Abstract 648: A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer
Authors: Altwegg Kristin Ann , Mann Monica , Chakravarty Dimple , Liu Zexuan , Liu Junhao , Pratap Uday P. , Ebrahimi Behnam , Sanchez John R. , Park Ben H. , Vankayalapati Hariprasad , Sareddy Gangadhara R. , Viswanadhapalli Suryavathi , Vadlamudi Ratna K. 
Published: June 2022

Abstract
A significant proportion of estrogen receptor positive breast cancers (ER+BC) will initially respond to treatment, but many eventually develop therapy resistance (TR-BC), and progress to incurable metastases. Oncogenic ER coregulators overexpressed in BC can contribute to constitutive, ligand-independent and ligand-dependent signaling which drives growth, resistance to therapy and metastasis. Proline-, glutamic acid, and leucine-rich protein 1 (PELP1), is a known coregulator that plays a critical role in ER oncogenic functions. Its expression is deregulated in BC and is a prognostic indicator of poor BC survival. The lack of a small molecule inhibitor that directly targets PELP1 represents a major knowledge gap. Therefore, we conducted a large scale peptide library screening and identified novel Peptide Inhibitor of PELP1 (PIP1). We demonstrated that PIP1 directly interacts with PELP1, promotes its degradation and has the potential to block PELP1 oncogenic functions in vitro. Using innovative peptidomimetic technology, we modeled PIP1 and synthesized several derivatives as Small Molecule Inhibitors of PELP1 (SMIPs). Using MTT assay and multiple BC cell lines, we identified a lead compound, SMIP34 with an IC50 of 5-10µM and with minimal effect on human mammary epithelial cells. SMIP34 in vitro activity was assessed by colony formation and Matrigel invasion assays. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, indicating target specificity. Further, MST and biotin-SMIP34 pulldown confirmed direct binding of SMIP34 to PELP1. Using ER+WT, mtER (mutant ER), and TR-BC cell lines we demonstrated that SMIP34 exhibits antiproliferative effects and reduces invasiveness. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation via the proteasome pathway. Thus MG132 treatment attenuated SMIP34 mediated degradation. RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. RNAseq analyses showed SMIP34 treatment altered the expression of genes associated with Estrogen response, Cell cycle and Apoptosis pathways. Cell cycle analyses revealed SMIP34 treatment promoted S phase arrest of BC cell lines. Using ER+WT, mtER, and PDX tumor tissues ex vivo, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Further, SMIP34 (20mg/kg/IP) treatment in vivo significantly reduced tumor progression in mouse models of ER+WT, mtER, and mtER patient-derived xenograft BC. Our results using in vitro, ex vivo, and in vivo models showcase SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling and may serve as a potential therapeutic molecule for treating ER+, mtER, and TR-BC. Supported by NIH 1F31CA257298 (KA) and VA I01BX004545 (RV).
Citation Format: Kristin Ann Altwegg, Monica Mann, Dimple Chakravarty, Zexuan Liu, Junhao Liu, Uday P. Pratap, Behnam Ebrahimi, John R. Sanchez, Ben H. Park, Hariprasad Vankayalapati, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer [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 648.