Generation of Novel Knockin Mouse Lines Harboring Leukemia-Associated Point Mutations at AML1/Runx1 Gene Locus.
Authors: Fukushime-Nakase Yoko , Nakao Mitsushige , Sugimoto Tohru , Okuda Tsukasa 
Published: April 2020

Abstract
Acute Myeloid Leukemia 1 (AML1; also called as Runt-related transcription factor 1: Runx1) encodes the DNA binding subunit of Core-Binding Factor (CBF) transcription factor complex which plays pivotal roles in several phases of hematopoietic regulation, including initial development of definitive hematopoiesis. AML1 is known as a frequent target of leukemia-associated chromosomal translocations, where chimeric AML1 genes with strong dominant-negative effects against normal CBF function are produced. Recently, point-mutations of the AML1 gene locus have also been reported to associate with sporadic cases of acute myelogenous leukemia or myelodysplastic syndromes, and pedigrees of Familial Platelet Disorder with Predisposition to Acute Myelogenous Leukemia (FPD/AML). Hot spots of the mutations are confined to within the Runt domain, which is the signature-motif of the AML1 molecules localized near the N-terminus and is responsible for both sequence-specific DNA binding and hetero-dimerization with CBFβ subunit. Biochemical examinations have so far demonstrated that these mutants lose their function as a transcription factor and that most of them also have dominant-negative effects at some extent. Indeed, three-dimensional analyses of the Runt domain structure revealed that frequent sites of the mutation, such as, R80, R139, R174, or R177, were mapped at residues important for the DNA-contact of the molecule while mutations of these residues left AML1’s ability to associate with CBFβ being un-affected, thus providing a molecular basis of their biochemical characteristics. In contrast, however, biological properties of the mutations are mostly left to be elucidated. In order to define the biologic consequences of the point-mutations at Runx1 gene locus within the context of entire mouse, we introduced each of the point-mutations, including R139Q, R174Q, or R177Q, into mouse germline by means of a gene-knockin approach. Heterozygous mice for each of the mutations were born healthy and grew up fertile as far as they were kept under specific pathogen-free conditions similarly to their wild-type siblings or to control mouse lines harboring a knockin allele with wild-type cDNA of Runx1. These observations indicate that the dominant-negative effects of the mutations, if any present, are not that strong as is the case for the AML1-MTG8 (ETO) chimeric gene which leads to mid-gestational death when transmitted to one allele of mouse germline. In addition, in contrast to the knockin of the wild-type cDNA which results in minimal phenotype in the homozygous progeny, homozygous mice for the R174Q allele were embryonic-lethal just like the simple disruption of this gene, clearly indicating that this leukemia-associated one amino acid substitution, arginine-to-glutamine at the 174th residue, abolished its biologic function completely. We are generating homozygous progeny by breeding for R139Q and R177Q alleles to further define their biologic consequences. In addition, examinations are currently undertaken to analyze if the heterozygous mice manifest any phenotype resulted from haplo-insufficiency along with possible dominant-negative effects by these mutations. These knockin mice should contribute to better understanding of the molecular mechanisms of AML1/Runx1 activity in normal and leukemic hematopoiesis.