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

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.

Targeting hedgehog signalling by arsenic trioxide reduces cell growth and induces apoptosis in rhabdomyosarcoma

“…Nevertheless, despite the efficacy of GANT61 in RMS cell lines as we and others could prove (33), it also compromised primary skeletal muscle cell viability and induced increased cell death.…”

“…Intriguingly, in RMS ligand-independent GLI1 activity has been observed, which may interfere with the efficiency of SMO inhibitors (14,15,32). Other groups showed, that both GLI1 knockdown and a combination of GLI1 and GLI2 siRNA reduced RMS cell survival (1,33).…”

Probing for a deeper understanding of rhabdomyosarcoma: insights from complementary model systems

“…These findings argue for dual-agent therapy in PI3K- and RAS–ERK-activated RMS. Similar combinatorial lethality in RMS results from dual inhibition of Hedgehog and PI3K–mTOR signalling using the GLI1 and GLI2 inhibitor GANT61 and the PI3K and mTOR inhibitor PI-103 in vitro and in vivo by caspase-dependent apoptosis 142 .…”

Suppression of GLI sensitizes medulloblastoma cells to mitochondria-mediated apoptosis

“…GLI has also been implicated in the mitochondrial apoptosis signalling pathways; for example, the apoptosis genes caspase-3 and caspase-9 were identified as direct transcriptional targets of GLI (Graab et al 2015). The role of GLI in the induction of apoptosis has also been investigated in the several human cancer cells expressing Bcl-2 (Wang et al 2010).…”