Write a comment

Does the introduction explain the objective of the research presented in the preprint?

Yes

The introduction explains the research objective by first establishing the clinical problem of apoptosis resistance in cancer, then presenting ferroptosis as a promising alternative cell death pathway. It identifies the poor understanding of ferroptosis resistance mechanisms as the key knowledge gap and introduces NRF2 and FSP1 as critical antioxidant regulators worth investigating. The introduction further justifies using TSC2-deficient models due to their relevant mTOR/iron dysregulation, ultimately culminating in a clear statement of purpose: to explore these survival mechanisms in TSC and cancer cells to identify targetable therapeutic vulnerabilities.

Are the methods well-suited for this research?

Highly appropriate

The methods are highly appropriate because they employ a rigorous, multi-faceted strategy that follows best practices in mechanistic cell biology. The use of isogenic cell line pairs allows for direct attribution of ferroptosis resistance to TSC2 loss, while the combination of pharmacological inhibitors and genetic knockdown for both NRF2 and FSP1 provides robust, complementary evidence that controls for off target effects. Crucially, the authors move beyond simple viability assays by directly quantifying the hallmarks of ferroptosis, lipid peroxidation and labile iron pools, and employing transcriptomic analysis to validate mechanistic changes, creating a compelling and well supported chain of evidence for their conclusions.

Are the conclusions supported by the data?

Highly supported

The conclusions are highly supported by the data, as they directly reflect the key experimental findings without overinterpretation. The demonstrated resistance of TSC2 deficient cells to ferroptosis inducers is robustly shown across multiple models and rescue experiments, while the roles of NRF2 and FSP1 are rigorously validated through both pharmacological and genetic approaches. The context dependent efficacy of these pathways particularly the differential response to FSP1 inhibition between cell types is well documented and supported by mechanistic insights from transcriptomic data. The authors forward looking therapeutic suggestions are reasonable extrapolations from these validated results, ensuring the conclusions remain firmly evidence based and realistic.

Are the data presentations, including visualizations, well-suited to represent the data?

Somewhat appropriate and clear

The data presentations and visualizations are generally effective and appropriate for the study, employing well-chosen graphical methods including bar graphs for viability, flow cytometry plots for cell death, volcano plots for transcriptomics, western blots for protein analysis, and microscopy for morphological assessment, all of which clearly communicate the respective data types and support the conclusions. However, the absence of integrated quantitative densitometry with the western blot images slightly limits immediate interpretation, as readers must refer to supplementary data or text for full quantitative analysis, preventing a highest rating for accessibility despite the overall clarity and logical multi-panel organization.

How clearly do the authors discuss, explain, and interpret their findings and potential next steps for the research?

Somewhat clearly

The authors discuss their findings and outline next steps with reasonable clarity. The discussion effectively summarizes the key results, particularly the context dependent roles of NRF2 and FSP1 in mediating ferroptosis resistance across different cellular models. The interpretation of results is generally logical and supported by the data, with appropriate acknowledgment of the finding that FSP1 inhibition does not resensitize all cell types examined. The proposed next steps are relevant and grounded in the experimental findings. Suggestions include exploring targeted delivery systems for ferroptosis inducers, repurposing existing treatments known to trigger ferroptosis, and developing predictive biomarkers through transcriptomic analysis. These recommendations directly follow from the research outcomes and identify concrete avenues for future investigation. However, the discussion could be enhanced by a more detailed exploration of the underlying mechanisms behind the differential dependency on FSP1. While compensatory upregulation of NRF2 targets is noted, a deeper mechanistic hypothesis could strengthen this section. Additionally, explicitly connecting the findings back to mTORC1 signaling a key pathway disrupted in the disease models studied would provide a more comprehensive conceptual framework. The language is generally clear and professional, though some sections could be more concise. Overall, the discussion and interpretation are somewhat clear, successfully highlighting the significance of the findings while proposing logical next steps, but would benefit from greater mechanistic depth and tighter integration with the established biological context of the study systems.

Is the preprint likely to advance academic knowledge?

Highly likely

This preprint is highly likely to advance academic knowledge due to its novel mechanistic insights into ferroptosis resistance, particularly in TSC2-deficient and cancer models. It compellingly demonstrates that context-dependent activation of NRF2 and FSP1 pathways underlies this resistance, challenging simpler narratives and providing a nuanced framework for understanding redox adaptation in disease states. The rigorous multi-model approach, combining genetic and pharmacological tools with transcriptomic validation, offers a robust foundation that will stimulate further research into targeting these pathways. The advance is somewhat tempered by the lack of in vivo validation and the aforementioned need to more deeply explore the link to mTORC1. However, the strength, novelty, and clarity of the cellular data are sufficient to predict this work will stimulate further research, replication attempts, and new experimental avenues, thereby advancing academic knowledge. By explicitly linking its findings to therapeutic strategies and predictive biomarker development, the work effectively bridges fundamental discovery and translational application, positioning it to influence both basic research and clinical innovation in ferroptosis-targeting therapies.

Would it benefit from language editing?

No

While the manuscript contains occasional minor grammatical imperfections and stylistic inconsistencies, these issues are infrequent and do not detract from the overall clarity, precision, or comprehension of the scientific content. The language is professional, the methodology is described with precision, and the results and conclusions are presented in a logical and unambiguous manner. The few existing minor errors are common in early preprint versions and do not pose a barrier to understanding the study's significant contributions. Therefore, while light proofreading could enhance polish, the manuscript does not require substantive language editing to be clearly understood by an academic audience.

Would you recommend this preprint to others?

Yes, it’s of high quality

Yes, I would highly recommend this preprint for its novel and robust examination of ferroptosis resistance mechanisms in TSC2-deficient and cancer models. The study compellingly demonstrates context-dependent roles for NRF2 and FSP1 using rigorous multi-model validation, combining genetic and pharmacological approaches with strong biochemical and functional assays. Its findings challenge simplified views of ferroptosis regulation and offer significant translational implications for targeting treatment-resistant cancers.

Is it ready for attention from an editor, publisher or broader audience?

Yes, after minor changes

While the preprint presents robust and novel findings with strong methodological rigor, the following revisions are recommended to enhance its clarity, mechanistic depth, and readiness for broader audience engagement: Address Key Mechanistic Gaps: Test the role of mTORC1 using inhibitors (e.g., rapamycin) to directly link TSC2 loss to NRF2/FSP1 activation and ferroptosis resistance. Validate compensatory NRF2-FSP1 interactions in resistant models (e.g., AML cells) using combinatorial inhibition assays. Improve Data Presentation: Integrate quantitative bar graphs with statistical annotations for western blots (e.g., HMOX1 upregulation in Figure 2). Replace overlayed histograms (e.g., LPO assays in Figure 4i) with summary bar graphs (mean ± SEM) for clarity. Add quantifiable insets to flow cytometry plots (Figure 1a–b) and clarify heatmap labels (Figure 4h). Refine Interpretations: Temper claims about NRF2-FSP1 regulation to reflect transcriptional-level evidence only, pending protein-level validation. Discuss context-dependent FSP1 efficacy (e.g., cell-type-specific factors) more explicitly in the discussion. Polish Language and Clarity: Correct minor grammatical errors and awkward phrasing to improve readability. Downplay less specific data (e.g., Necrostatin-1 rescue) to avoid confusion. These changes will solidify the narrative, ensure broader accessibility, and align the manuscript with standards for high-impact publication.

Competing interests

The author declares that they have no competing interests.