Specific loss of XIAP expression, not c-IAP1, is required for mediating apoptosis in high-risk neuroblastoma cells. A, Summary of IAP antagonists’ efficacy in killing neuroblastoma cells. The IAP antagonists were ranked on the basis of the average potency of killing (IC50) and specificity of killing toward only neuroblastoma cells: (i) A4, (ii) B3, (iii) BV6, (iv) LCL161, (v) CUDC-427, (vi) Debio 1143. B, Immunoblot analysis of c-IAP1, XIAP, PARP, and cleaved caspase-3 expression in neuroblastoma cell lines, BE(2)-C and KELLY, and noncancerous normal tissue cell line, HS5, after treatment with 10 µmol/L of either A4, B3, or BV6 at indicated timings (three independent replicates). C, Graphical representation showing the fold change of caspase-3/7 activity relative to vehicle controls after treatment with 10 µmol/L of either A4, B3, or BV6 at indicated timings (data were expressed as mean ± SD of three independent experiments conducted in duplicates; *, P < 0.05). D, Morphology of cells 48 hours posttreatment with 10 µmol/L of either A4, B3, or BV6. Images were taken using a light microscope with 10X magnification enlarged (scale: 1 cm: 200 µm). E, Immunoblot analysis of c-IAP1, XIAP, PARP, and cleaved caspase-3 expression in neuroblastoma cell lines, KELLY and NLF transfected with plasmid encoding either XIAP or empty vector, followed by treatment with 10 µmol/L of A4, B3, or BV6 at indicated timings (three independent replicates). β-actin is used as internal control.
Funding
VIVA Foundation for Children with Cancer (VIVA)
Children's Cancer Foundation (CCF)
Agency for Science, Technology and Research (A*STAR)
MOH | National Medical Research Council (NMRC)
ARTICLE ABSTRACT
XIAP, the most potent mammalian inhibitor of apoptosis protein (IAP), critically restricts developmental culling of sympathetic neuronal progenitors, and is correspondingly overexpressed in most MYCN-amplified neuroblastoma tumors. Because apoptosis-related protein in the TGFβ signaling pathway (ARTS) is the only XIAP antagonist that directly binds and degrades XIAP, we evaluated the preclinical effectiveness and tolerability of XIAP antagonism as a novel targeting strategy for neuroblastoma. We found that antagonism of XIAP, but not other IAPs, triggered apoptotic death in neuroblastoma cells. XIAP silencing induced apoptosis while overexpression conferred protection from drug-induced apoptosis. From a screen of IAP inhibitors, first-in-class ARTS mimetic A4 was most effective against high-risk and high XIAP-expressing neuroblastoma cells, and least toxic toward normal liver- and bone marrow–derived cells, compared with pan-IAP antagonists. On target engagement assays and nuclear magnetic resonance spectroscopy, A4 was observed to degrade rather than inhibit XIAP, catalyzing rapid degradation of XIAP through the ubiquitin-proteasome pathway. In MYCN-amplified neuroblastoma patient-derived xenografts, A4 significantly prolonged survival as a single agent, and demonstrated synergism with standard-of-care agents to reduce their effective required doses 3- to 6-fold. Engagement and degradation of XIAP by ARTS mimetics is a novel targeting strategy for neuroblastoma that may be especially effective against MYCN-amplified disease with intrinsically high XIAP expression. First-in-class ARTS mimetic A4 demonstrates preclinical efficacy and warrants further development and study.
XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells.
