Kanevetci, B. | Search bioRxiv

bioRxiv2026

Proximity labeling reveals cell cycle-specific NEK2 interactions and a regulatory axis controlling NUSAP1 stability

NEK2 is a cell cycle-regulated kinase best known for its role in centrosome separation, yet the phase-specific organization of its interaction network has remained unclear. Here, we combine a doxycycline-inducible TurboID system with mass spectrometry to generate a cell cycle-resolved NEK2 interactome in synchronized U2OS cells. Using generalized additive models (GAMs), we identified different enrichment trajectories of the NEK2 interacting proteins across G1/S, late S, and G2/M, linking NEK2 to chromosome and spindle regulation, RNA-ribonucleoprotein processes, vesicle/lysosome compartments, and ubiquitin-associated pathways. Targeted validations (streptavidin pull-down, co-immunoprecipitation, and immunofluorescence) confirmed the interaction and binding for selected partners. Focusing on NUSAP1, NEK2 induction led to rapid loss of NUSAP1 protein without changes in mRNA levels, and this decrease was blocked by the proteasome inhibitor MG-132. Consistently, NUSAP1 exhibited slower decay in cycloheximide chase assays and reduced ubiquitination in NEK2 knockout cells, indicating NEK2-dependent proteasomal turnover. Global proteomic analysis of NEK2-deficient cells revealed widespread remodeling of protein abundance, including increased NUSAP1 and decreased KIF2C, accompanied by coordinated changes in pathways governing mitotic progression, microtubule organization, and ubiquitin-mediated protein turnover. Together, these findings provide a dynamic map of the NEK2 interactome across the cell cycle and uncover a NEK2-NUSAP1 degradation pathway, offering a framework to study how kinase interactomes are remodeled by cell cycle progression.

cancer biology↗

bioRxiv2026

Stress adaptation pathways and HA-CD44 signaling maintain the survival of pancreatic cancer cells with centrosome amplification

Centrosome amplification (CA) is a hallmark of aggressive cancers, including pancreatic ductal adenocarcinoma (PDAC), and is linked to genomic instability and poor prognosis. While CA promotes tumor evolution, it also imposes substantial intracellular stress that cells must overcome to survive. However, the specific metabolic adaptations that enable cancer cells to tolerate stress induced by supernumerary centrosomes remain poorly understood. Here, we show that PDAC cells with CA acquire distinct metabolic dependencies that sustain survival. A metabolism-focused CRISPR-Cas9 screen, coupled with functional validations, identified critical vulnerabilities in three inter-connected axes: redox homeostasis, nucleotide sugar metabolism, and the unfolded protein response (UPR). Specifically, CA elevates intracellular reactive oxygen species (ROS), creating a reliance on glutamine metabolism and NRF2-driven antioxidant signaling. CRISPR screen hits in the hexosamine and uronic acid pathways revealed dependencies that converge on hyaluronic acid (HA) metabolism, and functional assays demonstrated that the HA-CD44 axis is required for centrosome clustering and mitotic fidelity, with its disruption increasing lethal multipolar divisions. In parallel, CA activated all branches of the UPR, and both hyper-activation and suppression of ER stress proved detrimental, indicating a finely tuned proteostatic equilibrium is essential for adaptation. Together, these findings show that, in a PLK4-driven model, centrosome-amplified cells rely on coordinated redox control, proteostatic buffering, and extracellular matrix signaling to tolerate CA-induced stress, revealing selective vulnerabilities that could be therapeutically exploited to target aggressive, therapy-resistant tumor subpopulations.

cancer biology↗