of enzymes critical for ISGylation which predict poorer survival outcomes in ER-positive breast cancer patients. Reduction of these proteins pharmacologically or by siRNA knockdown greatly impairs the viability, colony formation, and proliferation of the FOXM1i-resistant cells, with lesser impact on CDK4/6i resistant cells. Notably, CDK4/6i resistant cells and 3D-Matrigel cultures can still be growth inhibited by FOXM1i, and conversely the FOXM1i resistance can be overcome by palbociclib or abemaciclib, indicating that while the resistance mechanisms of these two classes of drugs have some similar features, they are sufficiently distinct so that sequential treatment approaches could be effective in supporting new options such as FOXM1 inhibitor use after progression on CDK4/6 inhibitors.

Abstract Purpose: Malignant peripheral nerve sheath tumors (MPNSTs) are deadly sarcomas that arise spontaneously or via transformation of benign tumors, called plexiform neurofibromas (PNFs) and atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBPs), in patients with Neurofibromatosis Type 1 (NF1). Understanding molecular events that cooperate to promote malignancy is a priority in the field. One putative driver, FOXM1, is a powerful transcription factor that heightens the activities of kinases, MEK and CDK4/6, known to drive MPNST growth. We sought to understand the role of FOXM1 in these deadly sarcomas and hypothesized it is a new druggable target that is essential for MPNST pathogenesis. Methods: FOXM1 mRNA and protein expression were evaluated in patient-matched PNFs, ANNUBPs, and MPNSTs via RNA-Seq and IHC. For in vitro studies, knockdown (KD) of FOXM1 was performed in MPNST cell lines (S462, sNF96.2, JH2-002). Proliferation, survival, and cell cycle progression were measured in response to FOXM1 inhibitors, including newly developed NB-55, NB-73 and NB-115 drugs. To directly test the in vivo role of FOXM1 in MPNST initiation and progression, de novo MPNSTs were initiated by Nf1/Ink4a/Arf editing in the sciatic nerve of Nf1+/-, DhhCre, Foxm1 floxed mice relative to Nf1+/-;DhhCre controls. Results: In patient-matched tumor sets, FOXM1 mRNA was significantly elevated in MPNSTs relative to PNF/ANNUBP precursor lesions. FOXM1 protein expression rose dramatically in a stepwise manner from normal nerve to PNFs, ANNUBPs, and MPNSTs. FOXM1 KD slowed MPNST cell growth. FOXM1 inhibitors (thiostrepton, FDI-6, and multiple NB drugs) effectively inhibited MPNST proliferation and induced apoptosis. Synergistic killing of MPNST cells was obtained by combining thiostrepton with a MEK inhibitor (mirdametinib), CDK4/6 inhibitor (palbociclib), or EGFR inhibitor (gefitinib), while NB drugs synergized best with gefitinib. Excitingly, targeted in vivo deletion of Foxm1 in the sciatic nerve significantly slowed tumor progression. Conclusion: Our data demonstrate FOXM1 is an important driver of MPNST pathogenesis. FOXM1 expression and transcriptional activity are greatly increased in MPNSTs compared to benign precursors from the same patients. In agreement, genetic and therapeutic inactivation of FOXM1 promoted MPNST cell arrest and death. Synergistic killing of MPNSTs was achieved by combined inhibition of FOXM1 with MEK, CDK4/6, or EGFR while Foxm1 ablation in the sciatic nerve significantly slowed tumor progression in vivo. Together, these findings reveal that FOXM1 inhibition in specific combination therapies represents a new treatment strategy for MPNST patients. Citation Format: Ellen M. Voigt, Quinn Hanigan, Joshua J. Lingo, Altay Koyas, Rebecca D. Dodd, Benjamin W. Darbro, Dragana Kopanja, Sung Hoon Kim, Benita S. Katzenellenbogen, John A. Katzenellenbogen, Dawn E. Quelle. FOXM1 as a drug target in NF1-associated malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3040.

High levels of exposure to di(2-ethylhexyl) phthalate (DEHP), a known endocrine disruptor, have been linked to adverse pregnancy outcomes, yet the mechanisms by which it impacts human uterine functions remain unclear. Here we report that exposure of differentiating primary human endometrial stromal cells (HESCs) to an environmentally relevant concentration of DEHP or its primary metabolite, mono(2-ethylhexyl) phthalate, markedly reduces the expression of the estrogen-regulated transcription factor hypoxia-inducible factor 2-α (HIF2α). We also noticed a simultaneous decrease in RAB27B expression, which is crucial for the trafficking and secretion of extracellular vesicles (EVs). EVs enhance communication among various cell types within the pregnant uterus, thereby ensuring reproductive success. We found that estrogen receptor α (ERα) could no longer bind to the HIF2α regulatory region following phthalate treatment, and epigenetic analysis suggested that this may be due to hypermethylation of nearby CpG islands. Further investigation revealed a potential interaction between ERα and the transcription factor specificity protein 1 (Sp1) within the HIF2α regulatory region, which is affected by the inhibition of Sp1 binding to the phthalate-induced hypermethylated DNA. Additionally, our results suggest that the abnormal DNA methylation is likely due to increased expression of the DNA methyltransferase 1 (DNMT1) gene in response to phthalate exposure. Overall, this study provides valuable mechanistic insights into how phthalate-induced differential DNA methylation disrupts estrogenic regulation of the HIF2α gene and, consequently, EV secretion during HESC differentiation. This knowledge is essential for understanding how phthalates may lead to adverse reproductive outcomes by disrupting hormonal regulation of cell-to-cell communication in the uterus.

The nonsteroidal estrogen, benzestrol, has potent estrogenic activity, and through a recent stereocontrolled synthesis, we have obtained all eight of its constituent stereoisomers. We find that only one of them, RSS benzestrol, has very high binding affinity for estrogen receptor alpha (ERα); the other seven isomers have 60 to 600-fold lower affinity. We now show that the potencies of the isomers in two cell activity assays, proliferation of ER-positive breast cancer cells and stimulation of estrogenic gene activity, reflect their varying binding affinities for ERα. The crystal structure of the RSS isomer itself is consistent with its presumed absolute configuration and also reveals its conformational flexibility within the solid crystal lattice. We modeled each of the 8 benzestrol stereoisomers bound to ERα. Their calculated binding energies and internal torsional energies grouped with their experimentally measured binding affinities and biological activities, and the conformation for the highest affinity RSS isomer bound to ERα maps closely onto the conformation of the ERα-bound potent nonsteroidal estrogen, trans-diethylstilbestrol. Hence, we now provide a structural context for this congeneric series of benzestrol stereoisomers by proposing energy-based conformations they adopt when bound to ERα that underlie their effectiveness as estrogens.

Current endocrine therapies for breast cancer are used to treat the 70 percent of estrogen receptor (ER) positive breast cancers, but de novo and acquired resistance drive progressive metastatic disease. Hormone therapy development efforts have used similar chemical targeting strategies such that the structural basis of ligand efficacy is not clear. More than a third of patients who develop endocrine therapy resistance have hotspot constitutively activating mutations in the ligand binding domain, most notably Y537S and D538G. While these mutants have been studied extensively in stabilizing agonist conformation and constitutive activity in the absence of estradiol, the structural basis for why these mutations cause loss of efficacy for antagonists is not as well studied. To improve our understanding of structural mechanisms of therapeutic efficacy in the ESR1 mutant setting, we synthesized a diverse series of compounds including ∼100 ligands based on a high affinity adamantyl scaffold. We diversified the ligand side chains with various pharmacophores to understand the structural basis of ligand potency and efficacy on wild type and mutant ERs. Crystal structures of 20 ligands bound to Erα-LBD revealed how the chemical side chain modulated receptor structure to stabilize different conformational states to drive anti-cancer activity. Previous work revealed how the ER mutants drive constitutive activity by stabilizing the active ER conformation, but this state does not occur with antagonist bound ER. Long timescale molecular dynamics simulations were used to understand conformational changes induced by these mutations in the context of the antagonist conformation of the key helix 12 (h12), which determines pathogenic versus therapeutic activity states. The Y537S mutation stabilized h12 in one of two stable antagonist substates with implications for generating treatment resistance. In contrast, the D538G mutation, which is characterized by a helix-breaking effect, generated multiple metastable conformational substates. These findings support a model where the ligand side chains alter the relative stability of different antagonist substates to drive efficacy, with conformations that are different in the wild type versus mutant ERα bound to endocrine therapies. Date of Presentation October 17, 2024

Background:Small cell lung cancer (SCLC) is a lethal lung malignancy and patients are often diagnosed with distant metastasis. Nearly all patients suffer from disease relapsing with inherent chemoresistance. Lack of targeted SCLC therapies further worsens disease outcomes, making it highly desirable to identify novel and effective therapeutic targets. Methods:To search for potential therapeutic targets in SCLC, we analyzed publicly available single-cell and bulk RNA-sequencing (RNA-seq) data from normal, lung adenocarcinoma, and SCLC tumor tissues. To assess the targeting potential of FOXM1, we developed various in vitro models, including DOX-On-shFOXM1 (Tet-ON) inducible stable knockdown systems. Cisplatin resistant human and murine SCLC cell lines were generated to assess the role of FOXM1 in chemotherapy resistance. Immunoblotting, immunohistochemistry (IHC), and immuno-fluorescence were used to analyze the expression of FOXM1 and target proteins. ChIP-assay was used to study protein-gene interactions. Further, multicolor flow cytometry was employed to study the effect of FOXM1 inhibition on human T cells activation and differentiation. Subcutaneous xenograft and SCLC spontaneous (RPM: RB fl/fl ;TP53 fl/fl ;LSL-MYC T58A ) mouse models were used to evaluate the efficacy of FOXM1 inhibitors. Results:Single-cell as well as bulk RNA-seq data revealed that FOXM1, an oncogenic transcription factor, is overexpressed in SCLC, and it was recapitulated in human and murine SCLC tissues and cell lines. Interestingly, chemo-resistant (CR) SCLC showed a substantially higher FOXM1 expression than naïve SCLC. Silencing FOXM1 genetically or pharmacologically by FOXM1 inhibitors revealed a marked reduction in cell viability, colony formation, migration and sphere formation in naïve and CR SCLC cells. Moreover, FOXM1 inhibition induced apoptosis and cell cycle arrest in SCLC cells. Furthermore, FOXM1 inhibition in combination with first-line platinum-based chemotherapy showed synergistic anticancer effects in both xenograft and RPM mouse models of SCLC. Our RNA-seq analysis revealed that FOXM1 inhibition altered the Aurora Kinase B (AURKB) signaling pathway, which is dysregulated in SCLC. Moreover, we found FOXM1 inhibition enhanced T cell activation and supported the differentiation of CD8 + cytotoxic T cells, and T cell-mediated killing of cancer cells. Conclusions:Our study demonstrates that FOXM1 targeting using small molecule inhibitors has the potential to be a novel therapeutic strategy to combat SCLC progression including chemotherapeutic resistance and reshaping the anti-tumor immune response.

Multiple sclerosis (MS) is a chronic autoimmune, demyelinating, and neurodegenerative disease that results in motor, visual, and cognitive deficits. While existing treatments can slow disease progression, they rarely restore lost neurological function or significantly enhance quality of life. Estrogen receptor β (ERβ) has emerged as a promising therapeutic target due to its ability to activate non-classical signaling pathways involved in neuroprotection, immune modulation, and remyelination. In this study, two chloroindazole-based ERβ-selective ligands, K102 and K110, were identified for their favorable pharmacokinetic profiles and performance in preclinical absorption, distribution, metabolism, and elimination (ADME) screening. These compounds demonstrated biological activity by promoting oligodendrocyte (OL) differentiation in both primary mouse and human OL cultures. In vivo, they enhanced axonal remyelination and improved functional electrophysiological outcomes in two mouse models of MS: experimental autoimmune encephalomyelitis (EAE) and cuprizone diet-induced demyelination. Additionally, K102 and K110 modulated immune responses, supporting OL survival and contributing to motor and visual recovery in EAE mice. These findings provide compelling preclinical evidence for advancing K102 and K110 to clinical development. By simultaneously addressing neurodegeneration and inflammation through ERβ-mediated signaling, these compounds offer a novel and potentially transformative approach to MS therapy.

Disclosure: J.C. Nwachukwu: None. C.K. Min: None. R.R. Kobylski: None. T.J. Kim: None. Y. Hou: None. S. Kim: None. G.R. Hancock: None. T. Izard: None. S.W. Fanning: None. B.S. Katzenellenbogen: None. J.A. Katzenellenbogen: None. K.W. Nettles: None. Drugs targeting the estrogen receptor-α (ER) fall into two chemical classes, selective estrogen receptor modulators and degraders (SERMs and SERDs), which are effective treatments for hormone-responsive breast cancer. Compounds in these chemical classes typically show similar optimized efficacy in pre-clinical models of breast cancer, including in the context of ER-activating mutations (e.g. ER Y537S) and other resistance pathways, limiting our ability to identify small differences that may be more impactful clinically. To understand structural and molecular drivers of ER antagonism, we expanded the repertoire of ER targeting strategies. We characterized 108 compounds derived from a high-affinity adamantyl scaffold, incorporating 8 distinct classes of pharmacophores, each featuring a diverse array of chemical groups. The chemically diverse ligands revealed that non-canonical targeting approaches produced complex structure activity relationships across a panel of hormone sensitive and resistant breast cancer cell lines and reporter systems, activity that was often more efficacious than existing ER-targeted therapies. We obtained X-ray crystal structures of 25 adamantyl ligands bound to ER resistance mutants. This revealed how the different ligand chemical groups generated diverse structural effects on the surface binding site for transcriptional regulatory proteins that regulate gene expression. Molecular dynamics simulations showed that ER Y537S and D538G stabilized different antagonist conformers, or substates, from the wild type receptor, suggesting a structural basis for resistance to ER antagonists. We selected a subset of 11 adamantyls for a computational approach called “ligand class analysis” (LCA). LCA leverages the wide range of growth inhibitory effects of these related compounds (i.e. a compound class) to provide the statistical robustness for machine learning the underlying mechanisms of action. RNA-seq from MCF-7 cells expressing the ER Y537S resistance allele revealed ∼1600 genes differentially regulated by the 11 adamantyl ligands. We used the growth inhibitory effect of each ligand as the dependent variable in machine learning and identified a gene set response pattern with >95% predictive power for ligand efficacy. Defining causal links from ligand to receptor structure and the cellular mediators of growth inhibition revealed basic principles of allosteric signaling and transcriptional regulation. LCA differentiated these outcome-focused ER signaling pathways from the full set of ligand-regulated cellular effects, while diversification of the ligand-receptor structures identified new ways of modulating ER activity for targeting endocrine resistant breast cancer. Supported by NIH R01CA275142, R01CA220284, R37CA27934, and the Breast Cancer Research Foundation BCRF-083 and BCRF-084 Presentation: Saturday, July 12, 2025

Relapsed and refractory multiple myeloma (RRMM) remains the leading cause of MM mortality. FOXM1 is strongly associated with RRMM, making it a compelling therapeutic target. Through three low-throughput screenings, we have identified nine FDA-approved drugs, including the BH3 mimetic Venetoclax, that synergize with FOXM1 inhibitor NB73 in killing MM cells. Venetoclax has shown effects in 6% of non-t(11;14) and 27% of t(11;14) MM cases. The NB73-Venetoclax combination barely induces acute toxicity in vivo and represses MM cells in vivo and ex vivo. NB73 enhances the ubiquitination and proteasomal degradation of FOXM1, an effect further amplified by Venetoclax. The NB73-Venetoclax combination abolishes FOXM1's binding to promoters of key MYC pathway genes, such as PLK1, leading to significant downregulation of their expression. Furthermore, the PLK1-specific inhibitor GSK461364 synergizes with NB73 to inhibit MM cell growth. Interestingly, NB73 does not sensitize U266 cells, a Venetoclax-resistant t(11;14) MM cell line expressing high FOXM1, to Venetoclax treatment, which is corrected by a new-generation BH3 mimetic Sonrotoclax and ALK inhibitor Ceritinib. Collectively, targeting FOXM1 demonstrates significant potential for enhancing the efficacy of FDA-approved drugs in RRMM. These findings shed new light on the discouraging outcomes of the Phase-III CANOVA study centering Venetoclax with an encouraging molecular clue.

Small cell lung cancer (SCLC) is a lethal lung malignancy, which is associated with distant metastasis and chemoresistance. Due to the limited availability of targeted therapies, identifying a potential therapeutic target is a pressing unmet need in SCLC. Single-cell and bulk-transcriptomic datasets were analyzed that revealed FOXM1 as a potential targeting candidate in SCLC. High FOXM1 expression was observed in human and murine SCLC tissues and cell lines. Interestingly, chemoresistant (CR) SCLC cells exhibited substantially higher FOXM1 expression compared to naïve SCLC. Furthermore, FOXM1 inhibition in combination with platinum-based chemotherapy showed synergistic anticancer effects in vitro and in vivo xenograft and spontaneous (RPM: RB1fl/fl; TP53 fl/fl; LSL-MYCT58A) mouse models of SCLC. Mechanistically, RNA-seq analysis revealed that FOXM1 inhibition altered the Aurora Kinase B (AURKB) signaling pathway. Notably, FOXM1 inhibition enhanced T cell activation, supported differentiation of CD8+ T cells, and T cell-mediated killing of SCLC cells. Additionally, FOXM1 inhibition enhanced CD8+ T cell and macrophage recruitment in the tumor microenvironment (TME) of immunocompetent RPM model. This study demonstrates that FOXM1 targeting small molecule inhibitors (FOXM1i) has the potential to be a novel therapeutic strategy to combat SCLC progression, including chemotherapeutic resistance and reshaping the anti-tumor immune response.

Baseline ESR1 mutations and fulvestrant efficacy. A, % Incidence of mutations in indicated genes at baseline in Cohort A (n = 79 assessable patients). B, Incidence of baseline ESR1 alterations within Cohort A (n = 79 assessable patients). C, PFS of patients in Cohort A, divided by baseline ESR1 Y537C mutation status (left) and ESR1 Y537S mutation status (right). P values from the log-rank test. HR >1 denotes worse PFS for that group. WT, wild-type; mt, mutant. D, MCF7 cells were cotransfected with the indicated ESR1 expression constructs and treated with the indicated concentration of fulvestrant in the presence of 1 nmol/L estradiol for 24 hours and estrogen response element-luciferase reporter activity determined. Two independent experiments.

Calculated IC50 and EC50 of 4OH tamoxifen and novel SERDs in ESR1-mutant models.

We report the development of an iterative Matteson homologation reaction with catalyst-controlled diastereoselectivity through the design of a new catalyst. This reaction was applied to the selective synthesis of each stereoisomer of benzestrol, a bioactive compound with estrogenic activity featuring three contiguous stereocenters. The different stereoisomers were assayed to determine their binding affinity for the estrogen receptor α (ERα), and the absolute configuration of the compound having uniquely high activity was determined. This research lays a framework for the catalytic synthesis and study of complete stereoisomeric sets of other bioactive molecules and chemical probes containing contiguous stereocenters.

Supplementary Table 1. Comparison of Potential Binding Energy and Distance of Pi-Pi Stacking Interaction with F404 and mutant modes. Supplementary Table 2. Clinicopathological features of PlasmaMATCH Cohort A.

F404 does not activate estrogen signaling. A, CRISPR clones of MCF7 cells expressing ESR1 F404L (1210T>C, CRISPR edit indicated by red arrows) or D538G (1613A>G; CRISPR edit indicated by black arrows) were identified by RT-PCR followed by Sanger sequencing (left-hand panels). Similarly, a second round of CRISPR was used to introduce ESR1 F404L (1210T>C) into a clone (D6C) that expressed D538G (1613A>G; right-hand panels). B, Estrogen-dependent growth was assessed in colony formation assay. Parental MCF7 cells and indicated ESR1-mutant models were grown in either the absence or presence of estradiol (1 nmol/L) for 14 days. C, Quantification of colony formation assays of ESR1-mutant models treated with and without estradiol (1 nmol/L). Sulforhodamine B (SRB)-stained colonies were dissolved, and absorbance at 565 nm was measured. Mean with SEM, n = 3 independent experiments, nonparametric one-way ANOVA with Dunn multiple comparisons test; **, P < 0.01. D, Expression of estrogen target genes, progesterone receptor (PgR), and trefoil factor-1 (TFF1), assessed by western blot in parental MCF7 cells and indicated ESR1-mutant models grown in either the absence or presence of estradiol (1 nmol/L) for 24 hours. E, MCF7 cells were transfected with ESR1 expression constructs with indicated ESR1 variants. Expression of ERα was determined by western blot. F, MCF7 cells were cotransfected with the indicated ESR1 expression constructs ERE-luciferase reporter and control construct. Cells were treated in either the absence or presence of estradiol (1 nmol/L) for 24 hours, and ERE-luciferase activity was assessed. Two-way repeated-measures ANOVA with Dunnett multiple comparisons test, n = 4 mean with SD; *, P < 0.05. WT, wild-type.

Despite significant improvements in the prognosis of Multiple Myeloma (MM), relapsed/refractory MM remains a major challenge. BCL2 inhibitor Venetoclax induced complete or very good partial responses in 6% of non-t(11;14) MM cases, compared to 27% in t(11;14) cases, when used as monotherapy in relapsed/refractory MM. Though Venetoclax was proposed to treat t(11;14) cases, the resistance became a concern. Furthermore, non-t(11;14) cases account for 80-85% of MM cases, which underscores the value of Venetoclax in non-t(11;14) MM. Here, we report a recently-invented small molecule inhibitor of FOXM1 NB73 synergizing with Venetoclax in killing MM cells. FOXM1, a critical forkhead box transcription factor in high-risk and relapsed/refractory MM, represents a promising therapeutic target of MM. We examined the mechanisms underlying the synergies of Venetoclax and NB73 using multi-omics and molecular and cellular biology tools in non-t(11;14) myeloma cell lines with high FOXM1 expression. NB73 induces immediate loss of FOXM1, decreases BCL2 expression, and increases Puma expression in myeloma cells. Venetoclax enhances NB73-induced FOXM1 ubiquitination and degradation. The NB73-Venetoclax combination abrogates the binding of FOXM1 to the promoters of genes in the MYC pathway, such as PLK1, MYC, CDC20, and CCNA2, leading to the repression of the transcription of these MYC pathway genes. The PLK1-specific inhibitor GSK461364 synergies with NB73 in suppressing myeloma cell growth. Therefore, NB73 synergizes with Venetoclax in killing myeloma cells. Conclusively, the NB73-Venetoclax combination abolishes FOXM1-mediated transcriptional activation of the MYC pathway, resulting in intensive apoptosis of myeloma cells without t(11;14) but with high FOXM1 expression. ### Competing Interest Statement B.S.K., J.A.K. and S.H.K. are co-inventors on patents filed by the University of Illinois to cover the FOXM1 inhibitor compounds described in this paper. B.S.K. and J.A.K, are members of the Scientific Advisory Board of Celcuity. The other authors declare no competing interests.

Di(2-ethylhexyl) phthalate (DEHP), a known endocrine-disrupting chemical, is a plasticizer found in many common consumer products. High levels of DEHP exposure have been linked to adverse pregnancy outcomes, yet little is known about how it affects human uterine functions. We previously reported that the estrogen-regulated transcription factor hypoxia-inducible factor 2 alpha (HIF2α) promotes the expression of Rab27b, which controls the trafficking and secretion of extracellular vesicles (EVs). EVs facilitate communication between multiple cell types within the pregnant uterus, ensuring reproductive success. In this study, we report that exposure of differentiating primary human endometrial stromal cells (HESC) to an environmentally relevant concentration (1 μg/mL) of DEHP or its primary metabolite mono(2-ethylhexyl) phthalate (MEHP) markedly reduces the expression of HIF2α . We also observed a concomitant decrease in RAB27B expression, reducing EV secretion from HESC. Interestingly, we found that DEHP or MEHP exposure disrupts estrogenic regulation of the HIF2α/Rab27b signaling pathway. Estrogen receptor alpha (ERα) could no longer bind to the HIF2α regulatory region following phthalate treatment, and epigenetic analysis suggested that this may be due to hypermethylation of nearby CpG islands. Further investigation revealed a potential interaction between ERα and the transcription factor Sp1 within the HIF2α regulatory region, which is affected by the inhibition of Sp1 binding to the phthalate-induced hypermethylated DNA. Additionally, our results suggest that the abnormal DNA methylation is likely due to increased expression of the DNA methyltransferase 1 ( DNMT1 ) gene in response to phthalate exposure. Overall, this study provides valuable mechanistic insights into how phthalate-induced differential DNA methylation disrupts estrogenic regulation of the HIF2α gene and, consequently, EV secretion during HESC differentiation. This knowledge is crucial for our understanding of how phthalates may cause adverse reproductive outcomes by disrupting the hormonal regulation of cell-to-cell communication within the pregnant uterus.

Abstract Fulvestrant is used to treat patients with hormone receptor–positive advanced breast cancer, but acquired resistance is poorly understood. PlasmaMATCH Cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Baseline ESR1 mutations Y537S are associated with poor outcomes and Y537C with good outcomes. Sequencing of baseline and EOT ctDNA samples (n = 69) revealed 3/69 (4%) patients acquired novel ESR1 F404 mutations (F404L, F404I, and F404V), in cis with activating mutations. In silico modeling revealed that ESR1 F404 contributes to fulvestrant binding to estrogen receptor–alpha (ERα) through a pi-stacking bond, with mutations disrupting this bond. In vitro analysis demonstrated that single F404L, E380Q, and D538G models were less sensitive to fulvestrant, whereas compound mutations D538G + F404L and E380Q + F404L were resistant. Several oral ERα degraders were active against compound mutant models. We have identified a resistance mechanism specific to fulvestrant that can be targeted by treatments in clinical development. Significance: Novel F404 ESR1 mutations may be acquired to cause overt resistance to fulvestrant when combined with preexisting activating ESR1 mutations. Novel combinations of mutations in the ER ligand binding domain may cause drug-specific resistance, emphasizing the potential of similar drug-specific mutations to impact the efficacy of oral ER degraders in development. This article is featured in Selected Articles from This Issue, p. 201