Contributions
Abstract: PB1599
Type: Publication Only
Background
Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood, around 75% of ALL occurs in children under 6 years. In spite of survival rate has increased over the last years, about 20% of patients continue to relapse. These cases are associated with a worse outcome, even in those genetic subtypes considered to have a good prognosis, such as the t (12.21) that fuses the ETV6/RUNX1 (TEL/AML1) genes.
ETV6/RUNX1 gene fusion as even initiating in the acquisition of leukemic phenotype has been demonstrated. However, its function in the pathology development and maintenance is still unclear. Several studies suggest that ETV6/RUNX1 promotes tumor survival through up- regulation of anti-apoptotic members of BCL-2 family protein.
In this study, we evaluated the ability of CRISPR/Cas9 genome editing system to eliminate the expression of ETV6/RUNX1, and therefore its effect on the expression of anti-apoptotic proteins.
Aims
To abrogate ETV6/RUNX1 expression by CRISPR/Cas9 technology in the REH cell line.
Methods
The REH cell line (DSMZ), from LAL and expressing the ETV6/RUNX1 fusion protein, was used. Based on the methodology of CRISPR/Cas9, sgRNAs directed towards the end of exon 5 of ETV6, were designed with the intention of producing indels that modify the ORF of the oncogene, and, therefore, the expression of the protein. These sgRNAs were cloned into a vector containing the Cas9 nuclease coding sequence and then were electroporated into the REH cells. The edition of the ETV6/RUNX1 coding sequence was checked by PCR and Sanger sequencing. Single-edited cell derived cell lines were established to check ETV6/RUNX1 mRNA expression and downstream targets expression such as BCL-2 by qPCR and Western Blot. Two single-edited cell clones with wt ETV6/RUNX1 sequence were used as controls.
Results
The ETV6/RUNX1 coding sequence in the REH cell line through the CRISPR/Cas9 system was successfully edited. The edition resulted in several frameshift mutations as insertions and deletions that modified the ORF of the oncogene, as well as a deletion between both sgRNAs. Two single-edited cell clones carrying a deletion and another with an insertion were selected. In silico analysis of their sequence reveal the generation of KO alleles in all of them. All single-edited clones showed a reduction up to 99,9% of mRNA level compared with REH parental cell line, showing an efficient abrogation of ETV6/RUNX1 expression.
To evaluate the effects of lack of oncoprotein expression in REH cell, we assessed the expression level of BCL-2 family member proteins. A decreased expression of BCL-2 and BCL-XL in all ETV6/RUNX1 KO clones compared with parental and control cell lines we observed, which demonstrates the reduction of the oncogenic potential of these cells.
Conclusion
For the first time, the CRISPR/Cas9 system to edit the sequence of ETV6/RUNX1 oncogene was using, inducing frameshift mutations, and leading to the eradication of the fusion product. The lack of ETV6/RUNX1 expression prevents pro-tumoral effects, such apoptosis suppression through BCL-2 family proteins overexpression. The use of CRISPR/Cas9 technology could be a promising new therapeutic option for ALL patients. Bone marrow leukemic stem cells could be edited by CRISPR-Cas9 technology to truncate ETV6/RUNX1 fusions, and truncated cells specifically selected to transplantation treatment.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Acute lymphoblastic leukemia, Gene therapy, TEL-AML1, Translocation
Abstract: PB1599
Type: Publication Only
Background
Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood, around 75% of ALL occurs in children under 6 years. In spite of survival rate has increased over the last years, about 20% of patients continue to relapse. These cases are associated with a worse outcome, even in those genetic subtypes considered to have a good prognosis, such as the t (12.21) that fuses the ETV6/RUNX1 (TEL/AML1) genes.
ETV6/RUNX1 gene fusion as even initiating in the acquisition of leukemic phenotype has been demonstrated. However, its function in the pathology development and maintenance is still unclear. Several studies suggest that ETV6/RUNX1 promotes tumor survival through up- regulation of anti-apoptotic members of BCL-2 family protein.
In this study, we evaluated the ability of CRISPR/Cas9 genome editing system to eliminate the expression of ETV6/RUNX1, and therefore its effect on the expression of anti-apoptotic proteins.
Aims
To abrogate ETV6/RUNX1 expression by CRISPR/Cas9 technology in the REH cell line.
Methods
The REH cell line (DSMZ), from LAL and expressing the ETV6/RUNX1 fusion protein, was used. Based on the methodology of CRISPR/Cas9, sgRNAs directed towards the end of exon 5 of ETV6, were designed with the intention of producing indels that modify the ORF of the oncogene, and, therefore, the expression of the protein. These sgRNAs were cloned into a vector containing the Cas9 nuclease coding sequence and then were electroporated into the REH cells. The edition of the ETV6/RUNX1 coding sequence was checked by PCR and Sanger sequencing. Single-edited cell derived cell lines were established to check ETV6/RUNX1 mRNA expression and downstream targets expression such as BCL-2 by qPCR and Western Blot. Two single-edited cell clones with wt ETV6/RUNX1 sequence were used as controls.
Results
The ETV6/RUNX1 coding sequence in the REH cell line through the CRISPR/Cas9 system was successfully edited. The edition resulted in several frameshift mutations as insertions and deletions that modified the ORF of the oncogene, as well as a deletion between both sgRNAs. Two single-edited cell clones carrying a deletion and another with an insertion were selected. In silico analysis of their sequence reveal the generation of KO alleles in all of them. All single-edited clones showed a reduction up to 99,9% of mRNA level compared with REH parental cell line, showing an efficient abrogation of ETV6/RUNX1 expression.
To evaluate the effects of lack of oncoprotein expression in REH cell, we assessed the expression level of BCL-2 family member proteins. A decreased expression of BCL-2 and BCL-XL in all ETV6/RUNX1 KO clones compared with parental and control cell lines we observed, which demonstrates the reduction of the oncogenic potential of these cells.
Conclusion
For the first time, the CRISPR/Cas9 system to edit the sequence of ETV6/RUNX1 oncogene was using, inducing frameshift mutations, and leading to the eradication of the fusion product. The lack of ETV6/RUNX1 expression prevents pro-tumoral effects, such apoptosis suppression through BCL-2 family proteins overexpression. The use of CRISPR/Cas9 technology could be a promising new therapeutic option for ALL patients. Bone marrow leukemic stem cells could be edited by CRISPR-Cas9 technology to truncate ETV6/RUNX1 fusions, and truncated cells specifically selected to transplantation treatment.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Acute lymphoblastic leukemia, Gene therapy, TEL-AML1, Translocation