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

QUANTIFICATION OF JAK2WT AND JAK2V617F ALLELE IN mRNA FROM PH(-) MYELOPROLIFERATIVE NEOPLASMS PATIENTS: CORRELATION WITH DNA ALLELE BURDEN AND DESEASE PROGRESSION

INTRODUCTION: Currently, the JAK2V617F allele quantification in Ph- MPN is performed on genomic DNA. However, compared to DNA, mRNA analysis may offer some advantages such as: 1)higher sensitivity; 2) inclusion of platelet mRNA; 3) mRNA transcriptional functionality. Therefore, we developed an absolute allele-specific RQ-PCR method to quantify JAK2WT, V617F and ABL (as housekeeping gene) transcript levels aiming to: 1) verify specificity and sensitivity of this assay; 2)evaluate whether gene expression levels correlate with disease phenotype and may mark disease progression (i.e. ET toward PV). METHODS: To construct reference curves, including five 10-fold serial dilutions, two plasmid standards were manufactured to contain 150 bp of either JAK2WT and JAK2V617F cDNA sequence. To perform an allelic discrimination we used primers described by Merker et al (JMD, 2010): reverse primers are complementary to the WT or the mutant cDNA sequence, respectively. Assay sensitivity was determined by serial 10-fold dilutions of the K562 (JAK2WT) and HEL (V671F homo) cell lines which were included in each experiment as negative and positive controls. RESULTS: We tested cDNA synthesized from peripheral blood buffy coat specimens of 48 MPN patients (16 PV; 25 ET, 7 PMF), 13 patients with secondary polycythemia (SP) and 23 healthy donors (CTRL). Respect to the qualitative PCR method (Baxter el al, Lancet 2005) our RQ-PCR assay shows a 100% concordance rate in identifying the presence of the JAK2V617F. V617F mutation was detected in 16/16 PV (100%), 19/25 ET (76%) and 5/7 (71%) PMF samples. The mean expression of JAK2WT allele, as absolute averaged copies+SEM was: healthy donors 12127+2181; SP 12244+2489; PV 4955+1642, WT ET 10876+1781; V617F ET 5693+830 and V617F PMF 3004+1442. Statistically significant differences were detected between CTRL and PV (p <0.005) and CTRL and V617F ET (p <0.005). The mean expression of V617F allele was PV 27107+12699, ET 6524 +1877 and PMF 29464+14587. A significant difference was found comparing PV and ET (p <0.005) and ET and PMF (p <0.005). Respect to the allele burden quantitative assay (IPSOGEN), the mutational burden (V617F/(V617F+WT) ratio detected by our assay is significantly higher (59% vs 36% burden in RQ-PCR and IPSOGEN, respectively; p <0.005). Three ET patients progressed to PV phase. The sequential analysis of the mutational burden ratio at diagnosis and during disease progression showed an increase in one patient’s sample at both DNA and mRNA level. By contrast, in the remaining 2 cases the ratio remained constant at the DNA level, but significantly increased when the mRNA was analysed. CONCLUSION: Present data demonstrated that our method is as specific but more sensitive than the allele burden determination. In addition, the complementary determination of mRNA transcript levels may help in elucidate the pathogenetic mechanisms of the Ph- MPN diseases

Realizing virtual research environments for the agri-food community: The AGINFRA PLUS experience

The enhancements in IT solutions and the open science movement are injecting changes in the practices dealing with data collection, collation, processing, analytics, and publishing in all the domains, including agri-food. However, in implementing these changes one of the major issues faced by the agri-food researchers is the fragmentation of the “assets” to be exploited when performing research tasks, for example, data of interest are heterogeneous and scattered across several repositories, the tools modelers rely on are diverse and often make use of limited computing capacity, the publishing practices are various and rarely aim at making available the “whole story” including datasets, processes, and results. This paper presents the AGINFRA PLUS endeavor to overcome these limitations by providing researchers in three designated communities with Virtual Research Environments facilitating the use of the “assets” of interest and promote collaboration. © 2020 John Wiley &amp; Sons Lt