Background:We had previously reported a novel small molecule, ERX-11, that directly interacts with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. To enhance the clinical translation of ERX-11, we sought to pursue both lead optimization and evaluate combinations of ERX-11 with other approved agents in breast cancer. Methods: We designed, synthesized and tested 500 derivatives of ERX-11 in multiple models of ER+ breast cancer. We also tested combinations of ERX-11 with multiple agents, including other ER targeting agents, chemotherapies and CDK4/6 inhibitors. We tested the effect of combination therapy using breast cancer cells with acquired resistance (Tamoxifen, Letrozole, Ribociclib resistant) and engineered models that express ER mutations. In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays and RNA-seq analysis. Xenograft, patient derived xenograft (PDX), patient derived explant (PDE) and xenograft derived explant (XDE) models were used for preclinical evaluation and toxicity. Result: Evaluation of 500 analogs of ERX-11 identified a number of leads with differential activity against ER+ and ER- breast cancer cells, identified several analogs including ERX-144, 208, 296, 315 with nanomolar potency against ER+ and therapy-resistant ER+ breast cancers. Validation of the mechanism of action of these analogs is ongoing. The combination of ERX-11 and palbociclib significantly blocked ER-mediated and ER-coregulators mediated oncogenic signaling and showed potent anti-proliferative activity against both endocrine therapy-sensitive and resistant breast cancer cells. In addition, ERX-11 inhibited ribociclib-resistant ER+ cell proliferation in a dose dependent manner. Mechanistic studies using IP-Mass spectrometry demonstrated that ERX-11 and palbociclib blocks the interaction between larger subset of coregulators with ER in therapy resistant breast cancer models. ERX-11 and palbociclib both exhibited potent anti-proliferative activity against therapy-sensitive and therapy-resistant ER+ve breast cancer cells, in xenograft models and in PDEs. Importantly, combination therapy of ERX-11 and palbociclib synergistically reduced the growth of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. Mass spec based DIA analyses and RNA-seq studies revealed that combinational treatment uniquely activated p53, unfolded response mediated apoptotic pathways, altered DNA damage response and suppressed E2F and Myc target genes. Biochemical studies confirmed combination therapy significantly altered E2F1, ER and DNA damage response pathways. Conclusion: We have successfully pursued two avenues to improving ERX-11 for clinical translation. We have developed ERX-11 analogs with higher potency against ER+ breast cancer. We have shown that combinational treatment with ERX-11 and palbociclib may overcome endocrine therapy resistance and CDK4/6 inhibitor (ribociclib) resistance. Citation Format: Viswanadhapalli S, Ma S, Lee T-K, Sareddy GR, Liu X, Ekoue D, Alluri A, Luo Y, Kassees K, Arteaga C, Alluri P, Weintraub SE, Tekmal RR, Ahn J-M, Raj GV, Vadlamudi RK. Enhancing the activity of a novel estrogen receptor coregulator binding modulator (ERX-11) against ER-positive therapy resistant 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 P5-04-23.
BACKGROUND: Recently,CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) is approved for the treatment of ER+ advanced breastcancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. Emerging studies indicate many therapy-resistant tumors retainER signaling, via interaction with critical oncogenic coregulatorproteins. Considering complex signaling interplay of ER and CDK4/6 axis, combination therapy of CDK inhibitor with other potent ER-targeted agents that block ER coregulatory signaling may extend the efficacy and may prevent the development of resistance to the CDK4/6 inhibitors. We recently developed a small organic molecule, ER coregulator binding modulator ERX-11 (EtiraRx-11). The objective of this study is to test the utility of novel combination therapy of ERX-11 with CDK4/6 inhibitor palbociclib in treating therapy resistant advanced BCa. METHODS: We have utilized multiple therapy sensitive and therapy-resistant BCa models with various genetic backgrounds. We tested efficacy using both acquired resistance and engineered models that express ER mutations or oncogenes. Efficacy of combination therapy was tested using established in vitro assays including, MTT, colony formation, apoptosis, and cell cycle progression. Mechanistic studies were conducted using reporter gene assays, gene expression, RNA-seq analysis and signaling alterations. Patient-derived BCa explant and Xenograft studies were used to determine the in vivo efficacy of the combination therapy. RESULTS: ERX-11 effectively blocked ER-mediated and ER-coregulator mediated oncogenic signaling and has potent anti-proliferative activity against both endocrine therapy-sensitive and therapy-resistant BCa cells. Mechanistic studies using IP-Mass spectrometry showed that ERX-11 blocks the interaction between a subset of coregulators with ER in resistant BCa models. ERX-11 exhibited potent anti-proliferative activity against therapy-sensitive and therapy-resistant ER-driven BCa cells in vitro, in xenograft models in vivo and in patient-derived breast tumor explants ex vivo. Co-treatment of ERX-11 with palbociclib synergistically reduced cell viability and induced apoptosis of therapy sensitive and resistant BCa model cells. Importantly, combination therapy of ERX-11 and the palbociclib synergistically reduced the growth and induced apoptosis of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. RNA-seq studies revealed that combinational treatment with ERX-11 and palbociclib uniquely activated p53 and unfolded response mediated apoptotic pathways and suppressed E2F and Myc target genes. Biochemical studies confirmed combination therapy significantly altered E2F1 and ER signaling pathways and promoted apoptosis. CONCLUSIONS: Our data support a critical role of blocking ER coregulator signaling in treating therapy resistance in advanced ER+ BCa. Combinational treatment with ERX-11 and palbociclib may overcome/delay endocrine therapy resistance. Citation Format: Viswanadhapalli S, Sareddy GR, Zhou M, Ali E, Li X, Ma S-H, Lee T-K, Tekmal RR, Ahn J-M, Raj GV, Vadlamudi RK. Blocking ER coregulator signaling enhances CDK4/6 inhibitor palbociclib therapy in ER-positive advanced breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-06.
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.
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