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The fusion of the genes ETV6 and RUNX1 cannot cause cancer on its own.

Immune system development linked to leukemia

Scientists have discovered a genetic signature that implicates a key mechanism in our immune system as a driving force behind Acute lymphoblastic leukaemia (ALL) the most common form of childhood leukaemia.

A key factor driving ALL leukaemia for one in four ALL patients is a mutation causing two of the victim's genes — ETV6 and RUNX1 — to fuse together. This genomic alteration happens before birth and kick starts the disease.

On its own the fused gene cannot cause cancer; it requires additional mutations before the leukaemia fully evolves.

This new study explores how this process occurs and was carried out by researchers from the Wellcome Trust Sanger Institute and The Institute of Cancer Research, London, with funding from the Kay Kendall Leukaemia Fund, Leukaemia and Lymphoma Research and the Wellcome Trust. Their work is published in Nature Genetics 12 Jan., 2014 DOI: 10.1038/ng.2874

Recombination activating genes (RAGs) encode enzyme proteins which rearrange the genome in normal immune cells to generate antibody diversity. In ALL patients with the ETV6 and RUNX1 fusion gene, researchers reveal how the proteins generated by this fusion can lead to the development of leukaemia.

"For the first time, we see the combined events that are driving this treatable but highly devastating disease. We now have a better understanding of the natural history of this disease, from the critical events of the fusion of ETV6-RUNX1, to the sequential acquisition of RAG-mediated genome alterations that ultimately result in this form of childhood leukaemia." 

Dr Elli Papaemmanuil, first author from the Wellcome Trust Sanger Institute.

The team sequenced the genomes of 57 ALL patients with the fusion gene and found that genomic rearrangement with specific deletions of DNA segments, are the predominant drivers of ALL cancer. All samples showed evidence of events involving RAG proteins.

RAG proteins use a unique sequence of DNA letters as a signpost to direct them to antibody regions. Researchers found that remnants of this unique sequence of DNA letters lay close to more than 50 per cent of cancer-driving genetic rearrangements.

Importantly, these genetic rearrangements led to the loss of the very genes required to control normal immune cell development.

It is the deletion of these RAG genes combined with the fusion gene, that leads to ALL leukaemia.

This striking genetic signature linking RAG proteins to genomic instability is not found in other common cancers such as breast, pancreatic and prostate cancer, or even other types of leukaemia.

"As we sequence more and more cancer genomes, we increasingly understand the mutation processes that support cancer's evolution," says Dr Peter Campbell, co-lead author from the Wellcome Trust Sanger Institute. "In ALL childhood leukaemia, we see the process required to make normal antibodies is co-opted by leukaemic cells which knock-out other genes specific for a normal immune response."

To better understand the genetic events that led up to the ALL cancer, the team used single-cell genomics, a state-of-the-art technique that can monitor DNA from an individual cell. Using blood samples from two patients, researchers followed the process which resulted in the DNA fusion and protein diversification leading to leukaemia.

"It may seem surprising that evolution should have provided a mechanism for diversifying antibodies that can collaterally damage genes that then contribute to cancer," said Professor Mel Greaves, co-senior author of the study from The Institute of Cancer Research, London, "But this only happens because the fusion gene (ETV6-RUNX1) that initiates the ALL disease 'traps' cells in a normally very transient period of cell development when RAG enzymes are active, allowing their imperfect specificity to become manifest."

The team will now investigate how the RAG-mediated genomic instability accrues in cells with the ETV6-RUNX1 fusion gene and what the role of this process is in patients that relapse.

"The more we understand about the genetic events that underlie leukaemia and other cancers, the better equipped we are to develop improved diagnostics and targeted therapy for patients with this disease," adds Dr Campbell.

Abstract
The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1–positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.

Funding
This work was supported by the Kay Kendall Leukaemia Fund, the Leukemia and Lymphoma Research and the Wellcome Trust.

Participating Centres
1. Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK.
2. Hospital Universitario 12 de Octubre, Madrid, Spain.
3. Institute for Cancer Research, Sutton, London, UK.
4. Department of Human Genetics, VIB and University of Leuven, Leuven, Belgium.
5. Northern Institute for Cancer Research, University of Newcastle, Newcastle-upon-Tyne, UK.
6. Centro Ricerca Tettamanti, Hospital San Gerardo, Monza, Italy.
7. Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
8. Paediatric Malignancy Unit, Molecular Haematology & Cancer Biology Unit, Camelia Botnar
Laboratories, Great Ormond Street Hospital for Children and University College London (UCL) Institute of Child Health, London, UK.
9. Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA.
10. Addenbrooke's National Health Service (NHS) Foundation Trust, Cambridge, UK. Department of Haematology, University of Cambridge, Cambridge, UK.

Selected Websites
The Kay Kendall Leukaemia Fund was established in 1984 under the Will of James Sainsbury CBE. It awards grants for research on aspects of leukaemia and for relevant studies on related haematological malignancies. Grants are awarded for first class research on innovative proposals, particularly those close to the care of leukaemia patients or the prevention of leukaemia or related diseases. Project grants are awarded twice yearly, and Intermediate and Junior Fellowships of 3 – 4 years are awarded annually. The Fund also considers support for capital projects that will have direct benefit to leukaemia patient care. http://www.kklf.org.uk

Leukaemia & Lymphoma Research is the only UK charity dedicated to improving the lives of patients with all types of blood cancer, including leukaemia, lymphoma and myeloma. Its life-saving research is focused on finding causes, improving diagnosis and treatments, and running ground-breaking clinical trials for all blood cancer patients. Around 30,000 people of all ages, from children to adults, are diagnosed with blood cancers every year in the UK. http://www.beatingbloodcancers.org.uk

The Wellcome Trust Sanger Institute is one of the world's leading genome centres. Through its ability to conduct research at scale, it is able to engage in bold and long-term exploratory projects that are designed to influence and empower medical science globally. Institute research findings, generated through its own research programmes and through its leading role in international consortia, are being used to develop new diagnostics and treatments for human disease. http://www.sanger.ac.uk

The Institute of Cancer Research, London, is one of the world's most influential cancer research institutes.

Scientists and clinicians at The Institute of Cancer Research (ICR) are working every day to make a real impact on cancer patients' lives. Through its unique partnership with The Royal Marsden NHS Foundation Trust and 'bench-to-bedside' approach, the ICR is able to create and deliver results in a way that other institutions cannot. Together the two organisations are rated in the top four cancer centres globally.

The ICR has an outstanding record of achievement dating back more than 100 years. It provided the first convincing evidence that DNA damage is the basic cause of cancer, laying the foundation for the now universally accepted idea that cancer is a genetic disease. Today it leads the world at isolating cancer-related genes and discovering new targeted drugs for personalised cancer treatment.

As a college of the University of London, the ICR provides postgraduate higher education of international distinction. It has charitable status and relies on support from partner organisations, charities and the general public.

The ICR's mission is to make the discoveries that defeat cancer. For more information visit http://www.icr.ac.uk.

The Wellcome Trust is a global charitable foundation dedicated to achieving extraordinary improvements in human and animal health. We support the brightest minds in biomedical research and the medical humanities. Our breadth of support includes public engagement, education and the application of research to improve health. We are independent of both political and commercial interests. http://www.wellcome.ac.uk