Fusion genes feature in many cancers, a form of mutation in which two genes are joined together, such as through deletion of the DNA sequences that normally separate the two genes. The resulting mutant fusion gene sequence encodes a fusion protein that can have novel effects, or in which both portions remain functional, but are now produced in at inappropriate times and in inappropriate amounts. This change in cell biochemistry can be important in driving cancerous behavior, and this appears to be the case in a meaningful fraction of cancer types.
Today's research materials discuss a clever use of CRISPR DNA editing techniques. CRISPR is used to induce targeted breaks in nuclear DNA at specific points relative to two well known fusion genes, with the result that the gene, if present, is skipped over and removed by the DNA repair mechanisms responsible for reassembling the broken chromosome. This same strategy could well be applied to a range of fusion genes in cancer. The most promising part of this approach is that it is very specific to the cells that exhibit this fusion gene mutation. Thus gene therapy vectors can be used deliver the CRISPR tools into tissues quite generally, with no detrimental effect on normal cells.
Fusion genes are the abnormal result of an incorrect joining of DNA fragments that come from two different genes, an event that occurs by accident during the process of cell division. If the cell cannot benefit from this error, it will die and the fusion genes will be eliminated. But when the error results in a reproductive or survival advantage, the carrier cell will multiply and the fusion genes and the proteins they encode thus become an event triggering tumour formation. Many chromosomal rearrangements and the fusion genes they produce are at the origin of childhood sarcomas and leukaemias. Fusion genes are also found in among others prostate, breast, lung and brain tumours: in total, in up to 20% of all cancers.
Because they are only present in tumour cells, fusion genes attract a great deal of interest among the scientific community because they are highly specific therapeutic targets, and attacking them only affects the tumour and has no effect on healthy cells. And this is where the CRISPR technology comes into play. With this technology, researchers can target specific sequences of the genome and, as if using molecular scissors, cut and paste DNA fragments and thus modify the genome in a controlled way. In a new study, researchers worked with cell lines and mouse models of Ewing's sarcoma and chronic myeloid leukaemia, in which they managed to eliminate the tumour cells by cutting out the fusion genes causing the tumour.
"Our strategy was to make two cuts in introns, non-coding regions of a gene, located at both ends of the fusion gene. In that way, in trying to repair those breaks on its own, the cell will join the cut ends which will result in the complete elimination of the fusion gene located in the middle." As this gene is essential for the survival of the cell, this repair automatically causes the death of the tumour cell.
In the context of cancer gene therapy, it is clear that targeting a single gene is often insufficient to eliminate cancer cells - yet, many types of cancers are addicted to the presence of a single oncogenic event that can reprogram cells and initiate tumorigenesis. This is the case for the so-called fusion oncogenes (FOs), which are chimeric genes resulting from in-frame fusions of the coding sequences of two genes involved in a chromosomal rearrangement. While the nature of the FOs may be diverse, they are primarily classified as involving transcription factors or tyrosine kinases. Silencing of FO transcripts has been shown to inhibit tumor cell growth in vitro and in vivo, demonstrating FO addiction in many human cancers.
FOs are ideal therapeutic targets for the development of new directed cancer treatments, owing to their cancer-driving roles, their restriction to cancer cells and the reliance of tumors on them. Unfortunately, FOs are challenging to target directly with candidate drugs. The ability to precisely manipulate cancer cell genomes to correct or eliminate cancer-causing aberrations by highly-efficient CRISPR/Cas9 genome editing opens new possibilities to develop FO-targeted options to eliminate cancer cells. In the present study, we describe a simple and efficient genome editing strategy specifically targeting FOs in cancer cells. Our CRISPR/Cas9-based approach induces two targeted intronic double strand breaks in both genes involved in a FO that, importantly, produces a cancer cell-specific genomic deletion that is dependent on the presence of the FO, and has no effect on wild-type gene expression in non-cancer cells.