A Subnuclear Targeting Defective Mutant of Runx1 Uncouples the Roles of Runx1 In Embryonic Development and Adult Definitive Hematopoiesis

Abstract 1387

As shown by gene ablation or replacement studies, Runx1, a master regulator of hematopoiesis, is critical for the emergence of the hematopoietic stem cell (HSC). Runx1 is also the most common target for point mutations and chromosomal translocations in human leukemia. Localization of Runx1 to distinct foci within the nucleus through a unique subnuclear targeting signal is linked to physiological roles of the protein. Many leukemia-related chromosomal translocations (e.g., 8;21 translocation that encodes for AML1-ETO protein) disrupt the Runx1 C-terminus and modify its biological activity as well as alter subnuclear localization. We hypothesize that altered subnuclear routing of leukemic proteins plays a key role and provides a common mechanism in human leukemias. We have previously developed a knock-in mouse model with a Runx1 C-terminal truncation (Runx1 Q307X), which similar to a complete ablation of Runx1 or a genetic knock-in of the leukemic AML1-ETO, causes mid gestation lethality. The Runx1 Q307X mutation also abolishes subnuclear targeting and alters Runx1-dependent myeloid gene expression. To further dissect the specific functional contributions of altered Runx1 subnuclear targeting, we developed a knock-in mouse model in which endogenous Runx1 is replaced with a triple point mutation (Runx1 HTY350-352AAA) that modifies the subnuclear targeting. In contrast to Runx1 Q307X or AML1-ETO knock-in mice, animals homozygous for Runx1 HTY350-352AAA bypass embryonic lethality. However, expression of key Runx1 target genes that regulate hematopoiesis is altered. In addition, bone marrow from Runx1 HTY350-352AAA animals shows increased proliferation ex vivo, resulting in an increase of myeloid and a decrease of B-lymphoid lineage cells. Importantly, red blood cell development, previously thought unrelated to Runx1 expression, is also modified in the Runx1 HTY350-352AAA homozygous animals, as shown by decreased mean corpuscle volume and selective effects on globin gene expression. Together, these findings show that altered subnuclear targeting uncouples the critical role of Runx1 during hematopoietic stem cell emergence and differentiation into various lineages in the adult.