Role of NFIB in normal hematopoiesis and MyeloproliferativeNeoplasms

Introduction: The three-canonical BCR-ABL negative myeloproliferative neoplasms (MPN), Polycythemia Vera (PV), Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF) are chronic diseases, which share the risk of disease evolution to an acute leukemia. In MPN, the most representative molecular lesion is a substitution of a valine for a phenylalanine (JAK2V617F) in the auto-inhibitory domain of JAK2, resulting in the constitutive expression of the gene.This point mutation has an incidence of about 96% in PV, 65% in PMF and 55% in ET. Microsatellite studies on chromosome 9 show the presence of a uniparental acquired disomy (UPD) of the short arm (9p), where JAK2 is located, as a common defect in MPN (Kralovics et al., ExpHematol. 2002). In a cohort study, all the samples with the 9pUPD were found positive for the JAK2V617F mutation (Klampfl et al, Blood 2011). The Nuclear Factor IB (NFIB) gene is present in the 9pUPD region, where a mutational hot spot takes place. NFIB belongs to the NFI family of CAAT box binding transcription factors, consisting of 4 separate genes (NFIA, -B, -C, -X). NFIA is a post-transcriptional target of myelopoiesis regulator miR-223, which plays a key role in directing the HSC/HPC maturation/differentiation into the erythroid or granulocytic lineages (Fazi et. al. Cell 2005; Starnes et al. Blood 2009). Genomic alterations of NFIA were detected in about 2% of MPN patients (Bernard, Leukemia 2009). The gene expression levels of NFIB are higher in CD34+HSC/HPC isolated from 5 JAK2V617F+PV patients than in normal controls (Berkofsky-Fessler, Clin Cancer Res 2010).However, the role of NFIB in normal and pathological hematopoiesis has not been yet investigated. Methods: DNA, mRNA, and proteins were isolated from human myeloid cell lines (K562, Hel and UKE-1) and buffy coat from peripheral blood (PB) and bone marrow (BM) cells isolated from MPN patients or healthy donors. Gene dosage and gene expression level were measured by qRT-PCR. Statistical analyses were used to calculate differences among and between groups by one way ANOVA and two-way t-test. Results: our preliminary data shows that: i) NFIB locus is amplified and its expression is increased in myeloid cell lines harboring the JAK2V617F; ii) NFIB is barely expressed in mononuclear cells isolated from healthy donors PB (n=23), while its expression increased in PB cells isolated from PV (n=18, p=0.034) and ET patients (n=27, p=0.005), independently from JAK2V617F status; iii) increased gene dosage of NFIB , paralleling JAK2 gene amplification is detected in MPN patients (n=27, p<0,001); iv) gene expression level of NFIB positively correlates with platelets count in ET patients (n=17, p=0.008). Conclusions: our preliminary data show the de-regulation of NFIB expression levels related to MPN, thus suggesting its role in MPN pathogenesis or disease evolution and usage as a marker for MPN diagnosis and/or prognosis.