Secondary Somatic Mutations in ETV6-RUNX1 Acute Lymphoblastic Leukemia Pathogenesis

ETV6-RUNX1, also known as TEL-AML1, is the most frequent chromosomal translocation in childhood Acute Lymphoblastic Leukemia (ALL) with an incidence of approximately 25%. This translocation occurs in hematopoietic stem cells and leads to the establishment of pre-leukemic progenitor B-cell clones that persist in the bone marrow for several years. It has been shown that the translocation itself is insufficient to generate ALL and that secondary somatic mutations are necessary to activate the leukemic phenotype. Recent genome analyses of patients exhibiting this type of leukemia have identified a host of mutated genes that may be working cooperatively to induce this hematologic malignancy. Previously, the cooperative action of these secondary mutations has been difficult to characterize due to a lack of ex vivo models that allow for targeted mutagenesis of multiple genes after the initial translocation. However, with the identification and development of CRISPR-Cas9 as a genome-editing tool, this is now a feasible undertaking and the crux of this project. Here we have generated a lentiviral CRISPR library composed of 59 unique guide RNAs (gRNA) to target 19 genes of interest that represent likely secondary hits responsible for activating the ETV6-RUNX1+ pre-leukemic clones. We will use this library to infect an ex vivo pro B-cell line and select for transformed mutants that have insertions/deletions at our loci of interest.