Ovarian Cancer Susceptibility and Chemosensitivity to KRAS Modulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates whether perturbing KRAS signaling can enhance sensitivity to cisplatin and paclitaxel in ovarian cancer, particularly in high-grade, aggressive tumors that frequently exhibit KRAS amplification and chemoresistance. The study uses multiple experimental models, and the overall concept is potentially impactful for the development of combination therapeutic strategies. However, several points require clarification and additional analyses to improve the strength of the paper

Major Comments

1. It would be good to validate the efficiency of KRAS knockdown/knockout (KD/KO) across the study. KRAS KD in Figure 1 is modest, and the validation is not shown in the other figures. Would be nice to check KRAS downstream pathway inhibition (e.g., KRAS and pERK by western blot, or DUSP6 expression by qPCR). Additionally, Tyler jacks paper published in Nature Communications (PMID: 29061961) shows good Kras KO. The author might want to consider using the sgRNA in the paper.

2. Statistical analyses are described in the text but are not shown in the figures, which makes reading challenging. Performing statistical analyses for all the comparisons and labeling significance directly on the figures would improve readability and clarity.

3. The manuscript discusses the additive or synergistic effects of KRAS perturbation with chemotherapy. However, these interactions are not quantified. Performing standard drug-interaction analyses such as ZIP or BLISS would allow a more objective assessment. 

4. Additional clarification about the rationale for using SKOV-3 cells would be helpful. Specifically, it would be important to state whether this line harbors KRAS amplification. Given that the study is motivated by the link between KRAS amplification and chemoresistance, experimentally comparing cisplatin/paclitaxel sensitivity in cell models with low versus high KRAS copy number would strengthen the rationale.

5. Figure 1C: Morphological changes are described and quantified in the text but not shown in the figures. Including the quantification and statistical analysis directly in the figures would improve clarity.

6. Figure 1D: It would be clearer to separate 2D and 3D experiments and directly compare parental, control, and KRAS-KO cells within each condition. Performing statistical analysis would make the results easier to interpret.

7. Figure 2: The normalization of cisplatin IC50 values is confusing. It would improve interpretation to show exact IC50 values for control and KRAS-inhibited cells across doxycycline concentrations without normalization.  Paclitaxel data are missing from this figure, and it would be helpful to include them.

8. The differences between control, scrambled, and KRAS-targeting oligonucleotides are not obvious. Including statistical analyses would help clarify the significance.

9. The enhancement of paclitaxel response appears relatively modest. ZIP or BLISS analyses would help determine the additive or synergistic effects.

10. The experimental design in Figure 5 is well conceived. BLISS or ZIP analyses would further strengthen the conclusion that combination effects are additive or synergistic.

11. BI2865 enhances chemosensitivity. However, the concentrations used in SKOV-3 cells (up to 100 µM) may limit clinical relevance. Testing other drugs, such as RAS(ON) inhibitors (RMC-6236), could strengthen translational implications and help address potential compensation by other wild-type RAS.

Author Response

Comment 1: It would be good to validate the efficiency of KRAS knockdown/knockout (KD/KO) across the study. KRAS KD in Figure 1 is modest, and the validation is not shown in the other figures. Would be nice to check KRAS downstream pathway inhibition (e.g., KRAS and pERK by western blot, or DUSP6 expression by qPCR). Additionally, Tyler jacks paper published in Nature Communications (PMID: 29061961) shows good Kras KO. The author might want to consider using the sgRNA in the paper.

Response 1: Thank you for your recommendations. We have added functional analysis of KRAS under conditions of pharmacological inhibition through ERK/pERK western blotting, in addition to the exising KRAS western blots and qPCR in the preious version. Our intent with the CRISPR study was partial knockdown to better represent possible effects with pharmacological agents that are unlikely to abolish expression or function.

Comment 2:Statistical analyses are described in the text but are not shown in the figures, which makes reading challenging. Performing statistical analyses for all the comparisons and labeling significance directly on the figures would improve readability and clarity.

Response 2: We have added references to statistical outcomes on the figures (1-3).

Comment 3: The manuscript discusses the additive or synergistic effects of KRAS perturbation with chemotherapy. However, these interactions are not quantified. Performing standard drug-interaction analyses such as ZIP or BLISS would allow a more objective assessment. 

Response 3: ZIP analyses were added to the manuscript, particularly for figures 4 and 5 as described under methodology.

Comment 4: Additional clarification about the rationale for using SKOV-3 cells would be helpful. Specifically, it would be important to state whether this line harbors KRAS amplification. Given that the study is motivated by the link between KRAS amplification and chemoresistance, experimentally comparing cisplatin/paclitaxel sensitivity in cell models with low versus high KRAS copy number would strengthen the rationale.

Response 4: The current work began with the most commonly utilized ovarian cancer cell line, SKOV3, with physiological to slightly elevated KRAS expression and was expanded into Kuramochi cells, as described, to highlight exactly what the reviewer mentions – Cisplatin and Paclitaxel sensitivity in a lower and higher KRAS expression set of cells (as is also described in the referenced literature), and the modification of their toxicity with varied KRAS inhibition in the setting of low and high expression.

Comment 5: Figure 1C: Morphological changes are described and quantified in the text but not shown in the figures. Including the quantification and statistical analysis directly in the figures would improve clarity.

Response 5: Quantification and statistical analysis was added to the figure

Comment 6: Figure 1D: It would be clearer to separate 2D and 3D experiments and directly compare parental, control, and KRAS-KO cells within each condition. Performing statistical analysis would make the results easier to interpret.

Response 6: The requested format was updated and statistics added to the graphs.

Comment 7: Figure 2: The normalization of cisplatin IC50 values is confusing. It would improve interpretation to show exact IC50 values for control and KRAS-inhibited cells across doxycycline concentrations without normalization.  Paclitaxel data are missing from this figure, and it would be helpful to include them.

Response 7: Normalization was removed, exact IC50s are included, and paclitaxel data is graphed.

Comment 8: The differences between control, scrambled, and KRAS-targeting oligonucleotides are not obvious. Including statistical analyses would help clarify the significance.

Response 8: Graphs were updated to show the IC50s and to include statistical analysis of their significance.

Comment 9: The enhancement of paclitaxel response appears relatively modest. ZIP or BLISS analyses would help determine the additive or synergistic effects. and The experimental design in Figure 5 is well conceived. BLISS or ZIP analyses would further strengthen the conclusion that combination effects are additive e or synergistic.

Response 9: ZIP analyses was added to figures 4 and 5.

Comment 10. BI2865 enhances chemosensitivity. However, the concentrations used in SKOV-3 cells (up to 100 µM) may limit clinical relevance. Testing other drugs, such as RAS(ON) inhibitors (RMC-6236), could strengthen translational implications and help address potential compensation by other wild-type RAS.

Response 10: Thank you for these salient points. Notes were added about the proof-of-concept concentrations of BI2865 used, and references were made to the RAS(ON) inhibitor in the discussion (lines 592-600) for future studies.

Reviewer 2 Report

Comments and Suggestions for Authors

Peer Review Report

Manuscript Title:
Ovarian Cancer Susceptibility and Chemosensitivity to KRAS Modulation

Journal: International Journal of Molecular Sciences (IJMS)

General Assessment

This manuscript presents a comprehensive and methodologically rigorous investigation into the role of KRAS modulation as a chemosensitization strategy in ovarian cancer. By integrating multiple orthogonal approaches—CRISPR/Cas9-mediated knockdown, inducible Tet-ON regulation, promoter-targeting PPRH oligonucleotides, and pharmacological KRAS inhibition—the authors provide a compelling and internally consistent dataset demonstrating that partial KRAS suppression enhances chemotherapeutic efficacy, particularly for taxane-based therapies.

The inclusion of both 2D and 3D culture systems, alongside validation in the high-grade serous ovarian cancer (HGSOC)–representative Kuramochi cell line, significantly strengthens the translational relevance of the study. The observed reversal of paclitaxel resistance in 3D spheroids following KRAS modulation is especially notable and represents a clear advance over prior work focused solely on monolayer cultures.

Overall, this is a strong and well-executed study that is well aligned with the scope and readership of IJMS. With targeted revisions to improve mechanistic depth, clarity around synergy analyses, and contextual framing, the manuscript would be suitable for publication.

Major Points for Consideration

1. Formal synergy analysis
• While the manuscript repeatedly refers to “synergistic” versus “additive” effects (particularly for paclitaxel combinations), synergy is inferred primarily from IC₅₀ shifts and curve behavior.

The authors’ use an extra sum-of-squares F test to evaluate statistically significant shifts in IC₅₀ values between single-agent and combination treatments. This approach is appropriate for determining whether best-fit dose–response parameters differ between conditions. However, it does not explicitly test for pharmacologic synergy relative to an additivity reference model; rather, it compares curve parameters across datasets without defining an expected additive response.

• Recommendation:
  Include a formal synergy analysis (e.g., Bliss independence, Loewe additivity, or Combination Index methods) for at least the key BI2865 + paclitaxel datasets. This would strengthen claims of true synergy.
1. Mechanistic validation
• The Discussion provides an excellent mechanistic framework linking KRAS signaling to microtubule dynamics, mitotic checkpoints, and taxane sensitivity; however, these mechanisms are not directly interrogated experimentally.
• Recommendation:
  While full mechanistic dissection may be beyond scope, the authors could strengthen the manuscript by:
• Adding limited validation (e.g., changes in ERK/AKT signaling, tubulin isotype expression, or apoptosis markers), or
• Explicitly clarifying that mechanistic conclusions are inferential and hypothesis-generating.
2. Doxycycline-related confounding in Tet-ON system
• The manuscript appropriately acknowledges doxycycline toxicity at higher concentrations, but interpretation of sensitization effects could still be confusing for some readers.
• Recommendation:
  Clarify more explicitly in the Results or figure legends that the observed optimal sensitization at intermediate doxycycline concentrations reflects a balance between KRAS knockdown and doxycycline-induced cytotoxicity.
3. Scope of PPRH conclusions
• PPRH oligonucleotides show chemosensitization in 2D but could not be evaluated in 3D due to delivery limitations.
• Recommendation:
  The Discussion could more clearly separate proof-of-principle biological effects from translational feasibility, emphasizing delivery challenges and the need for improved in vivo delivery systems.

Minor Comments

• Clarify whether BI2865 concentrations used in 3D cultures are within physiologically achievable ranges or are intended as proof-of-concept.
• Ensure consistent units (µM vs nM) are clearly labeled across figures to avoid reader confusion.
• A brief schematic summarizing how KRAS modulation alters spheroid architecture and drug response could improve accessibility.

Overall Recommendation

Recommendation: Minor Revision

This manuscript is scientifically sound and thoughtfully designed. The requested revisions primarily involve strengthening quantitative rigor and clarifying interpretation rather than addressing fundamental flaws.

Author Response

Comment 1: Include a formal synergy analysis (e.g., Bliss independence, Loewe additivity, or Combination Index methods) for at least the key BI2865 + paclitaxel datasets. This would strengthen claims of true synergy.

Response 1: ZIP analysis were performed and are included for figures 4 and 5.

Comment 2: While full mechanistic dissection may be beyond scope, the authors could strengthen the manuscript by adding limited validation (e.g., changes in ERK/AKT signaling, tubulin isotype expression, or apoptosis markers).

Response 2: Changes in ERK phosphorylation status were added for all KRAS inhibition studies in figures 4 and 5.

Comment 3: Clarify more explicitly in the Results or figure legends that the observed optimal sensitization at intermediate doxycycline concentrations reflects a balance between KRAS knockdown and doxycycline-induced cytotoxicity.

Response 3: Recommended text was added on lines 194-196

Comment 4: The Discussion could more clearly separate proof-of-principle biological effects from translational feasibility, emphasizing delivery challenges and the need for improved in vivo delivery systems.

Response4: See text on lines 625-628

Reviewer 3 Report

Comments and Suggestions for Authors

This study employs multiple complementary approaches to investigate KRAS modulation as a chemosensitization strategy in ovarian cancer. The experimental design is systematic, and the findings hold significant potential for overcoming drug resistance. However, several issues must be addressed to enhance the manuscript's rigor, clarity, and translational relevance.

 

1. The proposed mechanism that KRAS knockdown alters 3D architecture to improve drug penetration remains speculative. Direct experimental evidence (e.g., fluorescent drug uptake assays in 3D spheroids) should be provided to support this claim.

 

2. The authors only performed in vitro experiments. Validation in relevant animal models is critically needed to strengthen the translational impact of the findings.

 

3. Many figures have no statistic analysis, such as Figure 4A

 

4. There appears to be a citation error in line 300, where "Figure 3B" is referenced. Based on the context, this likely refers to "Figure 4B".

 

5. The statement confirmed the use of AI (Claude Sonnet 4.5). Please specify which sections received AI-assisted editing in detail.

 

6. To further support the conclusions regarding enhanced cell death, the authors should analyze apoptosis and cell cycle.

 

7. Western blot analysis of key signaling proteins (e.g., p-ERK, p-AKT, BCL-2, Survivin) after KRAS inhibition is necessary to elucidate the underlying molecular mechanisms driving the observed chemosensitization.

Author Response

Comment 1: The proposed mechanism that KRAS knockdown alters 3D architecture to improve drug penetration remains speculative. Direct experimental evidence (e.g., fluorescent drug uptake assays in 3D spheroids) should be provided to support this claim.

Response 1: As the reviewer correctly highlights, there is a limitation of our supposition. Neither paclitaxel nor cisplatin are fluorescent and allow for penetrance validation, however, and given financial constraints, the suggested studies are beyond the scope of the current works.

Comment 2: The authors only performed in vitro experiments. Validation in relevant animal models is critically needed to strengthen the translational impact of the findings.

Response 2: The findings presented, while thorough across an array of molecular and pharmacological modulations of KRAS, are in vitro only. 3D systems are more recapitulative of in vivo findings, but our work is unable to extend into orthotopic or xenograft models at this point due to both time and financial constraints.

Comment 3: Many figures have no statistic analysis, such as Figure 4A

 Response 3: We have amended the figures (all), and statistics have been added, as have ZIP analyses.

Comment 4: There appears to be a citation error in line 300, where "Figure 3B" is referenced. Based on the context, this likely refers to "Figure 4B".

Response 4: Thank you – that is fixed.

Comment 5: The statement confirmed the use of AI (Claude Sonnet 4.5). Please specify which sections received AI-assisted editing in detail.

Response 5: AI was used on the manuscript as a whole only to highlight any grammatical errors, no significant editing was performed.

Comment 6: To further support the conclusions regarding enhanced cell death, the authors should analyze apoptosis and cell cycle.

 Response 6: We agree that extensive examination into the mechanisms of cell death, as well as the mechanisms of synergy is an intriguing study suggested by the presented works. That work, however, is beyond the scope of the current manuscript.

Comment 7: Western blot analysis of key signaling proteins (e.g., p-ERK, p-AKT, BCL-2, Survivin) after KRAS inhibition is necessary to elucidate the underlying molecular mechanisms driving the observed chemosensitization.

Response 7: pERK/ERK analyses were added for SKOV and Kuramochi cells in figures 4 and 5 to examine functional changes.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the authors' responses to my comments. The revisions have improved the manuscript's clarity and provided stronger support for the conclusion.  I have no further major comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The author satisfactorily addressed most of the issues that the reviewers were concerned about in the revised manuscript. Key issues such as the lack of statistical analysis, citation errors, and disclosure of artificial intelligence usage have been corrected, and additional Western blot data have been incorporated to strengthen the mechanism discussion. For the remaining points, further experimental verification is not feasible. The author has appropriately acknowledged these limitations in the text and identified them as future research directions. The manuscript has now seen significant improvements in clarity and scientific rigor, and I support its publication.