Review Reports - Unraveling the Impact of KRAS Accessory Proteins on Oncogenic Signaling Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Vanshika Garget et al. submitted a manuscript entitled “Unraveling the Impact of KRAS Accessory Proteins on Oncogenic Signaling Pathways.” This work investigates the effects of the deletion of selected KRAS modulators (GAL3, PDEδ, NPM1, IQGAP1, and SHOC2) on the activation of MAPK and PI3K–AKT signaling pathways.The manuscript is well-structured and clearly written. The authors could have conducted the experiments more thoroughly to better support their hypothesis. The approaches used are appropriate, and the experimental design aligns with the research objectives. However, the authors could have briefly explained, in at least one sentence, why they employed different proliferation assays for the various cell lines. The data are clearly presented in the form of figures, graphs, and tables, which allow for well-supported conclusions.To further strengthen the quality of the article, I suggest considering the following comments:

For each cancer type (lung, pancreatic, and colorectal carcinoma), the authors could have used at least three KRAS(G12V)-mutant cell lines derived from patients of different ages and sexes to increase the robustness of the findings.
It would be beneficial if the authors deleted the selected genes in all cell lines and compared their behavior to determine whether the observed effects are specific to a particular cell line or represent a common feature of KRAS(G12V)-mutant cancer lines. For instance, GAL3 and PDEδ were deleted only in the SW-480 cell line and not in others. Since these two accessory proteins were proposed as potential targets for combination therapy, it would be valuable to assess the effects of their deletion on proliferation across multiple cell lines.

Author Response

Authors’ response: We thank the reviewer for the positive evaluation of the manuscript's structure, clarity, and experimental design. We also appreciate the comment regarding the depth of the experiments. In this study, we conducted a controlled comparative analysis of five KRAS-associated accessory proteins in KRAS(G12V)-associated adenocarcinoma cell models and a KRAS wild-type HEK-293 cell line. We validated each knockout at the protein level and assessed the functional consequences using defined signaling readouts (MAPK and AKT) and quantitative proliferation assays. All experiments were performed as three independent biological replicates. We believe this framework proves the principle of mapping the functional roles of KRAS-associated accessory proteins within the scope of this study. All changes to the revised manuscript are highlighted in yellow.

However, the authors could have briefly explained, in at least one sentence, why they employed different proliferation assays for the various cell lines.

Authors’ response: Different proliferation assays were employed based on the growth characteristics of each cell line and the required sensitivity for reliable quantification. Direct viable cell counting with trypan blue staining reliably measured the proliferation of GAL3, PDEδ, and NPM1 knockouts. However, manual counting was insufficient for detecting subtle differences in Capan-1 cell proliferation due to their slower growth rate. Thus, the CellTiter-Blue metabolic assay was used to increase sensitivity. The rationale behind this decision is explained in the Methods section.

The data are clearly presented in the form of figures, graphs, and tables, which allow for well-supported conclusions.

Authors’ response: We thank the reviewer for the positive feedback. Our goal was to present our figures and quantitative analyses in a clear and transparent manner, allowing readers to evaluate the robustness of our findings and conclusions.

To further strengthen the quality of the article, I suggest considering the following comments:

For each cancer type (lung, pancreatic, and colorectal carcinoma), the authors could have used at least three KRAS(G12V)-mutant cell lines derived from patients of different ages and sexes to increase the robustness of the findings.

Authors’ response: We thank the reviewer for this valuable suggestion. We agree that inclusion of multiple KRAS(G12V)-mutant cell lines derived from patients of different ages and sexes could further enhance the robustness of the study.

In this work, we selected well-established and extensively characterized KRAS(G12V)-mutant cell lines for lung, pancreatic, and colorectal carcinomas, which are widely used as representative models of KRAS-driven oncogenic signaling. These cell lines were chosen not only based on the availability of comprehensive genetic information, reproducibility, and relevance to the biological questions addressed but for the following reasons (see line 187): First, they all contain the G12V KRAS variant. Second, we tested various anti-KRAS antibodies and identified D2H12 (Cell Signaling) as one that exhibits high selectivity against KRAS(G12V) but not other KRAS variants, such as G12D and G12C (Supplementary Figure S1). This antibody is ideal for studying KRAS(G12V) complex formation with accessory proteins. To ensure reliability, all experiments were performed with multiple biological replicates and yielded consistent results across cancer types. While we acknowledge that patient age and sex may influence tumor heterogeneity, addressing this variable through a larger panel of cell lines is beyond the scope of the current study.

It would be beneficial if the authors deleted the selected genes in all cell lines and compared their behavior to determine whether the observed effects are specific to a particular cell line or represent a common feature of KRAS(G12V)-mutant cancer lines. For instance, GAL3 and PDEδ were deleted only in the SW-480 cell line and not in others. Since these two accessory proteins were proposed as potential targets for combination therapy, it would be valuable to assess the effects of their deletion on proliferation across multiple cell lines.

Authors’ response: We thank the reviewer for this valuable suggestion. GAL3 and PDEδ were selectively knocked out in SW-480 cells due to their higher expression levels compared to those in Capan-1 and SHP-77 cells (see Supplementary Figure S1). This strategy was chosen to obtain clear signaling and phenotypic readouts in a high-expression context. While extending these knockouts to additional KRAS(G12V) models could further explore context dependence, this is beyond the scope of the current study. This limitation is acknowledged in the Discussion section.

Reviewer 2 Report

Comments and Suggestions for Authors

Oncogene KRAS drives tumor/cancer growth by activating signaling pathways including MAPK,
PI3K-AKT, etc. In clinical practice, direct KRAS inhibitors could be used for treatments, but it is limited due to therapeutic resistance and toxicity. This research investigated the potential/alternative combinatorial therapeutic strategies for tumor/cancer by knocking out 5 KRAS-related accessory proteins in 5 cancer cell lines, and got very useful findings. Overall the experimental design, methodology, data collection and interpreation are all nice. A few minor issues may need to be improved in the reviewer's eyes.

Too many keywords and the Introduction is too long.
Representative cell culture images (especially immunostaining images) are needed except those Immunoblotting supplements.
Representative cell images in proliferation and migration are also needed.

Author Response

Oncogene KRAS drives tumor/cancer growth by activating signaling pathways including MAPK, PI3K-AKT, etc. In clinical practice, direct KRAS inhibitors could be used for treatments, but it is limited due to therapeutic resistance and toxicity. This research investigated the potential/alternative combinatorial therapeutic strategies for tumor/cancer by knocking out 5 KRAS-related accessory proteins in 5 cancer cell lines, and got very useful findings. Overall the experimental design, methodology, data collection and interpretation are all nice.

Authors’ response: We thank the reviewer for the positive evaluation of our study, especially with regard to the experimental design, methodology, and interpretation. We also appreciate the recognition of the relevance of our investigation of KRAS accessory proteins as modulators of combinatorial strategies in KRAS-driven cancers. All changes to the revised manuscript are highlighted in yellow.

A few minor issues may need to be improved in the reviewer's eyes.

Too many keywords and the Introduction is too long.

Authors’ response: We thank the reviewer for this suggestion. We have reduced the number of keywords and streamlined the introduction by improving its conciseness while preserving the essential conceptual framework.

Representative cell culture images (especially immunostaining images) are needed except those Immunoblotting supplements.

Authors’ response: We appreciate this comment. The primary focus of this study is the analysis of signaling outputs resulting from ablation of the accessory proteins. Accordingly, we prioritized immunoblot-based validation and quantitative phenotypic assays derived from independent biological replicates. Original immunoblots and corresponding quantifications are provided in the Supplementary Materials. Immunostaining-based imaging has been included in the revised manuscript. A validated anti-IQGAP1 antibody was used for this purpose (see Supplementary Figure S2). This could not be done for the other accessory proteins because appropriate, validated antibodies are not yet available.

Representative cell images in proliferation and migration are also needed.

Authors’ response: We thank the reviewer for this comment. We agree that representative images can be informative. In our study, however, we primarily assessed proliferation and migration using quantitative assays, such as cell counting and metabolic/fluorescence-based assays. We also used wound closure quantification and Transwell assays when applicable. These assays provide objective, reproducible measurements. Representative images were not systematically acquired during these experiments because validated antibodies against most of the studied accessory proteins were unavailable. In the revised manuscript, we added immunostaining-based imaging using a validated anti-IQGAP1 antibody (see Supplementary Figure S2). We also now include images of the scratch assay for wild-type (WT) and knockout (KO) Capan-1 cells.

Reviewer 3 Report

Comments and Suggestions for Authors

This study systematically investigates the functional roles of five KRAS-associated accessory proteins (GAL3, PDE6, NPM1, IQGAP1, SHOC2) in KRAS(G12V)-mutant cancer cells. By employing CRISPR-Cas9 knockout technology combined with signaling pathway and phenotypic analyses, the authors reveal the non-redundant functions of these proteins in modulating MAPK and PI3K-AKT signaling.

1-Please provide additional details in the CRISPR-Cas9 KO verification. Quantitative data on knockout efficiency (e.g., sequencing traces, % indel frequency) would strengthen the methodology.

2- Clarify the nature of replicates (N=3). State explicitly whether these are biological replicates (independent cell culture experiments) or technical replicates.

3- Consider adding a brief description of the cell counting method (automated counter type) and the specific lysis conditions for immunoblotting (duration, temperature).

Author Response

Authors’ response: We thank the reviewer for the positive assessment of our study. All changes to the revised manuscript are highlighted in yellow.

Please provide additional details in the CRISPR-Cas9 KO verification. Quantitative data on knockout efficiency (e.g., sequencing traces, % indel frequency) would strengthen the methodology.

Authors' response: We thank the reviewer for this suggestion. We verified knockout efficiency at the protein level by immunoblotting and found a loss of the target proteins. Since our primary objective was to evaluate the signaling and phenotypic consequences of functional protein loss, we deemed protein-level validation to be the most relevant form of confirmation. The CRISPR-Cas9 workflow and knockout verification strategy are described in detail in the Methods section. We used ribonucleoprotein (RNP) complexes delivered by the Lonza Nucleofector system, which is widely used for efficient genome editing in cancer cell lines. Initially, we assessed knockout efficiency by immunoblotting, confirming a marked reduction or complete loss of the target protein across independent experiments. Due to cost limitations, we did not perform quantitative genomic analyses, such as Sanger sequencing–based indel decomposition or next-generation sequencing. Nevertheless, the observed functional phenotypes were consistently reproduced across multiple transfections and correlated with the degree of protein ablation, supporting effective gene knockout (KO). Additionally, we performed immunostaining-based imaging with a validated anti-IQGAP1 antibody (see Supplementary Figure S2) to confirm IQGAP1 knockout in Capan-1 cells. This could not be done for the other accessory proteins because appropriate, validated antibodies are not yet available.

Clarify the nature of replicates (N=3). State explicitly whether these are biological replicates (independent cell culture experiments) or technical replicates.

Authors’ response: Thank you for requesting clarification. All experiments were performed using three biological replicates (N = 3). These replicates are defined as three independent cell culture experiments conducted on different days using cultures that were maintained separately. This information is now explicitly stated in the Methods section.

Consider adding a brief description of the cell counting method (automated counter type) and the specific lysis conditions for immunoblotting (duration, temperature).

Authors’ response: We thank the reviewer for this suggestion. The methods section now describes the cell counting approach, which uses a Bio-Rad TC20 automated cell counter and a Neubauer chamber. It also describes the lysis conditions for immunoblotting. These conditions include an initial five-minute incubation on ice, followed by centrifugation at 20,000 RPM for an additional five minutes at 4°C.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for clarifying my comments.