INHIBITION OF CHRONIC MYELOGENOUS LEUKEMIA-CELLS HARBORING A BCR-ABL B3A2 JUNCTION BY ANTISENSE OLIGONUCLEOTIDES TARGETED AT THE B2A2 JUNCTION

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder c haracterized by the BCR-ABL hybrid gene. Two types of hybrid BCR-ABL m RNA have been found, B2A2 and B3A2. As the BCR-ABL rearrangement is sp ecific to leukemic cells, selective inhibition of leukemic cell growth by BCR-ABL antisense oligonucleotides (ASO) has been reported in vitr o for CML patients and cell lines. However, controversial results have been obtained from preclinical studies using anti-BCR-ABL ASO, as non specific inhibition of leukemic cell growth was evidenced in some case s. B3 exon secondary structure uas deduced from its sequence and found to be a loop. According to this predictive structure of exon B3, a 56 -mer antisense oligonucleotide targeting the polypurine bases from the B2A2 junction was devised which would inhibit proliferation (MTT assa y) of B3A2 junction cell lines (K562 and a murine cell line Ba/F3 tran sfected with the B3A2 junctional sequence). This ASO had a hairpin-lik e secondary structure and was found to be much more resistant to the a ction of nucleases than control 18-mer standard oligonucleotides. Hybr idization to its target mRNA occurs via formation of a tripler structu re. A concentration of 5 mu M of specific 56-mer B2A2 ASO was necessar y to demonstrate 50% optical density (OD) reduction for K562 cell line and Ba/F3 transformed by B3A2 cDNA. Sense and non-sense 56-mer sequen ce or 18-mer linear ASO showed no effect for these concentrations. Wes tern blot showed a partial inhibition of P210 protein; expression of P 145(abl) remains unchanged. The 56-mer ASO also inhibited the prolifer ation of B2A2 junction cell line BV173 at the same concentration and s howed no effect on the HL60 cell line used as control.