GSK-3 and BCL-XL inhibition mitigates the competitive advantage of APC-mutant colorectal cancer cells

Abstract

BCL-XL is a crucial anti-apoptotic protein that supports survival of intestinal cells during the progression and in established colorectal cancer (CRC). While targeting BCL-XL with BH3 mimetics is effective, its significant toxicity highlights the need for alternative approaches. Importantly, the early steps in intestinal transformation are marked by a competition between normal and transformed stem cells in which the mutant cells gain a supercompetitive advantage due to the secretion of WNT inhibitors. Using multiple human and murine CRC models, we revealed that GSK-3 inhibition strongly sensitized to BH3 mimetic-induced killing. As expected, GSK-3 inhibition significantly upregulated the WNT pathway, but also led to marked enhancement of BH3 mimetic-induced apoptosis, as measured by mitochondrial BAX aggregation, Caspase-3 activation and Propidium Iodide exclusion. Furthermore, GSK-3 inhibition provided an advantage to wild-type intestinal organoids in competition with APC-mutant counterparts due to reactivation of the WNT pathway. More strikingly, combining GSK-3 and BCL-XL inhibition profoundly affected the supercompetition APC-mutant intestinal cells exert over the wild-types. In effect, the combination therapy enhanced the competitive fitness of wild-type cells and resulted in the killing of APC-mutant organoids, pointing to a novel combination therapy that can be further exploited in the treatment of adenomas and CRC.

Fig. 1. GSK-3 inhibitors sensitize CRC to BCL-XL inhibitor.

A Screening assay. Co01 cells were treated with 1 µM compounds from the library with or without 5 nM A-1155463. Targets of the hits that showed synergy were summarized. The targeting efficacy of five GSK-3 inhibitors was presented in the table. B–E Matrix titration and Bliss synergy scores for each concentration in the most synergistic area of two GSK-3 inhibitors CHIR-98014 (B, C) and CHIR-99021 (D, E) with A-1155463. Cell viability was measured by Cell Titer Blue. Inhibition rate was related to the untreated control (0,0 nM). Synergy scores were calculated by the online tool Synergyfinder. F, G Activation of Caspase-3 induced by 1 µM CHIR-98014 (F) or 2.5 µM CHIR-99021 (G) with or without 5 nM A-1155463 in Co01 was measured by flow cytometry. Percentage of the cells with active Caspase-3 was plotted.* :p ≤ 0.05,**: p ≤ 0.01. H, I Percentage of dead cells with PI influx induced by 1 µM CHIR-98021 (H) or 2.5 µM CHIR-99021 (I) with or without 5 nM A-1155463 in Co01 was measured by flow cytometry. *:p ≤ 0.05.

Fig. 2. GSK-3 inhibitors upregulate WNT signaling activity.

A Flow cytometry analysis of the TOP-GFP in Co01 cells treated with 1 µM CHIR-98021 or 2.5 µM CHIR-99021 for 24 h. * :p ≤ 0.05,**: p ≤ 0.01. B Gene Set Enrichment Analysis (GSEA) of WNT/β-catenin signaling pathways in Co01 cells treated with 1 µM CHIR-99021 for 48 h. p = 0.04338. C, D mRNA expression of LGR5 in Co01 cells treated with 1 µM CHIR-98014 (C) and 2.5 µM CHIR-99021 (D) for 24 h. E, F mRNA expression of CK20 in Co01 cells treated 1 µM CHIR-98014 (E) and 2.5 µM CHIR-99021 (F) for 24 h.

Fig. 3. BAX localization induced by combination of CHIR-99021 and A-1155463.

A Representative confocal microscopic images of Co01-BAX-KO-CFP-BAX-MitoDsRed. Cells were treated with 2.5 µM CHIR-99021 for 24 h before 5 nM A-1155463 was added to trigger CFP-BAX aggregation. The pictures were captured at 63× Oil magnification. B Statistics of the percentage of cells with BAX localization treated with 2.5 µM CHIR-99021 or/and 5 nM A-1155463, ***:p ≤ 0.001,****:p ≤ 0,0001.

Fig. 4. Competitive disadvantage of wild-type ISCs was lost upon combinatory inhibition on GSK-3β and BCL-XL.

A Schematic illustration of the setting of co-culture assay. B Representative images of the time course co-culture experiment. Co-cultures were treated with or without 10 µM CHIR-99021. Pictures were captured at 4× magnification on Days 1, 3, and 7 after treatment. C Representative images of the co-cultures treated with 5 or 10 µM CHIR-99021 with or/and 1, 2.5, 5 µM A-1155463. Pictures were captured at 4× magnification at Day 7 after treatment. D Statistics of the relative surface contribution of wild-type organoids (Red) and APC-mutant organoids (Green). E Mechanistic Illustration of the synergistic effect of combination of GSK-3β inhibitor and BCL-XL inhibitor on the competition between WT and APC-mutant ISCs.