The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas

Round 1

Reviewer 1 Report

In this manuscript, Elisabet Ampudia Mesias et al. investigated the role of the OTX2 gene in promoting tumor growth and triggering leptomeningeal metastasis in Group 3 medulloblastomas (MBs) by regulating the mTORC2 signaling pathway. Through experimentation with cell lines and mouse models, the study demonstrates that OTX2 overexpression enhances cell growth, motility, and polarization, leading to reduced survival and metastasis in mice. Mechanistically, OTX2 transcriptionally activates the mTOR gene's promoter and the mTORC2 pathway, correlating with increased expression of genes associated with cell motility and migration. Clinical analysis of human MB tumor samples confirms a positive correlation between OTX2 and mTOR mRNA expression, suggesting clinical significance. Furthermore, treatment with mTOR inhibitors reduces cell viability, suggesting potential therapeutic avenues through mTORC2 inhibition. Limitations include the focus on in vitro and murine models, highlighting the need for further clinical validation of therapeutic strategies targeting the OTX2-mTORC2 axis in human MB patients.

In this manuscript, Elisabet Ampudia Mesias et al. investigated the role of the OTX2 gene in promoting tumor growth and triggering leptomeningeal metastasis in Group 3 medulloblastomas (MBs) by regulating the mTORC2 signaling pathway. Through experimentation with cell lines and mouse models, the study demonstrates that OTX2 overexpression enhances cell growth, motility, and polarization, leading to reduced survival and metastasis in mice. Mechanistically, OTX2 transcriptionally activates the mTOR gene's promoter and the mTORC2 pathway, correlating with increased expression of genes associated with cell motility and migration. Clinical analysis of human MB tumor samples confirms a positive correlation between OTX2 and mTOR mRNA expression, suggesting clinical significance. Furthermore, treatment with mTOR inhibitors reduces cell viability, suggesting potential therapeutic avenues through mTORC2 inhibition.

The comprehensive approach employed, including in vitro and in vivo experiments, strengthens the validity of the conclusions drawn. Overall, this research significantly advances our understanding of medulloblastoma biology and has the potential to translate into improved clinical outcomes for patients. A really good paper.

Here are some suggestions.

1. It would be beneficial to include more discussion on the implications of the findings and their potential applications in drug discovery and development.

2. The manuscript still requires some editing work with respect to grammar and sentence construction. Some sentences are too long and complex. Consider breaking them into shorter, simpler sentences to improve clarity and readability.

Minor editing of English language required

Author Response

Reviewer 1:

The comprehensive approach employed, including in vitro and in vivo experiments, strengthens the validity of the conclusions drawn. Overall, this research significantly advances our understanding of medulloblastoma biology and has the potential to translate into improved clinical outcomes for patients. A really good paper.

 Here are some suggestions.

Comment 1: It would be beneficial to include more discussion on the implications of the findings and their potential applications in drug discovery and development.

Our Response: As suggested, we have included a section in the discussion highlighting the potential applications of our findings in drug discovery and development for MBs.

Comment 2: The manuscript still requires some editing work with respect to grammar and sentence construction. Some sentences are too long and complex. Consider breaking them into shorter, simpler sentences to improve clarity and readability.

Our Response: As suggested, the manuscript was now edited by Prof. Dr. Christopher L. Moertel renowned oncologist and expert on MBs.

Reviewer 2 Report

In this study, the authors investigate the role of OTX2 in malignancy transformation of Group 3 medulloblastoma. The findings demonstrate the essential role of OTX2 in medulloblastoma metastasis via OTX2-mediated mTORC pathway, suggesting its potential as alternative therapeutic strategy in treating group 3 medulloblastoma. The study is interesting, and the findings are attractive. The following are comments for the study:

1)     Please include the protein validation result (Western Blot) for the siRNA-mediated knock-down cell lines in Figure 7.

2)      Please replot the drug inhibition curve and obtain the IC50 in Figure 9.

Author Response

Reviewer 2:

In this study, the authors investigate the role of OTX2 in malignancy transformation of Group 3 medulloblastoma. The findings demonstrate the essential role of OTX2 in medulloblastoma metastasis via OTX2-mediated mTORC pathway, suggesting its potential as alternative therapeutic strategy in treating group 3 medulloblastoma. The study is interesting, and the findings are attractive. The following are comments for the study:

Comment 1: Please include the protein validation result (Western Blot) for the siRNA-mediated knockdown cell lines in Figure 7.

Our Response: We conducted validation studies for the siRNA-mediated gene silencing experiments presented in Figure 7. Our results demonstrated a significant reduction in the mTOR mRNA levels (~80-85 %), as confirmed by quantitative PCR (qPCR). Due to time constraints for this revision, we opted for qPCR validation instead of Western blot analysis. These validation experiments strengthen the reliability and robustness of our gene silencing data. This data is now presented as supplementary Fig. 3.

Comment 2: Please replot the drug inhibition curve and obtain the IC50 in Figure 9.

Our Response: We have reanalyzed the experiments presented in Figure 9. The data has been replotted to include IC50 curves, and the revised figures are now presented as Figure 9 and Figure 10 in the manuscript. These changes improve the clarity and interpretation of the experimental results, aligning with the reviewer's suggestion.

Author Response File: Author Response.pdf