DIFFERENTIAL GENE EXPRESSION AND PATHWAYS ENRICHMENT ANALYSIS IN TWO RNA-SEQ DATASETS OF JAK2V617F POSITIVE CELL LINE, TREATED WITH RUXOLITINIB, INDICATE FOCAL ADHESION PATHWAY MODULATION

Abstract

Objective: Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by activation of JAK/STAT and related pathways, leading to excessive proliferation, resistance to apoptosis and inflammation. The JAK2 inhibitor Ruxolitinib (Ruxo) is indicated for some groups of patients with MPN, reducing symptoms and improving overall survival. However, the factors that influence its effectiveness in reducing malignant clones and, consequently, the emergence of resistance, are not fully understood. Besides, the Focal Adhesion Kinase (FAK), a kinase involved in adhesion, migration, proliferation and apoptosis, participates in JAK2 V617F-positive cells'viability and apoptosis, and Ruxo seems to modulate genes related to the Focal Adhesion pathway in a public RNAseq dataset of SET-2 cells, as previously shown by this research group. The present work aimed to investigate whether the Focal Adhesion pathway is modulated after Ruxo treatment in JAK2 V617F-positive cells HEL cells, by analyzing two publicly available RNAseq datasets. Materials and methods: GSE184850 and GSE229712 were selected for analysis from the “Gene Expression Omnibus” (www.ncbi.nlm.nih.gov/geo/). Both datasets are from RNAseq in HEL cells (JAK2 V617F-positive) treated with DMSO and Ruxo 0.5 uM for 48h. The datasets were first analyzed separately for differentially expressed genes (DEGs) using DESeq2 package in R and Rstudio. Then, the common DEGs among top 250 DEGs were determined using ggVennDiagram package and analyzed in the STRING database (string-db.org) for KEGG pathways enrichment. Results: There were 153 common DEGs. Enrichment analysis in STRING database showed that Focal Adhesion pathway was among the 10 KEGG relevant pathways, with 10 DEGs: RELN, SHC1, ITGA5, IGFR1, FLT4, VASP, CCDN2, ACTN1, KDR, ZYX (strength 0.82, FDR: 0.00060). RELN and IGF1R were upregulated in Ruxo compared to DMSO, in both datasets, while SHC1, ITGA5, FLT4, VASP, CCDN2, ACTN1, KDR, ZYX were downregulated. Discussion: The downregulated genes are directly involved in migration and invasion processes. They are associated with cytoskeleton remodeling, which is important for disease progression. In addition, their expression is dysregulated in other types of cancers and leukemias. Upregulated genes RELN and IGF1R are genes involved in the activation of signaling pathways related to disease progression, such as those associated with proliferation and survival. Conclusions: The present results indicate a possible modulation of Focal Adhesion pathway after Ruxo treatment, which may be involved in treatment effectiveness and, therefore, further investigations regarding involvement of these genes in MPN may contribute to elucidate the influence of this pathway on patient treatment and prognosis.