Palbociclib targets β-catenin for degradation and synergizes with KRAS or ERK5 inhibition in colorectal cancer preclinical models.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality, largely driven by aberrant activation of the WNT/β-catenin and RAS pathways. Despite recent therapeutic advances, effective strategies to target these oncogenic signals remain limited. A multi-step chemical library screen was performed to identify compounds capable of suppressing β-catenin and RAS signaling. Drug mechanisms and combination effects were investigated through functional assays, in vitro and in vivo experiments, and transcriptomic profiling. Survival analysis of the Sidra-LUMC AC-ICAM CRC cohort (n = 303) was conducted to evaluate the prognostic significance of candidate target genes. We identified the CDK4/6 inhibitor palbociclib as an unexpected suppressor of β-catenin signaling. Uniquely, palbociclib promoted GSK3β-mediated β-catenin degradation by dampening AKT activity, revealing a previously unrecognized mechanism of action and broadening its role beyond canonical cell-cycle inhibition. Building on this mechanism, we found that combining palbociclib with the KRASG12D-specific inhibitor MRTX1133 elicited potent and selective anti-tumor effects in vitro and in vivo. Parallel screening also revealed the ERK5 inhibitor ERK5-IN-1 as a promising combination partner: co-administration with palbociclib strongly suppressed proliferation across multiple CRC models, showed minimal toxicity in normal cells, and produced durable tumor control in vivo. Transcriptomic profiling indicated that both combinations converge on a common program of cancer cell-state reprogramming, characterized by suppression of proliferative drivers and remodeling of metabolic and mitochondrial pathways. Underscoring the clinical relevance of our findings, survival analysis of the Sidra-LUMC AC-ICAM CRC cohort (n = 303) revealed that ERK5 target genes CREB5 and NUPR1, identified in our dataset, were consistently linked to poor prognosis, thereby connecting this signaling axis to unfavorable patient outcomes. Together, these findings position palbociclib as a versatile therapeutic backbone in CRC. By simultaneously targeting cell cycle and oncogenic signaling networks, palbociclib-based combinations induce synergistic and durable responses, offering a compelling rationale for tailored therapeutic strategies in molecularly defined CRC.