Comparing mTOR inhibitor Rapamycin with Torin-2 within the RIST molecular-targeted regimen in neuroblastoma cells

Abstract

The prognosis for patients with relapsed or refractory high-risk neuroblastoma remains dismal and novel therapeutic options are urgently needed. The RIST treatment protocol has a multimodal metronomic therapy design combining molecular-targeted drugs (Rapamycin and Dasatinib) with chemotherapy backbone (Irinotecan and Temozolomide), which is currently verified in a phase II clinical trial (NCT01467986). With the availability of novel and more potent ATP competitive mTOR inhibitors, we expect to improve the RIST combination therapy. By comparing the IC50 values of Torin-1, Torin-2, AZD3147 and PP242 we established that only Torin-2 inhibited cell viability of all three MycN-amplified neuroblastoma cell lines tested at nanomolar concentration. Single treatment of both mTOR inhibitors induced a significant G1 cell cycle arrest and combination treatment with Dasatinib reduced the expression of cell cycle regulator cyclin D1 or increased the expression of cell cycle inhibitor p21. The combinatorial index depicted for both mTOR inhibitors a synergistic effect with Dasatinib. Interestingly, compared to Rapamycin, the combination treatment with Torin-2 resulted in a broader mTOR pathway inhibition as indicated by reduced phosphorylation of AKT (Thr308, Ser473), 4E-BP (Ser65), and S6K (Thr389). Furthermore, substituting Rapamycin in the modified multimodal RIST protocol with Torin-2 reduced cell viability and induced apoptosis despite a significant lower Torin-2 drug concentration applied. The efficacy of nanomolar concentrations may significantly reduce unwanted immunosuppression associated with Rapamycin. However, at this point we cannot rule out that Torin-2 has increased toxicity due to its potency in more complex systems. Nonetheless, our results suggest that including Torin-2 as a substitute for Rapamycin in the RIST protocol may represent a valid option to be evaluated in prospective clinical trials for relapsed or treatment-refractory high-risk neuroblastoma.

Evaluating the RIST Molecular-Targeted Regimen in a Three-Dimensional Neuroblastoma Spheroid Cell Culture Model

“…The novel multimodal RIST treatment protocol combines two molecular targeted drugs, the mTOR inhibitor Rapamycin and the tyrosine-kinase inhibitor Dasatinib, as a ‘pre-treatment’, with conventional chemotherapeutics, the topoisomerase inhibitor Irinotecan and the alkylating agent Temozolomide ( Figure 4 ). Previous studies in monolayer cultures showed that ‘pre-treatment’ with Rapamycin plus Dasatinib (R+D) synchronizes NB cells in the cell cycle G1 phase and chemosensitizes them towards conventional ‘chemo-treatment’ with Irinotecan plus Temozolomide (I+T) [ 43 ].…”

The quest to develop an effective therapy for neuroblastoma

“…This study demonstrated the beneficial effects of combining FDA approved drugs in treating NB and confirmed that combination studies needed careful planning to provide optimal effectiveness of approved drugs. Another Phase 2 clinical study evaluated the effectiveness of Rapamycin and Dasatinib with Irinotecan and Temozolomide (Waetzig et al, 2021) in NB. This study indicated that combining advanced ATP competitive mTOR inhibitors with this therapeutic protocol resulted in improved outcomes (Waetzig et al, 2021;Xie et al, 2016).…”

“…Another Phase 2 clinical study evaluated the effectiveness of Rapamycin and Dasatinib with Irinotecan and Temozolomide (Waetzig et al, 2021) in NB. This study indicated that combining advanced ATP competitive mTOR inhibitors with this therapeutic protocol resulted in improved outcomes (Waetzig et al, 2021;Xie et al, 2016). In a Phase 2 clinical study anaplastic lymphoma kinase (ALK) was identified as a potential therapeutic target in NB.…”

Therapeutic Targeting of the Anaplastic Lymphoma Kinase (ALK) in Neuroblastoma—A Comprehensive Update

“…Crizotinib monotherapy down-regulated PI3K but not mTOR complex 1 (mTORC1) in cell lines harbouring MYCN amplifications [ 86 ]. The universal mTOR inhibitor Torin-2 effectively inhibited both mTOR complexes [ 87 ]; however, the loss of the feedback mechanisms rescued Akt phosphorylation in ultra-high-risk NBL [ 86 ]. Crizotinib in combination with the dual PI3K-mTOR inhibitor gedatolisib abrogated this Akt activation and also demonstrated effectiveness in less aggressive NBL, for instance, in ALK R1275Q mutated tumours [ 86 ].…”