Abstract 2854: Targeting error-prone alternative nonhomologous end joining (A-NHEJ) DNA repair pathway to treat ESR1-mediated therapy resistance

Abstract

Abstract Endocrine therapies for estrogen receptor (ESR1) positive breast cancer involve modulation of ESR1 signaling using either antiestrogens (AE) or aromatase inhibitors (AI). Despite initial positive response, most patients develop resistance to these drugs and therapy resistance is a major clinical problem. Emerging findings suggest that during the progression of ESR1 positive breast cancer, the cancer cell increasingly becomes dependent on the error-prone alternative-non-homologous end joining (A-NHEJ) DNA repair pathway for double strand break (DSB) repair leading to an increase in chromosomal abnormalities contributing to hormonal therapy resistance. The mechanism by which ESR1 pathway regulates A-NHEJ remains unknown. Recent studies from our lab discovered that ESR1 coregulator Proline-, Glutamic acid-, and Leucine-rich Protein-1 (PELP1) is phosphorylated by the DNA damage response kinases and plays a key role in the A-NHEJ pathway. In this study, we dissected the mechanism by which PELP1 regulates the A-NHEJ pathway and developed a novel inhibitor of PELP1-A-NHEJ axis. Using plasmid based repair assay, we provided evidence that PELP1 regulates degree of end-resection that is essential for A-NHEJ pathway mediated DNA repair. Immunoprecipitation assays revealed that PELP1 interacts with exonuclease protein, Mre11 that plays an essential role in A-NHEJ pathway. Using PELP1 domain deletions and GST pull down assays, we identified C-terminus of PELP1 as the region that interacts with key components of the A-NHEJ pathway. We have rationally screened a random peptide library composed of 10 million peptides, and identified a tight-binding peptide to the C-terminus of PELP1 that interferes with PELP1- Mre11 interaction. Using emerging stapled peptide technology, we converted this peptide to a stable cell permeable Peptide Inhibitor of PELP1 (sPIP3). sPIP3 bound to PELP1 with high affinity, interfered PELP1-Mre11 interaction, reduced A-NHEJ repair frequency in reporter based assays and significantly reduced frequency of chromosomal translocation. In MTT based assays, sPIP3 demonstrated significant cytotoxic effect on breast cancer cells with minimal effect on normal cells. Further, sPIP3 significantly reduced survival and promoted apoptosis of hormonal therapy resistant breast cancer model cells. Collectively, these findings demonstrate the therapeutic potential of sPIP3 which represents a novel drug for the treatment of therapy resistant breast cancer by inhibiting error prone A-NHEJ pathway. Citation Format: Samaya Krishnan, Binoj Nair, Gangadhara Sareddy, Monica Mann, Ratna K. Vadlamudi. Targeting error-prone alternative nonhomologous end joining (A-NHEJ) DNA repair pathway to treat ESR1-mediated therapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2854. doi:10.1158/1538-7445.AM2015-2854