Synthetic Lethality with FANCM inhibition in ALT-negative Cancer Cells

Abstract

Telomeres are nucleoprotein structures that protect chromosome ends from recognition by the DNA damage response machinery. Due to their high GC content and terminal location, telomeres are particularly prone to replication stress, which is a key contributor to genomic instability, and a defining feature of the Alternative Lengthening of Telomeres (ALT) pathway. This thesis investigates the molecular basis of telomere replication stress and explores how this vulnerability can be exploited for therapeutically.

The first part of this thesis focuses on the establishment of novel DNA labelling techniques to identify protein-DNA interactions at single molecule resolution. Combining proximity labelling with single molecule analysis, we aimed to investigate the dynamic behaviour of TRF1, a core shelterin telomere binding protein, during replication stress and oxidative stress. These experiments revealed the technical limitations of current labelling techniques and informed the design of alternate methods for elucidating protein-DNA interactions at single molecule level.

The second part of this thesis explores how replication stress can be targeted alongside other pathways to expand established therapeutics. A genome-wide CRISPR screen identified synthetic lethal interactors that sensitise telomerase positive cells to Ubexin-1 treatment, a small molecule inhibitor that targets the FANCM through the interaction between FANCM and the BTR complex, leading to ALT-specific FANCM degradation. The identified interactors promote selective cytotoxicity, sensitising telomerase positive cells to Ubexin-1 treatment and enhancing FANCM degradation. These findings expand the therapeutic potential of replication stress-targeting strategies beyond established contexts.

Overall, by exploring replication stress both mechanistically and therapeutically, this work advances our understanding of telomere maintenance, the replication stress response, and identifies new avenues for cancer therapy.