Glycosyltransferase engineering to probe the biology of O-linked protein glycosylation in metastatic breast cancer

Glycosylation is the most complex and abundant posttranslational modification of proteins.

Tumour-derived glycoproteins, including mucins such as the NHS-approved breast cancer biomarker CA 15-3 or mucin-1, are often highly modified with aberrant O(Ser/Thr)-linked glycan structures that modulate interactions with the microenvironment and components of the immune system.

Glycan biosynthesis is not directly encoded in the genome but conferred by >250 glycosyltransferases and glycosidases that are, in turn, often dysregulated during tumorigenesis. Glycans are the target of diagnostic and therapeutic (CAR-T, cancer vaccines) approaches.

It is thus of fundamental importance to profile cancer-derived glycoproteins to reveal mechanisms of disease and new targets.

However, we lack the methodologies to profile the activities of glycosyltransferases in the living cell and to distinguish tumour-derived from host (glyco-)proteins. Chemical tools can address both issues.

O-Glycosylation is primed by 20 GalNAc-T glycosyltransferase isoenzymes (T1…T20) that introduce the sugar GalNAc (N-acetylgalactosamine) to Ser/Thr side chains. The isoenzymes GalNAc-T4 and T14 are specifically associated with breast cancer upon dysregulation. However, the reasons for this association are unclear.

This project aims to develop chemical tools to profile breast cancer-derived glycoproteins, to understand the role of the microenvironment on glycoprotein signature and to track their destination by bioorthogonal chemistry and non-invasive in vivo imaging.

This proposal will be carried out in two ambitious Aims that are interdependent but individually underpinned by preliminary data to ensure feasibility: In Aim 1, we will generate reporter systems for the activity of GalNAc-T4 and T14 based on chemically armed sugars and rationally engineered GalNAc-T mutants accepting the chemical modification.

Breast cancer cells expressing engineered GalNAc-Ts will be conditioned in co-culture in 2D or 3D, the cancer glycoprotein signature assessed by mass spectrometry and compared with in vivo conditioned cells to shed light on the relevance of a functional microenvironment.

In Aim 2, the synthesis of new, cyclopropene-armed sugars will enable specific tracking of cancer-derived glycoproteins in vivo.

The cylopropene modification is selectively used by engineered GalNAc-Ts in cancer cells but not the host and allows for bioorthogonal reactions to occur in vivo.

Through attachment of fluorophores, we will non-invasively image the destination of both cancer cells and cancer-derived glycoproteins in distant sites and the tumour microenvironment, underpinned by glycoprotein profiling through MS.