A model to study the function of NPM-MLF1 in myelodysplasia.
Doctoral thesis, UCL (University College London).
Myelodysplastic syndromes (MDS) are bone marrow disorders characterized by ineffective haematopoiesis and peripheral cytopenias with frequent evolution to acute myeloid leukaemia (AML). Apoptosis is significantly deregulated in early MDS whereas advanced MDS is characterized by deregulation of the DNA damage response. As MDS proceeds to AML, the ratio of apoptosis to proliferation decreases, resulting in clonal outgrowth of abnormal cells. The t(3;5)(q25;q34) translocation, creating the NPM-MLF1 fusion, has been found as a sole cytogenetic abnormality in MDS/AML patients, although the sample numbers are limited. In these patients, inactivation of one functional NPM allele resulting from the t(3;5)(q25;q34) translocation may contribute to disease development. This concept is supported by the observations that Npm+/- mice exhibit disease phenotypes resembling human MDS (Grisendi, Bernardi et al. 2005). The t(3;5)(q25;q34) is recurrent, with poor prognosis, but the precise mechanism through which NPM-MLF1 induces malignant transformation remains unknown. We aimed to model this disease in vitro and in vivo by expressing NPM-MFL1 in mouse bone marrow hematopoietic stem/progenitor cells (HSPCs) and analyzing any changes in HSPC activity and response to DNA damage. The effect of NPM-MLF1 in vitro and in vivo was also examined in the Npm hemizygous context. Our data showed that NPM-MLF1 did not impair haematopoiesis in vitro and in vivo in wild-type HSPCs. FLT3/ITD is frequently associated with NPM mutations in AML patients. However, NPM-MLF1 did not show any collaboration with FLT3/ITD in our system. To recapitulate NPM hemizygosity in t(3;5)-MDS patients, we have expressed NPM-MLF1 in HSPCs derived from Npm+/- mice. A transient increase in the colony forming ability of the NPM-MLF1-expressing Npm+/- HSPCs was seen. Consistently, liquid culture experiments showed that NPM-MLF1-expressing Npm+/- HSPCs, but not NPM-MLF1-expressing Npm+/+ HSPCs, exhibited prolonged colony forming ability. This observation was accompanied by higher expression of the Meis1 gene in the NPM-MLF1-expressing Npm+/- HSPCs. Strikingly, expression of myeloid HPC-related genes, Hoxa9/10, which were undetectable in the NPM-MLF1-expressing Npm+/+ HSPCs, was maintained in the NPM-MLF1-expressing Npm+/- HSPCs. However, expansion of these cells was not seen in vivo. Such an effect was not seen in vivo, probably due to low reconstitution efficiency. In addition to prolonged myeloid HPC activity, NPMMLF1 was also found to alter radiation-induced H2AX phosphorylation, which is known to play a role in the DNA damage repair. Taken together, this study suggests that the ability of NPM-MLF1 to maintain HPC activity and interfere with DNA damage responses may favour the accumulation and outgrowth of the aberrant HSPCs. Secondary genetic abnormalities are most likely required to collaborate with NPM-MLF1 to transform the HSPCs, which may ultimately lead to overt AML. Thus, genome-wide analysis of MDS/AML patient samples harbouring NPM-MLF1 may uncover the mutations and/or genetic alterations crucial for disease development.
|Title:||A model to study the function of NPM-MLF1 in myelodysplasia|
|Additional information:||Permission for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Child Health|
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