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Published on: August 27, 2019
Comparing mTOR inhibitor Rapamycin with Torin-2 within the RIST molecular-targeted regimen in neuroblastoma cells
Rebecca Waetzig1, Marie Matthes1, Johannes Leister1
1Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, University Hospital of Regensburg, Franz-Josef-Strauss Allee 11, 93053, Regensburg, Germany.
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.
Insights
Novel mTOR inhibitor Torin-2 shows promise in treating high-risk neuroblastoma. Combining Torin-2 with Dasatinib synergistically reduced cancer cell viability and improved the RIST protocol, offering a potential new therapy.
Area of Science:
- Oncology
- Pharmacology
- Molecular Biology
Background:
- High-risk neuroblastoma with relapse or refractory disease has a poor prognosis, necessitating novel therapeutic strategies.
- The RIST protocol, a multimodal metronomic therapy, combines targeted drugs and chemotherapy and is under investigation in a Phase II clinical trial.
- Advancements in mTOR inhibitors offer opportunities to enhance existing combination therapies like RIST.
Purpose of the Study:
- To evaluate novel ATP-competitive mTOR inhibitors, specifically Torin-1, Torin-2, AZD3147, and PP242, as potential improvements to the RIST protocol.
- To assess the efficacy of Torin-2, alone and in combination with Dasatinib, in neuroblastoma cell lines, focusing on cell viability, cell cycle arrest, and pathway inhibition.
- To investigate the potential of substituting Rapamycin with Torin-2 in the RIST protocol for treating relapsed or refractory high-risk neuroblastoma.
Main Methods:
- Comparative analysis of IC50 values for four mTOR inhibitors against three MYCN-amplified neuroblastoma cell lines.
- Assessment of single and combination treatments (mTOR inhibitors + Dasatinib) on cell cycle progression and expression of key regulators (cyclin D1, p21).
- Evaluation of mTOR pathway inhibition by measuring phosphorylation of AKT, 4E-BP, and S6K.
- Inclusion of Torin-2 in a modified RIST protocol to assess its impact on cell viability and apoptosis compared to Rapamycin.
Main Results:
- Torin-2 demonstrated potent inhibition of neuroblastoma cell viability at nanomolar concentrations.
- Both Torin-1 and Torin-2 induced G1 cell cycle arrest, and their combination with Dasatinib showed synergistic effects.
- Torin-2 exhibited broader mTOR pathway inhibition compared to Rapamycin, with reduced phosphorylation of key signaling proteins.
- Substitution of Rapamycin with Torin-2 in the RIST protocol led to reduced cell viability and increased apoptosis, even at lower drug concentrations.
Conclusions:
- Torin-2 is a potent inhibitor of MYCN-amplified neuroblastoma cell lines and demonstrates synergistic effects when combined with Dasatinib.
- Replacing Rapamycin with Torin-2 in the RIST protocol may offer a more effective therapeutic option for relapsed or refractory high-risk neuroblastoma.
- Further clinical evaluation of Torin-2 in combination therapies for neuroblastoma is warranted, considering its potential for reduced immunosuppression and enhanced efficacy.
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