Dissecting Hydroxylase Biology Implicated in Development Disorders and Cancer

Hydroxylation is an emerging protein modification with largely unappreciated and poorly understood roles in fundamental cellular processes and a wide variety of human diseases. Protein hydroxylation in humans is generally catalysed by oxygenases that are dependent on a metabolite called 2-oxogulatarate (2OG, aka a-ketoglutarate). These so-called '2OG-oxygenases' are druggable enzymes with nutrient-sensing capabilities that can render their functions sensitive to (patho)physiological oxygen limitation (hypoxia) and metabolic alterations.

Jumonji-C (JmjC) protein hydroxylases are a poorly characterised sub-family of 2OG-oxygenases, several of which remain as functional 'orphans' with cellular targets and functions not clearly defined. Although isolated catalytic pockets are relatively well characterised, the molecular basis for regulated activity of physiologically-relevant JmjC hydroxylase complexes remains unclear.

Here, we propose to continue our investigation of a biomedically relevant JmjC enzyme that possesses a unique hydroxylase activity, to reveal the molecular mechanisms underlying its role in human disease. Based on a solid foundation of pre-existing data including protein interaction assignments, functional studies, and novel models (including patient-derived cells and cancer patient 'avatars' (organoids)), we will focus on understanding the structural basis and importance for hydroxylase activity of interaction with an obligate cancer-associated binding partner.

We aim to provide a comprehensive understanding of the role of the hydroxylase complex in DNA replication and DNA damage repair (DDR), including structural and functional analyses of its interaction with and hydroxylation of a newly identified substrate that has previously been implicated in similar cellular processes and diseases. The insights gained from this work will then provide a framework for exploration of the impact of hypoxia and cancer-associated metabolites ('oncometabolites') on regulation of this novel pathway.

In order to identify new actionable drug targeting opportunities, we will also leverage the information gathered to undertake a structure-guided analysis of the mechanisms by which the hydroxylase complex limits sensitivity to clinically-relevant DDR inhibitors. Importantly, we will continue and expand our published collaborative work developing first-in-class hydroxylase inhibitors, defining the importance of hydroxylase activity in tumour cell viability and the potential for broader application of DDR-focussed therapeutic strategies targeting this (and related) protein hydroxylase complexes.

Together, the work will significantly advance our understanding of enigmatic disease-associated protein hydroxylase complexes and assign the physiological target of this unique protein hydroxylase for the first time. This, in turn, will support studies into how the microenvironment can drive genome instability and therapy resistance, and a wider understanding of the importance of protein hydroxylation in health and disease.

Importantly, the work will also support the diagnoses of neurodevelopmental disorder patients and identify new actionable therapeutic strategies for the future benefit of cancer patients.