The receptor tyrosine kinase TIE2 and its ligands, angiopoietins (ANGPTs), promote angiogenesis. In addition to expression on vascular endothelial cells, TIE2 is expressed on M2-like pro-tumorigenic macrophages. Thus, the TIE2 inhibitor rebastinib was developed as a potential therapy to address multiple cancers. The objective of this study was to determine the effects of rebastinib alone and combined with chemotherapy in a syngeneic murine model of ovarian cancer. Female C57Bl6J mice were intraperitoneally injected with syngeneic ID8 ovarian cancer cells. Once tumors were established, mice were untreated (control) or treated with rebastinib, carboplatin plus paclitaxel (chemotherapy), or rebastinib plus chemotherapy. In one set of experiments, survival was followed for 140 days. In other experiments, ascites was harvested 24 h after the last treatment and analyzed by flow cytometry. In in vitro experiments, RNA sequencing was performed on ID8 cells and murine peritoneal macrophage cells (PMJ2R) after treatment with rebastinib, chemotherapy, or rebastinib plus chemotherapy. Tumor-bearing mice treated with rebastinib plus chemotherapy had longer median survival than mice treated with chemotherapy (132.5 vs. 127 days, P < 0.01). Ascites from mice treated with rebastinib had more CD45 + macrophages (P < 0.03) and cytotoxic T cells (P < 0.0001) than ascites from mice treated with chemotherapy. Rebastinib had no significant effect on the numbers of regulatory T cells, Tie2 + macrophages, or Tie2 + M2 macrophages. In ID8 cells, in vitro, rebastinib treatment upregulated 1528 genes and downregulated 3115 genes. In macrophages, in vitro, rebastinib treatment upregulated 2302 genes and downregulated 2970 genes. Rebastinib differentially regulated ANGPT-like proteins in both types of cells, including several ANGPT-like genes involved in tumorigenesis, angiogenesis, and proliferation. ANGPTL1, an anti-angiogenic and anti-apoptotic gene, was increased tenfold in ID8 cells treated with rebastinib (P < 0.001) but was not altered in macrophages. Rebastinib plus chemotherapy extends survival in a syngeneic murine model of ovarian cancer. Rebastinib alters proportions of immune cell subsets, increases cytotoxic T cells in ascites, and alters gene expression in tumor cells and macrophages.

Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ~25% surviving beyond 5 years. Evidence suggests that cancer stem cells contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. Moreover, low molecular weight hyaluronic acid, which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC.

Abstract Poly ADP ribose inhibitors (PARPi) are most effective in ovarian cancer tumors with homologous recombination (HR) deficiency. Our group has shown that histone deacetylase inhibitors (HDACi) sensitize HR proficient ovarian cancer cells to PARPi. Our current efforts are directed towards understanding how this therapeutic regimen alters tumor-associated macrophages (TAMs) in murine models of high grade serous ovarian cancer. To investigate the effects of Ola combined with Ent, we used HR-proficient ID8 P53 wild-type and ID8 P53−/− syngeneic murine models. Mice were randomized into 4 groups: control, Ola, Ent and Ola+Ent. Mice (Ent and Ola+Ent) were pre-treated with 15 mg/kg entinostat or vehicle (Ola and Ola+Ent) for one week via oral gavage and then treated with vehicle/Ent/Ola (100 mg/kg)/Ola+Ent for two weeks. Mice were sacrificed 24 h after the last dose to harvest tumors and ascites. Tumors were processed for histology to determine cell proliferation (Ki67) and immune cell markers (CCL2, M1 and M2-like macrophages). Ascites fluid was processed for flow cytometric analysis of immune cells. Tumors from parental ID8 P53 wild type mice showed significantly lower cell proliferation marker Ki67 (P<0.05), higher anti-tumorigenic M1-like CCL2 (P<0.05), and lower pro-tumorigenic M2-like mannose receptor (P<0.05) in Ent and Ola+Ent groups compared to vehicle and Ola. Ascites showed no significant change in anti-tumorigenic M1-like macrophages, but significantly increased pro-tumorigenic M2-like macrophages (P<0.005). Arginase-1, another marker of M1 macrophage, assessed by IF also confirmed significant decrease in pro-tumorigenic M2-like macrophage in Ent and Ola+Ent treated mice tumors. Additional immune cell markers identified in ascites are being analyzed. To summarize, in ID8 syngeneic mouse models, Ola and Ent treatment exerted anti-tumorigenic effects in tumors but potentially pro-tumorigenic effects in ascites. In conclusion, concomitant targeting of tumor TAMs and ascites TAMs may be a therapeutic regimen to investigate in the future. Citation Format: Vijayalaxmi Gupta, Katherine Roby, SImona Miceska, Andrew Wilson, Fiona Yull, Marta Crispens, Wendy Zhang, Dineo Khabele. Entinostat alters the M1/M2 ratio in ascites compared to tumors derived from an ID8 murine model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A100.

Poly ADP ribose inhibitors (PARPi) are most effective in ovarian cancer tumors with homologous recombination (HR) deficiency. Our group has shown that histone deacetylase inhibitors (HDACi) sensitize HR proficient ovarian cancer cells to PARPi. Our current efforts are directed towards understanding how this therapeutic regimen alters tumor-associated macrophages (TAMs) in murine models of high grade serous ovarian cancer. To investigate the effects of Ola combined with Ent, we used HR-proficient ID8 P53 wild-type and ID8 P53−/− syngeneic murine models. Mice were randomized into 4 groups: control, Ola, Ent and Ola+Ent. Mice (Ent and Ola+Ent) were pre-treated with 15 mg/kg entinostat or vehicle (Ola and Ola+Ent) for one week via oral gavage and then treated with vehicle/Ent/Ola (100 mg/kg)/Ola+Ent for two weeks. Mice were sacrificed 24 h after the last dose to harvest tumors and ascites. Tumors were processed for histology to determine cell proliferation (Ki67) and immune cell markers (CCL2, M1 and M2-like macrophages). Ascites fluid was processed for flow cytometric analysis of immune cells. Tumors from parental ID8 P53 wild type mice showed significantly lower cell proliferation marker Ki67 (P<0.05), higher anti-tumorigenic M1-like CCL2 (P<0.05), and lower pro-tumorigenic M2-like mannose receptor (P<0.05) in Ent and Ola+Ent groups compared to vehicle and Ola. Ascites showed no significant change in anti-tumorigenic M1-like macrophages, but significantly increased pro-tumorigenic M2-like macrophages (P<0.005). ID8 P53−/− mice tumors showed significantly lower Ki-67 (P<0.05) in Ola+Ent group compared to vehicle and mono-treatments. Ascites showed no significant change in total macrophage or pro-tumorigenic M2-like macrophages, but a significant decrease in anti-tumorigenic M1-like macrophages in Ent and Ola+Ent groups compared to vehicle. To summarize, in HR proficient ID8 P53 wildtype and ID8 P53−/− syngeneic mouse models, Ola and Ent treatment exerted anti-tumorigenic effects in tumors but potentially pro-tumorigenic effects in ascites. In conclusion, concomitant targeting of tumor TAMs and ascites TAMs may be a therapeutic regimen to investigate in the future. Citation Format: Vijayalaxmi G. Gupta, Tyler Woodard, Simona Miceska, Bisiyao Fashemi, Sangappa Chadchan, Wendy Zhang, Katherine Roby, Andrew Wilson, Fiona Yull, Marta Crispens, Sumanta Naik, Asya Smimov, Christina Stallings, Dineo Khabele. Olaparib combined with entinostat exerts differential effects on tumor-associated macrophages in tumors compared to ascites in syngeneic HR-proficient murine models of ovarian cancer. [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 3654.

Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ∼25% surviving beyond 5 years. Evidence suggests that cancer stem cells (CSCs) contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. IRAK1 and BRCA1/2 mutation status are mutually exclusive. Moreover, low molecular weight hyaluronic acid (LMW HA), which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC.

Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due in part to late-stage diagnoses. While the overall response rate to first line therapy is encouraging (~80%), the majority of women develop recurrent disease, characterized by resistance to standard chemotherapy. There is convincing evidence that cancer stem cells (CSCs) have implications in resistance and recurrence of OvCa. To understand the pathways involved in enhancing this stem-like phenotype, we performed RNA sequencing and identified TLR-ILR1 (TIR) pathways as highly activated in cisplatin resistant OvCa and stem-cell populations. Subsequently, using a tumor microarray, we observed that interleukin receptor-associated kinase 1 (IRAK1), a critical mediator of TIR signaling, is upregulated in OvCa patient tissues compared to normal. This expression further correlated with younger diagnosis age and shorter overall survival, suggesting a role in OvCa tumorigenesis. Knockdown of IRAK1 by specific shRNA in OvCa cells, significantly impaired CSC enriched spheroid growth and orthotopic tumor growth in mouse models replicating advanced peritoneal OvCa. RNA sequencing of IRAK1 knocked down cells identified downregulation of CSC related genes including STAT3, MYC, NOTCH1, and NOTCH3. In addition, there was a reduction in the cisplatin efflux transporter ABCC1 and stemness marker gene CD44. Advanced OvCa is often associated (~90% in stage III and IV) with the development of malignant ascites. We demonstrate for the first time that significant amounts of low molecular weight hyaluronic acid fragments (LMW HA) are present in ascites. LMW HA induced IRAK1 dependent activation of non-canonical signaling through PKC-β leading to induction of STAT3 phosphorylation and MYC. This was further accompanied by increased spheroid formation, demonstrating a critical role for IRAK1 in HA-induced stemness. We have further identified a selective IRAK1 inhibitor, TCS2210 (1,2-Dihydro-N-hydroxy-2-oxo-3-(3-phenylpropyl)-6-quinoxalinecarboxamide). TCS2210 abrogated LMW HA induced activation of IRAK1 and expression of stemness genes MYC, NOTCH1, and NOTCH3. Lastly, we found that TCS2210 effectively suppresses OvCa cell growth in vitro and in vivo, synergizing with standard-of-care cisplatin. Our data suggests a role of IRAK1 in OvCa, by enhancing cancer cell growth and stemness. It further suggests that IRAK1 is a putative target for advanced OvCa. Citation Format: David Standing, Prasad Dandawate, Jaimie Johnson, Sumedha Gunewardena, Michele T. Pritchard, Harsh Pathak, Dineo Khabele, Katherine Roby, Andrew Godwin, Roy Jensen, Scott Weir, Shrikant Anant. IRAK1 is a critical mediator of hyaluronic acid induced stemness. [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 5020.

Genetic alterations and response to licofelone or glafenine in ovarian MCTS.

This document contains supplemental figures 1-17. Figure legends corresponding to each SF are on the subsequent page. Supplemental Figure 1. MCTS formation across a panel of ovarian cancer cell lines. Supplemental Figure 2. The number of proliferating cells is universally reduced in ovarian cancer cells grown as spheroids. Supplemental Figure 3. HIO cell lines form small spheroids which lack stem-like properties. Supplemental Figure 4. JMJD1A protein expression is enhanced in ovarian cancer cells when grown in 3D. Supplemental Figure 5. Targeted therapies used in ovarian cancer clinical trials induce a variable pattern of expression of stem-like associated genes. Supplemental Figure 6. CellTiter-Glo measured cell viability and drug response in MCTS. Supplemental Figure 7. 3D drug screening in non-tumorigenic cell lines did not identify 3D Hits. Supplemental Figure 8. Secondary screening validated 3D hits across multiple cell lines. Supplemental Figure 9. Dosage response validation of a 2D hit and 2D and 3D hit. Supplemental Figure 10. Licofelone showed enhanced activity in paclitaxel resistant 2D cells. Supplemental Figure 11. Licofelone had more activity against ovarian cancer cell-derived MCTS than the COX-2 specific inhibitor celecoxib. Supplemental Figure 12. Combination index values show strong synergy between with paclitaxel following licofelone pretreatment. Supplemental Figure 13. Pretreatment of ovarian cancer cell-derived MCTS with paclitaxel synergistically enhances licofelone activity. Supplemental Figure 14. Combination of licofelone and paclitaxel leads to increases in apoptosis in ovarian cancer cell-derived MCTS. Supplemental Figure 15. Licofelone can also sensitize ovarian cancer cells grown as spheroids to carboplatin. Supplemental Figure 16. Licofelone treatment decreases mitochondrial metabolism in OVCAR8 spheroids. Supplemental Figure 17. Changes in Cell Proliferation in Tumor Cells Isolated from Recurrent PDX Ascites Fluid.

Supplemental Table 3. Mante-Haenszel hazard ratio shows licofelone combination increases the efficacy of paclitaxel.

Expression of ABC transporter genes in 2D and in 3D cultures.

Objectives: The Angiopoietin (ANGPT)/TIE2 kinase axis plays a critical role in cancer angiogenesis. Rebastinib, an investigational TIE2 inhibitor inhibits Tie2 receptor in endothelial cells and macrophages. M2-like Tie2+ macrophages, being pro-tumorigenic, are an attractive target for cancer therapy. Current clinical trials are testing rebastinib with chemotherapy in platinum-resistant ovarian cancer. However, the mechanism is not fully understood. The objective of this study was to determine the effects of rebastinib alone and combined with chemotherapy in preclinical models of ovarian cancer. Methods: RNA-Seq was carried out to determine rebastinib-induced gene expression in ID8 cells and PMJ2R murine peritoneal macrophages. To determine the pharmacodynamics of immune cells in ascites, syngeneic ID8 mice were pre-treated with 10 mg/kg/day rebastinib/control for a week, followed by rebastinib with or without 20 mg/kg carboplatin + 12 mg/kg paclitaxel (chemo) for two weeks. Mice were sacrificed 24h after the last treatment to harvest ascites for flow cytometry. Cohorts of mice from syngeneic ID8 murine model and from a patient derived xenograft (PDX) model of ovarian cancer were used for survival analysis. Results: RNA-Seq showed that in ID8 cells, 1528 genes were upregulated and 3115 genes were downregulated by rebastinib. In macrophages, 2302 genes were upregulated and 2970 genes were downregulated. Several ANGPT-like genes (ANGPTL2, ANGPTL4, ANGPTL6) involved in tumorigenesis, angiogenesis, proliferation were downregulated 2X-10X (p<0.001) in ID8 cells and macrophages. ANGPTL1, an anti-angiogenic and anti-apoptotic gene, was increased 10X in ID8 cells (p<0.001) but not altered in macrophages. ANGPT2, a context-dependent agonist/antagonist of TIE2 pathway, was increased 1.42X in ID8 (P<0.05) cells and 4.6X in macrophages (p<0.001). Flow studies showed that rebastinib significantly increased CD45+ macrophage (p<0.03 vs. chemo; p<0.02 vs. rebastinib+chemo), maintained Th leukocytes (p<0.03, vs. chemo) and significantly increased cytotoxic T cells (p<0.02 vs. control; p<0.0004, p<0.0001 vs. chemo and rebastinib+chemo, respectively). Rebastinib had no significant effect on regulatory T cells, Tie2+ macrophages or Tie2+ M2 macrophages. Rebastinib combined with chemotherapy improved median survival significantly in murine models of ovarian cancer - ID8 (p<0.01) and PDX (p<0.0001). Conclusions: Rebastinib exerts differential effects on tumor cells and macrophages that could contribute to its mechanism of action. Rebastinib alters immune cells and increases cytotoxic T cells in ascites. Rebastinib combined with chemotherapy extends survival in PDX and syngeneic ID8 murine models of ovarian cancer. Further investigation using rational combinations of rebastinib and chemotherapy are underway. Citation Format: Vijayalaxmi G. Gupta, Katherine F. Roby, Harsh B. Pathak, Andrew K. Godwin, Sumedha Gunewardena, Dineo Khabele. Rebastinib, a TIE2 antagonist improves chemotherapy response in homologous recombination proficient epithelial ovarian cancer murine models [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 3457.

OBJECTIVE:Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer.METHODS:We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model.RESULTS:Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability.CONCLUSION:Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erβnull ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERβ is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erβnull mutant model that fails to show follicle maturation was also included in order to identify the ERβ-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erβnull GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erβnull GCs. These latter ERβ-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERβ-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erβnull GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERβ.

Kisspeptin (KISS1) signaling in the hypothalamic-pituitary (H-P) axis plays an essential role in regulating gonadotropin secretion. KISS1 and KISS1 receptor (KISS1R) are also expressed in the ovary; however, the role of intraovarian KISS1 signaling remains unclear. Granulosa cell (GC)-specific expression of KISS1, and oocyte-specific expression of KISS1R indicate that GC-derived KISS1 may act on oocytes. Expression of KISS1 in GCs is induced by gonadotropins but it is absent in estrogen receptor β knockout (Erβnull) rat ovaries. We also observed that gonadotropin stimulation failed to induce maturation of Erβnull oocytes. Interestingly, KISS1 treatment of cumulus oocyte complexes (COCs) isolated from antral follicles promotes in vitro maturation of oocytes. Treatment of oocytes with KISS1 induced intracellular Ca2+ release, and increased activation of MAP kinase ERK1/2. KISS1 treatment also induced the expression of oocyte genes that are crucial for differentiation of GCs, and maturation of oocytes. Our findings suggest that ovarian KISS1-signaling plays an important role in gonadotropin induced follicle development and oocyte maturation.

BACKGROUND:Our environment is replete with chemicals that can affect embryonic and extraembryonic development. Dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are compounds affecting development through the aryl hydrocarbon receptor (AHR). OBJECTIVES:The purpose of this investigation was to examine the effects of TCDD exposure on pregnancy and placentation and to evaluate roles for AHR and cytochrome P450 1A1 (CYP1A1) in TCDD action. METHODS:Actions of TCDD were examined in wild-type and genome-edited rat models. Placenta phenotyping was assessed using morphological, biochemical, and molecular analyses. RESULTS:TCDD exposures were shown to result in placental adaptations and at higher doses, pregnancy termination. Deep intrauterine endovascular trophoblast cell invasion was a prominent placentation site adaptation to TCDD. TCDD-mediated placental adaptations were dependent upon maternal AHR signaling but not upon placental or fetal AHR signaling nor the presence of a prominent AHR target, CYP1A1. At the placentation site, TCDD activated AHR signaling within endothelial cells but not trophoblast cells. Immune and trophoblast cell behaviors at the uterine-placental interface were guided by the actions of TCDD on endothelial cells. DISCUSSION:We identified an AHR regulatory pathway in rats activated by dioxin affecting uterine and trophoblast cell dynamics and the formation of the hemochorial placenta. https://doi.org/10.1289/EHP9256.

Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of ESR2-signaling results in increased activation of primordial follicles in Esr2 -null ( Esr2-/- ) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wildtype and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2-signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1 indicating that the regulation of primordial follicle activation is ESR2-specific. Follicle activation was also increased in Esr2 -mutants lacking the DNA-binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2 suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles.