Fig 1: AXIN2 gene expression is a poor predictor of PORCN inhibitor sensitivityA. AXIN2 expression is widely suppressed in pancreatic cell lines treated with Wnt-C59 and ETC-159: Pancreatic cancer cell lines were treated with 100 nM ETC-159 or 100 nM Wnt-C59 for 24h. Total RNA was isolated and AXIN2 expression was analyzed by qRT-PCR. Expression of AXIN2 normalized to 18SrRNA in the ETC-159 or Wnt-C59 treated cells is represented as the percentage of respective DMSO treated controls. B. ETC-159 blocks foci formation in a subset of pancreatic cell lines: 1000-3000 cells of the indicated pancreatic cell lines were plated in 24 well plates and treated with ETC-159 (10 nM to 1 µM). Cell number and the duration for treatment were determined based on the growth rate of each line. Following methanol fixation the cells were stained with crystal violet. C. Expression of Notum is elevated in Gan mice: Total RNA was isolated from gastric tissue of K19-C2mE (C2mE) and K19-C2mE/Wnt1 (Gan) mice and Notum expression was measured by qRT-PCR. Expression was normalized to Hprt, n=5 mice /group. D. NOTUM inhibits Wnt/ß-catenin reporter activity induced by diverse Wnts: HT1080 cells were transiently transfected with the Super8xTOPFLASH (STF), NOTUM and the indicated Wnt expression plasmids. The reporter activity was normalized to mCherry expression, a control for transfection efficiency. Bars represent the mean ± SD. *** p =0.001, ** p =0.01, * p =0.05.
Fig 2: A. Combined inhibition of NOTUM and AXIN2 expression predicts sensitivity to PORCN inhibitors: AXIN2 and NOTUM expression was assessed in cell lines exposed to 100 nM ETC-159 for 24 hours. RNF43 mutant (filled symbols) and wild-type (open symbols) cell lines. ETC-159 sensitive (circle) or insensitive (diamond) cell lines. B. ETC-159 treatment decreases Notum expression in MMTV-Wnt1 allografts and HPAF-II tumors: RNA isolated from vehicle or ETC-159 treated MMTV-Wnt1 tumors (n=9 in each group) or HPAF-II tumors (n=6 in each group) was assessed by RT-qPCR. Data presented are mean ± SD, Data were analyzed using unpaired t-test, **** p =0. 0001, *** p =0. 001. C. NOTUM protein is decreased in plasma of ETC-159 treated mice: Control BALB/c nude mice or mice with established MMTV-Wnt1 tumors were treated daily for 10 days with ETC-159 (30 mg/kg). Blood was collected 6 hours after the last dose and plasma NOTUM levels were measured using ELISA. The graph shows group means ± SD. n=10 for tumor bearing mice in each group and n=4 for control BALB/c nude mice. D. Wnt-C59 treatment decreases NOTUM protein levels in MMTV-Wnt1 mice: Plasma NOTUM levels were measured in control (C57/Bl6, n=4) or transgenic MMTV-Wnt1 mice before initiating the treatment and after 3 days of treatment with 10 mg/kg Wnt-C59 as measured by ELISA. Data represents plasma NOTUM levels of individual mice. Dotted line represents NOTUM levels in control mice. V=vehicle-treated mice, C=Wnt-C59-treated mice. *** p =0.001, **** p =0.0001.
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