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Background: The obesity epidemic is rapidly increasing in the USA and obese women are at a higher likelihood of developing triple negative breast cancer (TNBC). Several studies implicated the importance of the breast microenvironment on the aggressive cancer biology especially obese microenvironment. However, the underlying mechanism(s) by which obesity contributes to the progression of TNBC remains unclear. The objective of this study is to test a novel concept that obesity upregulates leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and test whether LIFR inhibition blocks TNBC progression. Methods: Established TNBC cell lines were co-cultured with human primary adipocytes or incubated with adipocyte conditioned medium or with high glucose (HG) followed by treatment with LIFR inhibitor EC359. The effect of adiposity on TNBC cells was determined using cell viability, colony formation, and invasion assays. Mechanistic studies were performed using CRISPR/Cas9 KO of LIFR, Western blotting, RT-qPCR, and reporter gene assays. Utility of LIFR inhibitor EC359 was tested using xenografts, and patient derived organoid (PDO) models. Results: Treatment of TNBC cells with adipose conditions or HG increased the proliferation and invasion of TNBC cells. Western blot and RT-qPCR analyses confirmed that increased expression of LIFR correlated with enhanced downstream LIFR signaling such as STAT3 and subsequent activation of STAT3 target genes. CRISPR KO of LIFR or treatment of TNBC cells with EC359 significantly reduced the cell viability, colony formation and invasion under adipose conditions. Western blotting results showed that co-culture with adipocytes significantly enhanced LIFR downstream signaling in TNBC model cells and is effectively blocked by LIFR KO or EC359 treatment. Further, EC359 treatment blocked the adipose environment mediated growth of organoids. Importantly, co-implantation of adipocytes significantly enhanced TNBC xenograft tumor growth, however treatment with EC359 significantly attenuated adipocyte induced TNBC progression. Conclusions: Collectively, these results suggest that adiposity contributes to increased TNBC cell growth via upregulation of the LIF/LIFR pathway. The LIF/LIFR axis represents a potential therapeutic target for adiposity driven TNBC and the LIFR inhibitor EC359 could be used as a new therapeutic agent to treat obesity associated TNBC. Citation Format: Suryavathi Viswanadhapalli, Uday P Pratap, Behnam Ebrahimi, Logan Blankenship, Jaitri Joshi, Zexuan Liu, Kristin A Altwegg, Xiaonan Li, Gangadhara R Sareddy, Bindu Santhamma, Swapna Konda, Manjeet Rao, Edward Kost, Rajeshwar R Tekmal, Hareesh B Nair, Ratna K Vadlamudi. Leukemia inhibitory factor receptor inhibition reduces obesity driven progression of triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-01.

Background: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States. Despite initial response to chemotherapy, most OCa patients become chemo resistant and progress to metastatic disease. Here, we tested the hypothesis that the high basal level of endoplasmic reticulum stress (ERS) in OCa represents a critical vulnerability and drugs that further aggravate this already engaged system in OCa may exhaust its protective features and contribute to apoptosis induction. The objective of this proposal is to identify a hit compound that enhances ERS in OCa and to conduct mechanistic studies. Methods: We synthesized a small library of >200 chemically distinct oligobenzamide analogs with maintenance of the chemical backbone but altered R groups of ERX-11. We performed the primary screening of this library to evaluate the induction of mRNA levels of two canonical ERS/UPR (unfolded protein response) genes- sXBP1 and CHOP. Biological activity of ERX-208 was validated using multiple OCa cells. Mechanistic studies were conducted using CRISPR/Cas9 KO, Western blotting, reporter gene assays, IHC and RNA-seq analysis. PK (pharmacokinetics) and toxicity studies were done using C57BL/6 mice. Cell line-derived xenografts (CDXs), patient-derived xenografts (PDXs), patient-derived explants (PDEs), and patient-derived organoids (PDO) were used for preclinical evaluation. Results: From a screen of a curated ERX-11 derived oligobenzamide library, we identified a hit compound, ERX-208 that potently (IC50~100nM) induces ERS/UPR and apoptosis in multiple OCa cells in vitro. CRISPR KO screen identified the lysosomal acid lipase A (LIPA) protein as the critical target of ERX-208. LIPA KO abrogates response to ERX-208, while reconstitution of LIPA restores ERX-208 response. The time course studies showed a robust and consistent induction (>15-fold CHOP, and >10-fold sXBP1) by ERX-208 treatment within 24h. We confirmed induction of classic UPR components peIF2α, CHOP and LC3B using Western blotting in multiple OCa cells. Functionally, ERX-208 causes growth inhibition of OCa cells, as noted by MTT cell viability assays using 15 OCa cells with an IC50 of ~50-100nM. The activity of ERX-208 is distinct among oligobenzamides as ERX-11 has limited/no activity against OCa cells. RNA-seq analysis confirmed that ERX-208 induces significant ERS, UPR, and apoptosis. Further, ERX-208 reduced the growth of OCa PDO’s in vitro, PDEs ex vivo and CDXs and PDXs in vivo. ERX-208 treatment did not show any signs of toxicity and body weight of mice was not affected. IHC analyses showed increased activation of ERS/UPR markers such as GRP78, p-PERK and decreased proliferation measured by Ki67. Conclusions: Collectively, our results demonstrated the utility of ERX-208 and will establish a novel therapeutic paradigm in OCa that overcomes tumor heterogeneity by targeting LIPA and enhancing ERS leading to apoptosis. Citation Format: Suryavathi Viswanadhapalli, Tae-Kyung Lee, Kara Kassees, Gaurav Sharma, Rahul Gopalam, Karla Parra, Tanner Reese, Michael Hsieh, Uday P. Pratap, Xue Yang, Behnam Ebrahimi, Chia Yuan Chen, Scott Terry Elmore, Christian Cervantes, Zhenming Xu, Edward Kost, Gangadhara Reddy Sareddy, Rajeshwar Rao Tekmal, Jung-Mo Ann, Ganesh V. Raj, Ratna K. Vadlamudi. ERX-208 as a novel therapeutic for treating ovarian cancer by enhancing endoplasmic reticulum stress. [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 4813.

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.

Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States and a critical need exists for the development of novel therapies for the treatment of OCa. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Recent clinical studies showed that cancer cells elucidate feedback activation of LIFR that limits response to histone deacetylase (HDAC) inhibitors. Recently, we developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIF-LIFR interactions. Here, we examined whether LIFR inhibitor EC359 abrogate the side effects of histone deacetylase inhibitor SAHA (Vorinostat) for the treatment of OCa. Methods: The effect of EC359 and SAHA as a combination therapy on OCa cell viability was examined by MTT assays. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and caspase3/7 assays respectively. The efficacy of combination therapy on OCa stem cells was determined using extreme limiting dilution assays. Mechanistic studies were performed using western blotting, qRT-PCR and Mass Spectrometry analyses. The effect of combination therapy on STAT3 signaling was examined using reporter gene assays. The in vivo efficacy of combination therapy on tumors was examined using ex vivopatient derived explants and mouse xenograft models. Results: EC359 significantly enhanced the efficacy of SAHA in reducing cell viability, colony formation ability, and apoptosis compared to monotherapy of SAHA in multiple established and primary OCa cells. Further, EC359 enhanced SAHA ability to reduce self-renewal of OCa stem cells. As expected in STAT3 reporter assays, SAHA treatment activated STAT3 reporter and EC359 addition abrogated SAHA mediated STAT3 activation. Mechanistic studies using multiple OCa models and western blot analysis confirmed activation of LIFR signaling pathway upon SAHA treatment and its blockage by EC359 treatment. Treatment of human primary OCa tumor explants with EC359 enhanced ability of SAHA to decrease the proliferation (Ki-67 positivity) compared to monotherapy treated tumors. DIA based Mass Spectrometry analyses identified unique pathways modulated by combination therapy. Treatment of OCa xenografts with EC359 enhanced the ability of SAHA to reduce in vivotumor growth compared to monotherapy treated tumors. Conclusions: Our results suggest that EC359 has therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDAC inhibitors in treating OCa. Citation Format: Mengxing Li, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Bindu Santhamma, Hui Yan, Zhenming Xu, Edward Kost, Rajeshwar Rao Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Targeting LIFR overcomes HDAC inhibitor resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4316.