Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors identified that
(1) CDK4/6i treatment attenuates the growth of drug-resistant cells by prolongation of the G1 phase;
(2) CDK4/6i treatment results in an ineffective Rb inactivation pathway and suppresses the growth of drug-resistant tumors;
(3) Addition of endocrine therapy augments the efficacy of CDK4/6i maintenance;
(4) Addition of CDK2i with CDK4/6 treatment as second-line treatment can suppress the growth of resistant cell;
(5) The role of cyclin E as a key driver of resistance to CDK4/6 and CDK2 inhibition.
Strengths:
To prove their complicated proposal, the authors employed orchestration of several kinds of live cell markers, timed in situ hybridization, IF and Immunoblotting. The authors strongly recognize the resistance of CDK4/6 + ET therapy and demonstrated how to overcome it.
Weaknesses:
The authors need to underscore their proposed results from what is to be achieved by them and by other researchers.
Thank you for your thoughtful review and for highlighting both the strengths and weaknesses of our manuscript. We appreciate your recognition of the methodological rigor and the significance of our findings in addressing resistance to CDK4/6 inhibitors combined with endocrine therapy.
To address your concern regarding the need to delineate our results from those achieved by other researchers, we will incorporate clarifications in the revised manuscript. Specifically, we will:
(1) Clearly distinguish our novel contributions from prior findings in the field.
(2) Explicitly cite and discuss relevant studies to contextualize our work, ensuring that our contributions are appropriately framed within the broader body of knowledge.
These revisions will enhance the transparency and impact of our manuscript, as well as highlight the originality and significance of our findings. Thank you again for your constructive feedback.
Reviewer #2 (Public review):
Summary:
This study elucidated the mechanism underlying drug resistance induced by CDK4/6i as a single agent and proposed a novel and efficacious second-line therapeutic strategy. It highlighted the potential of combining CDK2i with CDK4/6i for the treatment of HR+/HER2- breast cancer.
Strengths:
The study demonstrated that CDK4/6 induces drug resistance by impairing Rb activation, which results in diminished E2F activity and a delay in G1 phase progression. It suggests that the synergistic use of CDK2i and CDK4/6i may represent a promising second-line treatment approach. Addressing critical clinical challenges, this study holds substantial practical implications.
Weaknesses:
(1) Drug-resistant cell lines: Was a drug concentration gradient treatment employed to establish drug-resistant cell lines? If affirmative, this methodology should be detailed in the materials and methods section.
We greatly appreciate the reviewer for raising this important question. In the revised manuscript, we will update the methods section to include a detailed description of how the drug-resistant cell lines were developed. Specifically, we will clarify whether a drug concentration gradient treatment was employed and provide step-by-step details to ensure reproducibility.
(2) What rationale informed the selection of MCF-7 cells for the generation of CDK6 knockout cell lines? Supplementary Figure 3. A indicates that CDK6 expression levels in MCF-7 cells are not notably elevated.
We appreciate the reviewer’s insightful question about the rationale for selecting MCF-7 cells to generate CDK6 knockout cell lines. This choice was guided by prior studies highlighting the significant role of CDK6 in mediating resistance to CDK4/6 inhibitors (1-4). Moreover, we observed a 4.6-fold increase in CDK6 expression in CDK4/6 inhibitor-resistant MCF-7 cells compared to their drug-naïve counterparts (Supplementary Figure 3A). While we did not detect notable differences in CDK4/6 activity between wild-type and CDK6 knockout cells under CDK4/6 inhibitor treatment, these findings point to a potential non-canonical function of CDK6 in conferring resistance to CDK4/6 inhibitors.
(3) For each experiment, particularly those involving mice, the author must specify the number of individuals utilized and the number of replicates conducted, as detailed in the materials and methods section.
We sincerely thank the reviewer for bringing this to our attention. In the revised manuscript, we will provide explicit details regarding the number of replicates and mice used for each experiment. This information will be included in the materials and methods section, figure legends, and relevant text to ensure transparency and clarity.
(4) Could this treatment approach be extended to triple-negative breast cancer?
We greatly appreciate the reviewer’s inquiry about extending our findings to triple-negative breast cancer (TNBC). Based on our data presented in Figure 1 and Supplementary Figure 2, which include the TNBC cell line MDA-MB-231, we anticipate that the benefits of maintaining CDK4/6 inhibitors could indeed be applied to TNBC with an intact Rb/E2F pathway.
Reviewer #3 (Public review):
Summary:
In their manuscript, Armand and colleagues investigate the potential of continuing CDK4/6 inhibitors or combining them with CDK2 inhibitors in the treatment of breast cancer that has developed resistance to initial therapy. Utilizing cellular and animal models, the research examines whether maintaining CDK4/6 inhibition or adding CDK2 inhibitors can effectively control tumor growth after resistance has set in. The key findings from the study indicate that the sustained use of CDK4/6 inhibitors can slow down the proliferation of cancer cells that have become resistant, and the combination of CDK2 inhibitors with CDK4/6 inhibitors can further enhance the suppression of tumor growth. Additionally, the study identifies that high levels of Cyclin E play a significant role in resistance to the combined therapy. These results suggest that continuing CDK4/6 inhibitors along with the strategic use of CDK2 inhibitors could be an effective strategy to overcome treatment resistance in hormone receptor-positive breast cancer.
Strengths:
(1) Continuous CDK4/6 Inhibitor Treatment Significantly Suppresses the Growth of Drug-Resistant HR+ Breast Cancer: The study demonstrates that the continued use of CDK4/6 inhibitors, even after disease progression, can significantly inhibit the growth of drug-resistant breast cancer.
(2) Potential of Combined Use of CDK2 Inhibitors with CDK4/6 Inhibitors: The research highlights the potential of combining CDK2 inhibitors with CDK4/6 inhibitors to effectively suppress CDK2 activity and overcome drug resistance.
(3) Discovery of Cyclin E Overexpression as a Key Driver: The study identifies overexpression of cyclin E as a key driver of resistance to the combination of CDK4/6 and CDK2 inhibitors, providing insights for future cancer treatments.
(4) Consistency of In Vitro and In Vivo Experimental Results: The study obtained supportive results from both in vitro cell experiments and in vivo tumor models, enhancing the reliability of the research.
(5) Validation with Multiple Cell Lines: The research utilized multiple HR+/HER2- breast cancer cell lines (such as MCF-7, T47D, CAMA-1) and triple-negative breast cancer cell lines (such as MDA-MB-231), validating the broad applicability of the results.
Weaknesses:
(1) The manuscript presents intriguing findings on the sustained use of CDK4/6 inhibitors and the potential incorporation of CDK2 inhibitors in breast cancer treatment. However, I would appreciate a more detailed discussion of how these findings could be translated into clinical practice, particularly regarding the management of patients with drug-resistant breast cancer.
We greatly appreciate this opportunity to further contextualize our findings within clinical practice. In the revised manuscript, we will expand the discussion to explore how the identified mechanisms can inform patient stratification and therapeutic combinations. We will also highlight the potential of integrating CDK2 inhibitors with continued CDK4/6 inhibition as a second-line strategy for HR+ breast cancer patients who exhibit resistance to CDK4/6 inhibitors, leveraging insights from current and ongoing clinical trials. This will provide a clearer framework for translating our findings into actionable therapeutic strategies.
(2) While the emergence of resistance is acknowledged, the manuscript could benefit from a deeper exploration of the molecular mechanisms underlying resistance development. A more thorough understanding of how CDK2 inhibitors may overcome this resistance would be valuable.
Thank you for this insightful suggestion. In the revised manuscript, we will delve deeper into the molecular mechanisms by which CDK2 inhibitors counteract resistance to CDK4/6 inhibitors and endocrine therapy. We will emphasize the role of the non-canonical Rb inactivation pathway and upregulated transcriptional activity in reactivating CDK2, which contribute to resistance under CDK4/6 inhibition. Furthermore, we will discuss how dual inhibition of CDK4/6 and CDK2 effectively suppresses this resistance pathway, offering a mechanistic rationale for the therapeutic potential of this combination strategy.
(3) The manuscript supports the continued use of CDK4/6 inhibitors, but it lacks a discussion on the long-term efficacy and safety of this approach. Additional studies or data to support the safety profile of prolonged CDK4/6 inhibitor use would strengthen the manuscript.
We greatly appreciate the reviewer for raising this important point. To address this, we will incorporate a discussion on the long-term safety and efficacy of CDK4/6 inhibitor maintenance therapy. Drawing from clinical trials and retrospective analyses (5-9), we will highlight data supporting the tolerability of prolonged CDK4/6i treatment, particularly in combination with endocrine therapy. We will also discuss its clinical benefits over chemotherapy or endocrine therapy alone, contextualizing these findings with our proposed therapeutic approach (6,8-11).
References:
(1) Yang C, Li Z, Bhatt T, Dickler M, Giri D, Scaltriti M_, et al._ Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence. Oncogene 2017;36:2255-64
(2) Li Q, Jiang B, Guo J, Shao H, Del Priore IS, Chang Q_, et al._ INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors. Cancer Discov 2022;12:356-71
(3) Ji W, Zhang W, Wang X, Shi Y, Yang F, Xie H_, et al._ c-myc regulates the sensitivity of breast cancer cells to palbociclib via c-myc/miR-29b-3p/CDK6 axis. Cell Death & Disease 2020;11:760
(4) Wu X, Yang X, Xiong Y, Li R, Ito T, Ahmed TA_, et al._ Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders. Nature Cancer 2021;2:429-43
(5) Martin JM, Handorf EA, Montero AJ, Goldstein LJ. Systemic Therapies Following Progression on First-line CDK4/6-inhibitor Treatment: Analysis of Real-world Data. Oncologist 2022;27:441-6
(6) Xi J, Oza A, Thomas S, Ademuyiwa F, Weilbaecher K, Suresh R_, et al._ Retrospective Analysis of Treatment Patterns and Effectiveness of Palbociclib and Subsequent Regimens in Metastatic Breast Cancer. J Natl Compr Canc Netw 2019;17:141-7
(7) Basile D, Gerratana L, Corvaja C, Pelizzari G, Franceschin G, Bertoli E_, et al._ First- and second-line treatment strategies for hormone-receptor (HR)-positive HER2-negative metastatic breast cancer: A real-world study. Breast 2021;57:104-12
(8) Kalinsky K, Accordino MK, Chiuzan C, Mundi PS, Sakach E, Sathe C_, et al._ Randomized Phase II Trial of Endocrine Therapy With or Without Ribociclib After Progression on Cyclin-Dependent Kinase 4/6 Inhibition in Hormone Receptor–Positive, Human Epidermal Growth Factor Receptor 2–Negative Metastatic Breast Cancer: MAINTAIN Trial. Journal of Clinical Oncology;0:JCO.22.02392
(9) Kalinsky K, Bianchini G, Hamilton EP, Graff SL, Park KH, Jeselsohn R_, et al._ Abemaciclib plus fulvestrant vs fulvestrant alone for HR+, HER2- advanced breast cancer following progression on a prior CDK4/6 inhibitor plus endocrine therapy: Primary outcome of the phase 3 postMONARCH trial. Journal of Clinical Oncology 2024;42:LBA1001-LBA
(10) Mayer EL, Wander SA, Regan MM, DeMichele A, Forero-Torres A, Rimawi MF_, et al._ Palbociclib after CDK and endocrine therapy (PACE): A randomized phase II study of fulvestrant, palbociclib, and avelumab for endocrine pre-treated ER+/HER2- metastatic breast cancer. Journal of Clinical Oncology 2018;36:TPS1104-TPS
(11) Llombart-Cussac A, Harper-Wynne C, Perello A, Hennequin A, Fernandez A, Colleoni M_, et al._ Second-line endocrine therapy (ET) with or without palbociclib (P) maintenance in patients (pts) with hormone receptor-positive (HR[+])/human epidermal growth factor receptor 2-negative (HER2[-]) advanced breast cancer (ABC): PALMIRA trial. Journal of Clinical Oncology 2023;41:1001-
