Abstract GMM-040: TARGETING WNT SIGNALING TO OVERCOME PARP INHIBITOR RESISTANCE
Authors: Yamamoto Tomomi M. , McMellen Alexandra , Watson Zachary L. , Aguilera Jennifer , Sikora Matthew J. , Ferguson Rebecca , Numammadov Elmar , Moldovan George-Lucian , Thakar Tanay , Kim Hyunmin , Cittelly Diana M. , Wilson Heidi , Behbakht Kian , Bitler Benjamin G. 
Published: June 2021

Abstract
PURPOSE: Epithelial ovarian cancer (EOC) has one of the highest death to incidence ratios of any cancer. High grade serous ovarian cancer (HGSOC) is the most common EOC and over 85% of HGSOCs are diagnosed at a late stage (III/IV). While about 80% of all HGSOC patients will respond to first line treatments (debulking surgeries and platinum-based therapies), the majority of patients will relapse. Poly(ADP)-ribose polymerase inhibitors (PARPi) are a promising treatment for recurring chemosensitive HGSOC. However, recurrence with resistance to both chemotherapies and PARPi is common, revealing a clinical need to understand the mechanisms underlying PARPi resistance. Canonical Wnt signaling is known to play a role in the tumorigenesis and chemoresistance of many cancers, including ovarian. Non-canonical Wnt signaling is able to inhibit beta-catenin dependent TCF/LEF transcription and loss of non-canonical Wnt signaling conveys a poor prognosis in HGSOC. Therefore, we sought to determine if aberrant canonical Wnt signaling plays a role in PARPi response/resistance in HGSOC.
METHODS: RNA sequencing was performed on PEO1 cells sensitive and resistant to a PARPi, olaparib, to determine differential expression of signaling pathways and transcription factors. This was validated with TOP/FOP-FLASH reporter assays and qPCR of genes associated with canonical Wnt signaling. Colony formation assays were performed to determine the effects of overexpressing a canonical Wnt ligand, Wnt3a, in sensitive cells treated with olaparib and to determine cell response to a Wnt inhibitor (pyrvinium pamoate; Pyr. Pam.). Levels of apoptosis after treatments with PARPi and/or Pyr. Pam. was measured with Annexin V/PI assays. DNA damage repair was assessed utilizing γH2Ax resolution and two-plasmid functional DNA repair assays. The effects of olaparib, Pyr. Pam. and combination treatments were assessed in vivo with a xenograft mouse model (intraperitoneal injection of PEO1-Wnt3a cells) and on ex vivo cultures of primary chemonaive tumors.
RESULTS: PARPi resistant cells had a significant enrichment of Wnt signaling and the TCF3/LEF transcription factors as compared to the sensitive cell lines. This also correlated with an increase in mRNA of canonical Wnt signaling activators, including the ligand WNT3A, and a decrease of Wnt repressors (SFRP1, WNT5A) in the resistant cells. Sensitive cells with Wnt3a overexpressed developed an increased resistance to olaparib. Treating PARPi resistant cells with Pyr. Pam. significantly decreased cell viability, suggesting a dependence on Wnt signaling. Combination treatment of olaparib and Pyr. Pam. resulted in a synergistic increase in apoptosis when compared to either treatment alone. PARPi resistant cells had a significant increase in the rate of γH2Ax resolution and higher DNA repair activity compared to sensitive cells. Pyr. Pam. exposure attenuated the DNA damage repair activity. In a xenograft mouse model combination olaparib and Pyr. Pam. resulted in significantly decreased tumor growth, tumor weight, and tumor nodule number as compared to control. In a primary HGSOC tumor ex vivo model, Pyr. Pam. significantly inhibited proliferation compared to control treatment.
CONCLUSION: Our data demonstrate that HGSOC cells upregulate canonical Wnt signaling to promote resistance to PARPi. Wnt inhibitors are a promising therapeutic approach for patients who develop PARPi resistance.
Citation Format: Tomomi M. Yamamoto, Alexandra McMellen, Zachary L. Watson, Jennifer Aguilera, Matthew J. Sikora, Rebecca Ferguson, Elmar Numammadov, George-Lucian Moldovan, Tanay Thakar, Hyunmin Kim, Diana M. Cittelly, Heidi Wilson, Kian Behbakht, and Benjamin G. Bitler. TARGETING WNT SIGNALING TO OVERCOME PARP INHIBITOR RESISTANCE [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-040.