Developing new cell models for brain tumours which use the Alternative Lengthening of Telomeres pathway

Background A hallmark of cancer cells is the ability to programmed cell death, ultimately resulting in uncontrolled growth of tumour cells. One of these mechanisms is immortalisation, whereby cancer cells elongate their telomeres.

Commonly, this involves re-expression of the enzyme telomerase – however, in some cancers, the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway has been implicated.

Although the molecular aetiology of ALT induction is still poorly understood, loss of function of the ATRX gene has been shown as a key factor.

However, loss of ATRX alone is insufficient to induce ALT, and recent work has highlighted elevated levels of reactive oxygen species in the ALT-pathway.

ALT-positive cancers are prevalent in cancers that typically affect children and young people, including a significant proportion of neuroblastomas, gliomas and sarcomas.

Furthermore, ALT-positivity is a very poor prognostic indicator – correlating with worse long-term outcomes and aggressive, metastatic behaviour.

Despite the clinical importance and recent advances in mechanistic understanding of ALT induction, there are no therapies targeting ALT-positivity in cancer.

Further research is limited by the lack of clinically relevant cellular systems available, as there are no ALT-positive/ALT-negative isogenic paired cell lines – which would allow for comparison between cells which are almost genetically identical, apart from ALT status. This prevents advancement within both molecular oncology and the testing of potential novel therapeutics.

Aims Our aim is to develop a pair of ALT-positive/ALT-negative isogenic glioma cell lines, starting from existing high-grade glioma cell lines. This will be an essential resource for use in future testing of new therapies.

Methods Multiple high-grade glioma cell lines will be characterised by various methods to determine telomere characteristics, including confirmation of ALT-negative status at baseline and telomere length, allowing selection of the optimal line for downstream genetic manipulation using CRISPR.

ATRX will be targeted, alongside genes that have been implicated in increasing reactive oxygen species (such as SETD2, SOD1 or DRG2). Successful knockout clones can then be re-assessed for ALT status and selection of an ALT-initiated clone.

How the results will be used Ultimately, successful development of this ALT-positive/ALT-negative cell line pairing will enable further research into ALT-cancers, including both basic and translational aspects.

The newly developed cell lines will be a useful resource for interrogating the molecular factors which drive the ALT-pathway, as well as providing an ideal cellular system in which to test novel targeted therapies for cancers that currently have very poor prognosis.