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This review reflects comments and contributions by Teena Bajaj, Emma Phillips, Veronica Busa, Arpita Ghosh, Akanksha, Garima Jain, Sanskriti Saxena, Pragadeeshwara Rao R, Ghada Tagorti, Rebecca A Shelley, Isaac Arddey & Ruchika Bajaj. Review synthesized by Arpita Ghosh and Garima Jain.

Brief summary of the study:

In their manuscript “Exploring G-quadruplex structure in PRCC-TFE3 fusion Oncogene: Plausible use as anti cancer therapy for translocation Renal cell carcinoma (tRCC)”, Neha et al., explore putative quadruplex forming sequences (PQS) in the PRCC-TFE3, which they identified in a previous study. They use various biophysical methods and a PCR-stop assay to show that one of these PQSs can be stabilized by pyridostatin (PDS) in vitro, and perform cellular assays to visualize G-quadruplex formation, show an impact of the G-quadruplex on PRCC-TFE3 expression, and demonstrate a general translational inhibitory effect using a luciferase reporter assay.

Overall, the study is valuable and sheds light on the potential of G-quadruplex stabilization as an anti-cancer strategy. However, several aspects of the article need further clarification and improvement

Major comments:

• The experiments support the claims made by the authors and show that the G4Q sequence can form a G-quadruplex that can affect the levels of PRCC-TFE3. However, as there are no experiments addressing the effect of PDS on cancer cell phenotype -  proliferation, viability etc, it remains to be seen whether this finding will have an impact on the treatment of translocation renal cell carcinoma. Similarly, it would be good to demonstrate the reversal of the cancer phenotype by inhibition of formation. The impact of this study requires the direct correlation between cancer phenotype and formation of G-quadruplex. Currently, the conclusions of the manuscript as a future cancer cure are too central in the narrative without additional experiments. 

• Although clearly written, there are many grammatical errors in the manuscript. 

• The introduction and discussion sections need to be streamlined. The introduction should be more focused and immediately relevant to the manuscript, and the discussion should avoid redundant repetition of the results.

• The article lacks a critical review of existing literature on G-quadruplex structures and their relevance in cancer therapy. Including a comprehensive literature review would enhance the context and significance of the study.

• The limitations of the study are not adequately discussed. The paper relies solely on in vitro experiments and cellular models, and a discussion of translational challenges in targeting G-quadruplexes in vivo would strengthen the credibility of the findings.

• The conclusion should provide a more nuanced summary of the study's findings and their implications for future research and clinical applications. Additionally, suggestions for addressing the study's limitations and potential areas for further investigation would be valuable.

• The discussion lacks depth in interpreting the findings, and a more comprehensive analysis of the implications of the results on G-quadruplex formation in the PRCC-TFE3 fusion gene as a therapeutic target is needed.

• The methodology section requires more specific details about the experimental procedures, especially regarding the biophysical techniques used. This should include information on primers, probes, experimental conditions, and controls for the PCR stop assay, dual luciferase assays, and confocal microscopy.

Minor comments:

• Utilize a sub-figure format and combine small results paragraphs into more comprehensive 'story-arcs' to improve the organization of figures and data presentation.

• Increase the text size for figure axes, labels, and legends in figures 1-5 and 8-10 for better readability.

• In Figure 6, include the quantification of bands along with statistics, in addition to the gel presentation.

• Provide a brief explanation of the tools (QGRS mapper and nBMST online server) mentioned in the abstract and their relevance in the study.

• Support the statement about the TFE3 fusion gene as a diagnostic marker for translocation renal cell carcinoma with more context and evidence in the introduction.

• Include a legend in Figure 1 explaining the abbreviations used (e.g., G4Q, G4M, TelQ) for better understanding.

• Make the x-axis label in Figure 8 more descriptive to specify the type of constructs being compared.

• Clarify what "relative expression" refers to in Figure 10 and explain its relevance.

• Elaborate on how G-quadruplex stabilization affects specific biological processes related to PRCC-TFE3 fusion gene expression.

• Be cautious with conclusive statements regarding G-quadruplex presence inside the PRCC segment of the fusion gene unless it is supported by stronger evidence.

• Each figure should have a comprehensive caption.

• Clearly state which figure is being referred to when discussing results in the text.

• Include information on the statistical analyses performed and report p-values or measures of statistical significance when appropriate.

• Consider adding controls using lower concentrations of KCl or LiCl to confirm the formation of parallel G-quadruplexes.

• Provide clarification about the use of Anti FLAG in the materials and methods section or results section.

• Consider giving subtopics titles reflecting major findings in the paragraph rather than naming the method used.

• Ensure clarity on whether the cell line used has the PRCC-TFE3 fusion gene and consider confirming this in another cell line.

• Label the treatments A, B, and C in Figure 9 for easier readability.

• Clarify the direct impact of G4Q27 motif on translation, as it has been shown to affect gene expression at the transcriptional level.

• Discuss any potential connection between PDS involvement in G-quadruplex stabilization and telomere disruption.

Suggestions for future studies:

• Investigate the effect of PDS on the phenotype of tRCC cell lines, particularly comparing cells with the PRCC-TFE3 fusion gene to their wild-type counterparts.

• Utilize in vivo studies with animal models (xenograft or transgenic mouse models) to understand the impact of G-quadruplex stabilization on the development and progression of tRCC.

• Explore the potential of combining G-quadruplex stabilization with existing or emerging targeted therapies for tRCC in combination therapies.

• Investigate the presence and impact of G-quadruplex structures in fusion oncogenes associated with other types of cancers beyond tRCC.

• By addressing these suggestions in future studies, the research can further enhance our understanding of G-quadruplexes and their potential as therapeutic targets in cancer treatment, particularly in the context of translocation renal cell carcinoma.

Competing interests

The author declares that they have no competing interests.