Contributions
Abstract: PB1666
Type: Publication Only
Background
Acute Myeloid Leukemia (AML) is a complex heterogeneous disease produced by clonal expansion of undifferentiated myeloid precursors, resulting in impaired hematopoiesis and bone marrow failure. Whole-genome sequencing and advances in next-generation sequencing have increased our knowledge of the molecular heterogeneity of AML, and have these insights been translated into improved disease classification. The IDH2R172 mutations have been proposed to constitute an independent subgroup into this classification, different from the rest of mutations in this gene. For understanding the role of these mutations in the leukemia progress and for the development of specific therapies is necessary to create new AML models. The CRISPR/Cas9 system has allowed a great advance in genome engineering due to its easy design and use in different model organisms. With this system is possible to develop AML models with single mutations or introduce them progressively to characterize the acquired driver mutations detected in patients.
Aims
We want sought to develop in vitro and in vivo models of AML using the CRISPR/Cas9 system to introduce IDH2 mutations. Objectives: Development of an easy and fast method to produce guide sequences; Introduce IDH2R172K mutation in leukemia cell lines; Introduce IDH2R172K and IDH2R140Q mutations in the nematode Caenorhabditis elegans. Due to the homology between the gene in model organism and humans this is feasible (the R172 and R140 amino acids are conserved).
Methods
To do the in vitro model, we have developed a fusion PCR system for generating constructs with the sgRNA sequences and the pU6 promoter, and optionally, adding the GFP reporter. These constructs were transfected in the broadly used HEK293 cells, and these constructs are able to cut the target genes. After a series of optimizations we have used these constructs in leukemia cell lines that express Cas9 to produce our model. Furthermore, we used ribonucleoprotein (RNP) complex in cells without Cas9 expression to compare the efficiency of the different procedures.
We have also introduced the IDH2R172K and IDH2R140Q mutations in C. elegans. To do so we prepared a mix with RNP and an oligonucleotide, as an editing template carrying the desired mutation, were injected along with a gRNA against a gene which produces easy-to-see phenotypes, as a marker of edition. With a PCR with specific primers we isolated homozygote worms with each mutation.
Results
With the optimization in HEK293 cells we selected the best sgRNA combination to modify IDH2 gene (20% of efficiency). Interestingly, we obtained the same efficiency in NB4 cell line which contained an insertion that induces constitutive expression of Cas9. The RNP complex was used in the NB4 cell line with 8% of efficiency. In future experiments we will introduce IDH2 R172K using these two methodologies. On the other hand, the marker phenotype let us isolate those worms that were efficiently edited, among which we were able to isolate worms carrying IDH2 R172K or IDH2R140Q mutations.
Conclusion
The CRISPR/Cas9 technology represents a high biomedical revolution that has already been used in multiple fields. Specifically, this system can be used to elucidate the functional and cooperating effect of the mutations detected in patients with AML.
We have used this technique to develop two AML models in which we can introduce mutations detected in patients in a sequential way, study the effect in the leukemic phenotype and could be a promising platform to test new drugs against this disease.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Genomics, Molecular
Abstract: PB1666
Type: Publication Only
Background
Acute Myeloid Leukemia (AML) is a complex heterogeneous disease produced by clonal expansion of undifferentiated myeloid precursors, resulting in impaired hematopoiesis and bone marrow failure. Whole-genome sequencing and advances in next-generation sequencing have increased our knowledge of the molecular heterogeneity of AML, and have these insights been translated into improved disease classification. The IDH2R172 mutations have been proposed to constitute an independent subgroup into this classification, different from the rest of mutations in this gene. For understanding the role of these mutations in the leukemia progress and for the development of specific therapies is necessary to create new AML models. The CRISPR/Cas9 system has allowed a great advance in genome engineering due to its easy design and use in different model organisms. With this system is possible to develop AML models with single mutations or introduce them progressively to characterize the acquired driver mutations detected in patients.
Aims
We want sought to develop in vitro and in vivo models of AML using the CRISPR/Cas9 system to introduce IDH2 mutations. Objectives: Development of an easy and fast method to produce guide sequences; Introduce IDH2R172K mutation in leukemia cell lines; Introduce IDH2R172K and IDH2R140Q mutations in the nematode Caenorhabditis elegans. Due to the homology between the gene in model organism and humans this is feasible (the R172 and R140 amino acids are conserved).
Methods
To do the in vitro model, we have developed a fusion PCR system for generating constructs with the sgRNA sequences and the pU6 promoter, and optionally, adding the GFP reporter. These constructs were transfected in the broadly used HEK293 cells, and these constructs are able to cut the target genes. After a series of optimizations we have used these constructs in leukemia cell lines that express Cas9 to produce our model. Furthermore, we used ribonucleoprotein (RNP) complex in cells without Cas9 expression to compare the efficiency of the different procedures.
We have also introduced the IDH2R172K and IDH2R140Q mutations in C. elegans. To do so we prepared a mix with RNP and an oligonucleotide, as an editing template carrying the desired mutation, were injected along with a gRNA against a gene which produces easy-to-see phenotypes, as a marker of edition. With a PCR with specific primers we isolated homozygote worms with each mutation.
Results
With the optimization in HEK293 cells we selected the best sgRNA combination to modify IDH2 gene (20% of efficiency). Interestingly, we obtained the same efficiency in NB4 cell line which contained an insertion that induces constitutive expression of Cas9. The RNP complex was used in the NB4 cell line with 8% of efficiency. In future experiments we will introduce IDH2 R172K using these two methodologies. On the other hand, the marker phenotype let us isolate those worms that were efficiently edited, among which we were able to isolate worms carrying IDH2 R172K or IDH2R140Q mutations.
Conclusion
The CRISPR/Cas9 technology represents a high biomedical revolution that has already been used in multiple fields. Specifically, this system can be used to elucidate the functional and cooperating effect of the mutations detected in patients with AML.
We have used this technique to develop two AML models in which we can introduce mutations detected in patients in a sequential way, study the effect in the leukemic phenotype and could be a promising platform to test new drugs against this disease.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Genomics, Molecular