gant-61 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

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Flow Cytometry; Fluorescent Antibody Technique; Hedgehog Proteins; Humans; Immunohistochemistry; Mice; Mice, Nude; Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma, Alveolar; Rhabdomyosarcoma, Embryonal; Signal Transduction; TOR Serine-Threonine Kinases; Trans-Activators; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1