Oncotarget

Research Papers:

Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma

Ulrike Graab _, Heidi Hahn and Simone Fulda

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Oncotarget. 2015; 6:8722-8735. https://doi.org/10.18632/oncotarget.2726

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Abstract

Ulrike Graab1, Heidi Hahn2, Simone Fulda1,3,4

1Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany

2Institute of Human Genetics, University Medical Center, Goettingen, Germany

3German Cancer Consortium (DKTK), Heidelberg, Germany

4German Cancer Research Center (DKFZ), Heidelberg, Germany

Correspondence to:

Simone Fulda, e-mail: simone.fulda@kgu.de

Keywords: apoptosis, hedgehog, PI3K, rhabdomyosarcoma

Received: October 15, 2014     Accepted: November 12, 2014     Published: March 06, 2015

ABSTRACT

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.


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