We investigated the conformational mechanisms underlying PPARγ activation in muscle-invasive urothelial carcinoma (MIUC) and sought to develop covalent inverse agonists to therapeutically reinforce a repressive state. The integration of mutational, structural, and biochemical analyses of PPARγ and RXRα guided the discovery of FX-909, a first-in-class clinical PPARγ inverse agonist that enforces a repressive conformational state, even in highly activated biological contexts. FX-909 is a potent, highly selective, and powerful suppressor of PPARγ transcriptional activity through the enhancement of PPARγ-nuclear co-repressor (NCOR) binding affinity. Treatment with FX-909 resulted in selective growth inhibition in PPARγ-activated MIUC cell lines and durable regressions in xenograft models of MIUC. FX-909 is capable of recapitulating PPARG genetic knockout phenotypes in vivo and is currently in clinical development for the treatment of intractable MIUC.

We present evidence of previously unrecognized allosteric connectivity across the TP53 DNA binding domain (DBD). Specifically, we have found evidence of explicit influence on the Zn2+-binding site from the region surrounding the hotspot Y220C mutation. This allosteric connectivity is intertwined with a temperature-dependent destabilization of Zn2+ binding in both the WT and Y220C DBDs. Our studies indicate that the Y220C mutation exacerbates this temperature-dependent destabilization of Zn2+-binding to result in overall destabilization of the Y220C variant. We provide detailed thermodynamic evidence that Rezatapopt, a small molecule reactivator of the Y220C DBD, engages Y220C through two distinct thermodynamic pathways and restores WT-level Zn2+-affinity to this oncogenic variant. A series of thermodynamic models describing the WT and Y220C conformational landscapes, as well as the Rezatapopt binding mechanisms, are proposed. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Significant unmet need exists in metastatic castration resistant prostate cancer (mCRPC). As the disease progresses, mutations, amplification and overexpression of the androgen receptor (AR), often accompanied by elevated intratumoral androgen levels, become increasingly prevalent, with 85% of mCRPC cases remaining AR-dependent. Current therapies target the cytoplasmic, hormone-unbound AR (AROFF); however, rising AR levels as prostate cancer (PC) advances represent a primary mechanism of resistance to these therapies. Nuclear, hormone-bound, transcriptionally active AR (ARON) is the main driver of androgen signaling and tumor progression in AR-dependent PC. Here we report the discovery of a selective ARON degrader that utilizes a novel allosteric binding site revealed upon hormone binding. We identified multiple x-ray validated ligands that bind to this novel ARON pocket. Conversion and optimization of these ligands into Cereblon-based heterobifunctional degraders resulted in the identification of FX-111. FX-111 achieves rapid and sustained degradation of AR (DC50 = 10 nM) and displays exquisite selectivity, with no observed degradation of other steroid hormone receptors or other proteins using proteome-wide analysis in multiple cell lines. FX-111 degrades AR and silences AR target genes effectively irrespective of increasing hormone as it is non-competitive with androgen binding, in contrast to the loss of potency seen with orthosteric degraders with increasing hormone levels. FX-111 degrades ARON when alternative agonists are used to stimulate its activity and can effectively degrade clinically relevant mutant forms of AR. RNA-seq analysis in LNCaP and VCaP cells treated with FX-111 reveals complete inhibition of androgen-driven transcriptional changes, with minimal gene expression impact in hormone-depleted conditions, underscoring FX-111’s specificity for AR degradation. In vivo, FX-111 induces robust AR degradation, prostate specific antigen suppression and significant tumor growth inhibition in VCaP xenograft models, with oral administration at 10 mg/kg BID. Addition of exogenous androgen in intact or castrated mice, mimicking intratumoral hormone, did not affect the pharmacodynamic profile or efficacy of FX-111, whereas orthosteric degraders lost both potency and the ability to suppress AR transcriptional activity. Potent suppression of AR function in prostate and testes tissues, with accompanying organ weight loss, was observed in mice. Targeting ARON in PC could potentially treat all forms of PC that are driven by AR signaling without the need for androgen deprivation. FX-111 is currently in IND-enabling studies with plans to initiate clinical studies in 2026. Citation Format: Jennifer A. Mertz, Thomas H. Graham, Brian J. Golbourn, Yong Li, Alex P. Rossi, Marco Chiumiento, Phuong A. Nguyen, Jeremy W. Setser, Gregg Chenail, Rainer Wilcken, Jonathan T. Goldstein, Hannah Nguyen, Christina S. Henderson, Kaylyn E. Williamson, Byron DeLaBarre, Christopher M. Bailey, Miljan Kuljanin, Matthew C. Koehler, James E. Audia, Jonathan E. Wilson, Jacob I. Stuckey, Robert J. Sims, . Discovery of FX-111, a first-in-class heterobifunctional degrader of transcriptionally active androgen receptor (ARON), to treat patients with AR-driven prostate cancer [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 5784.

We report our mechanistic investigation into the conformationally-driven ‘activation bias’ of PPARG in muscle-invasive urothelial cancer (MIUC) and our efforts to pharmacologically reverse this activation bias through covalent PPARG inverse agonism. We utilized studies into tumor-associated mutations in both PPARG and RXRA, as well as a combination of structure-based drug design merged with insights from biochemical mechanistic studies to discover FX-909, a first-in-class clinical PPARG inverse agonist that robustly enforces a conformationally ‘repressive’ state of PPARG, even in highly activated contexts such as RXRA S427F mutation and PPARG amplification. FX-909 is a potent, highly selective, and powerful suppressor of PPARG transcriptional activity through enhancement of PPARG nuclear corepressor binding (NCOR) affinity. Treatment with FX-909 resulted in selective growth inhibition in PPARG-activated MIUC cell lines. Further, FX-909 achieved durable regressions in xenograft models of MIUC through inverse agonism of PPARG. FX-909 is the first chemical tool available to the community that is capable of recapitulating PPARG genetic knockout in vivo and is currently in clinical development for the treatment of intractable MIUC. ### Competing Interest Statement Authors are shareholders in Flare Therapeutics.

Abstract In the last decade, genetic activation of PPARG in muscle invasive urothelial cancer (MIUC) has emerged as a potential therapeutic intervention point. More than two decades ago, the first PPARG covalent inverse agonist was described. While cellularly potent, subsequent drug discovery efforts to understand and improve the pharmacology of this initial inverse agonist were largely unsuccessful until quite recently. We have recently disclosed FX-909, a covalent inverse agonist of PPARG that shows durable tumor regressions in PPARG-activated xenograft models. Detailed structural, biochemical and medicinal chemistry studies were critical to both rational and rapid evolution from previously described covalent PPARG inverse agonists to FX-909, which exhibits both superior pharmacological and in vivo properties. Mechanistic understanding of the altered conformational landscape of PPARG in MIUC integrated with detailed insights into the covalent reaction coordinate of PPARG inverse agonists enabled the design of FX-909, specifically tailored to exhibit robust “conformational biasing” that counters the PPARG conformational “activation bias” observed in MIUC. Citation Format: Jacob I. Stuckey, Jennifer Mertz, Jonathan Wilson, Yong Li, Gregg Chenail, Miljan Kuljanin, James Audia, Robert Sims. Mechanistic insights for the advancement of PPARG inverse agonists in muscle invasive urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr IA014.

This file contains six supplementary figures: Figure S1 (related to Figure 1) provides additional characterization of the BETi tolerant cell lines; Figure S2 (related to Figure 1) provides additional information regarding gene expression changes in the BETi tolerant cell lines; Figure S3 (related to Figure 2) provides further support for the importance of BCL2 and BCL2L1 expression in the BETi tolerant cell lines; Figure S4 (related to Figures 3 and 4) quantifies Figure 3C, and provides supporting data and additional analysis for BCL2/BCL2L1/BAD as predictive biomarkers; Figure S5 (related to Figure 4) provides supporting data for BCL2 family expression as predictive biomarkers; Figure S6 (related to Figure 4) shows that BCL2/BCL2L1 protein expression can serve as predictive biomarkers for BETi response.

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin (Mb), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.

This file contains the following: source data for the graphs shown in Figure 3A (GI50, Z sub G1, and Z G1 increase); source data for the heatmap and graph shown in Figure 3B (expression fold change, log2 fold change, and gene score); qPCR source data for the heatmap and graph in Figure 4D; measurements of Western blotting band intensity from Figure S6.

This file contains source data for the graph in Figure 4A (GI50 and expression values for each of the genes in 245 cell lines).

This file contains the source of all cell lines used for the work described in the manuscript as well as the methods by which cell lines are authenticated at the repositories.

Abstract Although the treatment landscape of advanced urothelial cancer (UC) has changed dynamically over the last decade, metastatic UC still has amongst the worst 5-year recurrence rate of any cancer type, highlighting the need for new therapeutic options for these patients. Analogous to breast cancer, advanced UC is a heterogeneous disease, with luminal and basal subtypes. For luminal breast cancer, small molecule targeting of the lineage-defining transcription factor estrogen receptor (ER) is a demonstrated and effective therapeutic strategy. Two-thirds of advanced UC is classified as luminal, which is characterized by overexpression of the urothelial lineage-defining transcription factor PPARG. Thus, small molecule targeting of PPARG in luminal UC may serve as a therapeutic strategy analogous to ER in luminal breast cancer. While PPARG agonists have been well studied in the context of metabolic disease, PPARG inhibitors have received far less attention. Previously reported PPARG inhibitors display minimal phenotypic activity in pre-clinical UC models, calling into question the performance of these molecules, or alternatively, the role of PPARG as a survival lineage oncogene in UC. Leveraging reconstituted, multi-component PPARG biochemical systems, we first identified the limitations of previous tool compounds and then discovered a novel, covalent lead series with unique, context-dependent electrophilic properties. These efforts lead to the discovery of FX-909, a first-in-class covalent PPARG inverse agonist with powerful repressive conformational biasing activity. FX-909 is highly potent in cells (IC50=1 nM), demonstrates high specificity for PPARG (>2000-fold selective over PPARA/PPARD), and elicits robust in vitro growth inhibition in UC cell lines with activated PPARG signaling. Tumor regression was observed in UMUC9 (PPARG amplification) and HT1197 (RXRAS427F hotspot mutation) xenograft models of UC with oral dosing at 1 mg/kg. Predictable, on-target and reversible pharmacology was observed at FX-909 doses above 1 mg/kg, mimicking PPARG loss-of-function mutations with notable tissue remodeling in adipose tissue and the normal urothelium. These collective findings corroborate the role of PPARG as a key UC survival oncogene and suggest that FX-909 will be an effective therapy for patients with advanced UC harboring the luminal subtype. Citation Format: Robert Sims, Jennifer A. Mertz, Jonathan E. Wilson, James E. Audia, Kaylyn E. Williamson, Yong Li, Miljan Kuljanin, Will W. Motely, Byron DeLaBarre, Michaela Bowden, Jacob I. Stuckey. Discovery of FX-909, a first-in-class inverse agonist of the peroxisome proliferator-activated receptor gamma (PPARG) lineage transcription factor, to potentially treat patients with the luminal subtype of advanced urothelial cancer (UC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND08.

This file contains the legends for the six supplementary figures.

This file contains 4 supplementary tables: Table S1 provides the expression values of the indicated genes (RMA) in BETi sensitive and insensitive cell lines; Table S2 shows the enrichment of subtypes of cell lines following selection with the indicated criteria; Table S3 shows SNP analysis of parental and BETi tolerant A375 cells; Table S4 shows SNP analysis of parental and BETi tolerant NOMO-1 cells.

Targeting lineage-defined transcription factors with small molecules is a proven and effective therapeutic strategy, as demonstrated by estrogen and androgen receptor therapies for breast and prostate cancers. Expression of the transcription factor PPARG is associated with the luminal lineage subtype, which accounts for two-thirds of all advanced urothelial cancer (UC) patients. Taking into consideration the difficulty in obtaining sequential tumor biopsies in late-stage cancer patients, the feasibility of an accessible, less invasive surrogate tissue to evaluate PD biomarkers that support proof of mechanism of drug action, clinical proof of concept and ultimately the selection of optimal dosing, was evaluated. FX-909, a covalent PPARG inverse agonist is projected to initiate Phase 1 clinical studies in 2023 aimed at serving patients with advanced UC. In vivo UMUC9 UC xenograft model efficacy studies were performed in NGC mice treated with FX-909 at 0.03 mg/kg to 1 mg/kg BID for 21 days. Preclinical pharmacology studies were performed in Sprague Dawley male rats treated with FX-909 at 3, 10 and 30 mg/kg BID for 28 days to investigate potential biomarkers associated with on-target effects of FX-909. Ex vivo cultured human skin explant models derived from donors undergoing abdominoplasty procedures were utilized to bridge the interspecies translational gap to test the effect of FX-909 on human tissue. Tumor tissue from mouse xenografts, as well as inguinal white adipose tissue (iWAT) and skin tissue were collected. Changes in known PPARG target gene expression were measured by qPCR and transcriptomic differences by RNA-Seq. A subset of genes exhibited similar dose dependent trends in rat skin and adipose tissues, including known PPARG targets such as, Rbp4, Cidea and Ivd, fitting the expected decreasing exponential curve. Overall, 30% of all genes that responded to FX-909 treatment in skin displayed a similar dose-dependent response profile in iWAT with median Pearson correlations ranging from r=0.33-0.92, exemplified by the Ivd correlation, r=0.91 [p-value<0.0002], where approximately 50% gene suppression was observed at the lowest dose level. Importantly in UMUC9 models, dose-dependent suppression of FABP4/Fabp4 target gene showed strong correlation [r=0.98, p-value=0.003] between tumor and skin tissues. Based on these preclinical data and confirmatory experiments in the ex vivo setting, a novel FX-909 signature is under development to predict target modulation in the tumor. The consistent correlation of PPARG target gene suppression in tumor and normal tissues across species provide a strong rationale for a surrogate biospecimen collection, such as a skin punch biopsy, to establish the PK/PD relationship between the dose and response to FX-909 in the clinic. Citation Format: Jennifer A. Mertz, Stefan A. Kirov, Kaylyn E. Williamson, Marton Munz, Robert J. Sims, Michaela Bowden. Development of a surrogate tissue pharmacodynamic (PD) assay for clinical use with FX-909, a novel inhibitor of the urothelial luminal lineage transcription factor peroxisome proliferator-activated receptor gamma (PPARG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2802.

This file contains additional information regarding the methods used in the manuscript, including shRNA sequences, peptide and antibody information, and details about analysis methods.

Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for in vivo use.

Background: Small molecule targeting of cell lineage-defining transcription factors is a demonstrated and effective therapeutic strategy in oncology, exemplified by estrogen receptor (ER) agents in luminal breast cancer. Two-thirds of advanced UC is classified as luminal and overexpression of PPARG is characteristic of this molecular subtype. Currently, there is a poor understanding of how recurrent missense mutations in PPARG and its obligate heterodimer retinoid X receptor alpha (RXRA) impact PPARG function; previous tool compounds designed to inhibit PPARG have minimal phenotypic activity in UC cell lines. Material and Methods: The PPARG inhibitor FTX-6746 was evaluated in UC cell lines including UMUC9 (PPARG amplification) and HT1197 (RXRA-S427F hotspot mutation), which activate PPARG by different genetic mechanisms. In vitro analyses included clonogenic growth assays and assessment of target gene modulation after treatment. in vivo studies were performed in NCG or Balb/C nude mice with oral administration of FTX-6746 at doses indicated below. Results: Our biochemical studies indicate that patient-derived missense mutations in PPARG and RXRA bias an active conformation of PPARG, mimicking an agonist-bound state. Addressing the limitations of previous tool compounds, we discovered novel inhibitors that drive a powerful repressive conformation of PPARG with high specificity (>100X selective over PPARA/ PPARD). This results in robust PPARG target gene silencing in cells (IC50 = 5 nM), and in vitro growth inhibition preferentially observed in cell lines with activated PPARG signaling. Tumor growth inhibition or regression was observed in two xenograft models of UC at well-tolerated oral doses with no tumor regrowth upon cessation of treatment. Treatment of UMUC9 xenograft tumors with 3, 10, 30 and 60 mg/kg twice daily of FTX-6746 resulted in up to 80% target gene suppression in tumor tissue at day 2 and up to >100% tumor growth inhibition at day 21. Similar efficacy was observed in HT1197 xenograft studies treated with 30 and 60 mg/kg twice daily of FTX-6746, with >45% target gene suppression at day 2 and 85–112% tumor growth inhibition at day 42.Tabled 1ModelDose (mg/kg)Schedule%Tumor Growth Inhibition%Target Gene Suppression (D2)UMUC93BID*205916UMUC910BID*2070NDUMUC930BID*208373UMUC930BID*2110580UMUC960BID*21108NDHT119730BID*428546HT119760BID*4211259 Open table in a new tab Conclusions: These collective results suggest that PPARG is a lineage-defining transcription factor in UC and small molecule PPARG inhibition will be an effective therapy for patients with advanced urothelial cancer harboring the luminal subtype. Conflict of interest: Ownership: All authors are shareholders in Flare Therapeutics.

Background: About 20 000 patients are diagnosed with muscle-invasive UC annually, where the five-year survival rate is approximately 5% in metastatic cases. The transcription factor PPARG is associated with the luminal lineage subtype reflecting ∼65% of all advanced UC patients. Recurrent genetic alterations in PPARG, including focal amplification, missense mutations, and fusions, as well as hotspot mutations in its obligate heterodimer, retinoid X receptor alpha (RXRA) are characteristic of this molecular subtype. Materials and Methods: We analyzed tumor tissue collected from over 3000 patients with muscle-invasive UC sequenced using the Tempus xT assay (DNA-seq of 648 genes at 500x coverage; RNA-seq; 3262pt DNA, 2685pt both). Molecular classification as luminal (luminal papillary, luminal, and luminal infiltrated subtypes) or non-luminal (basal-squamous and neuronal subtypes) was performed using non-negative matrix factorization (NMF) rank 5 following the Robertson method, excluding PPARG from the gene set to eliminate the inference of bias. Mean expression was reported as Log2[TPM+1]. Mutations were reported as single nucleotide variants or insertion/deletions (SnvIndel). Results: We determined that luminal-papillary (669pt), luminal (449pt), and luminal-infiltrated (621pt) UC subtypes comprised 65% of the real-world cohort, consistent with subtype distribution as reported in the Robertson TCGA data set. Higher PPARG expression was associated with luminal subtypes (7.78 Log2[TPM+1], p < 0.00001) compared to non-luminal subtypes (5.47 Log2[TPM+1]). The prevalence of mutations in PPARG, RXRA, and fibroblast growth factor receptor 3 (FGFR3) was also significantly higher in the luminal subtype, where the frequency of PPARG amp (copy number 4–7) was 10% (p < 0.05), PPARG SnvIndel was 2.0% (p = 0.8), RXRA hotspot SnvIndel was 3.3% (p < 0.00005) and FGFR3 SnvIndel was 14% (p < 0.005). Collectively 1 in 3 advanced UC patients in a real-world population harbored mutations in PPARG, RXRA or FGFR3. A PPARG expression threshold (7.75 Log2[TPM+1]) was derived from logistic regression comparing amplified and diploid patients, where gain of even one copy of PPARG is associated with overexpression. Many diploid patients exhibit PPARG expression (7.49 Log2[TPM+1]) comparable to amplified patients. High PPARG expression was sustained in metastatic samples (7.55 Log2[TPM+1]), and expression did not significantly vary by metastatic tissue site (p > 0.05). Conclusion: To the best of our knowledge, this represents the largest real-world dataset from patients with advanced and metastatic UC evaluated to date. Stratification of advanced UC patients based upon PPARG status as a defining feature of the luminal phenotype supports targeting of this pathway with novel agents under development. Conflict of interest: Ownership: Flare-affiliated authors are shareholders of Flare Therapeutics.

Therapeutic targeting of the estrogen receptor (ER) is a clinically validated approach for estrogen receptor positive breast cancer (ER+ BC), but sustained response is limited by acquired resistance. Targeting the transcriptional coactivators required for estrogen receptor activity represents an alternative approach that is not subject to the same limitations as targeting estrogen receptor itself. In this report we demonstrate that the acetyltransferase activity of coactivator paralogs CREBBP/EP300 represents a promising therapeutic target in ER+ BC. Using the potent and selective inhibitor CPI-1612, we show that CREBBP/EP300 acetyltransferase inhibition potently suppresses in vitro and in vivo growth of breast cancer cell line models and acts in a manner orthogonal to directly targeting ER. CREBBP/EP300 acetyltransferase inhibition suppresses ER-dependent transcription by targeting lineage-specific enhancers defined by the pioneer transcription factor FOXA1. These results validate CREBBP/EP300 acetyltransferase activity as a viable target for clinical development in ER+ breast cancer.