A handful of childhood brain tumours keep their cells immortal by using a molecular workaround called the Alternative Lengthening of Telomeres (ALT) pathway, and researchers have no good way to study it in the lab. The problem is that no existing cell lines are genetically identical except for whether they use ALT. Without this clean comparison, scientists cannot pin down exactly which genes trigger the pathway or test drugs that might shut it down. ALT-positive tumours—common in gliomas, neuroblastomas, and sarcomas in children and young people—are aggressive and carry a very poor prognosis, yet no therapies target them. This project will create that missing tool. Using CRISPR, the team will knock out the ATRX gene and other genes linked to reactive oxygen species in high-grade glioma cell lines, then select clones that switch on ALT. The result will be a pair of isogenic cell lines—one ALT-positive, one ALT-negative—that are otherwise nearly identical. If successful, this resource will let researchers probe the molecular drivers of ALT and test new therapies in a clinically relevant system. The work is fundamental science: it builds the platform for future translational research, not a treatment itself. But without such tools, the biology of these deadly childhood cancers remains a black box.
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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.
Research Careers Committee - Training Intervention
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