In a major finding that could lead to development of new therapeutic targets for treatment of acute myeloid leukemia and Ewing's sarcoma, researchers at Centro Nacional de Investigaciones Cardiovasculares (CNIC) and the Centro Nacional de Investigaciones Oncológicas (CNIO) have successfully managed to reproduce the chromosomal translocations in human cells that lead to the development of the two types of cancer, a new study published in the journal Nature Communications reveals.
The study was led by Juan Carlos Ramírez and Raúl Torres, of the CNIC Viral Vectors Unit, and Sandra Rodríguez-Perales, of the Molecular Cytogenetics group at the CNIO, directed by Juan Cruz Cigudosa. The research team used innovative technology to demonstrate that it is possible to reproduce chromosomal modifications in human cells that are identical to the modifications observed in leukemias and other types of cancer. The new technology, which uses molecular tools to manipulate the genome, has two main advantages.
First, it allows researchers to generate previously unavailable cell models for the study of tumor biology. And second, future development will allow the study of new therapeutic targets and therapies. The development of tumors is caused by multiple changes in cell physiology and particularly in the genome. In leukemias and a group of cancers called sarcomas, large fragments of DNA are exchanged between different chromosomes, a phenomenon known as chromosomal translocation. These translocations are necessary steps in both the generation and the progression of many neoplastic processes. "Investigation of these kinds of cancers has been impeded until now by the lack of suitable cell and animal models," explains CNIC researcher Juan Carlos Ramírez, who adds that the difficulty in generating these chromosomal translocations in the laboratory has meant that researchers have lacked cell models with which to investigate the effect of a key marker of the disease: the presence of specific chromosomal translocations.
Using RGEN technology (for RNA-guided endonuclease), also known as CRISPR/Cas9, the team of CNIC and CNIO investigators demonstrated that it is possible to engineer cells to undergo these specific chromosomal translocations. Using human hematopoietic and mesenchymal stem cells, the researchers reproduced chromosomal translocations identical to those observed in patients with acute myeloid leukemia (a cancer of the blood and bone marrow) or Ewing's sarcoma (a type of bone cancer that affects children and adolescents). According to CNIO researcher Sandra Rodríguez-Perales "With this advance it is now possible to generate cell models with the same alterations detected in tumor cells from patients, and this will permit study of how these changes lead to the development of the tumor. Specifically, it will now be possible to experimentally recapitulate the processes subsequent to chromosomal translocation through which the cell transforms into a tumor cell".
The powerful RGEN tool was developed at the beginning of 2013 for gene manipulation in eukaryotic cells, including human cells. The technology is based on a small RNA (subgenomic RNA, or sgRNA) designed to complement and specifically bind a 20-nucleotide region of DNA. The bound sgRNA acts as a beacon for the enzyme Cas9, which introduces a cut at the edge of the targeted DNA. The system is very specific and effective, and allows investigators to cut the DNA double helix at any site desired. Torres, Rodríguez-Perales and Ramírez have shown that the technique can be applied in primary human cells to mark and introduce cuts in the chromosome regions that become translocated in some tumors. "As the cell's own DNA repair machinery attempts to repair the DNA cuts, material can be translocated between the two chromosomes, in many cases in a reciprocal manner," explained CNIC researcher Raúl Torres. The authors of the study are confident that this technology will help to clarify how and why these chromosomal translocations occur spontaneously, and that these advances will in time lead to new therapeutic approaches to the treatment of these cancers.