Abstract

Myeloproliferative disorders (MPDs) are characterized by abnormal proliferation of one or more myeloid lineages. Among MPDs, three distinct yet related disorders, PV, ET and IMF, harbor the same gain-of-function mutation JAK2V617F at a high frequency (~100%, 70%, 50%, respectively). Accumulating evidence suggest that other genetic lesions may cooperate with JAK2V617F to induce MPD. Analysis of microarray datasets from CD34+ cells isolated from a set of 9 PV patients as well as reanalysis of expression profiles of granulocytes from other sets of MPD patients reveals consistent up-regulation of the NFIB gene. NFIB is located ~9 Mb centromeric to the JAK2 locus (ch9p24) and encodes a nuclear transcription factor that binds to CAAT sequences. Examination of expression array databases (symatlas.gnf.org, www.oncomine.org) shows that NFIB is normally expressed at very low levels in human bone marrow and peripheral blood. Up-regulation of NFIB in MPD correlates with a recurrent amplification of the NFIB locus on chromosome 9p23. However, analysis of gene expression and chromosomal segment amplification in the same set of MPD specimens by cDNA microarray and SNP analysis showed that even in MPD patients without amplification of segments of chromosome 9, NFIB expression is upregulated. In JAK2V617F positive HEL cells quantitative genomic PCR showed that the JAK2 locus was amplifed 30 fold compared to control K562 cells and the NFIB locus amplified 10 fold. JAK2 expression in HEL cells measured by quantitative RT-PCR was elevated 5 fold above levels in K562 cells while NFIB expression was increased by a factor of 54. In another JAK2V617F containing leukemia cell line, UKE-1 there was a 6-fold amplification of JAK2 and 2-fold amplification of NFIB. However in UKE1 cells JAK2 expression was not elevated compared to K562 cells while NFIB expression was increased by a factor of 5. These data suggested that NFIB overexpression might be a common feature of JAK2V617-associated neoplasms, related in part to gene amplification, and that the regulation of JAK2 and NFIB expression was distinct. Supporting this idea, NFIB mRNA expression was not stimulated by lentivirus mediated overexpression of JAK2V617F in cytokine dependent TF1 cells nor repressed by blocking JAK2 kinase activity with a chemical inhibitor in HEL cells. To determine whether NFIB might play a role in malignant cell growth in MPD, a murine hematopoietic cell line transduced with the erythropoietin receptor, Ba/F3-EPOR, was transduced with a lentivirus harboring NFIB. Overexpression of NFIB in these cells promoted proliferation of these cells presence of erythropoietin (EPO) (1unit/ml). When grown in 0.1 or 0.01unit/ml of EPO, NFIB-expressing cells continued to show a 30–40% growth advantage with decreased cell death noted as well. To begin to understand the mechanisms of action of NFIB in hematopoitic cell growth, NFIB expressing and control cells were exposed to DNA damage in the form of ultraviolet radiation (UV). UV-induced transcription of the p21 cyclin dependent kinase inhibitor was repressed in Ba/F3-EPOR cells overexpressing NFIB compared to control cells. Furthermore engineered expression of NFIB in Ba/F3-EPOR cells consistently increased the number of cells entering the S-phase after addition of cytokine to starved cells. Mis-expresson of NFIB in MPD by chromosomal segment amplification or other mechanism may contribute to the development of MPD. Experiments are in progress to co-express NFIB and JAK2V61F in human and murine hematopoietic progenitor cells to ascertain the contribution of NFIB in vivo. NFIB and its target genes may represent additional therapeutic targets in MPD.

Disclosures: No relevant conflicts of interest to declare.

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