Contributions
Abstract: PB1907
Type: Publication Only
Background
CML is a disorder driven by hematopoietic stem cells that acquire the t(9;22)(q34;q11) traslocation resulting in the expression of the BCR-ABL oncogenic fusion protein, which increase cell survival, proliferation and induces an aberrant myeloid commitment. CML treatments are predominantly based on the use of tyrosine kinase inhibitors (TKI). However, CML eradication is still hindered by the emergence of TKI resistant cells. A definitive and effective therapeutic strategy is therefore still needed for these patients. Few studies have used genome edition CRISPR-based techniques to eliminate fusion oncogenes generated by chromosome translocations. BCR-ABL fusion is an ideal therapeutic target to be eliminated at the genomic level by using these new genome edition tools. We demonstrate that CRISPR/Cas9 system efficiently disrupts, at a genomic level, the BCR-ABLp210 gene fusion in an in vitro model of human BCR-ABL, avoiding its oncological effects.
Aims
To evaluate the ability of CRISPR/Cas9 technology to induce frameshift mutations on the BCR/ABL sequence avoiding its expression in K562 cell line.
Methods
The K562 cell line derived from a chronic myeloid leukemia (CML) patient and expressing B3A2 BCR-ABL fusion gene, is known to be particularly resistant to apoptotic cell death. Through CRISPR/Cas9-mediated genomic edition, we set to edit the coding sequence of BCR-ABL in K562 cells, inducing indels that modify the oncogene ORF, and therefore, the protein expression. We designed 3 sgRNAs against tyrosine kinase domain sequence of ABL1 and were introduced in a plasmid containing the Cas9 nuclease coding sequence. K562 cells were electroporated with the mix of sgRNAs. The genetic edition on ABL1 tyrosine kinase domain sequence was checked by PCR and Sanger sequencing. The BCR/ABL expression was analyzed by qPCR. To assess the edited cells viability and apoptosis, flow cytometry, measuring Annexin V staining and DNA content was used.
Results
We achieved to edit the coding sequence of BCR-ABL in K562 cells by CRISPR/Cas9 system, inducing mutations that modified the oncogene ORF. We found a 100 bp recurrent induced deletion in the sequence corresponding to the TK domain of ABL1. This deletion leads to a premature stop codon, which prevents the correct translation of the protein. As a result of this deletion, the edited K562 cells showed a decreased expression of ABL1, higher levels of apoptosis and an altered expression of downstream ABL1 targets, proof of lack of expression of the BCR-ABL oncoprotein.
Conclusion
We demonstrate the ability of CRISPR/Cas9 system to disrupt at genomic level, the coding sequence of BCR/ABL fusion oncogene resulting from translocation t (9;22) in established cell lines derived from patients of CML, avoiding its oncological effects. The use of CRISPR-Cas9 technology could be a promising new therapeutic option for CML patients who have developed TKI resistances. Bone marrow CML leukemic stem cells could be ex vivo edited by CRISPR-Cas9 technology and specifically selected to transplantation treatment.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): BCR-ABL, Chronic myeloid leukemia, Gene therapy
Abstract: PB1907
Type: Publication Only
Background
CML is a disorder driven by hematopoietic stem cells that acquire the t(9;22)(q34;q11) traslocation resulting in the expression of the BCR-ABL oncogenic fusion protein, which increase cell survival, proliferation and induces an aberrant myeloid commitment. CML treatments are predominantly based on the use of tyrosine kinase inhibitors (TKI). However, CML eradication is still hindered by the emergence of TKI resistant cells. A definitive and effective therapeutic strategy is therefore still needed for these patients. Few studies have used genome edition CRISPR-based techniques to eliminate fusion oncogenes generated by chromosome translocations. BCR-ABL fusion is an ideal therapeutic target to be eliminated at the genomic level by using these new genome edition tools. We demonstrate that CRISPR/Cas9 system efficiently disrupts, at a genomic level, the BCR-ABLp210 gene fusion in an in vitro model of human BCR-ABL, avoiding its oncological effects.
Aims
To evaluate the ability of CRISPR/Cas9 technology to induce frameshift mutations on the BCR/ABL sequence avoiding its expression in K562 cell line.
Methods
The K562 cell line derived from a chronic myeloid leukemia (CML) patient and expressing B3A2 BCR-ABL fusion gene, is known to be particularly resistant to apoptotic cell death. Through CRISPR/Cas9-mediated genomic edition, we set to edit the coding sequence of BCR-ABL in K562 cells, inducing indels that modify the oncogene ORF, and therefore, the protein expression. We designed 3 sgRNAs against tyrosine kinase domain sequence of ABL1 and were introduced in a plasmid containing the Cas9 nuclease coding sequence. K562 cells were electroporated with the mix of sgRNAs. The genetic edition on ABL1 tyrosine kinase domain sequence was checked by PCR and Sanger sequencing. The BCR/ABL expression was analyzed by qPCR. To assess the edited cells viability and apoptosis, flow cytometry, measuring Annexin V staining and DNA content was used.
Results
We achieved to edit the coding sequence of BCR-ABL in K562 cells by CRISPR/Cas9 system, inducing mutations that modified the oncogene ORF. We found a 100 bp recurrent induced deletion in the sequence corresponding to the TK domain of ABL1. This deletion leads to a premature stop codon, which prevents the correct translation of the protein. As a result of this deletion, the edited K562 cells showed a decreased expression of ABL1, higher levels of apoptosis and an altered expression of downstream ABL1 targets, proof of lack of expression of the BCR-ABL oncoprotein.
Conclusion
We demonstrate the ability of CRISPR/Cas9 system to disrupt at genomic level, the coding sequence of BCR/ABL fusion oncogene resulting from translocation t (9;22) in established cell lines derived from patients of CML, avoiding its oncological effects. The use of CRISPR-Cas9 technology could be a promising new therapeutic option for CML patients who have developed TKI resistances. Bone marrow CML leukemic stem cells could be ex vivo edited by CRISPR-Cas9 technology and specifically selected to transplantation treatment.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): BCR-ABL, Chronic myeloid leukemia, Gene therapy