Colorectal cancer (CRC) development is primarily driven by mutations in the gene APC, which disrupt WNT signaling and provide APC-mutant intestinal stem cells (ISCs) with a competitive advantage over their wild-type counterparts. This advantage is mediated by the secretion of WNT antagonists, allowing APC-mutant cells to outcompete the wild-types in the crypts and initiate tumor growth. In this study, we explored whether GSK-3 inhibition, which leads to upregulation of WNT signaling could enhance the effects of BCL-XL—a key regulator of cell survival—inhibition by BH3 mimetics and potentially restore the competitive fitness of normal ISCs over APC-mutants.

CRC spheroids and intestinal organoid models were treated with the GSK-3 inhibitors, alone or in combination with the BCL-XL inhibitor A-1155463. WNT pathway activity was assessed using the TOP-GFP reporter assay, quantitative PCR for WNT target gene expression, and RNA sequencing analysis to capture transcriptional changes following treatment. Apoptotic responses were assessed via caspase-3 activation, mitochondrial BAX aggregation, and propidium iodide exclusion assays. Competitive co-culture experiments between wild-type and APC-mutant ISCs were performed to evaluate the impact of combination therapy on cell fitness.

We found that GSK-3 inhibition significantly sensitized CRC cells to BCL-XL inhibition, leading to a strong synergistic induction of apoptosis. Mechanistically, GSK-3 inhibition amplified WNT signaling, potentially pushing APC-mutant cells beyond an oncogenic stress threshold. This was reflected in increased mitochondrial BAX aggregation and caspase-3 activation, indicating enhanced apoptotic priming. Gene expression analysis confirmed upregulation of WNT target genes upon GSK-3 inhibition, consistent with the observed increase in WNT pathway activity measured by the TOP-GFP reporter. Importantly, while APC-mutant cells underwent apoptosis, wild-type ISCs exhibited enhanced fitness, reversing the competitive imbalance in organoid models. This suggests that the combination therapy not only eliminates mutant cells but also restores wild-type ISC dominance, counteracting early tumor progression.

Our findings highlight the therapeutic potential of combining GSK-3 and BCL-XL inhibitors in CRC treatment. By simultaneously exploiting oncogenic stress in APC-mutant cells and restoring wild-type ISC fitness, this strategy may provide a targeted approach for early-stage CRC, particularly in patients with familial adenomatous polyposis (FAP).