CLL cells express relatively high-levels of XIAP, a principle downstream inhibitor of procaspase activation that also is expressed in many other types of cancer. Expression of XIAP may contribute to the resistance of CLL cells (and other cancers in general) to apoptosis induced by anti-cancer drugs and immune effector mechanisms. The anti-apoptotic activity of XIAP can be circumvented by SMAC, a natural inhibitor to the inhibitors of apoptosis (IAPs) that is released from mitochondria following activation of the intrinsic apoptotic pathway. SMAC inhibits XIAP by blocking its BIR domain(s), thereby precluding XIAP from inhibiting active caspases, such as caspase 9. Using mixture-based combinatorial libraries, we identified a series of polyphenylureas that selectively target the BIR2 domain of XIAP and that do not compete with SMAC for binding to XIAP (

Cancer Cell
). Structural activity studies identified analogs that had improved drug-like characteristics. We investigated whether an active (TPI 1540-14) XIAP-inhibitor or an inactive structural analog (TPI 1540-20) could influence the sensitivity of CLL cells to HLA class-I-restricted killing by allogeneic cytotoxic T lymphocytes (CTL) or to anti-CD20-directed antibody-dependent cell cytotoxicity (ADCC). For these studies we generated allogeneic CTL lines that could mediate specific killing of CLL cells in a HLA-class-I restricted manner. Moreover, the cytotoxicity of these CTL for CLL cells could be inhibited in a concentration-dependent fashion by W6/32, a mouse mAb that recognizes a framework determinant(s) common to all HLA class I molecules. Treatment of CLL cells with subsaturating amounts of W6/32 prior to the allogeneic CTL assay might mimic the situation commonly encountered by autologous CTL, which recognize cells that express relatively few class-I molecules bearing the target peptide-antigen. Treatment of CLL cells with TPI 1540-14, but not TPI 1540-20, significantly enhanced the specific killing of CLL cells by allogeneic CTL in a dose-dependent fashion. Moreover, the capacity of TPI 1540-14 to enhance CTL killing was more apparent when subsaturating concentrations of W6/32 mAb were used to treat the CLL target cells prior to the assay. With either compound, however, saturating amounts of W6/32 blocked CTL activity Similar effects were observed on the ADCC directed by the anti-CD20 mAb Rituximab using isolated allogeneic natural killer cells (NK cells) as effector cells. As noted in prior studies, NK cells failed to mediate high-level ADCC against Rituximab-treated CLL cells even at high effector:target ratios, conceivably due in part to the relatively low level expression of CD20 by CLL cells. However, treatment of CLL cells with TPI 1540-14, significantly enhanced Rituximab-directed ADCC by the allogeneic NK cells. We conclude that TPI 1540-14 can enhance CTL-mediated killing and Rituximab-directed ADCC of CLL cells in vitro. These studies suggest that inhibition of XIAP may enhance the activity of either active or passive immune therapeutic strategies in patients with this disease.

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