Abstract B059: Loss of Cullin-RING Ligase 5 mediates resistance to RAS inhibition in pancreatic cancer

Abstract

The proto-oncogene KRAS is mutated in greater than 90% of pancreatic ductal adenocarcinoma (PDAC). Although newly developed multi-RAS(ON) inhibitors (daraxonrasib) have significantly improved progression-free and overall survival compared with current standard-of-care chemotherapy in PDAC patients, both clinical and preclinical studies demonstrate that resistance inevitably emerges. Non-genetic (adaptive) resistance to RAS-targeted therapies accounts for up to 50% of cases of clinical resistance, but the underlying mechanisms remain incompletely understood. Proteostasis, in part mediated by the ubiquitin-proteasome system (UPS), a modular network of ubiquitin ligases that selectively targets proteins for degradation by the 26S proteasome, is essential for cancer cell survival during RAS pathway inhibition. We hypothesized that, because the UPS is modular, selective disruption of individual UPS components or complexes stabilizes specific proteins that promote growth or survival following RAS inhibition, thereby driving resistance without globally impairing proteostasis. Consistent with this hypothesis, genome-wide and UPS-focused pooled CRISPR screens, followed by validation using fluorescence competition and colony-forming assays, converged on loss of multiple components of the CRL5 (Cullin-RING ligase 5) complex—including CUL5, UBE2F, and ARIH2—as promoting selective outgrowth of PDAC models subjected to either genetic (CRISPR-mediated KRAS knockout) or pharmacologic RAS inhibition (daraxonrasib). Mechanistically, CRL5-deficient cells exhibited reduced G1 arrest without significant differences in apoptosis or senescence with chronic RAS inhibition, indicating that the observed growth advantage is driven by altered cell-cycle regulation rather than apoptotic escape. Consistent with the role of CRL5 in post-translational regulation, transcriptional profiling revealed nearly identical gene expression programs in CRL5-intact and CRL5-deficient PDAC cells, with no differentially expressed genes or enriched pathways under either basal conditions or following prolonged RAS inhibition. Likewise, pathway-level biochemical and transcriptional analyses detected no evidence of sustained rewiring of the canonical survival pathways examined following chronic RAS inhibition in CRL5-deficient cells, including MAPK, mTOR, YAP, NF-κB, and JAK/STAT signaling. Together, these findings suggest that CRL5-mediated resistance is driven by altered protein stability rather than transcriptional or signaling rewiring. Ongoing studies seek to evaluate the contribution of CRL5 loss to RAS inhibitor resistance in vivo and to identify CRL5 substrates responsible for diminished G1 arrest using integrated steady-state proteomic and phosphoproteomic analyses. Collectively, these findings and ongoing studies define how selective modulation of protein degradation governs adaptive resistance to RAS inhibition and provide a framework for developing rational combination strategies to improve the durability of RAS-targeted therapies in PDAC.