Protooncogene MYB Activates the Transcription of ROR1 in B Cell Malignancies

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a highly conserved gene that is associated with cell survival. ROR1 is uniquely expressed in some B cell malignancies and epithelial cancer cells although it is barely detected in normal adult tissues. The precise mechanisms of this ectopic expression have remained unknown. We checked ROR1 expression by FACS analysis and by quantitative RT-PCR. ROR1 was highly expressed in the samples from CLL,MCL,SMZL,and BL. Expression levels of ROR1 mRNA and cell surface ROR1 protein showed clear positive correlation (R2=0.55) suggesting that ROR1 expression is regulated mainly transcriptionally. Then, we cloned the 2.5-kb fragment upstream of ROR1 transcriptional start site and performed promoter assay using ROR1 positive and negative cell lines. The promoter activity was observed not only in ROR1 positive lines but also in negative lines, which suggests that this region may not be the only regulatory element. Thus, we hypothesized that conserved regions in intron 1, which is about 230 kb long, may play important roles in regulation of transcription. BLAST search identified 19 regions conserved between human and rat intron 1 of ROR1. Two regions, designated as CRS3 and CRS7, further showed homology with chicken ROR1 gene. Among them, CRS3 region had enhancer activity selectively in ROR1 positive B cell lines. A consensus nucleotide sequence for the binding of protooncogene MYB was found within CRS3 region. Actually, the protein binding to CR3S sequence was verified by EMSA assay using nuclear extract from MYB positive cells. Immunoblot analysis showed that ROR1 positive B cell lines had high expression of MYB. In addition, the expression levels of MYB protein and ROR1 mRNA were positively correlated in 7 CLL samples analyzed (R²=0.49). We introduced shRNA of MYB into ROR1 positive EW36 cell line. MYB shRNA could decrease CRS3 luciferase reporter activity. As a result, MYB shRNA decreased ROR1 expression as well as MYB expression. Collectively, MYB induced ROR1 expression and might contribute to tumorgenesis of B cell malignancies.