RECQL4; Linking DNA Replication and Bone Tumourigenesis
Creators: name: Author, Unknown
Publisher: name: University of Lincoln
Year: 2023

description: As part of cell division, the initiation of DNA replication is an important regulatorymechanism that maintains genomic stability over generations. RECQL4 belongs tothe RecQ DNA helicase family holding an important role in the initiation of DNAreplication. RECQL4 mutations can lead to disorders including type II RothmundThomson (RTS) syndrome. These patients demonstrate predisposition toosteosarcoma (OS) development. OS is a primary bone tumour showing extensivechromosomal instability. We hypothesise mesenchymal stem cells (MSC)differentiating to osteoblasts are particularly sensitive to RECQL4 mutations, whichmay lead to impaired differentiation and OS. Our aim is to establish a direct linkbetween the impairment of replication initiation, consequent chromosomalinstabilities and deregulation in osteoblast differentiation, and OS development.A model that phenocopies the effect of RECQL4 mutations was established inASC52telo cells to apply acute and chronic pressure on replication initiation usingPHA-767491, which inhibits DDK that acts upstream of RECQL4 in replicationinitiation (PHA cells). We monitored cell viability and chromosomal instabilitycharacteristics in these cells, and RTS cell lines. To establish if PHA cells sustaineddifferentiation capability, the cells were cultured using supplemented media forosteoblast and adipocyte differentiation, and were analysed by histochemicalstaining and immunofluorescence. Presence of DNA damage was quantified using?H2AX, and activation of the DNA damage response was assayed by westernblotting. The cells were cultured in ultra-low attachment plates to test for anchorageindependent growth, and further analysed by cell count, MTS and luminescenceassays. To identify protein-protein interactions of RECQL4, GFP-tagged RECQL4HeLa and U2OS cells were treated with SAHA, or hydroxyurea, pulled down withGFP-nanotrap, and analysed by mass spectrometry and western blot.We confirmed reduced proliferation rate while maintaining viability in PHA cells.Assaying mitochondrial membrane potential revealed no significant effect onmitochondrial function. Successful differentiation of PHA treated MSCs intoosteoblasts and adipocytes was confirmed. Expression of osteoblast differentiationmarkers: calcium, and RUNX2 was influenced by PHA. An increase was alsoobserved in chronic PHA cells under normal medium, indicating malignanttransformation. Sustained DNA damage was shown in chronic PHA-767491 treated ASC52telo cells, with a higher degree of CHK1 phosphorylation,anchorage-independent growth and reduced contact inhibition.We found that the RECQL4 mutated cell line AG05013tert was more sensitive toinhibition of replication initiation. Increase of DNA damage markers was observedin AG05013tert cells, but not in AG18375 and AG03587. Presence of MCM10 andPP2A in RECQL4 complexes was confirmed, and novel interactions with HDX andEN-2 were found.Overall, we demonstrated that chronic interference with DNA replication initiationleads to sustained DNA damage with characteristics of genomic instability, activatedDNA damage response that may become impaired over time, and may inducetransformation.To further these studies, RECQL4 knockdown using lentiviral transduction inosteoblasts would verify the cellular changes we propose, which lead tochromosomal instabilities and OS development. Novel protein interactions withRECQL4 could highlight new pathways with a direct and/or indirect role intumourgenesis.