Synergistic efficacy of inhibiting MYCN and mTOR signaling against neuroblastoma

Abstract

Background Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. Methods Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. Results Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. Conclusions Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.

The BRD4 inhibitor JQ1 suppresses tumor growth by reducing c-Myc expression in endometrial cancer

“…In addition to triggering apoptosis, JQ1 suppressed EC cell growth by inducing G1 cell cycle arrest. JQ1 has been reported to cause human neuroblastoma cell cycle arrest in G1 phase mainly by inhibiting the MYCN and mTOR signaling pathways [ 22 ]. JQ1 also decreases the proportion of cells in G2 phase and increases the share of cells in G1 phase in a preclinical model of pancreatic cancer [ 23 ].…”

Iron Chelator VLX600 Inhibits Mitochondrial Respiration and Promotes Sensitization of Neuroblastoma Cells in Nutrition-Restricted Conditions

“…Our study also observed several interesting phenomena. (1) Studies have shown that inhibiting mTOR signaling could deregulate MYCN-driven proliferation [14,18,19]. However, in this study, dual pan-class I PI3K and mTOR kinase inhibitor BEZ-235 [53] could deregulate mTOR activity but did not decrease the expression level of MYCN, indicating that regulating MYCN through mTOR may as well require another co-factor, such as mitochondrial inhibition.…”

“…Since the MYCN protein has not been directly druggable, an indirect inhibition of MYCN expression by targeting upstream or downstream components of the MYCN pathway is considered an alternative therapeutic option [13][14][15][16]. Oliynyk et al showed that MYCN induction upregulates the activity of glycolytic enzymes and mitochondrial oxidative phosphorylation in neuroblastoma [17], indicating that MYCN amplified childhood neuroblastoma could be more dependent on functional mitochondria.…”

“…Oliynyk et al showed that MYCN induction upregulates the activity of glycolytic enzymes and mitochondrial oxidative phosphorylation in neuroblastoma [17], indicating that MYCN amplified childhood neuroblastoma could be more dependent on functional mitochondria. In addition, several studies proved that inhibiting mTOR signaling to further downregulate cellular protein synthesis could deregulate MYCN-driven proliferation [14,18,19], suggesting that mTOR pathway is another key factor in MYCN-amplified children neuroblastoma.…”

MYCN in Neuroblastoma: “Old Wine into New Wineskins”

“…In the time course of research, it became evident that simultaneous inhibition of multiple targets could prove useful in the treatment of neuroblastoma. For example, a recent study has reported that the simultaneous inhibition of MYCN and mTOR using bromodomain extra-terminal protein inhibitors and temsirolimus manifested significant cell survival suppression [ 219 ]. Another study showed that the use of PARP and CHK1 inhibitors synergized to induce death in neuroblastoma cells and in primary cultures of MYCN amplified and MYCN overexpressing cells [ 220 ].…”