Investigation of cell signalings and therapeutic targets in PTPRK-RSPO3 fusion-positive colorectal cancer

Jae Heon Jeong; Jae Won Yun; Ha Young Kim; Chan Yeong Heo; Sejoon Lee

doi:10.1371/journal.pone.0274555

Peer Review History

Original SubmissionMarch 8, 2022
16 May 2022 Decision Letter - Suprabhat Mukherjee, Editor PONE-D-22-06899Investigation of cell signalings and therapeutic targets in PTPRK-RSPO3 fusion-positive colorectal cancer.PLOS ONE Dear Dr. Lee, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== The manuscript needs revision as suggested by the reviewer and myself. Please revise the manuscript and submit. However, submitting a revision does not guarantee the acceptance of the same. ============================== Please submit your revised manuscript by Jun 30 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Currently, your Funding Statement reads as follows: “This study was supported by a VHS Medical Center Research Grant, Republic of Korea (VHSMC 21038), grant no 18-2018-023 from the SNUBH Research Fund, and the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT)(No. 2022R1C1C1012986).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following financial disclosure: “This study was supported by a VHS Medical Center Research Grant, Republic of Korea (VHSMC 21038), grant no 18-2018-023 from the SNUBH Research Fund, and the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT)(No. 2022R1C1C1012986).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 5. One of the noted authors is a group or consortium “Chan Young Heo, Jae Won Yun”. In addition to naming the author group, please list the individual authors and affiliations within this group in the acknowledgments section of your manuscript. Please also indicate clearly a lead author for this group along with a contact email address. Additional Editor Comments (if provided): Oncogenesis events in CRC is indeed multifaceted and considering the impact of P:R fusion-positive CRC authors need to present a comparative view with the recently published papers citing the association of multiple signaling pathways in the course of CRC pathophysiology. Authors may follow and cite DOI: 10.3389/fgene.2021.608313 as well as relevant literature to improve the manuscript. Fig. 1 could be omitted and a scheme may be added at the last. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Jeong HJ et al. presented key indicators and clinically viable therapeutics for P:R fusion-positive CRC as well as inferred the actionable drugs in bioinformatics analysis using the Cancer Genome Atlas (TCGA) data. Through drug-target network analysis, several putative therapeutic candidates were shown effective as they were applied and involved in multiple cancer-pathways. The manuscript is generally well written and structured. However, in my opinion the data have some shortcomings in regards to some analyses, explanation, and logical sense. Comments: Line 82: the following sentences are not clear. 372 tumor samples with the barcode 01A were chosen, with other types of tumor samples, 11A (Normal) or 06A (Metastasized), being excluded. What do these numbers and barcodes referred to? - How was the permutation test calculated? As stated by the authors, the median of absolute R values, mostly affected 2,505 genes were selected by correlation cut-off (R > 0.2). What is the criteria to set a cut-off of R > 0.2? - What does median in Table S1 imply? Line 128: As stated, 7 patients demonstrated presence of the fusion mutation, whereas the remaining 416 patients were negative for the fusion based on the clinicpathological characteristics. These cases are not fully presented in the Table 1. The characteristics of PTPRK-RSPO3 fusion-positive and fusion-negative cases in TCGA colorectal cancer is not clear in Table 1. Moreover, what does median in Table S1 imply? All the supplementary Tables and figures should be renamed as indicated in Sup section including tittle of each and applied methods for data extraction. As Table 1 shows, none of the clinicopathological characteristics of selected cases is significant. Eighteen genes showed good correlation (R > 0.5) with RSPO3 in RNA expression. Is that correlation defines any significant RNA expression? Line 153: How come the ten different pathways were shown to be statistically significant? How was the significant means elaborated? Line 163: the following sentence “ten genes from highest R-values were as follows” is not clear. What is the implication of highest R-values? Fig 4 represents Drug-target relation obtained based on Civic and OncoKB databases. What was the intention to select these two databases? The VICC meta-database has recently been reported the most extensive source of information, featuring 92% of variants with a drug association (https://dx.doi.org/10.21873%2Fcgp.20250). Is there any explanation? Line 184: 4 genes (KRAS, FGFR2, ALK, and JAK2) were identified and they were all included in the inferred results using CIViC database. How the authors link the gene presence with their RNA expression involved in several pathways as well as disease conditions? Line 191: Of 19 druggable genes, five were involved in the multiple pathways. Have these genes as well as their related pathways experimentally reported? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Meysam Sarshar [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0274555.r001
Revision 1
29 Jun 2022 Author Response Response to Reviewer Comments We deeply appreciate the reviewer Meysam Sarshar’s attention to our paper. Below, we address each of the reviewer's comments point by point. We revised our manuscript using MS Word and highlighted the changes with yellow color. Point 1: Line 82: the following sentences are not clear. 372 tumor samples with the barcode 01A were chosen, with other types of tumor samples, 11A (Normal) or 06A (Metastasized), being excluded. What do these numbers and barcodes referred to? Response 1: Thank you for your comment. For better understanding of the TCGA data for readers, the following sentence has been modified as follows (Line: 78-79): “Of the 433 GDAC data downloaded, 53 normal and 1 metastasis data were omitted, and the remaining 379 data were analyzed.” The barcode contains information concerning the institution, patient, sample, and method of analysis. For instance, the two digits from 01A, 06A, 11A barcodes reveal information on the type of patient sample whether it be normal or tumor, and the alphabet indicates the vial containing the patient sample (https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/sample-type-codes). Although the majority of the TCGA data were tumor samples including 01-09, a portion of them, 10-19 were normal samples. Therefore, it was necessary to select and assess samples according to the purpose of analysis. We extracted primary solid tumor samples, which have barcodes 01, used for this study. Point 2: How was the permutation test calculated? As stated by the authors, the median of absolute R values, mostly affected 2,505 genes were selected by correlation cut-off (R > 0.2). What is the criteria to set a cut-off of R > 0.2? Response 2: Thank you for your detailed comment that improving our study. Of the 57 samples, we selected two genes and their RNA expression values were randomly ordered respectively, and Pearson correlation coefficient R value was obtained between two genes. This process was repeated 100,000 times. The resulting frequency of an R value higher than 0.2 by chance was 378 out of 100,000 times. Among the 2,505 genes that showed a correlation with RSPO3 of an R value of 0.2 or higher, approximately 0.378%, that is 20,531 gene multiplied by 0.00378 were estimated to yield 78 false positives. Hence, of the 2,505 genes, 2,427 genes may have a correlation with RSPO3. Point 3: What does median in Table S1 imply? (Moreover, what does median in Table S1 imply?) Response 3: Thank you for your comment. For instance, should KRAS mutation-positive samples occupy a majority of the 50 samples that were selected as control, there is a high likelihood for a large number of KRAS signature to be generated. In order to minimize the bias from undesired signature accumulation, 50 samples from the 186 samples expressing low RSPO3 expression were randomly selected as the control. Pearson R values were calculated between reference gene (RSPO3) and the other genes. Above process was repeated 100 times and the median R values were calculated in each gene. Point 4: Line 128: As stated, 7 patients demonstrated presence of the fusion mutation, whereas the remaining 416 patients were negative for the fusion based on the clinicopathological characteristics. These cases are not fully presented in the Table 1. The characteristics of PTPRK-RSPO3 fusion-positive and fusion-negative cases in TCGA colorectal cancer is not clear in Table 1. Response 4: Thank you for valuable comment. According to your comment, table 1 has been revised (Line: 138-140). The number of fusion and control samples were more clearly described in our study. The TCGA colorectal RNA samples used were extracted from the GDAC firehose. Of them, 379 were tumor solid samples, and of the 372 samples, excluding the 7 fusion-positive samples, 186 samples expressing less than 50% RSPO3 mRNA expression were selected as control (fusion-positive). The clinicopathological analysis was performed according to the criteria above and reflected in Table 1 and its pertinent text. We modified the sentences on line 123-126 as follows: “The clinicopathological characteristics in this study were described in Table 1. A total 379 tumor solid samples, and of the 372 samples, excluding the 7 fusion-positive samples, 186 samples expressing less than 50% RSPO3 mRNA expression were selected as control.” Point 5: All the supplementary Tables and figures should be renamed as indicated in Sup section including tittle of each and applied methods for data extraction. Response 5: Thank you for your comment. We revised the supplementary section on line 263-275 as follows: S1 Fig: Gene expression heatmap of cancer-related pathways enriched with genes correlated to RSPO3 in RNA expression. S2 Fig: The KEGG pathway maps for the human ERBB signaling pathway and pathways in cancer using the KEGG Mapper; genes correlated with RSPO3 expression are colored in pink. S3 Fig: Putative target genes involved in multiple pathways of PTPRK-RSPO3 fusion-positive cancer. S4 Fig: Inferred drug-target network in PTPRK-RSPO3 fusion-positive colorectal cancer based on VICC database. S1 Table: 2,505 genes correlated with RSPO3 (R >0.2). S2 Table: Putative target genes and actionable drugs involved in ten major cancer-related pathways. Point 6: As Table 1 shows, none of the clinicopathological characteristics of selected cases is significant. Eighteen genes showed good correlation (R > 0.5) with RSPO3 in RNA expression. Is that correlation defines any significant RNA expression? Response 6: Thank you for your comment. Since an overexpression of RSPO3 mRNA level is observed in PTPRK-RSPO3 fusion samples (PMID: 32631391 Figure 2B), we assumed that downstream genes affected by upregulated RSPO3 expression with fusion would have high R values in expression level by the Pearson correlation test. Due to the limited number (n=7) of RSPO3 fusion samples, it was not ideal to infer a statistically significant difference from clinical variable. Point 7: Line 153: How come the ten different pathways were shown to be statistically significant? How was the significant means elaborated? Response 7: Thank you for your comment. In summary, a P-value is calculated using the hypergeometric distribution. The P-value reflects the significance of the observed overlap between the input gene list and the module's members when compared to random expectations. A low P-value indicates that more of the module's members are present in the input list than would be expected by chance. The p-values are corrected for multiple testing using the false discovery rate method. Detailed explanation: PMID: 18940869 Point 8: Line 163: the following sentence “ten genes from highest R-values were as follows” is not clear. What is the implication of highest R-values? Response 8: Thank you for your comment. Since mRNA overexpression of RSPO3 occurs by PTPRK-RSPO3 fusion (PMID: 32631391 Figure 2B), it was determined that genes affected by expression downstream by upregulated RSPO3 would have high R values in the Pearson correlation test. The reason for selecting the 10 highest R-value genes is to find the most likely downstream pathway or target in the absence of a direct target therapy for RSPO3. Genes with the highest R values were extracted with the purpose of identifying therapeutic targets from the downstream pathway of RSPO3. Point 9: Fig 4 represents Drug-target relation obtained based on Civic and OncoKB databases. What was the intention to select these two databases? The VICC meta-database has recently been reported the most extensive source of information, featuring 92% of variants with a drug association (https://dx.doi.org/10.21873%2Fcgp.20250). Is there any explanation? Response 9: Thank you for your comment. The data provided by VICC meta-database is in the form of a JSON file. However, since the JSON files were broken and not unified in ordinary format, we had to manually parse every five databases (jax, brca, cgi, molecularmatch, pmkb) except CIViC and OncoKB. Then, we were only able to extract information about genes and drugs from jax, brca and molecularmatch databases. However, advanced information such as clinical significance, evidence direction and evidence type were limited to parse. For this reason, the analyzed results using VICC database were not included in the main figure but were included in the supplementary figures. We added explanation about VICC figures in our manuscript (Line: 270, 295). Point 10: Line 184: 4 genes (KRAS, FGFR2, ALK, and JAK2) were identified and they were all included in the inferred results using CIViC database. How the authors link the gene presence with their RNA expression involved in several pathways as well as disease conditions? Response 10: Thank you for your comment. Generally, there are three classes for activation signaling including hotspot mutation, amplification, and overexpression. For this study, RNA sequence-based overexpression was considered an activating signal. The drug database provides information on the relationship between the activating signaling (three classes mentioned above) and available drugs. For example, MET activating mutations are including amplification, over-expression, and activating point mutations and the three class of mutations are mostly sharing target-drug sensitivity (capmatinib, tepotinib). So, the three types of mutations were considered as showing similar target-drug sensitivity in silico level. Although the sensitivity of the drugs may differ according to the various types of signal activation, the purpose of this study is to enroll as many drugs with high potential as possible. It would be ideal for these hypotheses to be validated with further additional experimentations. However, the scope of this study does not encompass validation experiments and will take into consideration for future studies. Point 11: Line 191: Of 19 druggable genes, five were involved in the multiple pathways. Have these genes as well as their related pathways experimentally reported? Response 11: Thank you for your comment. While reviewing the content, five additional druggable genes involved multiple pathways were found and added to the manuscript (Line:217-230). The Consensus Path Database (CPDB, http://cpdb.molgen.mpg.de/CPDB) we utilized is a database combined by integrating 177 biological pathways based on experimentally reported studies: INOH (PMID: 2212066), KEGG (PMID: 27899662), NetPath (PMID: 20067622), PID (PMID: 18832364), Reactome (PMID: 26656494), and Wikipathways (PMID: 26481357). We attached a table of CPDB sources and 10 druggable genes involved in multiple pathways on below: Gene Source Pathway PIK3R1 KEGG Apoptosis PIK3R1 KEGG VEGFR Related Pathway PIK3R1 KEGG ErbB Related Pathway PIK3R1 KEGG JAK-STAT Pathway PIK3R1 KEGG EGFR PIK3R1 KEGG Tyrosine Kinases PIK3R1 KEGG Pathways In Cancer PIK3R1 KEGG SCF-KIT PIK3R1 Reactome Apoptosis PIK3R1 Reactome VEGFR Related Pathway PIK3R1 Reactome ErbB Related Pathway PIK3R1 Reactome JAK-STAT Pathway PIK3R1 Reactome EGFR PIK3R1 Reactome Tyrosine Kinases PIK3R1 Reactome Pathways In Cancer PIK3R1 Reactome SCF-KIT PIK3R1 Wikipathways Apoptosis PIK3R1 Wikipathways VEGFR Related Pathway PIK3R1 Wikipathways ErbB Related Pathway PIK3R1 Wikipathways JAK-STAT Pathway PIK3R1 Wikipathways EGFR PIK3R1 Wikipathways Tyrosine Kinases PIK3R1 Wikipathways Pathways In Cancer PIK3R1 Wikipathways SCF-KIT PIK3R1 PharmGKB Apoptosis PIK3R1 PharmGKB VEGFR Related Pathway PIK3R1 PharmGKB ErbB Related Pathway PIK3R1 PharmGKB JAK-STAT Pathway PIK3R1 PharmGKB EGFR PIK3R1 PharmGKB Tyrosine Kinases PIK3R1 PharmGKB Pathways In Cancer PIK3R1 PharmGKB SCF-KIT PIK3R1 PID Apoptosis PIK3R1 PID VEGFR Related Pathway PIK3R1 PID ErbB Related Pathway PIK3R1 PID JAK-STAT Pathway PIK3R1 PID EGFR PIK3R1 PID Tyrosine Kinases PIK3R1 PID Pathways In Cancer PIK3R1 PID SCF-KIT PIK3R1 INOH Apoptosis PIK3R1 INOH VEGFR Related Pathway PIK3R1 INOH ErbB Related Pathway PIK3R1 INOH JAK-STAT Pathway PIK3R1 INOH EGFR PIK3R1 INOH Tyrosine Kinases PIK3R1 INOH Pathways In Cancer PIK3R1 INOH SCF-KIT KRAS Reactome Pathways In Cancer KRAS Reactome Tyrosine Kinases KRAS Reactome ErbB Related Pathway KRAS Reactome SCF-KIT KRAS Reactome VEGFR Related Pathway KRAS Reactome EGFR KRAS KEGG Pathways In Cancer KRAS KEGG Tyrosine Kinases KRAS KEGG ErbB Related Pathway KRAS KEGG SCF-KIT KRAS KEGG VEGFR Related Pathway KRAS KEGG EGFR KRAS Wikipathways Pathways In Cancer KRAS Wikipathways Tyrosine Kinases KRAS Wikipathways ErbB Related Pathway KRAS Wikipathways SCF-KIT KRAS Wikipathways VEGFR Related Pathway KRAS Wikipathways EGFR KRAS PharmGKB Pathways In Cancer KRAS PharmGKB Tyrosine Kinases KRAS PharmGKB ErbB Related Pathway KRAS PharmGKB SCF-KIT KRAS PharmGKB VEGFR Related Pathway KRAS PharmGKB EGFR JAK2 KEGG SCF-KIT JAK2 KEGG Tyrosine Kinases JAK2 KEGG Pathways In Cancer JAK2 KEGG VEGFR Related Pathway JAK2 KEGG JAK-STAT Pathway JAK2 Reactome SCF-KIT JAK2 Reactome Tyrosine Kinases JAK2 Reactome Pathways In Cancer JAK2 Reactome VEGFR Related Pathway JAK2 Reactome JAK-STAT Pathway JAK2 INOH SCF-KIT JAK2 INOH Tyrosine Kinases JAK2 INOH Pathways In Cancer JAK2 INOH VEGFR Related Pathway JAK2 INOH JAK-STAT Pathway JAK2 Wikipathways SCF-KIT JAK2 Wikipathways Tyrosine Kinases JAK2 Wikipathways Pathways In Cancer JAK2 Wikipathways VEGFR Related Pathway JAK2 Wikipathways JAK-STAT Pathway TP53 KEGG Direct p53 effectors TP53 KEGG ErbB Related Pathway TP53 KEGG Wnt Related Pathway TP53 KEGG Apoptosis TP53 KEGG Pathways In Cancer TP53 Wikipathways Direct p53 effectors TP53 Wikipathways ErbB Related Pathway TP53 Wikipathways Wnt Related Pathway TP53 Wikipathways Apoptosis TP53 Wikipathways Pathways In Cancer TP53 PID Direct p53 effectors TP53 PID ErbB Related Pathway TP53 PID Wnt Related Pathway TP53 PID Apoptosis TP53 PID Pathways In Cancer MAP2K1 Wikipathways JAK-STAT Pathway MAP2K1 Wikipathways VEGFR Related Pathway MAP2K1 Wikipathways Pathways In Cancer MAP2K1 Wikipathways EGFR MAP2K1 Wikipathways ErbB Related Pathway MAP2K1 KEGG JAK-STAT Pathway MAP2K1 KEGG VEGFR Related Pathway MAP2K1 KEGG Pathways In Cancer MAP2K1 KEGG EGFR MAP2K1 KEGG ErbB Related Pathway MAP2K1 PharmGKB JAK-STAT Pathway MAP2K1 PharmGKB VEGFR Related Pathway MAP2K1 PharmGKB Pathways In Cancer MAP2K1 PharmGKB EGFR MAP2K1 PharmGKB ErbB Related Pathway MAP2K1 INOH JAK-STAT Pathway MAP2K1 INOH VEGFR Related Pathway MAP2K1 INOH Pathways In Cancer MAP2K1 INOH EGFR MAP2K1 INOH ErbB Related Pathway FGFR2 KEGG Pathways In Cancer FGFR2 KEGG VEGFR Related Pathway FGFR2 KEGG Tyrosine Kinases FGFR2 Reactome Pathways In Cancer FGFR2 Reactome VEGFR Related Pathway FGFR2 Reactome Tyrosine Kinases FGFR2 PharmGKB Pathways In Cancer FGFR2 PharmGKB VEGFR Related Pathway FGFR2 PharmGKB Tyrosine Kinases FGFR2 INOH Pathways In Cancer FGFR2 INOH VEGFR Related Pathway FGFR2 INOH Tyrosine Kinases ALK KEGG VEGFR Related Pathway ALK KEGG Pathways In Cancer ALK INOH VEGFR Related Pathway ALK INOH Pathways In Cancer HIF1A PID VEGFR Related Pathway HIF1A PID Pathways In Cancer HIF1A KEGG VEGFR Related Pathway HIF1A KEGG Pathways In Cancer CDKN1B KEGG Pathways In Cancer CDKN1B KEGG ErbB Related Pathway CDKN1B Wikipathways Pathways In Cancer CDKN1B Wikipathways ErbB Related Pathway PTEN KEGG Direct p53 effectors PTEN KEGG Pathways In Cancer PTEN PID Direct p53 effectors PTEN PID Pathways In Cancer PTEN Wikipathways Direct p53 effectors PTEN Wikipathways Pathways In Cancer Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0274555.r002
4 Jul 2022 Decision Letter - Suprabhat Mukherjee, Editor PONE-D-22-06899R1Investigation of cell signalings and therapeutic targets in PTPRK-RSPO3 fusion-positive colorectal cancer.PLOS ONE Dear Dr. Lee, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Aug 18 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Suprabhat Mukherjee, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments (if provided): Authors need to address the following comments. Oncogenesis events in CRC is indeed multifaceted and considering the impact of P:R fusion-positive CRC authors need to present a comparative view with the recently published papers citing the association of multiple signaling pathways in the course of CRC pathophysiology. Authors may follow and cite DOI: 10.3389/fgene.2021.608313 as well as relevant literature to improve the manuscript. Fig. 1 could be omitted and a scheme may be added at the last. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0274555.r003
Revision 2
18 Jul 2022 Author Response July 18th, 2022 Editor, Plos One Dear Editor We deeply appreciate your attention to our paper. Below, we address each of the reviewer's comments point by point. We revised our manuscript using MS Word and highlighted the changes with yellow color. Point 1: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Response 1: Thank you for your comment. We corrected a miscited 20th reference from “Wilson RG, Smith AN, Bird CC. Immunohistochemical detection of abnormal cell proliferation in colonic mucosa of subjects with polyps. J Clin Pathol. 1990;43(9):744-747. doi:10.1136/jcp.43.9.744” to “Yamamoto S, Sakai N, Nakamura H, Fukagawa H, Fukuda K, Takagi T. INOH: ontology-based highly structured database of signal transduction pathways. Database (Oxford). 2011;2011: bar052. Published 2011 Nov 26. doi:10.1093/database/bar052”. We also checked and confirmed other references. Point 2: Oncogenesis events in CRC is indeed multifaceted and considering the impact of P:R fusion-positive CRC authors need to present a comparative view with the recently published papers citing the association of multiple signaling pathways in the course of CRC pathophysiology. Authors may follow and cite DOI: 10.3389/fgene.2021.608313 as well as relevant literature to improve the manuscript. Response 2: We appreciate your valuable comment. We reviewed the recent articles regarding integrated bioinformatics approach of colorectal cancer and presented a comparative view in the discussion section on line 241-252 as follows: “Previous studies that systematically explore gene biomarkers with bioinformatics analysis in colorectal cancer have focused on discovering prognosis-related biomarkers using differentially expressed genes (DEGs) analysis and machine learning techniques. (26, 27). To our best knowledge, our study differs from previous studies in two respects. First, the purpose of this study is to discover novel targets and therapeutics related to original mutations by analyzing downstream pathways and genes affected by target mutations that cannot be directly targeted. Second, our study is based on a structural variation (P:R fusion by DNA structural variation) that is a driver mutation in colorectal cancer. As consequence, almost all genes correlated with P:R fusion are downstream-level genes affected by fusion. In this aspect, our study is different from other studies, and, for example, it is not clear whether COL11A1 is a primary driver or is affected by other drivers in the study by Ritwik et al (27).” Point 3: Fig. 1 could be omitted, and a scheme may be added at the last. Response 3: Thank you for your comment. We omitted Fig.1 and generated Fig.4 regarding a scheme of this study on line 228-238. Additionally, we removed all the funding-related information in our manuscript and would like to change the funding information in “Financial Disclosure” section as follows: “This study was supported by a VHS Medical Center Research Grant, Republic of Korea (VHSMC22057), grant no 18-2018-023 from the SNUBH Research Fund, and The National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2022R1C1C1012986).” Thank you again for your thoughtful consideration. Best regards, Sejoon Lee Department of Pathology and Translational Medicine Clinical Precision Medicine Center Seoul National University Bundang Hospital Tel:+82-31-787-8124 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0274555.r004
31 Aug 2022 Decision Letter - Suprabhat Mukherjee, Editor Investigation of cell signalings and therapeutic targets in PTPRK-RSPO3 fusion-positive colorectal cancer. PONE-D-22-06899R2 Dear Dr. Lee, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Suprabhat Mukherjee, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0274555.r005
Formally Accepted
8 Sep 2022 Acceptance Letter - Suprabhat Mukherjee, Editor PONE-D-22-06899R2 Investigation of cell signalings and therapeutic targets in PTPRK-RSPO3 fusion-positive colorectal cancer. Dear Dr. Lee: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Suprabhat Mukherjee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0274555.r006

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