Wnts regulate the expression of HR and FA pathway genes via MYBL2 Wnt-activated genes show TFBS enrichment for FOXM1 and MYBL2.Figure 5Wnt-activated genes show TFBS enrichment for FOXM1 and MYBL2. Promoters of Wnt-activated HR and FA pathway genes were scanned for transcription factor binding site (TFBS) motifs using ENCODE, ReMAP, and Literature ChIP-seq databases using the web-based tool, CHEA3. Temporal regulation of MYBL2 in HPAF-II orthotopic xenografts treated with ETC-159. Each data point represents an individual tumor. The horizontal lines represent median of 4-6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1.5x interquartile range. MYBL2 protein levels are reduced upon ETC-159 treatment in HPAF-II xenografts. Tumor lysates from vehicle or ETC-159-treated (56 h) mice were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Each lane represents an individual tumor Expression of MYBL2 is regulated by β-catenin. Expression of MYBL2 (i) protein levels as measured by immunoblotting and (ii) transcripts as measured by qRT-PCR in HPAF-II tumors with or without stabilized β-catenin treated with vehicle or 100 nM ETC-159 for 56 hours. Stabilized β-catenin prevents the ETC-159 induced decrease in both MYBL2 transcripts and protein levels. The horizontal lines represent mean of replicates. β-catenin regulates the expression of MYBL2 in HPAF-II cells. HPAF-II cells were either treated with ETC-159 (100 nM) or transfected with siRNA against β-catenin alone or in the presence of ETC-159 for 48 hours. The relative expression of MYBL2 as measured by qRT-PCR is shown. The horizontal lines represent mean of replicates. Expression of DNA repair genes in Wnt-addicted cells is regulated by MYBL2. HPAF-II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC-159 (100 nM) for 48 hours. Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT-PCR. Data are representative of three independent experiments. The horizontal lines represent mean of replicates. Enrichment of MYBL2 binding on the promoters of DNA repair genes. HPAF-II cells were treated with DMSO or ETC-159 (100 nM) for 48 hours. Chromatin immunoprecipitated with IgG or MYBL2 antibodies was analyzed using primers specific for the DNA binding sites in the indicated DNA repair genes. Data presented are fold enrichment normalized to input. Data are representative of three independent experiments. H-K. Expression of MYBL2 is downregulated upon ETC-159 or G007-LK treatment in multiple Wnt-high tumors. Expression of MYBL2 in AsPC-1 xenograft tumors and colorectal cancer PDX (as measured by RNA-seq) or in pancreatic cancer PDX with RNF43 mutation (measured by qRT-PCR) is reduced with ETC-159 treatment. G007-LK reduces MYBL2 expression in COL320HSR cells (measured by qRT-PCR). p-values were calculated with Mann-Whitney U test. Each data point represents an individual tumor or replicate. n = 4-6 samples/group. The horizontal lines represent median of 4-6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1.5x interquartile range. L. PORCN inhibitor ETC-159 creates a BRCA like state: Wnt ligand binding to the cell surface receptors Frizzled (Fzd) and LRP5/6 leads to the activation of Wnt/β-catenin target genes such as MYBL2 that in turn activates the HR and FA pathway genes. ETC-159 treatment inhibits this HR and FA pathway and consequently enhances sensitivity to olaparib.