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Expression Of Constitutively Active Raf-1 In The Mitochondria Restores Antiapoptotic And Leukemogenic Potential Of A Transformation-deficient BCR/ABL Mutant

Paolo Salomoni, Mariusz A. Wasik, Richard F. Riedel, Krzysztof Reiss, John K. Choi, Tomasz Skorski, Bruno Calabretta

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The oncogenic BCR/ABL protein protects hematopoietic cells from 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 (p185ΔBCR) 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, p185ΔBCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185ΔBCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185ΔBCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185ΔBCR/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, p185ΔBCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185ΔBCR 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.