JAK2V617F mRNA metabolism in myeloproliferative neoplasm cell lines

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of leukemias with defective regulation of myeloid stem cell proliferation. They include four distinct diseases: chronic myeloid leukemia, polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In 2005, four independent studies have concurred to the identification in MPN patients of a specific mutation in the Janus kinase 2 (JAK2) protein (1849 G-T in exon 14; 617Val-Phe) in the majority of MPNs. Indeed, this JAK2V617F variant was identified in 95% of PV, 55% of ET and 65% of PMF patients. The amino-acid change triggers a constitutive activation of the JAK2 protein, independently of cytokines. Whether JAK2V617F is the initiating event in these MPNs remains under debate; however, JAK2V617F seems to drive the phenotype of the disease. JAK2 gene is located on human chromosome 9, at p24.1 locus. It is composed of 25 exons and encodes a 1132 amino-acid protein of 130.7 kDa apparent molecular weight. The gene is expressed only in blood cells, bone marrow and lymph nodes. Occurrence of JAK2V617F in association with phenotypically different classes of MPNs raised a controversy as to the real function of the mutated JAK2 and the possible involvement of additional genetic determinants in these phenotypic discrepancies. Recent whole genomic studies have identified few single-nucleotide polymorphisms (SNPs) in the vicinity of exon 14 (Figure 1a). Notably, it has been anticipated that the 46/1 haplotype predisposes to the JAK2V617F mutation. Other studies have suggested that several SNPs could be implicated in MPN phenotype disparity. The question remains as to the possible role of these SNPs in MPN phenotypic discrepancies. This 46/1 haplotype is a 280-kb-long region on chromosome 9 that includes JAK2, INSL4 and INSL6 genes. Four SNPs define the ‘GGCC’ part of this haplotype and are located in the JAK2 gene: rs3780367 T/G in intron 10, rs10974944 C/G in intron 12, rs12343867 T/C in intron 14 and rs1159782 T/C in intron 15 (Figure 1a). All four SNPs are in complete linkage disequilibrium. The 46/1 haplotype includes the sequences frequently mutated in MPNs like mutations in exons 12 and 14. On another hand, the rs10974944G minor allele was significantly more common in PV than in ET conditions. In addition, it was shown that other JAK2 SNPs, namely rs7046746, rs10815148 and rs12342421, were also significantly associated with PV and ET but not with PMF. In this work, we hypothesized that the JAK2V617F and/or the 46/1 haplotype could have an impact on JAK2 mRNA level or/and pre-mRNA maturation. To provide new insights into the JAK2 mRNA expression and splicing, six cell lines, derived from patients with MPNs, were chosen on the basis of their JAK2 status (Figure 1b), and cultured in appropriate media (Supplementary Table 1). It has been suggested that both in human and mouse, the phenotypic expression of the disease depends on the intensity of the signaling, which is thought to be modulated only by the number of JAK2V617F copies. Moreover, it was recently emphasized that the number of JAK2 genes in UKE-1 cell line increases with time in culture. We therefore determined the actual number of JAK2 genes in the six cell lines used in our laboratory before further investigation (Figure 1b). The number of JAK2 genes in SET-2, UT-7 and UKE-1 cell lines was as previously reported, whereas only eight JAK2 gene copies were found in HEL cell line. KU-812 cell line was found to have two copies of the gene. As for TF-1, the cell line was described with two copies of the JAK2 gene, however, the subclone in our laboratory displayed only one copy. It is possible that a loss of heterozygosity occurred, leading to a haploid cell line. Comparative data are gathered in Figure 1b. We next established the genotypes and haplotypes around the JAK2 gene for the six JAK2V617-negative or -positive cell lines (Figure 1b). To define the JAK2-associated haplotypes, genomic DNA obtained from the six cell lines was amplified with specific primers: one set of primers allows the amplification of a 3047-bp fragment containing the two intron 14 SNPs rs12340895 and rs12343867, in 30 of the JAK2V617F mutation, along with the JAK2V617F mutation itself (Figure 1a). Another set of primers was used to characterize the intron 12 SNP rs10974944, in 50 of the JAK2V617F mutation. PCR fragments were then fully sequenced and sequence polymorphisms characterized. The HEL, TF-1, UT-7 and UKE-1 cell lines were then chosen to test whether the JAK2V617F mutation affects the steady-state level of JAK2 mRNA. These cells have the same ‘TCTT’ haplotype; two of them, HEL and UKE-1, are homozygous for the JAK2V617F mutation, whereas the two others, TF-1 and UT-7, contain only the wild-type allele (Figure 1b). Quantitative analyses revealed that accumulated JAK2 mRNA level differs widely from one cell line to another (Figure 2a). However, once correlated to JAK2 gene copy number, these data showed no difference between cell lines, suggesting that each copy of the gene yields about the same amount of JAK2 mRNA molecules accumulating in each cell line, independently of the JAK2V617F mutation (Figure 2b). Collectively, JAK2 mRNA steady-state level correlates with JAK2 gene copy number, regardless of the presence or absence of exon 14 singlenucleotide mutation. To identify potential exonic and intronic motifs and predict the effects of mutations on pre-mRNA splicing, a preliminary bioinformatics study was performed using ‘Human Splicing Finder’ algorithm http://www.umd.be/HSF/. This analysis showed a differential binding of several splicing factors between the wild-type and the mutated sequence (Supplementary Figure 1). In particular, one potential site for the serine–arginine-rich (SR) protein SRSF6 is lost in the mutated sequence compared with the wild type, whereas a putative site for SRSF2, another member of the SR protein family, appears (Supplementary Figure 1). In silico analysis also revealed that the G to C mutation favors the occurrence of two exonic splicing silencers (ESSs): a putative ESS (PESS) octamer, and Fas-ESSs (Supplementary Figure 1). On the basis of this preliminary in silico analysis, we hypothesized that the JAK2V617F mutation itself or the associated haplotype could affect the JAK2 mRNA splicing or/and stability, and therefore modulate the protein expression and activity, which would affect the phenotypic discrepancies of JAK2V617Fassociated MPNs. Citation: Blood Cancer Journal (2014) 4, e222; doi:10.1038/bcj.2014.43 & 2014 Macmillan Publishers Limited All rights reserved 2044-5385/14