Abstract
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by ineffective and disordered hematopoiesis with an increased risk of transformation to acute myeloid leukemia (AML). Interstitial deletion of 5q (del 5q) is the most frequent chromosomal abnormality seen in MDS. Two subgroups of MDS with del 5q have been described. The first, the so called “5q-syndrome” is defined by isolated del 5q and no excess blasts in the bone marrow, female predominance, typical dysmegakaryopoiesis, thrombocytosis and a favorable outcome. The second subgroup involves MDS with del 5q associated with an excess of marrow blasts and/or chromosomal abnormalities in addition to del 5q, that usually do not have the typical features of the 5q-syndrome and carry poorer prognosis. Haploinsufficiency for the ribosomal protein RPS14 gene has recently been identified as a compelling candidate gene for causing the 5q- syndrome, but the genes responsible for poor prognosis del 5q MDS remain to be determined. NPM-1, a highly conserved, ubiquitously expressed nucleolar phosphoprotein that belongs to the nucloeplasmin family of nuclear chaperones, maps to a region on chromosome 5q that is the target of deletions in both denovo and therapy-associated MDS in humans.. NPM-1 has been implicated in ribosome biogenesis and the transport of pre-ribosomal particles, maintenance of genomic stability by the control of cellular ploidy, in DNA repair and in the regulation of DNA transcription by controlling chromatin condensation/decondensation. NPM1−/− mice die at mid-gestion (E11.5) due to severe anemia. NPM-1 has been shown to be deleted or involved with chromosomal translocations in hematologic malignancies, and is one of the most frequently mutated genes in AML. NPM-1+/− mice develop a hematological syndrome very similar to that observed in MDS patients. The haploinsufficient NPM-1 model therefore provides an excellent platform to examine not only MDS but also to study AML progression. Studies on the molecular mechanisms underlying 5q- syndrome have been hampered by the lack of in vitro model cell lines. We have generated a factor dependent myeloid cell line from the bone marrow of NPM-1+/+ and NPM-1+/− mice using retroviral transduction of a truncated retinoic acid receptor alpha gene. The resulting cell lines are IL-3-dependent and can be induced to undergo neutrophil maturation by the addition of GM-CSF and/or all- trans retinoic acid (ATRA). We have shown that, like neutrophils derived from the bone marrow of NPM1+/− mice, the NPM-1+/− cell line-derived neutrophils display both defective neutrophil-specific gene expression and abnormal neutrophil function. We found in particular, that the expression of the myeloid master regulator C/EBPα but not that of the related myeloid-specific C/EBPε gene, was significantly decreased in NPM-1+/− cells as compared to their wild type counterparts (NPM-1+/+). While neutrophils derived from NPM1+/− cells appear morphologically normal, the expression of both neutrophil primary granule protein genes (defensins) and secondary granule protein genes (lactoferrin, MMP8 and MMP9) was significantly decreased. The relationship between NPM-1 and C/EBPα is currently being investigated. Additionally, the function of NPM-1+/− derived neutrophils was compromised as assessed by ROS production as well as by chemotaxis assays. The defective oxidative burst may be the result of decreased expression of a key component of the neutrophil NADPH oxidase complex i.e. gp91phox, which is critical for ROS production. We confirmed that these neutrophil-associated defects were also seen in primary neutrophils from the NPM-1+/− mice. We are currently examining the effect of knocking down RPS14 in our factor-dependent NPM-1+/− cells to determine if the two genes cooperate to render the cells factor independent. This cell line will help to delineate the functions of NPM-1 and provide a platform for examining the potential role of haploinsufficiency of NPM-1 in the propensity for 5q- MDS to progress to AML.
Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability