Generación de modelos de leucemia pediátrica con expresión de NUP98-KDM5A en células pluripotentes humanas

Abstract

Pediatric acute myeloid leukemia (AML) is a hematologic disorder characterized by uncontrolled proliferation of myeloid progenitor cells in bone marrow. It is caused by genomic alterations, most frequently chromosomal translocations, which occur during fetal development. Current treatments are not effective in all patients and often cause long-term side effects, so it is of utmost importance to develop new, more specific, and effective treatments. Much knowledge about the mechanisms and development of pediatric AML is lacking due in part to low availability of patient samples and limited fidelity of disease models. One of the major unknowns is the cell in which the mutation originates during fetal development. For these reasons, in this thesis we have proposed the use of human pluripotent stem cells (hPSC) as the basis for the generation of a model for the study of pediatric AML. hPSC are cells capable of differentiating into any cell type and can be used to study embryonic hematopoiesis. The chromosomal translocation t(11;12)(p15;p13) producing the NUP98-KDM5A (NK5A) fusion gene is found exclusively in pediatric AML patients. In this thesis, several models with NK5A expression in hPSC have been developed. Using the CRISPR/Cas9 system we generated hPSC with the chromosomal translocation t(11;12)(p15;p13), however in culture there was a negative selection of the edited cells and expansion, and characterization was not possible. Through constitutive expression of NK5A with lentiviral vectors, clonal hPSC lines with different levels of NK5A expression were generated, which allowed the study of the phenotype of chromosomal aberration accumulation due to errors in the mitosis process. We suggest that these aberrant mitoses are caused by NK5A interference with endogenous NUP98 functions, by NK5A interacting with the RAE1 protein, in addition to increased DNA damage during mitosis. In this model we further observed a block of mesodermal differentiation in hPSC expressing NK5A, without producing hematopoietic cells. Finally, a model of doxycycline-inducible expression of NK5A in hPSC was generated. This last model allowed the expression of NK5A at different time points of hematopoietic differentiation. Depending on the population in which the fusion protein is expressed, a blockage in different populations. This model could be useful to identify the cell of origin and the mechanisms of transformation of pediatric AML caused by the expression of NK5A.