NPM1 Haploinsufficiency Results in Increased Numbers of Hematopoietic Stem Cells and Progenitor Cells.

Abstract 738

Myelodysplastic syndromes (MDS) are characterized by defective hematopoietic stem/progenitor cell maturation, resulting in ineffective hematopoiesis. This group of disorders is characterized by cytogenetic abnormalities and approximately 25% of cases progress to acute myeloid leukemia (AML). NPM1 is frequently mutated in AML and translocations involving NPM1 occur in a number of hematopoietic malignancies including MDS. NPM1 heterozygous mice (NPM1 +/−) have been shown to have a MDS-like phenotype. Taken together, these data suggest an important role for NPM1 in the function of hematopoietic stem cells (HSC) and/or committed progenitors. In order to evaluate NPM1 function in early hematopoiesis, we have evaluated NPM1 expression in both the mouse and human hematopoietic systems. Using quantitative RT-PCR, we show that NPM1 expression levels are 2-3-fold higher in normal CD34+ bone marrow progenitor cells compared to total bone marrow in humans. Furthermore, NPM1 expression levels are decreased by ∼50% in 9/37 MDS CD34+ cells when compared to normal controls. Of interest, NPM1 expression is reduced primarily in patients with poor or intermediate prognosis. Consistent with a functional role for NPM1 in HSC, NPM1 +/− mice (developed by gene trapping and obtained from the MMRRC at UC-Davis) contained significantly increased numbers of HSC (Lin-cKit+Sca+CD34-CD150+) within the Lin-cKit+Sca+ population compared to those from the littermate controls (52 ± 2.6% vs,74 ± 12%, p < 0.01). Consistent with prior reports, NPM +/− mice contained significantly fewer mature erythrocytes (Ter119+CD71lo) in the bone marrow compared to WT controls (6.5 ± 1.8% vs 10 ± 0.5% p < 0.01). In order to study NPM +/− HSC function, we tested the ability of these HSCs to form colonies in methylcellulose. NPM1 +/− HSCs formed increased numbers of both CFU-GM and CFU-GEMM colonies and decreased numbers of CFU-E colonies compared to WT HSC. Flow cytometric analysis of pooled day 14 colonies from individual mice revealed a >2 fold increase in cKit+ progenitor cells from NPM1 +/− colonies (2.0 ± 1.0% vs. 0.2 ± 0.2%, p = 0.02), suggesting that the differentiation potential of NPM+/− HSCs is impaired. To characterize HSC function in vivo, equal numbers of double-sorted HSCs from WT and NPM1 +/− mice were transplanted in triplicate into lethally irradiated C57B6 (CD45.2) recipients. Analysis of peripheral blood donor chimerism (CD45.1+CD45.2+) 21 days post-transplantation showed that NPM1 +/− HSC-transplanted recipients exhibited markedly lower granulocyte chimerism than WT HSC recipients (5.5 fold reduction, 2 ± 2% vs. 11 ± 5%, p < 0.01). This finding suggests that although NPM1 +/− mice have increased numbers of HSC, these HSC exhibit either altered myeloid fate decisions or decreased bone marrow homing capacity. We are currently investigating long-term engraftment potential to further elucidate the function of HSC in NPM1 +/− mice in vivo. In aggregate, these data demonstrate a functional role for NPM1 in early myeloid differentiation and strongly suggest that NPM effects may be exerted as early as at the level of the HSC.