Simultaneous Activation of p53 and Inhibition of XIAP Enhance the Activation of Apoptosis Signaling Pathways in AML. (2008)

Abstract p53, a key regulator of apoptosis, functions primarily upstream in the apoptotic cascade by directly and indirectly modulating Bcl-2 family of proteins. XIAP, a potent antiapoptotic protein, functions primarily downstream by suppressing caspases. Activation of p53 by MDM2 antagonist nutlin3a or inhibition of XIAP by small molecule inhibitors such as phenylurea 1396-11 was found to induce apoptosis in AML cells. Since the functions of XIAP and p53 are mediated and their activities controlled by a network of numerous components, some of which cross-regulate each other, we hypothesized that simultaneous activation of p53 and inhibition of XIAP would be a more effective at activating apoptotic signaling in AML cells. To test this idea, we treated AML cells with nutlin3a and 1396-11 and found that the combination synergistically induced cell death at 24 hours in OCIAML3 cells (combination index CI=0.200±0.047) and Molm13 cells (CI=0.565±0.082), two cell lines harboring wild type p53. Knocking down p53 expression by shRNA blunted the synergistic effect and downregulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin3a-induced apoptosis in OCI-AML3 cells, suggesting that the synergy was mediated by both p53 activation and XIAP inhibition. The specificity was further supported by data showing that inhibition of MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Although nutlin3a alone induced apoptosis in OCI-AML3 cells, the cell death was not robust and caspase-3 activation was minimal by itself even at 48 hours with 10 μM of nutlin3a. Immunoblot analysis showed increased expression of p53 and its downstream target p21. Of note, because p21 not only induces G1 cell cycle block, it additionally exhibits antiapoptotic activity that diminishes the effects of p53 activation, we also studied effects of these agents on p21 levels. When nutlin3a and 1396–11 were combined, caspase-3 activation was greatly increased and nutlin3a-induced p21 expression was significantly diminished. Moreover, in these experiments, caspase inhibition restored p21 levels and diminished apoptosis enhanced by 1396-11, suggesting that XIAP inhibition-mediated caspase activation eliminates p21, enhancing nutlin3a-induced apoptosis. Furthermore, activation of p53 by nutlin3a increased caspase-6 protein levels and induced mitochondrial release of SMAC, an antagonist of XIAP, suggesting that p53 activation shifts the balance toward apoptosis, promoting the effect of XIAP inhibition. Most importantly, p53 activation and XIAP inhibition greatly enhanced apoptosis in primary blasts from AML patients. Five out of six samples treated showed synergistic killing at 24 hours (CI=0.73±0.13), even when the cells were protected from drug-induced and spontaneous apoptosis by MS-5 stroma cells (CI=0.45±0.06). In conclusion, results demonstrate that simultaneous activation of p53 by antagonizing MDM2 and inhibition of XIAP synergistically activate apoptotic signaling pathways and promote death of AML cells, in part by modulating p21, caspases, and cytosolic SMAC levels. Since both, XIAP and p53, are presently being targeted by ongoing clinical trials in leukemia patients, the combination strategy holds promise for expedited translation into the clinic.