Abstract
Abstract Patients with high-risk neuroblastoma (NB) frequently succumb to chemoresistant disease that we hypothesize results from deregulation of Bcl2 family proteins. We have shown that NB mitochondria demonstrate specific cytochrome (cyto) c release in response to diverse BH3 peptides. Such “BH3 profiles” identified Bcl2 protein addiction patterns and predicted NB sensitivity to the Bcl2-family antagonists ABT-737 (targeting Bcl2, Bclxl, Bclw) and AT-101 (> Mcl1 avidity) in vitro. We have further assessed whether BH3 profiles are preserved in xenografts and correlate with in vivo therapeutic responses. Heirarchical clustering of BH3 responses from >15 NB profiles identified three groups- “Mcl1 dependence” with a Noxa predominant cyto c release, “Bclxl/Bclw dependence” with a Bik-dominant response, and “BH3 resistance” with markedly blunted cyto c release. BH3 profiles from xenografts tended to co-cluster with their parent monolayer responses. Notably, BH3 resistant NBs are from cell lines derived at relapse after cytotoxic therapy. While most NBs have activated Bim sequestered to pro-survival proteins at steady state, co-IPs show no activated Bim on Mcl1, Bcl2 or Bclxl in relapsed NB cells. This “loss of priming” may explain the extreme therapy resistance seen clinically. In vitro, Bclxl/w dependent SMS-SAN is exquisitely sensitive to combinations of ABT-737 (10 nM) and melphalan, doxorubicin, or etoposide, with cytotoxic IC50s decreased by >1 log. Mcl1 dependent IMR5 cells were also sensitive to cytotoxics plus ABT-737 at 1 uM. BH3 resistant NBs were insensitive to ABT-737 combinations. Real-Time PCR for p53 targets following melphalan treatment show increased Puma, Noxa, and p21 expression in Mcl1 dependent IMR5, but not in BH3 resistant (p53 mutant) SK-N-AS or Bcl-xl/w dependent SMS-SAN, explaining ABT-737/melphalan synergy in IMR5 through possible Noxa/Puma neutralization of Mcl1. Lastly, NB xenografts (XG) from the 3 distinct BH3 profile groups were treated with cyclophosphamide (CTX, 75 mg/kg IPx4), ABT-737 (100 mg/kg IP daily x14), or both drugs. ABT-737 alone partially regressed SMS-SAN XGs, while the ABT-737/CTX combination completely regressed 6/9 SMS-SAN XGs, with cure of 3/9 mice following a single treatment cycle. BH3 profiles from XGs that re-grew showed stable Bclxl/w dependence, and re-treatment led to regression again. Mcl1 dependent XGs were insensitive to ABT-737 combination therapy. The same combination therapy using AT-101 was largely ineffective in all but Mcl1 dependent XGs where marginal benefit was seen. Intact mitochondrial apoptosis is critical to cytotoxic therapy effectiveness. BH3 profiles from NB mitochondria not only reveal mechanisms of relapsed tumor chemoresistance, but accurately identify NBs that benefit from small molecule Bcl2 family inhibition to re-instate chemotherapy potency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5252.
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