Expression of constitutively active Raf-1 in the mitochondria restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant

The oncogenic BCR/ABL protein protects hematopoietic cells horn apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood A BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185 Delta BCR) failed to protect interleukin 3-deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185 Delta BCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fi-action of p185 Delta BCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185 Delta BCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released fi-om the mitochondria into the cytosol. p185 Delta BCR/M-Raf-transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185 Delta BCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185 Delta BCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemia-inducing effects of BCR/ABL.