WNT inhibition creates a BRCA-like state in Wnt-addicted cancer (2021)
Figure 2

Figure 2: Homologous recombination (HR) and Fanconi anemia (FA) repair pathway genes are regulated by Wnt signaling

Heatmap of selected temporal clusters containing the Wnt-activated genes that are enriched for DNA repair pathways. Transcriptomic data from HPAF-II orthotopic pancreatic tumors (dataset originally reported in Madan et al, 2018) was assessed at multiple time points during treatment with the PORCN inhibitor 37.5 mg/kg bid ETC-159 (Figure EV1C). Genes that were differentially expressed over time (FDR < 10%) following PORCN inhibition were clustered based on their pattern of transcriptional response. Clusters 1, 5 and 12 refer to temporal clusters defined in (Madan et al, 2018) and TI50 is the time (in hours) after start of therapy to achieve 50% inhibition in gene expression for each cluster.

Gene Ontology (GO) biological process enrichment of each cluster of Wnt-activated genes. Analysis of Wnt-activated genes in clusters 1, 5 and 12 from HPAF-II orthotopic pancreatic tumors (Figure 2A) and colorectal cancer (CRC) patient-derived xenograft (PDX) highlights enrichment of genes involved in multiple DNA repair pathways including interstrand cross-link repair and double strand break (DSB) repair via homologous recombination (HR).

DNA repair genes involved in HR and FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF-II orthotopic xenografts. Comparison of log2 fold change (FDR < 10%) in the expression of Wnt-activated genes over time across multiple DNA repair pathways shows that genes regulating HR and FA pathways have higher fold changes compared to genes regulating BER, NER or other pathways. BER: base excision repair; HR: homologous recombination; FA: Fanconi anemia; MMR: mismatch repair; NER: nucleotide excision repair; NHEJ: non-homologous end joining; TLS: translesion DNA synthesis. The horizontal line represents median of 4-6 replicates with the upper and the lower edges of the box representing the 75th and 25th percentile of the data respectively and the whiskers representing 1.5 x interquartile range.

D, E. ETC-159 treatment of HPAF-II orthotopic xenografts reduces the expression and protein levels of multiple HR and FA pathway genes. (D) Temporal expression of selected Wnt-activated HR and FA pathway genes are shown. n = 7-10 tumors/group. (E) Tumor lysates from HPAF-II xenografts treated with vehicle or ETC-159 for 8 or 56 hours were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. Each lane represents an individual tumor.

F, G. Wnt inhibition reduces the expression and protein levels of HR and FA pathway genes in a Wnt-addicted colorectal cancer patient-derived xenograft. Mice with CRC PDX driven by a RSPO3 translocation were treated with vehicle or ETC-159 for 56 hours before harvesting. (F) Gene expression was analyzed by RNA-seq (dataset reported in (Madan et al, 2016)). The graph shows the normalized expression of the indicated genes. The horizontal line represents mean of 4 tumors/group. (G) Tumor lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. Each lane represents an individual tumor.

H, I. Wnt inhibition reduces the expression of HR and FA pathway genes in a RNF43-mutant pancreatic cancer patient-derived xenograft and AsPC-1 cells. (H) Pancreatic canc­er PDX with G371fs RNF43 mutation treated with vehicle or ETC-159 (30 mg/kg) for 21 days were analysed for changes in the expression of the indicated genes measured by qRT-PCR. Each data point represents an individual tumor. The horizontal lines represent mean of 3 tumors/group. (I) AsPC-1 cells were seeded in low adherence plates, treated with DMSO or ETC-159 (100 nM) for 72 hours and the expression of indicated genes was measured by qRT-PCR.

J. Wnt inhibition reduces homologous recombination. AsPC-1 or Panc 08.13 cells with a stable integration of HR reporter construct DR-GFP (encoding a modified GFP gene with the I-SceI site and a donor GFP sequence that restores GFP sequence upon HR-mediated DNA repair) were transfected with a plasmid expressing I-SceI endonuclease, followed by treatment with DMSO or ETC-159 (100 nM) for 24 hours. The percentage of GFP positive cells as measured by flow cytometry indicates that the extent of repair via HR pathway in the presence of ETC-159 treatment is reduced in Wnt high AsPC-1 cells or but not in Wnt-insensitive Panc 08.13 cells. Data shows the values from three independent experiments with two to three replicates each. p-values were calculated using Mann-Whitney U test.