benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Rhabdomyosarcoma--Alveolar

We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Line, Tumor; Chick Embryo; Drug Screening Assays, Antitumor; Drug Synergism; Furans; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Molecular Targeted Therapy; Neoplasm Proteins; Nuclear Proteins; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Rhabdomyosarcoma, Alveolar; Rhabdomyosarcoma, Embryonal; Signal Transduction; TOR Serine-Threonine Kinases; Transcription Factors; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2