Integrated Genomic-Transcriptomic Study Highlights Accumulation of Genetic Variants and Activation of Inflammatory Pathways

INTRODUCTION: The molecular basis of Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) is unclear. So-called "driver" mutations in JAK2, CALR, or MPL are present in the vast majority of cases, but there is no compelling evidence to explain how each mutant gene can lead to phenotypically distinct and/or overlapping disease phenotypes. In an attempt to understand the molecular events that underlay clinical characteristics of MPNs, we studied gene expression profiles and sequenced hematopoietic cells of MPN patients with a focus on myelofibrosis (MF) landscape.

METHODS: Patients were consented to MI-ONCOSEQ study approved by the University of Michigan IRB. Peripheral blood or bone marrow aspirates were either enriched for CD34 expressing cells or peripheral blood or bone marrow mononuclear cells (PBMC/BMMC) were analyzed. MI-ONCOSEQ is a Next Generation Sequencing platform to identify genetic aberrations in 1,711 genes and gene expression and fusion analysis of 24,774 capture targets by transcriptome sequencing. Gene expression data was analyzed between various sub-groups of MF based upon clinical (spleen size) or molecular (mutations in Ras pathway genes) characteristics. Gene set enrichment analysis was conducted for the MF cohorts (23 CD34 enriched, 76 PBMC/BMMC) and ET/PV/PrePMF cohort (12 CD34, 35 PBMC/BMMC).

RESULTS: We analyzed the genetic landscape of 163 patients with MPNs: 113 with overt MF and 50 with ET (18), PV (23), or prePMF (9). In addition to driver genes, 183 other gene variants were observed. The number of gene variants was higher in older patients (median 4 and 5 variants in those aged 70-80 and >80 yrs respectively) with MF as compared to ET/PV/PrePMF where the median number of variants did not exceed 3. Mutations in ASXL1, TET2, RAS and SRSF2 increased in frequency with age (Fig 1). Gene expression profiles of sub-groups of MF were further analyzed to understand aberrantly regulated molecular pathways. Hierarchical clustering of all MF patients showed that CD34 enriched samples to be distinct from the PBMC/BMMC cohort and therefore these cohorts were analyzed separately. Moreover, hierarchical clustering suggested differences in patients with large spleens. In the comparison of MF to ET/PV/PrePMF within the CD34 population gene enrichment highlights hemopoiesis, leukemia related pathways as well as endoplasmic reticulum and Golgi transport pathways. Previously, we saw that RAS pathway mutations predicted proliferative disease with high WBC counts. Therefore, we focused on RAS pathway mutated cohorts versus RAS wild type cohorts and identified dysregulated pathways by gene set enrichment analysis (Fig 2). The RAS mutated MF cohort (PBMC/BMMC fraction) showed up-regulation of cytokines IL6 (p-value 9.39E-06), IL8 (p-value 1.16E-04), and IL1beta (p-value 5.24E-04) and down-regulation of the TNF superfamily (p-value 9.98E-04). Most notably, there was up regulation of NFkB transport to the nucleus (p-value 8.69E-05) and transcription factor activity. In general, several metabolic pathways were affected and inflammatory pathways were up-regulated. Since spleen size is an indicator of disease severity, progression and response to therapy, gene set enrichment between cohorts of patients with different spleen size (>6cm by physical exam versus <6cm) was analyzed (Fig 3). The data suggest dysregulation of megakaryocyte differentiation (p-value 2.05E-05), cytokine production (p-value 9.09E-05) and signaling, JAK-STAT pathway (p-value 5.10E-05), RAS signaling (p-value 7.67E-05) and NFkB pathway (p-value 1.31E-04) in patients with larger spleens.

CONCLUSIONS: Age is a high risk for many hematological malignancies. We analyzed the number of genetic variants by age and determined that accumulation of higher number of mutations might indicate why disease progresses rapidly in older patients. Not only genetic variants, but gene expression changes also contribute to the pathogenesis of MF. Analysis of gene expression changes show enrichment of genes regulating JAK-STAT pathway activity and cytokine production as anticipated, but also implicates epigenetic regulators and the RAS signaling pathway in disease biology. Moreover, enriched pathways in gene expression analysis underscore the dysregulation of NFkB, perhaps as a result of inflammatory response. Thus these pathways are promising candidates for intervention in patients with MF.

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