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ETV6::FRK gene expression analysis in Ba/F3 cells by whole transcriptome sequencing

ABSTRACT: ETV6::FRK is a rare kinase-related fusion gene which was identified only in AML but not in ALL. Herein, we firstly identified ETV6::FRK fusion gene in a patient with pediatric B-ALL. Because FRK is Src family tyrosine kinase, we performed functional analysis of ETV6::FRK to establish molecular targeting therapy. Our case with B-ALL was refractory to conventional chemotherapy and received allogeneic bone marrow transplantation following administration of blinatumomab. Cytogenetic analysis demonstrated 46,XY,t(6;12)(q21;p13) and target capture mRNA sequencing revealed ETV6::FRK. Ba/F3 cells expressing ETV6::FRK generated by retroviral transduction (Ba/F3-ETV6::FRK) and analyzed for IL-3 independent growth. Gene expression analysis using whole transcriptome sequencing and gene set enrichment analysis (GSEA) was performed for comprehensive analysis of gene expression profile related to ETV6::FRK. It was also analyzed whether dasatinib, which is Src-kinase inhibitor, suppressed the growth of Ba/F3-ETV6::FRK in vitro and in vivo. Ba/F3-ETV6::FRK proliferated without IL-3, suggesting ETV6::FRK had proliferation activity. Western blot revealed that constitutive phosphorylation of tyrosine residue of ETV6::FRK and STAT3/STAT5, suggesting constitutive activation of FRK-STAT3/STAT5 pathway. GSEA of oncogenic gene sets (C6) from the GSEA Molecular Signatures Database revealed that, compared with control cells, Ba/F3-ETV6::FRK cells were enriched for up-regulation of SNF5 target genes and down-regulation of RB target genes involved in cell cycle regulation. In vitro killing assay showed that dasatinib killed efficiently Ba/F3-ETV6::FRK. Dasatinib also suppressed the growth of Ba/F3-ETV6::FRK in vivo and extended the survival time of the xenografted NSG mice. These findings suggest that activation of FRK-STAT3/STAT5 pathway contributes aberrant growth promotion of Ba/F3-ETV6::FRK. Our study demonstrated that dasatinib might be effective for the patient with B-ALL harboring ETV6::FRK.

ORGANISM(S): Mus musculus

PROVIDER: GSE220610 | GEO | 2022/12/31

REPOSITORIES: GEO

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Project description:STAT5A and STAT5B proteins belong to the family of signal transducers and activators of transcription. They are encoded by 2 separate genes with 91% identity in their amino acid sequences. Despite their high degree of conservation, STAT5A and STAT5B exert non-redundant functions, resulting at least in part from differences in target gene activation. To better characterize the differential contribution of STAT5A and STAT5B in gene regulation, we performed single or double knock-down of STAT5A and STAT5B using small interfering RNA. Subsequent gene expression profiling and RT-qPCR analyses of IL-3-stimulated Ba/F3-beta cells led to the identification of putative novel STAT5 target genes. Chromatin immunoprecipitation assays analyzing the corresponding gene loci identified unusual STAT5 binding sites compared to conventional STAT5 responsive elements. Some of the STAT5 targets identified are upregulated in several human cancers, suggesting that they might represent potential oncogenes in STAT5-associated malignancies. Experiment Overall Design: Ba/F3-beta cells transfected with either control (scramble I) or Stat5A and Stat5B siRNAs were stimulated with IL-3 for either 30 minutes or 2 hours. Unstimulated Ba/F3-beta cells transfected with scramble I siRNA were used as a control. Experiment Overall Design: Total RNA was isolated using the RNeasy Maxi kit, including an on-column DNase I treatment to eliminate genomic DNA contamination, according to the manufacturerâs protocol (Qiagen, Valencia, CA). DNase-treated total RNA (5 Âµg) was synthesized into biotinylated cRNA probe using one-cycle target labeling and IVT labeling (Affymetrix, Inc., Santa Clara, CA) according to manufacturerâs instructions. Fifteen Âµg of biotinylated cRNA probe from each sample was hybridized onto murine U74A, U74B, U74C Affymetrix Microarray chips. The hybridized chips were then washed using the Affymetrix GeneChip Fluidics 400 station and scanned in the Affymetrix GeneChip Scanner 3000 (Affymetrix, Inc., Santa Clara, CA) according to manufacturerâs instructions. The quality of cRNA probe synthesis and efficiency of hybridization was analyzed in the GeneChip Operating System for each Affymetrix chip once scanning was complete. Microarray data were normalized using MAS5 and then analyzed using the Genespring (Agilent, Palo Alto, CA). Experiment Overall Design: Out of the 36,767 genes, 14,553 passed our noise and P/M/A call filters. Genes were further selected for being stimulated at least 2-fold by IL-3 after 30 minutes (164 genes) and 2 hours (553 genes) stimulation compared to the unstimulated cells (ScI-transfected cells). Then, IL-3 induced genes that were down-regulated at least 2-fold in the STAT5A/B siRNA-transfected cells at 30 minutes and 2 hours (12 and 29 respectively) were identified as putative STAT5 target genes and further investigated.

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ETV6::FRK gene expression analysis in Ba/F3 cells by whole transcriptome sequencing

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