New players in the regulation of DNA replication fork speed

Project Information

META-CYCLE

Grant agreement ID: 101108184

DOI

10.3030/101108184

Project closed

EC signature date 8 May 2023

Start date 1 January 2024

End date 31 December 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 230 774,40

Coordinated by

KRAEFTENS BEKAEMPELSE
Denmark

Periodic Reporting for period 1 - META-CYCLE (New players in the regulation of DNA replication fork speed)

Reporting period: 2024-01-01 to 2025-12-31

Summary of the context and overall objectives of the project

Faithful DNA replication is essential for cell division and organismal health. When this process goes wrong, replication stress arises—a defining hallmark of cancer cells—leading to stalled replication forks, DNA damage accumulation, and genomic instability. Understanding the molecular mechanisms that govern how fast DNA is copied, and how this is coordinated with cell cycle progression, represents a critical frontier in cancer biology with direct therapeutic implications.
The META-CYCLE project was designed to uncover the intricate connections between cellular metabolism, cell cycle regulation, and DNA replication fidelity. The central hypothesis was that metabolic rewiring in rapidly dividing cancer cells is not simply a passive supply chain for building blocks, but an active regulatory layer directly controlling replication fork speed and genomic stability.
Primary objectives:
• Identify metabolic signatures that predict dysregulated DNA synthesis in response to cell cycle alterations (WP1)
• Characterise the molecular mechanisms by which metabolic enzymes regulate replication fork dynamics and genome stability through direct modulation of replisome components (WP2)
• Discover new metabolic regulators of S-phase progression and DNA synthesis speed through systematic screening (WP3)
The project focused on glioblastoma—one of the most aggressive and treatment-resistant brain cancers (~3–4 per 100,000 people annually; median survival <15 months)—while generating insights broadly applicable across cancer types.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The META-CYCLE project evolved strategically from its original design, successfully integrating bioinformatics, metabolomics, cell biology, and advanced imaging to achieve breakthrough discoveries.
Phase 1 — Bioinformatics Discovery (Months 1–8): Rather than beginning with planned metabolomic characterisation, the project initiated with an unbiased computational screening of the DepMap database. A sophisticated bioinformatics pipeline was developed integrating gene expression data from cancer cell lines and patient samples, protein interaction databases, replication fork proteomics, and CRISPR dependency datasets. Two novel metabolic enzymes were identified as strong candidates for direct involvement in DNA replication control, with predicted functional connections to the core replisome components PCNA (Proliferating Cell Nuclear Antigen) and POLA1 (DNA Polymerase α).
Phase 2 — Functional Validation (Months 6–18): Experimental validation in multiple cancer cell lines (U2OS, HeLa, U87-MG, T98G) and normal controls (RPE1) revealed that glycolytic candidates directly regulate DNA synthesis. Key discoveries included: (i) direct modulation of PCNA activity—the first demonstration of metabolic enzyme involvement in core replisome function; (ii) a quality-control mechanism whereby these enzymes limit ssDNA accumulation at replication forks; (iii) impaired Okazaki fragment processing upon their depletion; and (iv) disrupted RPA phosphorylation dynamics. A dual perturbation strategy (combining pharmacological and genetic glycolysis modulation with direct replication machinery manipulation) and a sophisticated single-cell resolution imaging pipeline were developed and validated.
Phase 3 — Integration and Metabolomic Validation (Months 10–24): Bidirectional communication between replication stress and glycolytic flux was established, revealing a sophisticated regulatory circuit rather than simple one-way control. Preliminary glioblastoma data indicate heightened dependency of cancer cells on glycolytic-replication coupling relative to normal cells.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

META-CYCLE delivered three paradigm-shifting results:
1. Metabolic Enzymes as Core Replisome Regulators: Prior to this project, metabolism and DNA replication were viewed as parallel processes responding to common upstream signals. META-CYCLE demonstrated for that metabolic enzymes directly regulate core replisome machinery (PCNA), establishing metabolism as an active, integral regulatory layer of replication fork function. This fundamentally challenges textbook understanding of these two processes as independent.
2. Metabolic Control of Lagging-Strand Fidelity: No metabolic role had previously been recognised in Okazaki fragment maturation or lagging-strand ssDNA management. META-CYCLE revealed that metabolic regulators prevent excessive ssDNA accumulation during lagging-strand synthesis—a preventive rather than responsive quality-control mechanism, operating upstream of canonical checkpoint pathways.
3. Integrated Regulatory Circuit: The project positioned metabolic regulation as the missing link connecting cell cycle commitment, replication fork speed, and genomic stability. The discovery of bidirectional communication—whereby replication stress itself feeds back to alter glycolytic flux—establishes a sophisticated regulatory loop with implications for understanding cancer-specific replication vulnerabilities.
These results provide mechanistic rationale for targeting the glycolytic-replication axis in cancer, particularly in glioblastoma where these dependencies appear amplified relative to normal cells.