Background: Mutations in ESR1 genes (30-40% frequency) play an important role in acquired endocrine therapy resistance and metastases. The most commonly observed are two ESR1 LBD mutations, D538G andY537S. These mutant ERα (MT) proteins have high constitutive transcriptional activity leading to therapy resistance. Furthermore, the ability of the constitutively active mutants to interact with coregulators is associated with the promotion of tumor growth. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1), an oncogenic coregulator of ERα, plays a critical role in ERα signaling, and its dysregulated expression is a prognostic indicator for poorer breast cancer (BCa) survival. The objective of this study was to test the utility of Small Molecule Inhibitor of PELP1 (SMIP34) for treating ESR1 mutant (MT-ER) driven BCa. Methods: Four BCa models that express either ESR1 mutation D538G or Y537S and their respective wild-typeERα (WT-ER) controls were used to test the utility of targeting the PELP1 axis using PELP1-specific shRNA orSMIP34. Celltiter Glo, MTT, colony formation, and Boyden chamber invasion assays were used to test the efficacy of SMIP34. Western blot, RNA-Seq, and reporter gene assays were utilized to uncover the mechanistic insights. Pre-clinical evaluation was performed using MT-ER expressing xenograft explant (XDEX) and patient-derived explant (PDEX) assays. Results: BCa model cells expressing MT-ER showed increased cell proliferation, whilst PELP1 knock-down significantly reduced their proliferation. Immunoprecipitation results confirmed that PELP1 constitutively associates with MT-ER. PELP1 knock-down or treatment with PELP1 inhibitor SMIP34 significantly reduced proliferation of the four MT-ER models with an IC50 of 3-5μM. PELP1 knock-down or SMIP34 treatment significantly reduced the constitutive ERE reporter activity observed in MT-ER models. RTqPCR assays confirmed the downregulation of MT-ER target genes in PELP1 knock-down and SMIP34 treated cells. Furthermore, PELP1 knock-down or SMIP34 treatment significantly reduced the invasiveness and colony formation of MT-ER BCa models. Mechanistic studies using Western blot revealed that SMIP34 contributes to PELP1 degradation by its direct binding to PELP1. SMIP34 significantly decreased proliferation of MT-ER BCa cells in XDEX andPDEX assays, as measured by Ki67 staining. Conclusion: Our results suggest that PELP1 associates with MT-ER and targeting the PELP1 axis with SMIP34will have therapeutic utility in treating MT-ER driven BCa. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545(R.K.V)

Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Junhao Liu, Zexuan Liu, Uday P. Pratap, Benham Ebrahimi, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Targeting the PELP1 axis for treating ESR1 mutant driven breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-15.