Wnt high cells that are resistant to PORCN inhibition can be sensitized by combination with olaparib RNF43 mutant cells have high cell surface abundance of FZDs.Figure 3RNF43 mutant cells have high cell surface abundance of FZDs. Endogenous cell surface FZD levels was assessed by flow cytometric analysis in PaTu8988T cells and PL45 cells (with RNF43 inactivating mutations) and Panc 08.13 cells with wild-type RNF43. Cells were stained with pan-FZD antibody clone F2.A or an isotype control followed by anti-human Fc fragment APC-conjugated secondary antibody. MFI = median fluorescence intensity. Wnt inhibition using siRNA against β-catenin or ETC-159 treatment reduces the expression of HR and FA pathway genes in PaTu8988T cells. PaTu8988T cells were transfected with indicated control or β-catenin siRNAs or treated with ETC-159 (1 µM) for 48 hours, total RNA was isolated and expression of β-catenin (CTNNB1), AXIN2 and DNA repair genes was measured by qRT-PCR. The horizontal lines represent mean of replicates. C, D. ETC-159 and olaparib synergistically inhibit the growth of ETC-159 resistant pancreatic cancer cell line PaTu8988T. (C) PaTu8988T cells were seeded in soft agar and treated with ETC-159, olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A. Representative image of soft agar colonies from PaTu8988T cells combination study in a 48-well plate is shown. (D) The Combination Index (CI) values of olaparib and ETC-159 calculated from two independent experiments using the Chou-Talalay CompuSyn software. Olaparib and ETC-159 combination is more effective in preventing the growth of PaTu8988T xenografts in mice. NSG mice with established PaTu8988T orthotopic xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (30 mg/kg), olaparib (50 mg/kg) or a combination of ETC-159 and olaparib. Treatment was initiated after the tumors were established. Tumor weights in the respective groups at the end of the 18 days treatment are shown. Each dot represents an individual tumor and the horizontal lines represent the mean. p-values were calculated with Mann-Whitney U test. ETC-159 and olaparib synergistically inhibit the growth of ETC-159 resistant pancreatic cancer cell line PL45. PL45 cells seeded in 48 well plates were treated with ETC-159, olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A. A representative image of the wells from the combination study is shown. Wnt inhibition does not synergize with olaparib in Wnt-low Panc 08.13 cells. Panc 08.13 cells were seeded in soft agar and treated with increasing doses of olaparib alone or in combination with ETC-159 as indicated and allowed to form colonies for 2 weeks. The data is representative of two independent experiments with each point representing an average colony count ± SD of duplicates. Wnt inhibition through ETC-159 does not change the expression of HR and FA pathway genes in Panc 08.13 cells. Panc 08.13 cells were cultured in low adherence plates and treated with ETC-159 for 72 hours. Total RNA was then isolated and expression of indicated genes was measured by qRT-PCR. The horizontal lines represent mean of replicates.