Discovery and functional investigation of how ROS can regulate the cell cycle

Background: Aberrant cell cycle and reduction/oxidation (redox) control are hallmarks of cancer, yet their crosstalk is poorly understood.

The importance of phosphorylation and ubiquitylation in cell cycle regulation has long been recognised, but emerging evidence indicates that the redox system also has an important role in promoting cell proliferation by virtue of reactive oxygen species (ROS). ROS oxidize cysteines to sulfenic acids creating reversible PTMs found on key cell cycle regulators.

While this implies that redox changes affect proliferation, the potential of cysteine oxidation as a key cell cycle- regulatory PTM and accordingly as a target for innovative cancer therapies remains unexplored.

Redox control of cell proliferation has clear implications for understanding tumour growth, where the redox state of a tumour often differs from normal tissue, and response to radiotherapy that generates ROS.

Aims: We aim to interrogate the influence of ROS on cell cycle control by investigating how sulfenic acids act as cell cycle-regulatory PTMs in normal and cancer cells.

We will focus on three objectives: i) identification and functional characterization of cell cycle-dependent cysteine PTMs, ii) investigation of coordination and feedback between cell cycle control and ROS signalling, and iii) discovery of mechanism(s) involved in cysteine-oxidation reversal during mitosis and its importance for cell division and genome stability.

Methods: We have developed cutting-edge quantitative mass spectrometry approaches to generate cell cycle stage-dependent sulfenic acid proteomes, including information on the stoichiometry of cysteine oxidation (Objective i).

We will validate and functionally characterize these PTMs on cell cycle regulators by employing genetically-encoded sensors and chemical probes to monitor cell cycle and redox states in response to specific mutations, chemical and metabolic perturbations of cells grown in 2D and 3D cultures (Objective i-iii).

We will employ CRISPR/CRISPRi screening to identify redox regulators involved in resetting the redox state during mitosis and evaluate their role for chromosome segregation and genome stability in cancer cells with higher and lower levels of ROS (Objective iii).

How the results of this research will be used: The cell cycle stage-specific sulfenic acid proteomes and their subsequent functional investigations will greatly improve our understanding of how redox signalling modulates cell cycle control.

Research on all objectives is driven by our ambition ultimately to translate our mechanistic findings into the development of cancer-relevant leads, as previously performed by my laboratory.

By uncovering proliferative nodes between cell cycle and redox control, our work may indicate new avenues for treatments.