RET Signaling Pathway in Human Cancer: Oncogenic Mechanisms, Selective Inhibitors, and Emerging Resistance Strategies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review addresses an important subject: the biology of RET signaling, its role in oncogenesis, and the clinical development of RET-targeted therapies. The topic is highly relevant for both translational researchers and clinicians given the rapid therapeutic advances in this field. The manuscript is comprehensive, well-referenced, and structured to cover molecular biology, oncogenic alterations, and therapeutic strategies.

Nevertheless, the manuscript would benefit from some revisions to improve clarity, depth, and presentation. Some issues relate to content (critical discussion, clinical integration), while others involve formatting and consistency (excessive paragraph breaks, gene nomenclature, and font usage).

Major comments:

-While the manuscript is thorough in summarizing known mechanisms and clinical data, it largely reads as a descriptive compilation. The authors should expand their critical perspective on some open questions (e.g., RET’s dual role in colorectal cancer as oncogene vs. tumor suppressor, or RET’s impact on immune evasion and immunotherapy response).

-The future directions section could better highlight combination therapies, biomarker-based patient selection, and next-generation inhibitors targeting resistance mutations.

-The molecular signaling details (Sections 2.1–2.2) are very dense. A schematic figure already accompanies this; thus, some of the repetitive residue-by-residue phosphorylation descriptions could be streamlined for clarity.

-Conversely, the clinical sections (thyroid, NSCLC, others) would benefit from deeper integration of real-world outcome data, resistance rates, and ongoing clinical trial perspectives.

-Figure legends should be clearer, and ideally, at least one summary table should be added comparing selective vs. multi-kinase inhibitors (targets, FDA approval year, clinical outcomes, common toxicities).

-A visual overview of mechanisms of resistance would strengthen the clinical relevance.

Minor comments:

-The manuscript is currently fragmented by an excessive number of short paragraphs, particularly in Section 2.2. Related concepts should be merged into coherent paragraphs to improve readability.

-On pages 18-19, certain sections appear in bold font without justification. This should be corrected for consistency.

-Gene symbols (e.g., RET, BRAF, NCOA4, CCDC6, KIF5B) should be consistently italicized throughout, in accordance with standard nomenclature guidelines. Protein names should remain in regular font.

-Consider shortening the abstract slightly and highlighting the clinical challenges more clearly (e.g., resistance and therapeutic sequencing).

-Abbreviations (e.g., MKIs, TKIs, ORR, PFS) should be defined once and used consistently thereafter.

-Ensure that the most recent clinical trial updates for selpercatinib and pralsetinib are cited (e.g., LIBRETTO-431, ARROW long-term follow-up).

-Conclusion: A more concise concluding section summarizing therapeutic implications and research gaps would make the review more impactful.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comment 1: While the manuscript is thorough in summarizing known mechanisms and clinical data, it largely reads as a descriptive compilation. The authors should expand their critical perspective on some open questions (e.g., RET’s dual role in colorectal cancer as oncogene vs. tumor suppressor, or RET’s impact on immune evasion and immunotherapy response).

Response 1: We thank the reviewer for this thoughtful comment. We respectfully note that the manuscript does address RET’s dual role in colorectal cancer, highlighting both the oncogenic function of RET fusions (e.g., NCOA4-RET and CCDC6-RET) and the potential tumor-suppressive effects of RET mutations associated with methylation and downregulation. These contrasting findings are explicitly discussed to emphasize the current uncertainty surrounding RET’s role in CRC. However, because mechanistic data explaining this context-dependent behavior remain limited, we intentionally refrained from speculative interpretation. We believe our summary accurately reflects the evidence base and provides readers with a balanced understanding of this controversy.

Comment 2: The future directions section could better highlight combination therapies, biomarker-based patient selection, and next-generation inhibitors targeting resistance mutations.

Response 2:

Thank you for this valuable suggestion. In response, we substantially revised the Future Directions section to incorporate detailed discussion on biomarker-based patient selection, combination therapy strategies, and next-generation RET inhibitors addressing resistance mechanisms.

Specifically, we added:

• A comprehensive overview of biomarker-driven patient selection, emphasizing the importance of next-generation sequencing (NGS)—particularly RNA-based NGS—for sensitive detection of RET fusions and the role of broad genomic profiling in guiding therapy selection.
• Expanded discussion on acquired resistance mechanisms, including secondary RET mutations (e.g., solvent front G810C/S/R and gatekeeper V804M/L mutations) and bypass signaling pathway activations involving MET, EGFR, FGFR, and Hedgehog-Gli.
• Inclusion of next-generation RET inhibitors (e.g., APS013118) designed to overcome resistance and improve CNS penetration.
• Detailed examples of combination therapies under investigation, such as selpercatinib plus crizotinib for MET amplification–driven resistance, RET inhibitors with EGFR or MET inhibitors for bypass activation, and arsenic trioxide plus pralsetinib to inhibit Hedgehog-Gli signaling.
• Clarification on therapeutic adaptation in cases of histologic transformation, including a shift to small-cell lung cancer chemotherapy regimens when indicated.

Collectively, these revisions directly address the reviewer’s comment by integrating current and emerging approaches—biomarker-guided selection, combination therapy, and next-generation inhibitor development—that are shaping the evolving landscape of RET-targeted cancer therapy.

Comment 3: The molecular signaling details (Sections 2.1–2.2) are very dense. A schematic figure already accompanies this; thus, some of the repetitive residue-by-residue phosphorylation descriptions could be streamlined for clarity.

Response 3: In response to the reviewer’s comment, Sections 2.1–2.2 were streamlined by removing redundant residue-by-residue phosphorylation details that were already depicted in the accompanying schematic figure. Repetitive descriptions of RET isoforms, domains, and overlapping signaling cascades were consolidated for clarity, while all original references and scientific content were retained. The revised version improves flow and readability, highlights key structural and signaling concepts, and maintains technical accuracy without unnecessary repetition.

Comment 4: Conversely, the clinical sections (thyroid, NSCLC, others) would benefit from deeper integration of real-world outcome data, resistance rates, and ongoing clinical trial perspectives.

Response 4:

We respectfully disagree with the reviewer’s comment that the clinical sections lack integration of real-world outcome data, resistance rates, and ongoing clinical trial perspectives. These aspects were explicitly incorporated throughout multiple sections of the manuscript. For instance, in the Thyroid Cancer section, we highlighted real-world correlations between specific RET mutations (e.g., M918T in sporadic medullary thyroid carcinoma) and worse clinical outcomes, including lower survival and higher recurrence rates, directly reflecting prognostic and outcome data [55–58]. We also discussed the therapeutic relevance of these findings through the demonstrated clinical efficacy of selective RET inhibitors such as selpercatinib in advanced MTC, emphasizing real-world applicability [59].

Similarly, in the Non-Small Cell Lung Cancer section, we incorporated clinical and epidemiologic data showing RET fusions as oncogenic drivers in 1–2% of NSCLC cases and detailed their association with higher rates of brain metastases (27% at diagnosis, increasing to 49% over disease course), which represent real-world outcome statistics [3,71]. We also discussed emerging resistance patterns—such as RET fusion-mediated resistance to osimertinib—and the ongoing development of RET-specific tyrosine kinase inhibitors as part of current clinical research efforts [71].

Furthermore, resistance mechanisms and ongoing clinical trial perspectives are explored in detail in the dedicated Resistance Mechanisms section, where we outline solvent-front and gatekeeper mutations (e.g., G810C/S/R, V804M/L) as key causes of acquired resistance, and describe next-generation RET inhibitors and combination therapy trials under active investigation to address these challenges [167–170]. Together, these sections provide an integrated view of molecular alterations, clinical outcomes, resistance dynamics, and therapeutic advancements in RET-altered cancers.

Comment 5: Figure legends should be clearer, and ideally, at least one summary table should be added comparing selective vs. multi-kinase inhibitors (targets, FDA approval year, clinical outcomes, common toxicities).

Response 5: We fully agree that such an illustration would enhance clarity and clinical relevance. We are in the process of developing a concise, high-quality schematic that accurately summarizes these pathways. However, generating an original and precise figure requires additional time to ensure scientific accuracy and design quality. We respectfully request a brief extension to finalize this figure, which will be included in the following version. 

Comment 6: A visual overview of mechanisms of resistance would strengthen the clinical relevance.

Response 6: As per response 5.

Comment 7: The manuscript is currently fragmented by an excessive number of short paragraphs, particularly in Section 2.2. Related concepts should be merged into coherent paragraphs to improve readability.

Response 7: As noted in our response to Comment 3, we have substantially revised Section 2.2 to improve flow and readability by merging conceptually related paragraphs and ensuring smoother transitions between topics.

Comment 8: On pages 18-19, certain sections appear in bold font without justification. This should be corrected for consistency.

Response 8: We agree with the reviewer’s observation and have corrected the formatting inconsistencies on pages 18–19 by removing the unintended bold font to ensure uniformity throughout the manuscript.

Comment 9: Gene symbols (e.g., RET, BRAF, NCOA4, CCDC6, KIF5B) should be consistently italicized throughout, in accordance with standard nomenclature guidelines. Protein names should remain in regular font.

Response 9: We agree with the reviewer’s comment and have revised the manuscript to ensure that all gene symbols (e.g., RET, BRAF, NCOA4, CCDC6, KIF5B) are consistently italicized, while corresponding protein names remain in regular font, in accordance with standard nomenclature guidelines.

Comment 10: Consider shortening the abstract slightly and highlighting the clinical challenges more clearly (e.g., resistance and therapeutic sequencing).

Response 10: We agree with the reviewer’s comment and have revised the abstract to improve clarity, flow, and conciseness. The updated version is now a single cohesive paragraph that better integrates key points on RET biology, therapeutic advances, and mechanisms of resistance while maintaining scientific precision and readability.

Comment 11: Abbreviations (e.g., MKIs, TKIs, ORR, PFS) should be defined once and used consistently thereafter.

Response 11: We agree with the reviewer’s comment and have ensured that all abbreviations (e.g., MKIs, TKIs, ORR, PFS) are defined upon first use and applied consistently throughout the manuscript.

Comment 12: Ensure that the most recent clinical trial updates for selpercatinib and pralsetinib are cited (e.g., LIBRETTO-431, ARROW long-term follow-up).

Response 12: We agree with the reviewer’s comment and have incorporated the most recent clinical trial updates for both selpercatinib and pralsetinib. Specifically, information from the LIBRETTO-431 phase III trial has been added to the section on selpercatinib, and data from the ARROW long-term follow-up analysis have been integrated into the section discussing pralsetinib to reflect updated efficacy and safety outcomes.

Comment 13: A more concise concluding section summarizing therapeutic implications and research gaps would make the review more impactful.

Response 13: The updated section now provides a clear summary of therapeutic implications and key research gaps, emphasizing resistance mechanisms, biomarker refinement, and future directions for RET-targeted therapies.

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Reviewer 2 Report

Comments and Suggestions for Authors

The reviewed manuscript provides a detailed analysis of the available data on the RET receptor, including its structure, activation mechanisms, and pro-oncogenic mutations. The authors thoroughly examine the types of cancers associated with mutant RET receptors and analyze available anti-cancer drugs with both broad and more targeted actions. Additionally, they discuss the mechanisms of resistance to these treatments.

While the manuscript is well-written, there are multiple minor revisions that could be made to improve the readability and clarity of the text. The review can be recommended for publication in IJMS after a minor revision.

Major comment

Why didn’t authors discuss specific mutations associated with HSCR? Subsection HSCR looks oversimplified.

Minor comments

Some abbreviations are introduced several times in the text. For example, RET, ORR, GDNF, NRTN, ARTN, PSPN, etc.  Authors are kindly requested to check all abbreviations and exclude their re-introduction.

In the text of manuscript, abbreviations should be introduced, when they are first time used. For example, cysteine-rich domain (lines 113, 118).  It is incorrect to use an abbreviation and then introduce it several paragraphs later. For example, NRTN, ARTN, etc.

If abbreviation is introduced, it should be further used.  For example, see page 7 for cysteine-rich domain/CRD.

Line 32 Please, correct: GDNF is the glial cell line-derived neurotrophic factor.

Line 95. Please, delete the sentence “The following sections describe the function of RET signaling in human cells:”

Legend to Figure 1. Please delete “Description:”

Lines 120-121 The sentence is unclear. “The tyrosine kinase domain is divided into two sections by the sequence of amino acids”.

Abbreviation TK from Figure 1 is not described either in Figure 1 legend or in the corresponding text.  

Lines 126-127 Please, delete or modify sentence “The intracellular regions of RET51 and RET9, along with the signaling 126 cascades initiated by phosphotyrosines.”

Why didn't the authors include the RET43 isoform in Fig. 1?

Line 153. Please, correct numbering of amino acids to “658-1114”.

Line 192 Please, cite Figures in brackets like (Figure 1), and not as “Refer to figure 1”.

Lines 259-260 and lines 263-264.  These two statements are similar in meaning and can be combined. 

Line 289 and next.  Glu768Asp (E768D), Leu790Phe (L790F), Tyr791Phe (Y791F) – these are two equally used styles to describe mutations (three-letter and one-letter abbreviations).  Please, select one style.

Line 318. Perhaps, title “RET fusion” or “RET gene fusion” is better than “Gene mutation”.

Lines 342-343 The statement “the Japanese atomic bomb explosion” is not correct. Change it to either “the USA atomic bomb explosion in Japan” or “the atomic bomb explosion in Japan”

Lines 538-540 “A study by Plaza-Menacho et al indicated…” -reference to the corresponding manuscript should be indicated.

Please, cite correctly works in subsection Sorafenib. For example, not [95], [96], but [95,96], etc.

Please consider typing the drug names in lowercase in the text.

Line 732. Check grammar in “… approval metastatic…”

Lines 739-740; 813-839; 841-850; 852-862.  Change the bold font to a plain one.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comment 1: Why didn’t authors discuss specific mutations associated with HSCR? Subsection HSCR looks oversimplified.

Response 1: We appreciate the reviewer’s comment. While the subsection on Hirschsprung’s disease provides a concise overview, specific RET mutations—such as those resulting in loss of function through deletions, insertions, nonsense, and missense variants—are already discussed in the broader section addressing RET mutations and their biological consequences. Given that the manuscript includes a dedicated and comprehensive discussion of RET mutation types and mechanisms across disease contexts, we opted to maintain brevity in the HSCR subsection to avoid redundancy while ensuring clarity and focus.

Comment 2: Some abbreviations are introduced several times in the text. For example, RET, ORR, GDNF, NRTN, ARTN, PSPN, etc.  Authors are kindly requested to check all abbreviations and exclude their re-introduction.

Response 2: Thank you for that observation. We agree and have revised the abbreviations and excluded their re-introduction.

Comment 3: In the text of manuscript, abbreviations should be introduced, when they are first time used. For example, cysteine-rich domain (lines 113, 118).  It is incorrect to use an abbreviation and then introduce it several paragraphs later. For example, NRTN, ARTN, etc. If abbreviation is introduced, it should be further used.  For example, see page 7 for cysteine-rich domain/CRD.

Response 3: Thank you for that observation. We agree and have revised the abbreviations and excluded their re-introduction.

Comment 4: Line 32 Please, correct: GDNF is the glial cell line-derived neurotrophic factor.

Response 4: We agree, the correction has been applied.

Comment 5: Line 95. Please, delete the sentence “The following sections describe the function of RET signaling in human cells:”

Response 5: Thank you, the line has been deleted.

Comment 6: Legend to Figure 1. Please delete “Description:”

Response 6: Thank you, we hae deleted “description” from the legend.

Comment 7: Lines 120-121 The sentence is unclear. “The tyrosine kinase domain is divided into two sections by the sequence of amino acids”.

Response 7: Thank you, we agree. The sentence has been deleted.

Comment 8: Abbreviation TK from Figure 1 is not described either in Figure 1 legend or in the corresponding text.  

Response 8: Thank you, we agree. We have added a clarification in the corresponding text.

Comment 9: Lines 126-127 Please, delete or modify sentence “The intracellular regions of RET51 and RET9, along with the signaling 126 cascades initiated by phosphotyrosines.”

Response 9: Thank you, we have deleted the sentence.

Comment 10: Why didn't the authors include the RET43 isoform in Fig. 1?

Response 10: We appreciate the reviewer’s insightful observation. The RET43 isoform was not included in the initial version of Figure 1 as our primary focus was on the two major functional isoforms, RET9 and RET51, which are the most extensively characterized in the context of signaling and oncogenic activity.

Comment 11: Line 153. Please, correct numbering of amino acids to “658-1114”.

Response 11: Thank you, we have corrected the numbering.

Comment 12: Line 192 Please, cite Figures in brackets like (Figure 1), and not as “Refer to figure 1”.

Response 12: We respectfully agree. The figures citations have been updated.

Comment 13: Lines 259-260 and lines 263-264.  These two statements are similar in meaning and can be combined. 

Response 13: Thank you, we agree. The sentence is updated and can be revised in lines 168-169.

Comment 14: Line 289 and next.  Glu768Asp (E768D), Leu790Phe (L790F), Tyr791Phe (Y791F) – these are two equally used styles to describe mutations (three-letter and one-letter abbreviations).  Please, select one style.

Response 14: Thank you, we have selected the three-letter style. This can be found in line 195 onwards

Comment 15: Line 318. Perhaps, title “RET fusion” or “RET gene fusion” is better than “Gene mutation”.

Response 15: Thank you, we agree. This has been updated in line 223 to RET fusion.

Comment 16: Lines 342-343 The statement “the Japanese atomic bomb explosion” is not correct. Change it to either “the USA atomic bomb explosion in Japan” or “the atomic bomb explosion in Japan”

Response 16: Thank you, we agree. This has been edited in line 248.

Comment 17: Lines 538-540 “A study by Plaza-Menacho et al indicated…” -reference to the corresponding manuscript should be indicated.
Response 17: We thank the reviewer for this observation. The statement is indeed referenced at the end of the sentence as citation [72], which corresponds to the study by Plaza-Menacho et al. We have double-checked to ensure that the citation is correctly placed and properly formatted in the revised manuscript. This can be found in Line 440-444

Comment 18: Please, cite correctly works in subsection Sorafenib. For example, not [95], [96], but [95,96], etc.

Response 18: Thank you for this observation. We have corrected the citation.

Comment 19: Please consider typing the drug names in lowercase in the text.

Response 19: We agree with this observation and the corrections have been made.

Comment 20: Line 732. Check grammar in “… approval metastatic…”

Response 20: 637-639 Thank you, we have corrected the grammar of this sentence.

Comment 21: Lines 739-740; 813-839; 841-850; 852-862.  Change the bold font to a plain one.

Response 21: 645-646; 717-741, 742-751; 752-786 We have unbolded the font. Thank you.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made substantial improvements to the manuscript and have satisfactorily addressed the majority of the previous comments. Nevertheless, with regard to comment 5, which requested the inclusion of a summarizing table (rather than an illustration), and comment 6, which recommended the addition of a visual overview depicting the mechanisms of resistance, these points remain unaddressed. I strongly encourage the authors to incorporate these elements, as they would significantly improve the readability, clarity, and overall impact of the manuscript.

Author Response

Comment1:

Thank you for the thoughtful feedback and for highlighting the remaining points regarding comments 5 and 6. We appreciate the reviewer’s suggestions and fully agree that incorporating a summarizing table and a visual overview of the resistance mechanisms will strengthen the manuscript.

To ensure these elements are completed with the appropriate level of accuracy and quality, we kindly request an extension to submit the revised manuscript by mid-January. Please let us know if this timeline is acceptable.

Thank you again for your consideration and for the constructive guidance throughout this process.