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

Ulrike Graab,Heidi Hahn,Simone Fulda

doi:10.18632/oncotarget.2726

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.

Highlights

RMS, the most common pediatric soft-tissue sarcoma, can be classified into two major subtypes, i.e. alveolar (ARMS) and embryonal (ERMS), according to the mutation status and histological features [1, 2]
We discovered that the GLI1/2 inhibitor GANT61 and the dual PI3K/mTOR inhibitor PI103 synergistically induced apoptosis in all RMS cell lines (Fig. 1A)
Parallel experiments showed that GANT61 and PI103 cooperated to suppress mRNA levels of HH target genes GLI1, GLI2 and patched homolog (PTCH) (Fig. S2A) and to reduce phosphorylation of key components of the PI3K/AKT/mTOR pathway such as AKT, S6 and 4E-BP1 (Fig. S2B)

Summary

Introduction

RMS, the most common pediatric soft-tissue sarcoma, can be classified into two major subtypes, i.e. alveolar (ARMS) and embryonal (ERMS), according to the mutation status and histological features [1, 2]. PTCH is an essential component of the HH signaling pathway, which is activated in RMS i.e. via loss of chromosomal region 9p22 or by www.impactjournals.com/oncotarget amplification of the 12q13–15 region including the GLI1 gene [3]. Canonical HH signaling pathway is activated via binding of one of the ligands, e.g. sonic hedgehog (SHH), to the transmembrane receptor PTCH. This leads to the inactivation of PTCH and subsequently to the release of the second transmembrane receptor smoothened (SMO) [8]. The balance between GLI activator and repressor forms results in expression of HH target genes, including GLI1 and PTCH [10]. Besides the canonical HH pathway, GLI proteins can be activated in a non-canonical and SMO-independent manner via phosphorylation by PI3K/AKT [11, 12], mTOR/ S6 [13], RAS [11, 14] or MAPK/ERK [15]

Methods

Results

Conclusion