Sorafenib-mediated Mcl-1 and cFLIP_L down-regulation synergistically increase TRAIL lethality in human leukemia cells

B42

In the present study, interactions between the Raf and multi-kinase inhibitor Sorafenib and TRAIL (TNF-related apoptosis-inducing ligand) were investigated in malignant hematopoietic cells. Pretreatment (24h) of U937 leukemia cells with 7.5 μM sorafenib, a concentration that was minimally toxic, dramatically and synergistically potentiated apoptosis induced by sub-lethal concentrations of TRAIL/Apo2L (e.g., 75ng/ml). Similar interactions were observed in other malignant hematopoietic cells including Raji, Jurkat, Karpas, K562, U266 cells and primary AML blasts. Notably, the combination was minimally toxic to normal CD34⁺ bone marrow cells. Exposure of sorafenib-pretreated cells to TRAIL potently and rapidly induced mitochondrial injury and release of cytochrome c, Smac and AIF into the cytosol, and caspase -9, -3, -7, and -8 activation. Sorafenib pretreatment also downregulated Bcl-xL and abrogated Mcl-1 expression, while co-administration of TRAIL strikingly increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade (i.e., in U937 cells ectopically expressing a caspase-8 dominant-negative construct) significantly blocked Sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. No changes were observed in levels of DR4/DR5 or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC); however, sequential administration of sorafenib/TRAIL strikingly increased activation of DISC-associated procaspase-8. Attention then focused on cFLIP (FLICE-inhibitory protein), a potent procaspase-8 inhibitor. Pre-exposure of cells to sorafenib led to marked cFLIP_L down-regulation, but no changes in expression of the short form of this protein (cFLIP_S). Mooreover, sorafenib-mediated cFLIP_L downregulation involved a translational mechanism associated with diminished eIF4E phosphorylation. Notably, ectopic expression of cFLIP_L significantly reduced sorafenib/TRAIL lethality. Together, these results, taken in conjunction with earlier findings, suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP_L, most likely at the translational level, and these events cooperate to promote simultaneous engagement of the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis. They also suggest that a strategy combining sorafenib with TRAIL warrants attention in leukemia and possibly other hematologic malignancies.