A “canonical” Npm1 mutation Knock-in Mouse Model Revealed Subtle but Definitive Myeloid Expansion with Poor HSC Niche Interaction

Abstract 762

Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein, which has nucleocytoplasmic shuttling activity. Somatic mutations in NPM1 gene result in cytoplasmic dislocation of NPM1 (NPM1c) and are frequently associated with acute myeloid leukemia (AML). The pathogenetic effects of mutated NPM1 protein have been explored by animal models including transgenic or “humanized” knock-in mouse models. Here, we demonstrate the first “canonical” mouse Npm1 mutant knock-in model. Different from the previously report of humanized NPM1 mutant knock-in model, we inserted TCTG after nucleotide c.857 of murine Npm1 coding sequence (c.854–857dupTCTG), a pattern identical to human NPM1 mutation, without any “humanized” sequence. This mutation caused a shift of peptide sequence from WQWRKSL* (amino acid 286–292) to LCLAVEEISLRKGFKQFEIFCLHFCNS* (amino acid 285 to 311), a pattern mildly different from the change in human NPM1 mutation, but is still predicted to generate a nuclear export signal. NPM1c+ genotype and protein accumulation in cytoplasm were confirmed with PCR and immunocytochemistry, respectively. We found that the homozygous NPM mutant (NPM^c+/c+) mice were embryonic lethal before E10.5 day, while hetrozygote (NPM^wt/c+) mice survived and were fertile, and born with Mendelian ratio. Most NPM^wt/c+ mice had normal hematologic parameters and remained disease-free, however, these mice developed a delayed-onset aberration on the distribution of granulocyte-monocye progenitors (GMP), monocytes, and B lymphocytes in blood, spleen, and bone marrow. Colony forming unit assay showed normal hematopoietic development of marrow hematopoietic stem cells (HSC), but poor cobblestone formation while HSCs contacted with stroma microenvironment in NPM^wt/c+ mice, suggesting the NPMc mutant may affect the ability of HSCs on contact signal expression. Three (12.5%) of 24 NPM^wt/c+ mice developed leukocytosis and splenomegaly mimicking myeloproliferative neoplasm of human. Microscopic examination showed panmyelosis of the bone marrow and existence of hematopoiesis in the spleen. In addition, the immune activities of NPM^wt/c+ mice's splenocytes and thymocytes with mitogen stimulation were decreased. In summary, our “canonical” NPM^wt/c+ mouse model demonstrated subtle but definitive phenotypes in hematopoietic cells and provided insight into the pathogenesis of NPM1 mutation in human acute myeloid leukemia. A second hit may be necessary for the development of AML in NPM^wt/c+ mice since no AML was detected in these mice till 20 months of age.