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. 2016 Oct 25;7(43):69816-69828.
doi: 10.18632/oncotarget.12002.

Large Variety in a Panel of Human Colon Cancer Organoids in Response to EZH2 Inhibition

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Free PMC article

Large Variety in a Panel of Human Colon Cancer Organoids in Response to EZH2 Inhibition

Martijn A J Koppens et al. Oncotarget. .
Free PMC article

Abstract

EZH2 inhibitors have gained great interest for their use as anti-cancer therapeutics. However, most research has focused on EZH2 mutant cancers and recently adverse effects of EZH2 inactivation have come to light. To determine whether colorectal cancer cells respond to EZH2 inhibition and to explore which factors influence the degree of response, we treated a panel of 20 organoid lines derived from human colon tumors with different concentrations of the EZH2 inhibitor GSK126. The resulting responses were associated with mutation status, gene expression and responses to other drugs. We found that the response to GSK126 treatment greatly varied between organoid lines. Response associated with the mutation status of ATRX and PAX2, and correlated with BIK expression. It also correlated well with response to Nutlin-3a which inhibits MDM2-p53 interaction thereby activating p53 signaling. Sensitivity to EZH2 ablation depended on the presence of wild type p53, as tumor organoids became resistant when p53 was mutated or knocked down. Our exploratory study provides insight into which genetic factors predict sensitivity to EZH2 inhibition. In addition, we show that the response to EZH2 inhibition requires wild type p53. We conclude that a subset of colorectal cancer patients may benefit from EZH2-targeting therapies.

Keywords: GSK126; Tp53; colorectal cancer; organoids; polycomb.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Organoids from different CRC tumors respond differently to chemical EZH2 inhibition
(A) EZH2 expression in normal colonic organoids and CRC organoids. Both the mean and the variance of EZH2 expression are higher in tumor organoids as compared to normal organoids. (B) EZH2 expression in normal colon and the different molecular CRC subtypes. Expression data were taken from CRC RNA-sequencing samples from the TCGA database. CMS4 tumors express EZH2 significantly lower than other subtypes: comparing CMS1 and CMS4, p-value: 1.2 * 10−7, comparing CMS2 and CMS4, p-value: 1.6 * 10−7, and comparing CMS3 and CMS4, p-value: 0.0277. EZH2 expression in normal colon tissue was lower than tumor tissue (compared with CMS1-4 combined, p-value: 2.0 * 10−15). The numbers below the x-axis indicate the number of samples. (C) Western blot of four organoid lines that were treated with 4 μM GSK126 and harvested after fourteen days. H3K27me3 is strongly reduced in GSK126-treated versus DMSO-treated samples. (D, E) Box plot showing the trapezoidal AUCs for all replicates of 20 CRC organoids in passage 1 (D) and eight CRC organoids in passage 2 (E). (F, G) Box plot showing the trapezoidal IC50s for all replicates of 20 CRC organoids in passage 1 (F) and eight CRC organoids in passage 2 (G). Horizontal gray lines within boxes demarcate the medians, boxes delineate the middle 50% of the data, and whiskers mark 25% and 75% quartiles. The horizontal blue lines in (E and G) demarcate the medians from passage 1 for the same organoid line.
Figure 2
Figure 2. Adherently cultured GSK126-responsive organoids remain sensitive to EZH2 inhibition
(A) Western blot confirming reduction of H3K27me3 in adherent organoid cultures upon 14 days of GSK126 treatment. (B) Left panel: microscopy images of adherently cultured P7T and P19TA, treated with either 4 μM of GSK126 or DMSO for fourteen days. Right panel: bar plot showing the average fold expansion in number of cells after fourteen days of DMSO or GSK126 treatment. Error bars indicate standard deviation. Scale bar: 100 μm, same magnification applies to all images in (B).
Figure 3
Figure 3. Response to EZH2 inhibition associates with mutation status and BIK gene expression
(A) PAX2-mutant organoid lines are significantly more sensitive to GSK126 according to all four viability measures in passage 1. (B) ATRX-mutant organoid lines are significantly more sensitive to GSK126 for two out of four viability measures for passage 1. In A and B, four panels of box plots are shown (AUC: left two panels, IC50: right two panels; median: white background, fitted: gray background) comparing organoid lines that are wild type (left group, green dots) with organoids that are mutant (right group, red dots) for the indicated genes. Horizontal gray lines within boxes demarcate the medians, boxes delineate the middle 50% of the data, and whiskers mark 25% and 75% quartiles. (C) BIK expression inversely correlates with GSK126 response for three out of four viability measures (fitted IC50, and median and fitted AUC) in passage 1.
Figure 4
Figure 4. Sensitivity to EZH2 inhibition is correlated with activity of WT-p53 signaling
(A) The four highest correlating drug responses with response to GSK126 (heatmap of Pearson correlations between all pairs of 84 drugs is presented in Supplementary Figure 4A). (B) Scatter plot showing the high correlation between the AUCs of Nutlin-3a and GSK126. (C) Western blot showing efficient Ezh2 KD and increased levels of Trp53 in Ezh2 KD organoids. (D) Average relative growth of VAR, VAPR and VAR-sh-Trp53 tumor organoids with or without Ezh2 KD, normalized to not-induced samples (”-Dox”, black bars). Error bars indicate standard deviation. (n = three independent experiments; 1-way ANOVA followed by Post hoc tests; *P = 6.7*10−4, only P-values < 0.05/12 are depicted).

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