Nuclear CK1α as an integral component of the WNT/β-catenin transcriptional complex is required in WNT-driven tumor-initiating cells

Alternative splicing of the CSNK1A1 gene generates multiple CK1α isoforms with distinct subcellular localizations and regulatory properties. While cytoplasmic CK1α is a well-established negative regulator of canonical WNT signaling, the role of nuclear CK1α remains poorly understood. Here, we identify L-insert-containing nuclear CK1α isoforms as functional components of the β-catenin/TCF transcriptional complex in gastrointestinal cancer cells. Using isoform-specific assays, we demonstrate that CK1α isoforms containing the L-insert exhibit greater nuclear accumulation than isoforms lacking the L-insert and support WNT-driven transcription. Knockdown of L-insert-containing CK1α isoforms in esophageal adenocarcinoma (EAC) and colorectal cancer (CRC) models reduces β-catenin recruitment to TCF-bound chromatin and attenuates WNT target gene expression. Kinase-inactive CK1α mutants retained their association with the β-catenin/TCF complex but showed reduced WNT reporter activity, indicating that CK1α catalytic activity is dispensable for complex association but is required for full transcriptional activation. Consistent with the canonical negative regulatory function of CK1α, pharmacological activation of CK1α with SSTC3 increased β-catenin Ser45 phosphorylation, reduced active β-catenin abundance, and limited its nuclear availability and binding to TCF sites, thereby attenuating WNT transcriptional activity and reducing tumor-initiating cell (TIC)-enriched populations in gastrointestinal tumors. These effects translate into reduced tumorigenic potential and delayed tumor regrowth in vivo. Together, our findings identify L-insert-containing nuclear CK1α isoforms as positive regulators of WNT transcription, in contrast to the negative regulatory function of cytoplasmic CK1α within the β-catenin destruction complex, and provide a rationale for pharmacological CK1α activation as a strategy to target WNT-driven TICs.