Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis

Abstract

Genomic studies have identified somatic alterations in the majority of myeloproliferative neoplasms (MPN) patients, including JAK2 mutations in the majority of MPN patients and CALR mutations in JAK2-negative MPN patients. However, the role of JAK-STAT pathway activation in different MPNs, and in patients without JAK2 mutations, has not been definitively delineated. We used expression profiling, single nucleotide polymorphism arrays, and mutational profiling to investigate a well-characterized cohort of MPN patients. MPN patients with homozygous JAK2V617F mutations were characterized by a distinctive transcriptional profile. Notably, a transcriptional signature consistent with activated JAK2 signaling is seen in all MPN patients regardless of clinical phenotype or mutational status. In addition, the activated JAK2 signature was present in patients with somatic CALR mutations. Conversely, we identified a gene expression signature of CALR mutations; this signature was significantly enriched in JAK2-mutant MPN patients consistent with a shared mechanism of transformation by JAK2 and CALR mutations. We also identified a transcriptional signature of TET2 mutations in MPN patent samples. Our data indicate that MPN patients, regardless of diagnosis or JAK2 mutational status, are characterized by a distinct gene expression signature with upregulation of JAK-STAT target genes, demonstrating the central importance of the JAK-STAT pathway in MPN pathogenesis.

Figure 1

Gene expression profiling distinguishes patients with MPN from normal controls and homozygous JAK2V617F-mutant MPN patients from others. (A) Hierarchical clustering was performed on gene expression microarray data from the granulocytes of 55 MPN patients and 11 normal subjects. MPN patients were characterized by a distinct gene expression profile compared with normal subjects. Correlation of patient samples with clinical MPN subtype and JAK2 genotype showed that MPN patients with homozygous JAK2V617F mutations were characterized by a unique cluster of differentially expressed genes among MPN patients. (B) Heatmap representation of significant differentially expressed genes between normal subjects and MPN patients with homozygous JAK2V617F (265 genes FC >3 and FDR <0.01), heterozygous JAK2V617F (222 genes FC >2 and FDR <0.05), and JAK2 WT genotypes (209 genes FC >2 and FDR <0.05). A red-blue color scale depicts normalized gene expression levels (red: high; blue: low).

Figure 2

MPN patients are characterized by a transcriptional signature of increased JAK2 activity regardless of JAK2 genotype. (A) Heatmap representation of differentially expressed genes (FC >3 and FDR <0.01) among MPN patients. A red-blue color scale depicts normalized gene expression levels (red: high; blue: low). Displayed are the top 100 differentially expressed genes derived from a supervised analysis comparing transcript expression in granulocytes from JAK2V617F homozygous mutant granulocytes vs normal subjects. The transcripts encoding JAK2, STAT5B, CD177 (PRV1), and MAPK214 are displayed. (B) GSEA showing enrichment of JAK2 shRNA signature in MPN patients relative to normal subjects regardless of JAK2 mutational status.

Figure 3

CALR-mutant MPN patients are characterized by a gene signature associated with activated JAK2 signaling. (A) Mutational status of JAK2, CALR, and MPL mutational status as well as clinical MPN diagnosis in 290 MPN patients. An individual column represents each patient. (B) GSEA showing enrichment of JAK2 shRNA signature in MPN patients with CALR mutations relative to normal subjects. (C) Heatmap representation of the 433 significantly differentially expressed genes (413 genes upregulated and 20 downregulated; FDR <0.01 and FC >2) in granulocytes from CALR-mutant MPN patients relative to normal subjects (21 MPN patients and 11 normal subjects). A red-blue color scale depicts normalized gene expression levels (red: high; blue: low). (D) GSEA showing significant enrichment of CALR-mutant MPN signature in MPN patients with homozygous JAK2V617F mutations relative to normal subjects.

Figure 4

Integrative genomic analysis shows impact of mutations coexisting with CALR and JAK2 mutations on transcriptome of MPN patients. (A) Circos plots showing mutational frequencies and cooccurrences in 97 MPN patient samples. Genetic data regarding JAK2V617F allele burden, cytogenetic alterations, and mutations in CALR, TET2, ASXL1, and IDH1/2 as well as mass spectrometric-based genotyping data are displayed. (B) Integration of somatic genetic alterations, recurrent copy number alterations from SNP array data, and gene expression of key altered transcripts in MPN patients. Each patient is represented in an individual column in the top heatmap and relative level of gene expression of STAT5B, JAK2, MAPK14, and MET is shown in the bottom heatmap. (C) Significantly differentially expressed genes (FDR <0.05) based on supervised analysis of gene expression of TET2-mutant vs WT MPN patients (93 samples). Sixty-one genes were significantly differentially expressed.