The majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and its expression is commensurate with tumor grade and resistance to numerous therapies. MethodsThe effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using a combination of immuno uorescence and subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. ResultsTVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant cells compared to the parental cells. Additionally, TVB-3166 signi cantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition signi cantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. ConclusionOur preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

Glioblastoma (GBM) is the most prevalent and aggressive type of adult brain tumors with low 5-year overall survival rates. Epidemiologic data suggest that estrogen may decrease brain tumor growth, and estrogen receptor beta (ERβ) has been demonstrated to exert anti-tumor functions in GBM. The lack of potent, selective and brain permeable ERβ agonist to promote its anti-tumor action is limiting the therapeutic promise of ERβ. In this study, we discovered that Indanone and tetralone-keto or hydroxyl oximes are a new class of ER agonists. Due to its high activity in ERβ reporter assays, specific binding to ERβ in polar screen assays, and potent growth inhibitory activity in GBM cells, CIDD-0149897 was discovered as a possible hit by screening a library of compounds. CIDD-0149897 is more selective for ERβ than ERα (40-fold). Treatment with CIDD-0149897 markedly reduced GBM cell viability with an IC50 of ~7-15 µM, while having little to no effect on ERβ-KO cells and normal human astrocytes. Further, CIDD-0149897 treatment enhanced expression of known ERβ target genes and promoted apoptosis in established and patient-derived GSC models. Pharmacokinetic studies confirmed that CIDD-0149897 has systemic exposure, and good bioavailability in the brain. Mice tolerated daily intraperitoneal treatment of CIDD-0149897 (50 mg/kg) with a 7-day repeat dosage with no toxicity. Additionally, CIDD-0149897 treatment significantly decreased tumor growth in U251 xenograft model and extended the survival of orthotopic GBM tumor-bearing mice. Collectively, these findings pointed to CIDD-0149897 as a new class of ERβ agonist, offering GBM patients a potential means of improving survival.

Background: Glioblastoma (GBM) is the most prevalent and aggressive type of adult brain cancers with low 5-year overall survival rates. Epidemiologic data suggest estrogen may decrease brain tumor growth. The lack of powerful, selective brain permeable estrogen receptor beta (ERβ) ligands to promote its anticancer action is limiting the therapeutic promise of ERβ. The goal of this project is to create a new class of brain permeable ERβ agonists for the treatment of GBM. Method: We have used four different assays to identify potent leads. (1) ERβ and estrogen receptor alpha (ERα) reporter assays, (2) Polar Screen Estrogen Receptor (ER) Competitive Binding Assays, (3) Cell viability assays using GBM cells, and (4) Activity in WT and ERβ-KO GBM cells. CellTiter-Glo Cell Viability and colony formation assays were used to test the efficacy of ERβ agonists. Invasion was measured by matrigel invasion chamber assays and apoptosis was measured by Caspase-Glo® 3/7 and Annexin V assays. Flow cytometry was used to evaluate the cell cycle. Western blotting, RNA-Seq, RT-qPCR, and ERβ KO cells were used in the mechanistic studies. Both orthotopic patient-derived xenografts and GBM cell line-derived (CDX) xenografts were used to test the ERβ agonist's activity in vivo. Results: Indanone and tetralone-oximes or keto-oximes have been hypothesized, produced, and evaluated as new ER agonists. Due to its high action in ERβ reporter assays, specific binding to ERβ in polar screen assays (EC50 91 nM), and potent growth inhibitory activity (IC50 7.4) in GBM cells, CIDD-97 was discovered as a possible hit by screening a library of ~60 compounds. CIDD-97 is significantly more selective for ERβ than ERα (40-fold). Treatment with CIDD-97 markedly reduced U251 cell viability while having little to no effect on U251-ER-KO cells and normal human astrocytes. Further, CIDD-97 treatment decreased the expression of stemness markers in patient derived GSC lines and promoted apoptosis. Additionally, CIDD-ERβ agonist improved TMZ's ability to reduce GBM cell survival. The activation of ERβ target genes in cells treated with CIDD-97 was verified by using RNA-Seq and RTqPCR. PK studies confirmed that CIDD-0149897 has systemic exposure, was found in the brain, and its maximal detectable levels suggested that it has good BBB permeability. Mice tolerated daily intraperitoneal treatment of CIDD-0149897 (50 mg/kg) with a 7-day repeat dosage with no evidence of toxicity. Additionally, when compared to vehicle CIDD-97 (50 mg/kg/i.p.) treatment significantly decreased tumor growth in xenograft models and extended the survival of tumor-bearing mice in orthotopic GSC models. Conclusion: Collectively, these findings pointed to CIDD-97 as a possible ERβ agonist and is easily adaptable to clinical use alongside current chemo- and radiation-therapy regimens, offering GBM patients an additional means of improving survival. Citation Format: Uday P. Pratap, Michael Tidwell, Henriette U. Balinda, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Stanton McHardy, Andrew Brenner, Ratna K. Vadlamudi. Development of potent estrogen receptor beta agonists for treating glioblastoma [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 1718.

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