Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins
David S. Ziegler, … , Leigh Zawel, Andrew L. Kung
David S. Ziegler, … , Leigh Zawel, Andrew L. Kung
Published August 1, 2008
Citation Information: J Clin Invest. 2008;118(9):3109-3122. https://doi.org/10.1172/JCI34120.
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Research Article Oncology

Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

Abstract

Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.

Authors

David S. Ziegler, Renee D. Wright, Santosh Kesari, Madeleine E. Lemieux, Mary A. Tran, Monish Jain, Leigh Zawel, Andrew L. Kung

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Figure 1

Structure and function of LBW242.

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Structure and function of LBW242. (A) Schematic of the apoptotic pathway...
(A) Schematic of the apoptotic pathway with structure and site of action of LBW242, which binds to the IAPs and prevents their neutralization of the caspases. (B) LBW242 competes with full-length Smac for occupancy of the XIAP-BIR3 surface groove, as assessed by a time-resolved fluorescence energy transfer assay. (C) LBW242 overcomes XIAP-BIR3–mediated repression of caspase-3 activity in a cell-free extract, resulting in activation of caspase-3 and cleavage of a fluorogenic substrate. (D) LN827 cells treated with the indicated concentrations of LBW242 for 4 hours reveal no change in cellular levels of XIAP or caspase-9 (input). Immunoprecipitation of caspase-9 followed by immunoblot analysis revealed dose-dependent decrease in associated XIAP (IP caspase-9). (E and F) Densitometric analysis with data expressed relative to vehicle controls. (G) LBW242 concentrations in plasma, brain, and tumor of 3 SK-OV-3 tumor–bearing nude mice after 14 days of daily parenteral dosing of LBW242 (50 mg/kg), measured 4 hours following the final dose. Data represent mean ± SEM for plasma and tumor, and the average of pooled samples for brain. (H) U87 cells were treated in triplicate with the indicated concentrations of LBW242. Relative cell numbers were assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (see Methods). Data points represent the mean ± SEM.