Biology subjects

Ozcan, S. C.

Publications and source records attributed to Ozcan, S. C..

7 recordsLinked to original sources

bioRxiv2026

GLproxScape reconstructs spatial chromatin occupancy landscapes from tiled genomic locus proteomics

Genomic locus proteomics combines proximity labeling with mass spectrometry to identify the proteins associated with user-defined genomic loci. However, per-region enrichment values from tiled guide designs are typically pooled before hit calling, collapsing the latent spatial structure encoded by overlapping measurements. Here, we describe GLproxScape, an R package that treats per-region enrichments as indirect spatial measurements and reconstructs latent chromatin occupancy landscapes through a Gaussian labeling-kernel forward model. Sequence-specific transcription factors are resolved by motif-anchored non-negative least-squares deconvolution against JASPAR or HOCOMOCO position weight matrices, while chromatin regulators which lack defined DNA-binding motifs are inferred as broad occupancy zones, enabling recovery of overlapping members of multi-subunit complexes. Applied to published genomic locus proteomics datasets at the human TERT, MYC, FOXP2, and FOXQ1 loci and the mouse Ripk3 locus, GLproxScape recovered known regulators with predicted positions independently supported by ChIP-Atlas peaks, reconstructed candidate co-binding relationships, and identified chromatin complexes inaccessible to pooled analyses. Systematic sgRNA-ablation experiments further showed that densely tiled designs improve event recovery and positional stability, providing concrete experimental guidance for future genomic locus proteomics studies.

bioinformatics↗

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↗

bioRxiv2025

RESTRICT-seq enables time-gated CRISPR screens and uncovers novel epigenetic dependencies of SCC resistance

Cancer cell evasion of therapy is a highly adaptive process that undermines the efficacy of many treatment strategies. A significant milestone in the study of these mechanisms has been the advent of pooled CRISPR knockout screens, which enable high-throughput, genome-wide interrogations of tumor dependencies and synthetic lethal interactions, advancing our understanding of how cancer cells adapt to and evade therapies. However, the utility of this approach diminishes when applied to dynamic biological contexts, where processes are transient and sensitive to routine cell culture manipulations that introduce noise and limit meaningful discoveries. To overcome these limitations, we present RESTRICT-seq, a next-generation pooled screening methodology that restricts Cas9 nuclear activation in controlled, repeated cycles. By confining Cas9 catalytic activity to strict temporal windows, RESTRICT-seq mitigates undesired fitness penalties that accumulate throughout CRISPR screens. When benchmarked against conventional pooled screens and standard inducible CRISPR protocols, RESTRICT-seq revealed significantly fewer divergent cell clones and increased signal-to-noise ratio, overcoming a key limitation of traditional methods. Leveraging RESTRICT-seq, we conducted a comprehensive functional survey of the druggable mammalian epigenome, uncovering several elusive epigenetic drivers of treatment resistance in cutaneous squamous cell carcinoma (cSCC). This revealed PAK1 as a previously unrecognized mediator of cSCC resistance in human and mouse SCC, offering new insights into a prognostic marker and therapeutic target of high clinical significance. Our findings establish RESTRICT-seq as a powerful tool for extending the applicability of pooled CRISPR screens to dynamic and previously intractable biological contexts.

cancer biology↗

bioRxiv2024

Epidrug Screening Identifies Type I PRMT Inhibitors as Modulators of Lysosomal Exocytosis and Drug Sensitivity in Cancers

Epigenetic changes drive differential gene expression, contributing to oncogenic transformation and drug resistance. Lysosomes are crucial in cell signaling and the sequestration of toxins and chemotherapeutic agents. This sequestration followed by expulsion through lysosomal exocytosis is a factor in drug resistance. The epigenetic regulation of lysosomal exocytosis remains poorly understood. Our research focuses on this regulation, hypothesizing that epigenetic modifier drugs (epidrugs) capable of inhibiting lysosomal exocytosis and could serve as potential therapeutics. Additionally, we investigate their potential synergy with drugs known to be sequestered in lysosomes. To examine this concept, we screened approximately 150 epigenetic drugs targeting various reader, writer, or eraser proteins. These drugs were assessed for their combined cytotoxic effects with cisplatin, their impact on lysosomal exocytosis, and on lysosomal biogenesis. Our findings reveal that among the epidrugs showing synergy with cisplatin and further reducing cell viability in combination, two type I PRMT inhibitors, MS023 and GSK3368715, inhibited lysosomal exocytosis. Notably, neither of these drugs altered the expression of the CLEAR lysosomal biogenesis network of genes, suggesting the involvement of novel regulators in lysosomal functions. To explore the specific components of the trafficking machinery affected by PRMT inhibitors, we conducted an RNA-seq analysis, uncovering several differentially expressed genes (DEGs). In addition to previously described functions such as methylation activity, or DNA repair; these DEGs included those involved in vesicular trafficking, lysosomal enzyme activity and lysosome dynamics, offering potential insights into the mechanism of reduced exocytosis and identifying a novel mode for its regulation. Additionally, both inhibitors exhibited synergy with other drugs known to be sequestered in lysosomes, such as carboplatin, oxaliplatin, sunitinib, and doxorubicin, indicating that inhibition of lysosomal exocytosis may be a common phenomenon for such drugs. These findings underscore the potential of Type I PRMT inhibitors as therapeutic agents in cancer treatment. Consistently, analysis on the publicly available patient data revealed that lower levels of type I PRMTs (PRMT1 and 6) were associated with better patient response to these drugs, further suggesting their potential as drug candidates for combination therapy to enhance chemotherapy efficacy and improve cancer patient survival rates.

cancer biology↗

bioRxiv2023

Less is More: Nek2A Unclusters Extra Centrosomes and Induces Cell Death in Cancer Cells via KIF2C Interaction

Unlike normal cells, cancer cells frequently exhibit extra centrosomes, leading to formation of multipolar spindles that can trigger cell death. Nevertheless, they manage to divide successfully and escape the deadly consequences of unequal segregation of genomic material by coalescing their extra centrosomes into two poles. This unique trait of cancer cells presents a promising target for cancer therapy, focusing on selectively attacking cells with supernumerary centrosomes. Nek2A is a kinase involved in mitotic regulation, including the centrosome cycle, where it phosphorylates linker proteins to separate centrosomes. In this study, we investigated if Nek2A also unclusters extra centrosomes, akin to its separation function. Reduction of Nek2A activity, achieved through knockout, silencing, or inhibition, promotes centrosome clustering, whereas its overexpression results in unclustering. Significantly, this unclustering activity induces cell death, but only in cancer cells with extra centrosomes, both in vitro and in vivo. Notably, none of the known centrosomal (e.g., CNAP1, Rootletin, Gas2L1) or non-centrosomal (e.g., TRF1, HEC1) Nek2A targets were implicated in this unclustering activity. Additionally, Nek2A operated via a mechanism distinct from other unclustering factors like HSET and NuMA. Through TurboID proximity labeling analysis, we identified novel proteins associated with the centrosome or microtubules, expanding the known interaction partners of Nek2A. KIF2C, in particular, emerged as a novel interactor, confirmed through coimmunoprecipitation and localization analysis. The silencing of KIF2C diminished the impact of Nek2A on centrosome unclustering and rescued cell viability. Additionally, elevated Nek2A levels were indicative of better patient outcomes, specifically in those predicted to have excess centrosomes. Therefore, while Nek2A is a proposed target, its use must be specifically adapted to the broader cellular context, especially considering centrosome amplification. Discovering partners such as KIF2C offers fresh insights into cancer biology and new possibilities for targeted treatment.

cancer biology↗

bioRxiv2023

Prolonged over-expression of PLK4 amplifies centrosomes through formation of inter-connected centrosome rosette clusters

The centrosome cycle is a tightly regulated process to ensure proper segregation of chromosomes. Not surprisingly, centriole number is tightly controlled via multiple mechanisms, one of which involves PLK4, an upstream kinase facilitating centriole biogenesis and duplication. Aberrations in this process can result in supernumerary centrosomes, which are frequently observed in a variety of cancers due to high levels of PLK4. Interestingly, extra centrosomes induced by PLK4 over-expression go through unique intermediate structures called the centrosome rosettes (CRs), where the mother centriole is surrounded by numerous daughter centrioles. The maturation and molecular nature of these CRs have not been investigated in detail. Upon prolonged PLK4 over-expression, cells exhibited large centrosomes that were clustered and contained more than two CRs, which we defined as centrosome rosette clusters (CRCs). As expected, these structures required high PLK4 levels at two consecutive cell cycles and were still interconnected with canonical centrosomal linker proteins such as C-Nap1, Rootletin, and Cep68. Knockout of these linker proteins resulted in distancing of CRs and CRCs as observed by increased diameter of the CRCs in interphase. In contrast, Nek2 knockout inhibited the separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. These results suggest a cell cycle dependent model for PLK4 induced centrosome amplification, which occurs in two consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle. Author summaryThe overexpression of PLK4 can lead to the formation of centrosome rosette structures, which harbor two centrioles around the mother centriole. Although the generation of centrosome rosettes by PLK4 overexpression has been previously investigated, little is known about the cell cycle-dependent maturation and linking of these structures. Here, we report that prolonged PLK4 overexpression results in amplification of centrosomes through the generation of centrosome rosette clusters (CRCs). These CRCs are interconnected via canonical centrosomal linker proteins such as C-Nap1, Rootletin, and CEP68 and are regulated by mechanisms controlling centrosome linking and separation. We also describe two different spatial binding types of amplified centrosomes following PLK4 induction: planar-oriented and circular-oriented. Since PLK4-associated centrosome amplification occurs naturally in both cancer and multiciliated cells, we believe that this research will contribute to a better understanding of the canonical mechanism of PLK4-induced centrosome amplification.

cell biology↗