Discovery of a fusion kinase in EOL-1 cells and idiopathic hypereosinophilic syndrome

Abstract

Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disease of unknown etiology. Recently, it has been reported that imatinib mesylate (Gleevec), an inhibitor of Bcr-Abl kinase useful in the treatment of chronic myeloid leukemia, is also effective in treating HES; however, the molecular target of imatinib in HES is unknown. This report identifies a genetic rearrangement in the eosinophilic cell line EOL-1 that results in the expression of a fusion protein comprising an N-terminal region encoded by a gene of unknown function with the GenBank accession number NM_030917 and a C-terminal region derived from the intracellular domain of the platelet-derived growth factor receptor alpha (PDGFRalpha). The fusion gene was also detected in blood cells from two patients with HES. We propose naming NM_030917 Rhe for Rearranged in hypereosinophilia. Rhe-PDGFRalpha fusions result from an apparent interstitial deletion that links Rhe to exon 12 of PDGFRalpha on chromosome 4q12. The fusion kinase Rhe-PDGFRalpha is constitutively phosphorylated and supports IL-3-independent growth when expressed in BaF3 cells. Proliferation and viability of EOL-1 and BaF3 cells expressing Rhe-PDGFRalpha are ablated by the PDGFRalpha inhibitors imatinib, vatalanib, and THRX-165724.

Fig. 1.

Structures of inhibitors of platelet-derived growth factor tyrosine kinases imatinib (Gleevec), vatalanib, and THRX-165724.

Fig. 2.

Inhibition of EOL-1 cell viability by imatinib, vatalanib, and THRX-165724 as measured in the MTT assay. The data shown are representative of three experiments.

Fig. 3.

(A) NM_030917-PDGFRα fusion transcript sequence from EOL-1. The underlined sequences are alternatively spliced exons observed in the NM_030917 segment. The asterisk indicates the fusion junction. (B) Predicted amino acid sequence of the NM_030917-PDGFRα fusion protein based on cDNA sequences. The underlined peptides are derived from alternatively spliced exons. Blue, tryptic peptides from NM_030917 identified by MS; red, tryptic peptides from PDGFRα identified by MS; purple, tryptic peptide identified by MS that spans the fusion junction between NM_030917 and PDGFRα. The asterisk indicates the fusion junction. (C) Schematic of the deletion on 4q12 that leads to the fusion of the 5′ end of the NM_030917 gene and exon 12 of PDGFRα.

Fig. 4.

(A) Fusion points of the Rhe-PDGFRα mRNAs. The Rhe exon numbering is based on the NCBI annotation of the Rhe genomic locus (positions 1583318–1666068, GenBank accession no. NT_022853). EOL-1 and patient 3: Rhe exon 11 followed by PDGFRα sequence within exon 12 (nucleotide 1866, GenBank accession no. M22734). Patient 1: Rhe exon 8 followed by PDGFRα sequence within exon 12 (position 1829). (B) Genomic structure of Rhe-PDGFRα fusions in EOL-1 and patients 1 and 3. Breakpoints (BP) are indicated by arrows and by the position of the breakpoint in the Rhe gene according to NT_022853. Splice sites are underlined. The Rhe exon sequence is shown in uppercase, the Rhe nontranslated intron sequence is shown in lowercase, and the PDGFRα exon sequence is shown in uppercase. (C) Point mutation in Rhe-PDGFRα in patient 1 at relapse. The nucleotide sequence is shown in blue, and the amino acid sequence is shown in black. A C → T point mutation in Rhe-PDGFRα corresponding to nucleotide 2149 in PDGFRα is highlighted in red. The codon change from ACA to ATA (bold) results in a T674I substitution. T674 corresponds to T315 in c-Abl (GenBank accession no. M14752), which is part of the imatinib binding pocket and establishes a hydrogen bond with imatinib based on crystal structure data.

Fig. 5.

(A) Inhibition of Rhe-PDGFRα phosphorylation in EOL-1 cells by imatinib, vatalanib, and THRX-165724. (B) Inhibition of Rhe-PDGFRα phosphorylation in BaF3 cells by imatinib. Immunoprecipitation was performed with a rabbit anti-PDGFR serum, and Western blotting was performed with anti-phosphotyrosine antibody 4G10. Subsequently, the blots were reprobed with anti-PDGFR serum.

Fig. 6.

Inhibition of viability of BaF3 cells expressing Rhe-PDGFRα by imatinib, vatalanib, and THRX-165724 as measured in the MTT assay carried out in the absence (A) and presence (B) of IL-3. The data shown are representative of three experiments.