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Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Here, we describe a rationally designed first-in-class inhibitor of LIFR, EC359, which directly interacts with LIFR to effectively block LIF/LIFR interactions. EC359 treatment exhibits antiproliferative effects, reduces invasiveness and stemness, and promotes apoptosis in triple-negative breast cancer (TNBC) cell lines. The activity of EC359 is dependent on LIF and LIFR expression, and treatment with EC359 attenuated the activation of LIF/LIFR-driven pathways, including STAT3, mTOR, and AKT. Concomitantly, EC359 was also effective in blocking signaling by other LIFR ligands (CTF1, CNTF, and OSM) that interact at LIF/LIFR interface. EC359 significantly reduced tumor progression in TNBC xenografts and patient-derived xenografts (PDX), and reduced proliferation in patient-derived primary TNBC explants. EC359 exhibits distinct pharmacologic advantages, including oral bioavailability, and in vivo stability. Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling.See related commentary by Shi et al., p. 1337

Supplementary Figure S1 shows SPR studies and Biotin EC359 pull down assays; Supplementary Figure S2 shows superimposition of hLIFR onto the mLIFR -hLIF complex; Supplementary Figure S3 shows sequence alignment of human (P42702) and mouse (P42703) LIFR ; Supplementary Figure S4 shows five prominent sites identified through sitemap program Schrödinger; Supplementary Figure S5 shows binding poses of EC359 at Site-3; Supplementary Figure S6 shows MM-GBSA scores for different poses obtained from the IFD; Supplementary Figure S7 shows ligand induced conformational changes in the best scored pose; Supplementary Figure S7 shows ligand induced conformational changes in the best scored pose; Supplementary Figure S8 shows the RMSD of protein and ligand; Supplementary Figure S9 shows pharmacological features of EC359; Supplementary Figure S10 shows effect of dox inducible CRISPR/Cas9 mediated KO of LIFR on STAT3 signaling and effect of EC359 on self-renewal of stem cells; Supplementary Table 1 shows primer sequences used for RT-qPCR analysis.

Supplementary methods section contain description of synthesis and characterization of EC330 and EC359, SPR studies, structural and sequence comparison of human and mouse LIFR, energy minimization of hLIFR, Protein and ligand preparation, molecular docking and MM-GBSA calculations, and molecular dynamics simulation.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease. TNBC lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elucidate feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. Here, we examined whether the novel LIFR inhibitor, EC359, has the ability to counteract negative effects of LIFR signaling to enhance HDACIs therapeutic efficacy in the treatment of TNBC. Methods: We tested multiple HDACIs currently in clinical trials including vorinostat, panobinostat, romidepsin, and givinostat using multiple TNBC models. The effect of combination therapy of HDACIs and EC359 on TNBC cell viability and invasion was examined using MTT assays and matrigel invasion assays respectively. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and Caspase 3/7 assays, respectively. Mechanistic studies were performed using Western blotting, qRT-PCR, and reporter gene assays. The efficacy of combination therapy in vivo was examined using Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models. Results: We demonstrated that the treatment of TNBC models with HDACIs increased the expression of LIFR. Immunohistochemistry analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC samples. Knockdown of LIFR or treatment with a small molecule inhibitor of LIFR (EC359) significantly enhanced the efficacy of HDACIs in reducing cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy of HDACIs or EC359 in TNBC cell lines. Mechanistic studies, reporter gene assays and biochemical studies using multiple TNBC models exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359+HDACI combination therapy. Treatment of human breast tumors utilizing PDEX assays showed that EC359 enhanced the ability of HDACIs to decrease the proliferation (Ki-67 positivity) compared to monotherapy. Furthermore, using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides greater therapeutic efficacy than monotherapy. In addition, treatment with EC359 can overcome the feedback activation of LIFR currently observed in the treatment of TNBC with HDACIs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P Pratap, Yiliao Luo, Junhao Liu, Kristin A Altwegg, Xiaonan Li, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R Sareddy, Rajeshwar R Tekmal, Hareesh B Nair, Klaus J Nickisch, Ratna K Vadlamudi. Targeting LIFR enhances the activity of HDAC inhibitors for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-08.

Histone deacetylase inhibitors (HDACi) are identified as novel therapeutic agents, however, recent clinical studies suggested that they are marginally effective in treating triple negative breast cancer (TNBC). Here, we show that first-in-class Leukemia Inhibitory Factor Receptor (LIFRα) inhibitor EC359 could enhance the therapeutic efficacy of HDACi against TNBC. We observed that both targeted knockdown of LIFR with CRISPR or treatment with EC359 enhanced the potency of four different HDACi in reducing cell viability, cell survival, and enhanced apoptosis compared to monotherapy in TNBC cells. RNA-seq studies demonstrated oncogenic/survival signaling pathways activated by HDACi were attenuated by the EC359 + HDACi therapy. Importantly, combination therapy potently inhibited the growth of TNBC patient derived explants, cell derived xenografts and patient-derived xenografts in vivo. Collectively, our results suggest that targeted inhibition of LIFR can enhance the therapeutic efficacy of HDACi in TNBC., Li, Viswanadhapalli et al utilized multiple in vitro, ex vivo and in vivo models of TNBC to investigate LIFR inhibition. The authors reported that HDAC inhibition in TNBC cells led to an increase of LIFR expression and over activation of the downstream signaling elements (e.g., STAT3, mTOR, AKT), enhancing the aggressive potential of TNBC cells, and an LIFR inhibitor, EC359, synergistically enhanced the efficacy of HADC inhibitors in suppressing TNBC in vitro and in vivo.

Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Here, we describe a rationally designed first-in-class inhibitor of LIFR, EC359, which directly interacts with LIFR to effectively block LIF/LIFR interactions. EC359 treatment exhibits antiproliferative effects, reduces invasiveness and stemness, and promotes apoptosis in triple-negative breast cancer (TNBC) cell lines. The activity of EC359 is dependent on LIF and LIFR expression, and treatment with EC359 attenuated the activation of LIF/LIFR-driven pathways, including STAT3, mTOR, and AKT. Concomitantly, EC359 was also effective in blocking signaling by other LIFR ligands (CTF1, CNTF, and OSM) that interact at LIF/LIFR interface. EC359 significantly reduced tumor progression in TNBC xenografts and patient-derived xenografts (PDX), and reduced proliferation in patient-derived primary TNBC explants. EC359 exhibits distinct pharmacologic advantages, including oral bioavailability, and in vivo stability. Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling. See related commentary by Shi et al., p. 1337

Background: Leukemia inhibitory factor (LIF) and its receptor LIFR are over-expressed in multiple solid tumors and play a key role in tumor growth, progression, and resistance to standard anti-cancer treatments. Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of breast cancer (BCa) mortality. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BCa patients. LIF signaling also promotes maintenance of stem cells. Therefore, targeting the LIF/LIFR axis may have therapeutic utility in TNBC. Methods: We rationally designed a small organic molecule (EC359) that emulates the LIF/LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Direct binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and microscale thermophoresis technique (MST) assays. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models were used for preclinical evaluation and toxicity. Results: Molecular docking studies showed that EC359 interacts at the LIF/LIFR binding interface. SPR and MST studies confirmed direct interaction of EC359 to LIFR. EC359 reduced the growth of TNBC cells with high potency (IC50 50-100nM) and promoted apoptosis. Further, EC359 treatment reduced invasion and stemness of TNBC cells. EC359 activity is dependent on the expression levels of LIFR and showed little or no activity on TNBC cells that have low levels of LIFR or ER+ve BCa cells. Further, EC359 significantly reduced the viability of cisplatin and taxane-resistant TNBC cells and enhanced the efficacy of HDAC inhibitors. Mechanistic and biochemical studies showed that EC359 interacts with LIFR and effectively blocking LIF/LIFR interactions. EC359 also blocked LIFR interactions with other LIFR ligands such as oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. EC359 treatment attenuated the activation of LIF/LIFR driven pathways including STAT3, mTOR, AKT, and MAPK. RNA-seq analysis identified regulation of apoptosis as one of the important pathway modulated by EC359. In TNBC xenograft and PDX assays, EC359 significantly reduced tumor progression. Further, using human primary BCa PDEX cultures, we demonstrated that EC359 has the potential to substantially reduce the proliferation of human BCa. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window. Conclusions: EC359 is a novel targeted therapeutic agent that inhibits LIF/LIFR oncogenic signaling in TNBC via a unique mechanism of action. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. (DOD BCRP grant #BC170312) Citation Format: Viswanadhapalli S, Luo Y, Sareddy GR, Santhamma B, Zhou M, Li M, Pratap UP, Altwegg KA, Li X, Srinivasan U, Ma S, Chang A, Riveros AC, Zhang KY, Dileep KV, Pan X, Murali R, Bajda M, Raj G, Brenner A, Manthati V, Rao M, Tekmal RR, Nair HB, Nickisch KJ, Vadlamudi RK. Development of a first-in-class small molecule inhibitor (EC359) targeting oncogenic LIF/LIFR signaling for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-02.

// Hareesh B. Nair 1 , Robert Baker 2 , Michael A. Owston 2 , Renee Escalona 2 , Edward J. Dick 2 , John L. VandeBerg 2 and Klaus J. Nickisch 1 1 Evestra, Inc., San Antonio, TX, USA 2 Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA Correspondence to: Hareesh B. Nair, email: // Keywords : endometriosis, baboon, endometrial stromal cells, attachment, invasion, Pathology Section Received : November 23, 2015 Accepted : February 09, 2016 Published : February 19, 2016 Abstract Endometriosis is a chronic estrogen-dependent disease that occurs in approximately 10% of reproductive age women. Baboons offer a clear benefit for studying the initiation and progression of endometriosis since baboon is very close to humans phylogenetically. Progestins are used in the treatment of endometriosis. The therapeutic window of progestins depends on the ratio of its affinity towards progesterone receptor agonism verses antagonism. The present study is to determine the role of pure antiprogestin in baboon endometriosis. We hypothesize that pure antiprogestin will induce unopposed estrogenicity and spontaneous endometriosis in baboons. The rate of endometrial invasion and attachment through modeled peritoneum in the presence and absence of progesterone and antiprogestin was evaluated in this study. A baboon model of endometriosis induced by unopposed estrogenicity using progesterone receptor antagonist (EC304) was used in this study. We observed EC304 has induced unopposed estrogenicity that deregulated proteins involved in attachment, invasion, cell growth, and steroid hormone receptors in this model. Our data suggest that depleting progesterone levels in the endometrium will increase estrogen hyper-responsiveness that leads to increased endometriotic lesion progression in the baboon ( Papio anubis) model. This study reports a refined model of human endometriosis in baboons that could potentially be used to develop new diagnostic and therapeutic strategies for the benefit of women suffering from endometriosis.

Background: Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BC patients. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elicit feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. The objective of this study is to examine the therapeutic efficacy of combination therapy using preclinical and patient-derived xenograft (PDX) models. Methods: We tested utility of combination therapy using multiple HDACIs that are currently in clinical trails along with EC359. The effect of combination therapy was evaluated using MTT, invasion, colony formation, and Caspase3/7 assays. Mechanistic studies were performed using Western blotting, qRT-PCR, and STAT3 reporter assays. The efficacy of combination therapy in vivo was examined using xenograft, PDX, and patient-derived explant (PDEx) models. Results: Immunohistochemical analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC tissues. Treatment of TNBC model cells with four different HDACIs increased the expression of LIFR. LIFR inhibitor EC359 at nM concentration is additive to HDACIs in reducing cell viability. Knockdown of LIFR or treatment with EC359 significantly enhanced the efficacy of HDACIs in reducing the cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy in TNBC model cells. On the contrary, treatment with STAT3 inhibitor requires µM concentrations to reduce the cell viability of TNBC cells and is not additive to HDACIs. Mechanistic studies utilizing STAT3 reporter gene assays and biochemical studies using multiple TNBC model cells exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359 therapy. Treatment of human TNBC utilizing PDEx assays showed that EC359 enhanced the ability of HDACIs to decrease proliferation (Ki-67 positivity) compared to monotherapy. Using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDACIs in treating TNBC. Supported by DOD BCRP grant W81XWH-18-1-0016 (R.K. Vadlamudi; K.J. Nickisch) Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P. Pratap, Yiliao Luo, Junhao Liu, Kristin A. Altwegg, Xiaonan Li, Ahmed Gulzar, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R. Sareddy, Manjeet K. Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors [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 562.

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.

Background: Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a major critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Recent studies in breast cancer have shown that feedback activation of LIFR limits response to histone deacetylase (HDAC) inhibitors and induce resistance. We rationally designed a small molecule (EC359) that emulates the LIF-LIFR binding site and functions as a LIFR inhibitor from a library of compounds. Here, we tested the utility of EC359 as a monotherapy and to effectively block LIF-LIFR interactions in overcoming resistance to HDAC inhibitors. Methods: We have used multiple triple negative breast cancer (TNBC) models that represent all six types of TNBC. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDeX) models were used for preclinical evaluation and toxicity. Results: EC359 treatment exhibited anti-proliferative effects, reduced invasiveness and stemness, and promoted apoptosis in all six TNBC cell lines. The activity of EC359 is dependent on LIF and LIFR expression and CRISPR mediated knockdown of LIFR significantly abolished EC359 activity. Treatment with EC359 attenuated the activation of LIF-LIFR driven pathways including STAT3, mTOR, and AKT. EC359 significantly reduced tumor progression in TNBC xenografts, PDX models and reduced proliferation in patient derived primary TNBC explants. In MTT based cell viability assays, addition of EC359 enhanced efficacy of SAHA compared to monotherapy of SAHA. In clonogenic survival assays, EC359 significantly enhanced ability of SAHA to reduce the colony formation compared to monotherapy. Mechanistic studies using three different TNBC models using western blot analysis and reporter gene assays confirmed activation of LIFR signaling pathway upon SAHA treatment and its blockage by EC359. Treatment of TNBC PDX explants with EC359 enhanced ability of SAHA to substantially decrease the proliferation (Ki-67 positivity) compared to monotherapy treated tumors. Conclusions: Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling as a monotherapy or in combination with HDAC inhibitors. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Yiliao Luo, Gangadhara R Sareddy, Bindu Santhamma, Mei Zhou, Shihong Ma, Rajni Sonavane, Uday P. Pratap, Kristin A. Altwegg, Annabel Chang, Alejandra Chávez-Riveros, Kalarickal V. Dileep, Kam Y. Zhang, Marek Bajda, Ganesh V. Raj, Andrew Brenner, Vijaya Manthati, Manjeet Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Therapeutic utility of EC359 for targeting oncogenic LIFR signaling in triple negative breast 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 4716.

Progesterone receptor (PR) plays a key role in reproductive functions, and compounds that inhibit progesterone action (antiprogestins) have potential use in the treatment of estrogen- and progesterone-dependent diseases, including uterine leiomyomas and breast cancer. In the present study, we chemically synthesized novel 17-fluorinated steroids and evaluated the cytotoxicity profiles of these compounds in T47D breast cancer cells compared to the activity of known antiprogestins, including ZK230 211, RU-486, CDB2914, CDB4124 and ORG33628. We analyzed in vitro receptor-binding assays and PR-transactivation assays to establish the antiprogestational activity of these molecules. The representative antiprogestin EC304 was found to inhibit in vitro tumorigenicity in a dose-dependent fashion in T47D cells by a colony formation assay at 1 and 10 nM concentrations. The potent in vivo antiprogestational activity of EC304 was also demonstrated in an antinidation assay for the interruption of early pregnancy in rats. The strong antiprogestational activity and absence of antiglucocorticoid activity in EC compounds may demonstrate their utility in the treatment of leiomyoma, endometriosis and breast cancer.

Background: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers. OCa patients initially respond to standard combinations of surgical and cytotoxic therapy; however, ~80% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. OCa stem cells are implicated in the tumor initiation and therapy resistance. LIFR signaling plays a critical role in OCa progression and stemness. Further, high circulating LIF levels correlate with tumor recurrence and chemoresistance. The autocrine loop involving LIF, LIFR and STAT3 axis drives sustained fibroblast production of inflammatory mediators. This represents a significant problem and a critical need exists for development of novel therapies targeting the LIFR axis for treating OCa. Methods: We have rationally designed and synthesized a small organic molecule (EC359) that emulates the LIF-LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and IP assays. Mechanistic studies were conducted using Western, RT-qPCR, and RNA-Seq analysis. Xenograft models were used for preclinical evaluation and toxicity. The efficacy of EC359 was tested using Patient-Derived eXplants (PDeX). Results: Global analysis of online databases revealed negative correlation of OCa survival with LIFR expression. Molecular docking studies showed EC359 interacts at the LIF-LIFR binding interface. SPR studies confirmed interaction of EC359 to LIFR. Western analysis of eight cells that represent four subtypes of OCa confirmed higher expression of LIF and LIFR. EC359 reduced the growth of eight OCa cells with high potency (IC50 10-50 nM) and promoted apoptosis. EC359 treatment reduced stemness of OCa cells. EC359 activity is dependent on the level of expression of LIFR with little activity on cells that do not express LIFR. EC359 significantly reduced the viability of carboplatin- and taxol-resistant OCa cells. Mechanistic studies showed EC359 interacts with LIFR and block its interaction with LIF. EC359 treatment reduced the STAT3 phosphorylation, mTOR and downstream survival signaling cascades. RNA sequencing revealed unique pathways blocked by EC359. Treatment of xenograft tumors with EC359 significantly reduced the tumor volume compared to control. Further, using PDeX of OCa, we demonstrated that EC359 has potential to reduce the proliferation. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window. Conclusions: EC359 is a novel agent that targets LIF-LIFR axis and has activity against chemotherapy-resistant and primary OCa tumors. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Citation Format: Suryavathi Viswanadhapalli, Hareesh B Nair, Bindu Santhamma, Gangadhara R Sareddy, Yiliao Luo, Xinlei Pan, Edward R Kost, Ramachandran Murali, Rajeshwar Rao Tekmal, Klaus J Nickisch, Ratna K Vadlamudi. Development of LIFR inhibitor EC359 as a novel therapeutic for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5875.

Background: An IL-6 family member, leukemia inhibitory factor (LIF), is a pleotropic cytokine involved in multiple cellular signaling and pathophysiology of various malignancies including cancer. Overexpression of LIF is significantly correlated with advanced tumor stage, larger tumor size and worse relapse free survival rate. The objective of present study to develop and characterize next generation steroidal LIF/LIFR inhibitor based on our first LIF inhibitor, EC330. In this study, we optimized EC330 by synthetic modifications and generated EC359 to reduce its binding to classical steroid receptors and to improve its oral bioavailability. Material and methods: In silico docking studies were used to identify putative interaction of EC359 and LIF/LIFR receptor complex. We confirmed binding of EC359 to LIFR using surface plasmon resonance (SPR). Biophysical and biological characterization was performed by measuring cytotoxicity in various cancer cell lines and anchorage-independent colony assays. Specificity to LIF/LIFR was measured by GST pull-down assay and by SPR using commercially available recombinant proteins of LIF and LIFR. In vivo efficacy and toxicity was tested using syngeneic mice tumors and xenograft models. Results: EC359 showed cytotoxicity in various cancer cells at low nano-molar range, blocked formation of colonies in soft agar and inhibited triple negative breast cancer (TNBC) stem cells. Physical direct-interaction was confirmed by SPR which showed EC359 binding to LIFR with an affinity of 81μM. EC359 showed cytoskeletal disruption and targeting cancer-associated fibroblasts (CAFs) through inhibition of alpha-SMA but not beta-tubulin. Blockade of LIF-LIFR interaction reduced the STAT3 phosphorylation, mTOR and further downstream signaling cascades. In vivo, EC359 treatment (1 and 5mg/kg) dose dependently reduced tumor burden in both TNBC xenograft and murine syngeneic MM51 models. Pharmacologically, EC359 exhibits a high oral bioavailability and long half-life in rats with a wide therapeutic window. Conclusions: Our findings establish EC359 as a novel LIF/LIFR targeting drug with therapeutic perspectives for patients with advanced primary tumors. LIF/LIFR targeting may result in the blockade of JAK- STAT signaling pathway as well as cancer fibroblast associated pro invasive tumor microenvironment in regular as well as therapy resistant tumors. Citation Format: Hareesh B. Nair, Bindu Santhamma, Suryavathi Viswanadhapalli, Gangadhara R. Sareddy, Xinlei Pan, Vijaya Manthati, Ratna K. Vadlamudi, Murali Ramachandran, Klaus J. Nickisch. Development of a first-in-class leukemia inhibitory factor (LIF)/LIFR inhibitor, EC359 for targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B04.

The leukemia inhibitory factor (LIF), an IL-6 family member, is a pleotropic cytokine has been demonstrated as an oncogene and involved in multiple cellular mechanisms during the initiation and progression of various human malignancies. Overexpression of LIF was significantly correlated with advanced tumor stage, larger tumor size and worse relapse free survival rate. In the present study EC330, a novel steroidal LIF inhibitor showed cytotoxicity in various cancer cell lines and NCI-60 cell line panel at low nano-molar range. EC330 blocked formation of colonies in soft agar and inhibited angiogenesis (tube formation) in human umbilical vein endothelial cells. EC330 binds to LIF/LIFR complex by insilico docking studies and reduced the STAT3 phosphorylation in a dose dependent manner through blocking LIF-JAK-STAT signaling. EC330 showed marked specificity in MCF-7 cells overexpressing LIF verses MCF-7 cells. The compound further showed cytoskeletal disruption and targeting cancer associated fibroblasts (CAFs). EC330 treatment (0.5 and 2.5mg/kg) dose dependently reduced tumor burden in ovarian (IGROV-1) and triple negative breast cancer (MDA-MB-231) cell xenografted mouse models. These findings establish EC330 as a novel LIF inhibitor having targeted therapeutic perspectives for patients with aggressive primary tumors. LIF targeting may result in the blockade of cancer fibroblast associated pro invasive tumor microenvironment in regular as well as therapy resistant tumors. Citation Format: Hareesh B. Nair, Bindu Santhamma, Surya Viswanadhapa, Klaus J. Nickisch. First-in-class steroidal leukemia inhibitory factor (LIF) inhibitor in targeted cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-208.

The concept of portfolio management has been widely used in the pharmaceutical industry. It is used to evaluate the commercial value and the risk structure of development projects. The final goal is to select a portfolio of projects that addresses the strategic objectives of the organisation optimally and that leads to the highest overall portfolio value. Companies now start to apply the portfolio management concept on their research portfolios. Although the basic principle remains the same, the methodology applied has to be adapted to the greater uncertainty that early research projects carry. Commonalities and differences of the portfolio management process in research and development are described and recommendations are given how to harmonise the two different approaches.

Progesterone receptor (PR) is a ligand activated transcription factor which plays a crucial role in female reproduction. PR antagonists have been shown to repress estrogen dependent proliferation in the uterus and mammary gland. Even though PR antagonists are potent therapeutic candidates for breast cancer, partial agonism towards glucocorticoid receptor (GR) and androgen receptor (AR) lead to undesirable side effects. Evestra's rational design and development approach in the synthesis of EC304 was aimed at minimizing these side effects as well as to deliver more potent and selective antiprogestins. We used select screen® (Invitrogen) to determine relative binding affinity of EC304 with AR,GR and PR. The binding affinity of EC304 showed strong antagonism towards PR with mild or negligible agonism with AR or GR. PR transactivation was inhibited by EC304 with IC50 of 0.04nM in the presence of progesterone (5nM). EC304 showed superior cytotoxicity over known antiprogestins ZK230211, ORG33628, CDB2914, CDB4124 and RU486 in T47D cells. IC50 of EC304 was found to be 0.5nM in a 6-day cytotoxicity assay (MTT) in T47D cells stimulated with 1nM estrogen (E2). In a 21-day in vitro tumorigenicity assay, EC304 inhibited 80 and 100% of colony formation of T47D cells in the presence of E2 at 1 and 10 nM respectively. Further studies revealed that EC304 induced apoptosis and G1-phase arrest in cell cycle progression when co-incubated with E2 for 24 h. Treatment with EC304 down regulated PR target genes and up regulated cell cycle dependent kinase (CDK) inhibitor P21 in vitro. Ongoing studies address whether EC304 is specifically acting on PR-A or PR-B isoform at gene expression, protein levels and PR isoform specific knockdowns in T47D and other metastatic breast cancer cells. Considered together, these data indicate that EC304 might be a safer but efficacious novel antiprogestin for hormone sensitive postmenopausal breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-02.