Figure 2. WNT-induced sensitization to asparaginase is mediated by WNT/STOP.

A, HCT15 cells were treated with vehicle or asparaginase (100 U/L) together with human RSpondin3 (75 ng/ml) and WNT3A protein (100 ng/ml) (referred to collectively as WNT ligands here) for 10 days. Cell size was assessed by forward scatter height (FSC-H) by flow cytometry (left). Scatter plot depicts results of individual biologic replicates, with horizontal bars indicating mean, and error bars indicating SEM (right). Differences between groups were analyzed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons.

B, Mouse intestinal organoids of the indicated genotypes were treated with vehicle or asparaginase (100 U/L), and cell size was assessed by forward scatter height (FSC-H) by flow cytometry on a BD FACS DIVA instrument. Scatter plots (left) depict results of individual biologic replicates, with horizontal bars indicating mean, and error bars indicating SEM. Differences between groups were assessed by two-sided ANOVA with Tukey adjustment for multiple comparisons. Histograms (right) show results from a representative organoid of the indicated genotype treated with asparaginase.

C, Mouse intestinal organoids of the indicated genotypes were incubated with a pulse of the methionine analog 5-azidohomoalanine (AHA) for 18 hrs. Organoids were then released from AHA and treated with asparaginase (100 U/L) during the chase period. The degree of AHA label retention was assessed by flow cytometry at the indicated timepoints. Results are normalized to time 0 for each condition. See also Supplementary Fig. S2. Error bars indicate SEM.

D, Apc deficient; Kras; p53 organoids were cultured in basal medium, transduced with the indicated constructs and treated with vehicle, asparaginase (100 U/L), BRD0705 (1 μM) or combo (100 U/L asparaginase + 1 μM BRD0705) for 10 days. Viability was assessed as in (1G). Images were taken from a representative of three experiments. Scale bar, 100 μm. Differences between groups were analyzed using a one-way ANOVA with Tukey adjustment for multiple comparisons.

E, Apc deficient; Kras; p53 organoids were cultured in basal medium, transduced with the indicated constructs and treated with vehicle, asparaginase (100 U/L), BRD0705 (1 μM) or combo (100 U/L asparaginase + 1 μM BRD0705) for 10 days. Viability was assessed as in (1G). Images were taken from a representative of three experiments. Scale bar, 100 μm. Differences between groups were analyzed using a one-way ANOVA with Tukey adjustment for multiple comparisons.

* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001, n.s., p > 0.05.

All error bars represent SEM.