Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms

Abstract

Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3-23.3 (n=1), 9q33.1-34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31-36.33 (n=6), 17q21.2-q21.31 (n=5) and 17q25.1-25.3 (n=5) and deletions affecting 18p11.31-11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a 'HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal.

Figure 1

CNAs in MPN (a). Ideogram representing regions of chromosomal gains (red) and loss (green) identified using Affymetrix GeneChip Mapping 250K arrays in 87 patients with MPN. Individual lines represent a single patient (b). Top: genomic profile of chromosome 17 in one patient (s531) showing a gain at 17q21.1–21.31 encompassing 144 genes including EZH1 and ITGA2B, which are also transcriptionally upregulated in MPN patients (Supplementary Table S6). Bottom: genomic profile of chromosome 6 in one patient (s242) showing a gain at 6p22.3 encompassing JAR1D2. Red lines represent the mean log₂ ratio of the intensity of the samples relative to 60 unrelated HapMap normal controls.

Figure 2

Chromosome 9 aberrations are associated with elevated JAK2V617F allele burden and gene expression signature. Gene expression differences in patients with normal (n=25) versus abnormal (+9, 9pLOH) chromosome 9 cytogenetics (n=13) (Supplementary Table S7). A subset of genes upregulated (>2.7-fold, P<0.05; 26 genes) or downregulated (>3-fold, P<0.05; 26 genes) in patients with chromosome 9 anomalies compared with normal chromosome 9 cytogenetics are displayed in a heatmap.

Figure 3

PMF and ET patients display higher frequencies of chromosomal aberrations compared with PV. Frequency plots of chromosomal gains (red) or loss (green) in individuals with PV (n=23), PMF (n=15) and ET (n=25). Y axis represents the frequency of copy number change in each patient subgroup. T-test was used to identify significant differences in the frequency of chromosomal aberrations between these three patient subtypes.

Figure 4

NFIB protects cells from cytokine withdrawal-induced cell death and promotes cell growth (a). Baf3/EPOR cells transduced with control (EF) or EF-NFIB lentivirus for 48 h and GFP+ cells were replated in RPMI with 1 U/ml, 0.1 U/ml and 0.01 U/ml EPO for 48 h. Cell viability was determined by Trypan blue exclusion (b). Cumulative cell counts were performed over 10 days. Data are expressed as the average of three independent experiments±s.e.m. (^**P<0.05).