BH3-domain peptides synergize with cytotoxic agents to restore apoptosis sensitivity in chemoresistant neuroblastoma

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Neuroblastoma (NB) is a common and highly lethal pediatric tumor. Children with high-risk NB often have initial near-complete therapy responses yet ultimately succumb to progression of resistant disease. Cytotoxics induce tumor response through programmed cell death (PCD) activation and alterations in these pathways have been implicated in therapy-resistance. We hypothesize that understanding PCD pathway deregulation in NB may elucidate targets for novel pro-death therapeutics. Cytotoxic agents induce genotoxic stress that activates BH3 proteins. Pro-death multidomain members (Bak, Bax) are engaged by these activated BH3 proteins to execute mitochondrial PCD. Anti-death members (Bcl2, BclW, BclX, Mcl1) oppose this process through competition for BH3 proteins. Data support that NBs have competent latent mitochondrial death machinery but inefficiently activate PCD following cell stress. Our data from primary tumors and cell lines demonstrate elevated Mcl1 expression as a potential cause of defective PCD in NB. BH3 mimetic peptides (BH3mps) include minimal BH3 death domains with a cell transduction motif (r8) and are sufficient for PCD induction. We have shown that BH3mps from BID and BAD (r8BIDBH3 and r8BADBH3) induce apoptosis in NB cells in vitro and inhibit tumor growth in vivo (AACR 2004) at low micromolar concentrations nontoxic to normal cells. We have extended these studies to BH3mp’s mimicking BAD, BID, BIM, PUMA and NoxA to define their relative potency to engage PCD in chemoresistant NB cell lines alone and with cytotoxic agents. Assays for biomass, caspase activation and AnnexinV-EGFP showed that specific BH3mps potently restored chemosensitivity to diverse cytotoxics for multiple cell lines tested. For example, mafosfamide at concentrations up to 50 mcg/ml had no cytotoxic activity against IMR5, a MYCN-amplified cell line. However, exposure to as little as 2.2 mcg/ml mafosfamide in the presence of sublethal r8BADBH3 (2 mcM) induced marked PCD (65% cytotoxicity at 72 hours). r8BADBH3 given concurrently with doxorubicin demonstrated similar re-establishment of sensitivity (decreasing the IC50 by one log) but required higher peptide doses (5 mcM). We propose that r8BADBH3 functions as an enabler BH3mp, re-engaging PCD signaling in chemoresistant NB by binding to anti-death Bcl2 members and competitively displacing sequestered activated BH3 proteins. Ongoing studies include co-immunoprecipitation and MS/MS to query Bcl2 protein-protein interactions before and after BH3 peptide and cytotoxic exposure as well as assessments in vivo using genetically engineered NB prone mouse models. Emerging technologies to deliver and stabilize peptides for clinical use encourages further development of BH3 peptides as novel anti-cancer agents that may be used as adjuncts to conventional cytotoxics to re-engage apoptosis in refractory malignancies.