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 apopto sis induced by growth factor deprivation, but the mechanisms are only partially understood A BCR/ABL mutant lacking amino acids 176-426 in t he BCR domain (p185 Delta BCR) failed to protect interleukin 3-deprive d 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MA P kinase pathway. Compared to p185 wild-type transfectants, p185 Delta BCR-transfected cells showed markedly reduced levels of Bcl-2 and exp ressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 express ion in the mitochondrial fi-action of p185 Delta BCR cells was also ma rkedly diminished and mitochondrial RAF was undetectable. In p185 Delt a BCR cells transfected with a mitochondria-targeted, constitutively a ctive RAF (M-Raf) BAD was expressed in the hyperphosphorylated form an d released fi-om the mitochondria into the cytosol. p185 Delta BCR/M-R af-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 32D cl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combine d immunodeficiency (SCID) mice, p185 Delta BCR/M-Raf double transfecta nts were leukemogenic, whereas cells expressing only p185 Delta BCR sh owed no leukemogenic potential. Together, these data support the exist ence of a BCR/ABL-dependent pathway that leads to expression of an act ive RAF in the mitochondria and promotes antiapoptotic and leukemia-in ducing effects of BCR/ABL.