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Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest ovarian cancer stem cells (OCSCs) contribute to chemotherapy resistance and tumor relapse. Recent studies demonstrated estrogen receptor beta (ERβ) exerts tumor suppressor functions in OCa. However, the status of ERβ expression in OCSCs and the therapeutic utility of the ERβ agonist LY500307 for targeting OCSCs remain unknown. OCSCs were enriched from ES2, OV90, SKOV3, OVSAHO, and A2780 cells using ALDEFLUOR kit. RT-qPCR results showed ERβ, particularly ERβ isoform 1, is highly expressed in OCSCs and that ERβ agonist LY500307 significantly reduced the viability of OCSCs. Treatment of OCSCs with LY500307 significantly reduced sphere formation, self-renewal, and invasion, while also promoting apoptosis and G2/M cell cycle arrest. Mechanistic studies using RNA-seq analysis demonstrated that LY500307 treatment resulted in modulation of pathways related to cell cycle and apoptosis. Western blot and RT-qPCR assays demonstrated the upregulation of apoptosis and cell cycle arrest genes such as FDXR, p21/CDKN1A, cleaved PARP, and caspase 3, and the downregulation of stemness markers SOX2, Oct4, and Nanog. Importantly, treatment of LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopic OCa murine xenograft models. Our results demonstrate that ERβ agonist LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and shows significant promise as a novel therapeutic agent in treating OCa.

Endometrial cancer (EC) often exhibit aberrant activation of PI3K/Akt/mTOR signaling and targeted therapies using mTOR inhibitors showed limited success. The epigenetic modifier, lysine-specific histone demethylase-1A (KDM1A/LSD1) is overexpressed in EC, however, the mechanistic and therapeutic implications of KDM1A in EC are poorly understood. Here, using 119 FDA-approved drugs screen, we identified that KDM1A inhibition is highly synergistic with mTOR inhibitors. Combination therapy of KDM1A and mTOR inhibitors potently reduced the cell viability, survival, and migration of EC cells. Mechanistic studies demonstrated that KDM1A inhibition attenuated the activation of mTOR signaling cascade and abolished rapamycin induced feedback activation of Akt. RNA-seq analysis identified that KDM1A inhibition downregulated the expression of genes involved in rapamycin induced activation of Akt, including the mTORC2 complex. Chromatin immunoprecipitation experiments confirmed KDM1A recruitment to the promoter regions of mTORC2 complex genes and that KDM1A inhibition promoted enrichment of repressive H3K9me2 marks at their promoters. Combination therapy of KDM1A inhibitor and rapamycin reduced the tumor growth in EC xenograft and patient derived xenograft models in vivo and patient derived tumor explants ex vivo. Importantly, in silico analysis of TCGA EC patients data sets revealed that KDM1A expression positively correlated with the levels of PI3K/Akt/mTOR genes. Collectively, our results provide compelling evidence that KDM1A inhibition potentiates the activity of mTOR inhibitors by attenuating the feedback activation of Akt survival signaling. Furthermore, the use of concurrent KDM1A and mTOR inhibitors may be an attractive targeted therapy for EC patients.

Background: Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest that ovarian cancer stem cells (OCSCs) contribute to tumor relapse and chemotherapy resistance. Recent studies demonstrate that OCa cells express estrogen receptor beta (ERβ), which functions as a tumor suppressor. However, the status of ERβ expression in OCSCs and the therapeutic utility of ERβ agonist LY500307 for targeting OCSCs remain unknown. In this study, we tested the hypothesis that OCSCs express ERβ and that treatment with ERβ agonist reduces stemness and promotes apoptosis of OCSCs. Methods: We isolated subpopulations of OCSCs from SKOV3, A2780, and patient-derived ascites cells using ALDEFLUOR kit and examined the expression of ERβ in OCSCs using RT-qPCR. The effect of ERβ agonist LY500307 on OCSCs was examined utilizing cell viability, cell cycle, sphere formation, self-renewal, and apoptosis assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, and Western blot analysis. The efficacy of LY500307 on the tumor-initiating capacity of OCSCs was determined via in vivo limiting dilution assay studies using orthotopic intrabursal xenograft murine models. Results: RT-qPCR assay showed that ERβ, particularly ERβ isoform 1, is highly expressed in OCSCs compared to non-OCSCs. ERβ agonist LY500307 significantly reduced the viability of OCSCs compared to non-OCSCs. Treatment of OCSCs with LY500307 significantly reduced the sphere formation and self-renewal of OCSCs. Further, LY500307 treatment resulted in G2/M cell cycle arrest and promoted the apoptosis of OCSCs. Mechanistic studies using RNA-seq analysis demonstrated that ERβ agonist treatment resulted in the modulation of pathways related to cell cycle and apoptosis. Western blots and RT-qPCR assays confirmed the upregulation of genes involved in apoptosis and cell cycle arrest, such as FDXR and CDKN1A. Importantly, treatment of LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopically implanted OCa murine models. Conclusions: Our results demonstrate that ERβ agonist LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and may serve as a novel therapeutic agent in treating OCa Citation Format: Yi He, Salvador Alejo, Prabhakar P. Venkata, Jessica D. Johnson, Ilanna Loeffel, Julie Ann Martel, Uday P. Pratap, Suryavathi Viswanadhapalli, Rajeshwar R. Tekmal, Ratna K. Vadlamudi, Edward Kost, Gangadhara R. Sareddy. Therapeutic targeting of ovarian cancer stem cells using estrogen receptor beta agonist [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 891.

Background: Ovarian cancer (OCa) is the deadliest gynecologic cancer. Recent studies suggest that OCa cells express estrogen receptor beta (ESR2), which functions as a tumor suppressor. However, ESR2 expression decreases during tumor progression and under the selection pressure of chemotherapy; this decrease occurs via epigenetic mechanisms. The lysine-specific histone demethylase 1A (KDM1A), an epigenetic regulator, is overexpressed in OCa. We reason that agents that restore the expression/functions of ESR2 may provide a novel therapeutic opportunity for suppression of OCa. In this study we tested the hypothesis that KDM1A suppresses ESR2 expression/functions and that inhibition of KDM1A potentiates ESR2 mediated tumor suppression. Methods: We examined the utility of combination therapy of KDM1A inhibitor and ESR2 agonist on established and patient derived OCa cells using cell viability, survival, apoptosis and invasion assays. KDM1A knockout (KDM1A-KO) cells were generated using the CRISPR/Cas9 system. Mechanistic studies were conducted using RNA-seq, ERE-Luc reporter assays, RT-qPCR, co-IP, ChIP and Western blot analysis. The efficacy of combination therapy on primary ovarian tumors was examined using patient derived explant (PDEx) models. In vivo efficacy of KDM1A inhibitor and ESR2 agonist was studied using orthotopic OCa and patient derived xenograft models. Results: Analysis of the gene expression profiles of OCa patients showed that KDM1A negatively correlates with ESR2. Combination of KDM1A inhibitor NCD-38 and ESR2 agonist LY500307 synergistically reduced cell viability, survival and invasion and increased apoptosis of established and patient derived primary OCa cells. Inhibition or knockout of KDM1A sensitized OCa cells to ESR2 agonist treatment and significantly increased the expression of ESR2 and its target genes. Further, we found that KDM1A is recruited to the ESR2 0N promoter and interacts with ESR2. In addition, inhibition of KDM1A increased the active histone methylation mark H3K4-me2 at the ESR2 0N promoter. RNA-seq analysis demonstrated that ESR2 agonist treatment resulted in modulation of pathways related to cell cycle, apoptosis and DNA damage. Importantly, combination treatment significantly reduced the tumor growth in orthotopic and patient derived xenograft models and reduced the proliferation of OCa cells in PDEx models. Conclusions: Our results demonstrate that KDM1A inhibition enhances ESR2 expression/functions and combination therapy of KDM1A inhibitor and ESR2 agonist is an attractive therapy for treating OCa. Citation Format: Prabhakar Pitta Venkata, Bridgitte E. Palacios, Yihong Chen, Salvador Alejo, Yi He, Ilanna Loeffel, Uday Pratap, Kristin Altwegg, Suryavathi Viswanadhapalli, Takayoshi Suzuki, Rajeshwar Tekmal, Edward Kost, Gangadhara Sareddy. KDM1A inhibition activates estrogen receptor beta pathway to suppress ovarian cancer progression [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 1764.