Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma.
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.
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.
Ulrike
U
Graab
Institute of Human Genetics, University Medical Center, Goettingen, Germany.
Heidi
H
Hahn
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.|German Cancer Consortium (DKTK), Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
Simone
S
Fulda
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002642:Chick Embryo; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D005663:Furans / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D058990:Molecular Targeted Therapy; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D018232:Rhabdomyosarcoma, Alveolar / Q000473:pathology*; D018233:Rhabdomyosarcoma, Embryonal / Q000473:pathology*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2
D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D005663:Furans; C551027:GANT 61; C400551:GLI1 protein, human; C082593:GLI2 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C522973:PI103; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; C509239:SHH protein, human; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C546842:MTOR protein, human; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020169:Caspases
PI3K; apoptosis; hedgehog; rhabdomyosarcoma