PARP Inhibitors Combined with Abiraterone Overcome Resistance in Metastatic Castration-Resistant Prostate Cancer Independently of Androgen Receptor

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This preclinical study addresses an important clinical challenge in metastatic castration-resistant prostate cancer (mCRPC): therapeutic options following resistance to androgen receptor signaling inhibitors (ARSIs), particularly Abiraterone.

It evaluates the combination of PARP inhibitors (PARPi) with Abiraterone in Abiraterone-resistant prostate cancer models and demonstrates enhanced antitumor activity compared with single-agent treatments.

While clear in the title the abstract does not clearly distinguish this work from existing clinical studies demonstrating PARPi–ARSI synergy so It may be interpreted as another combination-therapy study in mCRPC.

Showing benefit of maintaining Abiraterone in PC3 and DU145 cell line models despite resistance and low AR signaling is unexpected and therefore novel.

Given the growing body of literature supporting upfront PARPi–ARSI combinations, the manuscript risks being perceived as confirmatory and it clearly would make sense that the authors not only in the title but also in the abstract articulate how their work addresses an unmet knowledge gap.

The experiments are not surprising, adequate, classical elegant and well done. The paper concisely and well written. Some reviewers may argue that two cell lines are not sufficient to draw conclusions, but the data would convince me to pursue this avenue further in clinical and experimental settings. In particular to get a more mechanistic  in sight into the observed phenomenon, which is not included and would be an important next step. 

Author Response

Comments and responses to Comments of Reviewer 1:

Overall, we greatly thank Reviewer 1 for his his/her appreciation of our work and for the comments raised concerning the Abstract section. Undoubtedly these comments have favorably impacted the new manuscript and will surely interest all actors of this scientific domain.

We made changes in the Abstract section according to one of the comments in the revised manuscript. We have highlighted all the changes in yellow in the text of the revised manuscript.

This preclinical study addresses an important clinical challenge in metastatic castration-resistant prostate cancer (mCRPC): therapeutic options following resistance to androgen receptor signaling inhibitors (ARSIs), particularly Abiraterone.

It evaluates the combination of PARP inhibitors (PARPi) with Abiraterone in Abiraterone-resistant prostate cancer models and demonstrates enhanced antitumor activity compared with single-agent treatments.

Comment 1: While clear in the title the abstract does not clearly distinguish this work from existing clinical studies demonstrating PARPi–ARSI synergy so it may be interpreted as another combination-therapy study in mCRPC.

Response: Thank you for this important comment and suggestion. As mentioned in the Abstract section ‘Combining PARPi with ARSIs has improved progression-free (PFS) and overall survival (OS), especially in ARSI-naïve patients, but limited data exist for resistant disease’ and it is more especially on resistance that we have focused the present work and not only on a potential synergistic effect between both therapeutic compounds. In other words, we have revised the abstract to clearly articulate the unmet knowledge gap addressed by our study. While prior clinical trials (e.g., PROPEL and MAGNITUDE) have evaluated PARPi–ARSI combinations in treatment-naïve, AR-positive, or BRCA/HRR-mutant patients, our work focuses on intrinsically hormone-insensitive, AR-negative, BRCA-wildtype models, representing a clinically distinct population with limited therapeutic options. These revisions highlight that our study addresses a critical gap in understanding how PARP inhibition can enhance abiraterone efficacy in resistant and AR-independent prostate cancer, rather than simply confirming existing combination strategies. The Abstract section was modified in the revised manuscript to include this statement: This work focuses on intrinsically hormone-insensitive, AR-negative, BRCA-wildtype models, representing a clinically distinct population with limited therapeutic options. We thus investigated the therapeutic potential of combining Abiraterone with PARPi (Niraparib or Olaparib) in Abiraterone-resistant prostate cancer. We hope that the Reviewer will agree this modification.

Comment 2: Showing benefit of maintaining Abiraterone in PC3 and DU145 cell line models despite resistance and low AR signaling is unexpected and therefore novel.

Response: Thank you.

Comment 3: Given the growing body of literature supporting upfront PARPi–ARSI combinations, the manuscript risks being perceived as confirmatory and it clearly would make sense that the authors not only in the title but also in the abstract articulate how their work addresses an unmet knowledge gap.

Response: We made the expected changes as precised above. We did not change the Title in the revised manuscript since the Reviewer stated above that the Title was clear enough considering the work done here. We hope that the Reviewer will agree with our statement. 

Comment 4: The experiments are not surprising, adequate, classical elegant and well done. The paper is concise and well written. Some reviewers may argue that two cell lines are not sufficient to draw conclusions, but the data would convince me to pursue this avenue further in clinical and experimental settings. In particular to get a more mechanistic insight into the observed phenomenon, which is not included and would be an important next step.

Response: Yes, the Reviewer is right. The next step will be to get deeper insights into the cellular mechanisms underlying the observed effects.

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors explored the combination of abiraterone with PARP inhibitors in androgen receptor independent models of metastatic castration resistant prostate cancer. The combination of abiraterone with PARP inhibitors has already demonstrated clinical benefit in mCRPC, and therefore the therapeutic strategy itself is not novel. For example, a randomized phase III trial showed that abiraterone combined with olaparib significantly prolonged radiographic progression free survival compared with abiraterone plus placebo in mCRPC patients irrespective of HRR mutation status (PMID: 38319800).

However, the potential contribution of this study lies instead in exploring the therapeutic potential of this combination, particularly in abiraterone resistant and androgen receptor (AR) independent disease settings, which occur in advanced prostate cancer when it becomes resistant to AR targeted therapies. Therefore, this work will be of interest to cancer biologists. Using AR negative PC3 and DU145 models, the authors show that the addition of PARP inhibition enhances antitumor activity compared with either agent alone, suggesting that abiraterone may exert AR independent effects that sensitize tumors to PARP inhibition.

• I suggest the authors describe the overall high level findings in 1-2 lines toward the end of the Introduction as a bridge toward the Results section.
• Results section: 4.1, Fig. 1A-B: please mention the type of statistical tests performed, and include this information in all figure legends.
• Results section 4.3: The opening paragraph is incorrect as written in the current manuscript: “The results of 2D experiments showed that Niraparib, but not Olaparib, was most effective in decreasing cell viability in combination with Abiraterone in PC3 cells, while Olaparib, but not Niraparib, was most effective in combination with Abiraterone in DU145” Please correct this. Based on the findings (Fig. 2A, right), in 2D culture, PC3 cells were sensitive to Niraparib but not Olaparib, with the observed reduction in viability largely driven by Niraparib monotherapy rather than a synergistic interaction with Abiraterone (i.e., there is no difference between Niraparib alone vs Niraparib + Abiraterone). In contrast, DU145 cells showed evidence of a synergistic reduction in viability when treated with Abiraterone and Olaparib, whereas the effects of Niraparib were predominantly attributable to single agent activity. If the authors believe this reviewer misinterpreted the data, please include a statistical test comparing Niraparib vs Niraparib + Abiraterone in Fig. 2A, right.
• End of Results 4.3: The statement “Taken together, these results show that the effects on cell viability of each agent alone and in combination are very similar between 2D and 3D models, arguing clearly the robustness of the results obtained in vitro” is not accurate. In PC3 2D cultures there was no synergy with Abiraterone + Niraparib, whereas there is synergy in the 3D model. In DU145, there is synergy with Abiraterone + Olaparib in both 2D and 3D models. Please correct this.
• Discussion section: The authors state “However, there is a severe lack of data regarding the efficacy of combining second-generation hormone therapy and PARPi after patients with mCRPC have progressed in the disease following androgen deprivation” This is not entirely accurate as there are published clinical trials showing benefit of abiraterone + PARP inhibitor combinations in mCRPC (e.g., PMID: 38319800).
• The authors state “But tomorrow, do we have arguments for adding the PARPi to Abiraterone in order to overcome acquired resistance based on HRR recombination and BRCA mutations?” They likely mean HRR deficiencies?
• I suggest the authors tone down this statement at the end of the Discussion: “From all these results we can now respond positively to the above-mentioned question: ‘Do we have arguments for adding the PARPi to Abiraterone in order to overcome acquired resistance based on HRR recombination and BRCA mutations?’. That will probably have a strong echo to the clinical setting …..…” As noted earlier, there are clinical papers on this (e.g., PMID: 38319800). Instead, they should discuss how they have investigated the therapeutic potential of this combination, particularly in abiraterone resistant and AR independent disease settings seen in advanced prostate cancer. The authors should also cite the important clinical study (PMID: 38319800) demonstrating the efficacy of abiraterone combined with olaparib in mCRPC.

Author Response

Comments and responses to Comments of Reviewer 2:

First of all, we greatly thank Reviewer 2 for his/her appreciation of our work and for the comments raised. We responded to them the best we can. As for Reviewer 1 and 3, undoubtedly, all these comments have favorably impacted the new manuscript and will surely interest all actors of this scientific domain.

We made changes according to the comments in the revised manuscript. We have highlighted all the changes in yellow in the text of the revised manuscript.

In this study, the authors explored the combination of abiraterone with PARP inhibitors in androgen receptor independent models of metastatic castration resistant prostate cancer. The combination of abiraterone with PARP inhibitors has already demonstrated clinical benefit in mCRPC, and therefore the therapeutic strategy itself is not novel. For example, a randomized phase III trial showed that abiraterone combined with olaparib significantly prolonged radiographic progression free survival compared with abiraterone plus placebo in mCRPC patients irrespective of HRR mutation status (PMID: 38319800).

Comment 1: However, the potential contribution of this study lies instead in exploring the therapeutic potential of this combination, particularly in abiraterone resistant and androgen receptor (AR) independent disease settings, which occur in advanced prostate cancer when it becomes resistant to AR targeted therapies. Therefore, this work will be of interest to cancer biologists. Using AR negative PC3 and DU145 models, the authors show that the addition of PARP inhibition enhances antitumor activity compared with either agent alone, suggesting that abiraterone may exert AR independent effects that sensitize tumors to PARP inhibition.

Response: We thank the Reviewer for this insightful comment and for highlighting the potential therapeutic significance of our study. We agree that the main contribution of this work lies in exploring the efficacy of combining abiraterone with PARP inhibition in abiraterone-resistant and AR-independent prostate cancer settings. As noted by the Reviewer, the use of AR-negative PC3 and DU145 models allowed us to demonstrate that the addition of PARP inhibitors significantly enhances antitumor activity compared with either agent alone. These findings support the hypothesis that abiraterone may exert AR-independent effects that sensitize prostate cancer cells to PARP inhibition. We have revised the manuscript to more clearly emphasize this point in the Introduction section, and to highlight the relevance of our findings to advanced, AR-independent prostate cancer and to the cancer biology community.

Comment 2: I suggest the authors describe the overall high-level findings in 1-2 lines toward the end of the Introduction as a bridge toward the Results section.

Response: We thank the Reviewer for this helpful suggestion. We have revised the end of the Introduction in the revised manuscript to include a concise 1–2 sentence summary of the overall findings, highlighting the enhanced antitumor activity observed with the combination of abiraterone and PARP inhibition in AR-independent prostate cancer models. This addition provides a clearer transition to the Results section.

Comment 3: Results section: 4.1, Fig. 1A-B: please mention the type of statistical tests performed and include this information in all figure legends.

Response: Thank you for this comment. Please note that the paragraphs numbering has been changed when the Journal formatted the text. We agree with the Reviewer and have now specified the statistical tests used in Section 4.1. In addition, we have updated the legends of Fig. 1A–B and all other figures throughout the revised manuscript to clearly indicate the statistical tests performed, the definition of error bars, and the significance thresholds.

Comment 4: Results section 4.3: The opening paragraph is incorrect as written in the current manuscript: “The results of 2D experiments showed that Niraparib, but not Olaparib, was most effective in decreasing cell viability in combination with Abiraterone in PC3 cells, while Olaparib, but not Niraparib, was most effective in combination with Abiraterone in DU145” Please correct this. Based on the findings (Fig. 2A, right), in 2D culture, PC3 cells were sensitive to Niraparib but not Olaparib, with the observed reduction in viability largely driven by Niraparib monotherapy rather than a synergistic interaction with Abiraterone (i.e., there is no difference between Niraparib alone vs Niraparib + Abiraterone). In contrast, DU145 cells showed evidence of a synergistic reduction in viability when treated with Abiraterone and Olaparib, whereas the effects of Niraparib were predominantly attributable to single agent activity. If the authors believe this reviewer misinterpreted the data, please include a statistical test comparing Niraparib vs Niraparib + Abiraterone in Fig. 2A, right.

Response: We thank the Reviewer for carefully evaluating these data and agree that the original wording of the opening paragraph in Section 4.3 was inaccurate. We have revised this paragraph to accurately reflect the results shown in Fig. 2A (right).

Specifically, the revised text now clarifies that in 2D cultures, PC3 cells were sensitive to Niraparib but not Olaparib, and that the reduction in viability was primarily driven by Niraparib monotherapy, with no statistically significant difference between Niraparib alone and Niraparib combined with Abiraterone. In contrast, DU145 cells exhibited a synergistic reduction in viability when treated with abiraterone and Olaparib, whereas the effects observed with Niraparib were largely attributable to single-agent activity.

To further address the Reviewer’s point, we have also included a direct statistical comparison between Niraparib alone and Niraparib plus Abiraterone in PC3 cells in Fig. 2A, confirming the absence of a significant synergistic effect in 2D culture.

Comment 5: End of Results 4.3: The statement “Taken together, these results show that the effects on cell viability of each agent alone and in combination are very similar between 2D and 3D models, arguing clearly the robustness of the results obtained in vitro” is not accurate. In PC3 2D cultures there was no synergy with Abiraterone + Niraparib, whereas there is synergy in the 3D model. In DU145, there is synergy with Abiraterone + Olaparib in both 2D and 3D models. Please correct this.

Response: We agree with the Reviewer that this statement was overly general and did not accurately capture the differences observed between 2D and 3D models. We have revised this section to more precisely describe the findings. The corrected text in the revised manuscript now states that while DU145 cells demonstrate consistent synergy between Abiraterone and Olaparib in both 2D and 3D models, PC3 cells show synergy with abiraterone and Niraparib only in the 3D model, but not in 2D culture. This revision more accurately reflects the experimental data and avoids overstatement regarding the similarity between model systems.

Comment 6: Discussion section: The authors state “However, there is a severe lack of data regarding the efficacy of combining second-generation hormone therapy and PARPi after patients with mCRPC have progressed in the disease following androgen deprivation” This is not entirely accurate as there are published clinical trials showing benefit of abiraterone + PARP inhibitor combinations in mCRPC (e.g., PMID: 38319800).

Response: We thank the Reviewer for this important clarification. We agree that clinical trials evaluating Abiraterone plus PARPi combinations in mCRPC have been reported. However, the specific clinical scenario addressed in our study can be clearly distinguished from these prior clinical evidence.

Specifically, we now note that recent clinical trials, including PROPEL (Abiraterone + Olaparib) and MAGNITUDE (Abiraterone + Niraparib), have demonstrated clinical benefit of these combinations primarily in treatment-naïve mCRPC patients or in patients without prior exposure to Abiraterone. While these studies establish the therapeutic potential of PARP inhibition in the first-line mCRPC setting, they do not directly address mechanisms of resistance or therapeutic strategies in patients who have already progressed on Abiraterone therapy. In contrast, our study models Abiraterone-resistant prostate cancer using intrinsically hormone-insensitive cell lines and evaluates whether PARP inhibition can enhance antitumor activity in this resistant, AR-independent context. We believe this distinction highlights the complementary and translational relevance of our findings for a clinically high-need population.

Comment 7: The authors state “But tomorrow, do we have arguments for adding the PARPi to Abiraterone in order to overcome acquired resistance based on HRR recombination and BRCA mutations?” They likely mean HRR deficiencies?

Response: We agree with the reviewer that the wording was imprecise. We have corrected this sentence to replace “HRR recombination” with “homologous recombination repair (HRR) deficiencies”, including BRCA1/2 alterations, to accurately reflect the underlying biology. This revision improves clarity and ensures consistency with current terminology used in the field.

Comment 8: I suggest the authors tone down this statement at the end of the Discussion: “From all these results we can now respond positively to the above-mentioned question: ‘Do we have arguments for adding the PARPi to Abiraterone in order to overcome acquired resistance based on HRR recombination and BRCA mutations?’. That will probably have a strong echo to the clinical setting …..…” As noted earlier, there are clinical papers on this (e.g., PMID: 38319800). Instead, they should discuss how they have investigated the therapeutic potential of this combination, particularly in abiraterone resistant and AR independent disease settings seen in advanced prostate cancer. The authors should also cite the important clinical study (PMID: 38319800) demonstrating the efficacy of abiraterone combined with olaparib in mCRPC.

Response: We thank the Reviewer for this constructive suggestion, and we agree that the original concluding statement overstated the translational implications of our findings. We have therefore revised the final paragraph of the Discussion into two paragraphs to tone down claims of clinical readiness and to more appropriately position our results within the context of existing clinical evidence. Specifically, we have rephrased this section to emphasize that our study investigates the therapeutic potential and biological rationale of combining abiraterone with PARP inhibition in abiraterone-resistant and AR-independent prostate cancer models, rather than providing definitive arguments for clinical implementation. This revised framing highlights the relevance of our findings to advanced disease settings that are not directly addressed in current first-line clinical trials. In addition, we have now cited the important clinical study demonstrating the efficacy of abiraterone combined with olaparib in mCRPC (PMID: 38319800, New Reference 47), as well as discussing how our preclinical data complement these clinical observations by addressing resistance mechanisms and AR-independent disease contexts.

Reviewer 3 Report

Comments and Suggestions for Authors

Major Comment:
The manuscript presents technically sound preclinical data supporting enhanced antitumor effects of PARP inhibitors combined with abiraterone; however, the central claims require refinement. The study frames its findings as overcoming abiraterone resistance and demonstrating synergistic activity, yet the experimental models used are intrinsically AR-negative and no formal synergy analysis is provided. In addition, mechanistic insight remains limited, and the translational relevance to clinically studied AR-positive or BRCA-altered populations is not fully reconciled. Addressing these conceptual, mechanistic, and interpretive issues is necessary to strengthen the manuscript.

1. The manuscript positions the study as addressing abiraterone resistance; however, resistance in this work is defined by lack of response at ≤10 µM abiraterone in PC3 and DU145 cells, both of which are intrinsically AR-negative. As shown in Figures 1A–D and Figure 6, these models lack detectable AR expression at baseline. The authors should explicitly clarify that the study interrogates intrinsic hormone insensitivity rather than acquired abiraterone resistance, as this distinction materially affects interpretation of the conclusions.
2. In Figures 2A–D, the combination of niraparib or olaparib with abiraterone is described as synergistic; however, the dose–response matrices shown do not include combination index calculations or reference models for additivity. In several conditions (e.g., niraparib ≥10 µM in PC3 cells), the observed loss of viability is comparable between monotherapy and combination treatment, suggesting dominant PARP inhibitor cytotoxicity rather than combination-specific effects.
3. In the 3D organoid assays (Figures 3 and 4), combination effects are only apparent within a narrow intermediate dose range. At higher PARP inhibitor concentrations (20–50 µM), organoid viability approaches baseline-independent toxicity, making it difficult to distinguish combination effects from monotherapy-driven cell death. The authors should explicitly delineate this effective window and avoid extrapolating synergy across the full concentration range tested.
4. The mechanistic interpretation relies heavily on TUNEL staining (Figure 8) to infer apoptosis as the primary driver of tumor suppression. However, Ki67 staining (Figure 7) shows minimal differences between treatment groups, and no DNA damage or repair markers are assessed. Without evidence of increased DNA damage or impaired homologous recombination, the mechanistic link between PARP inhibition and enhanced abiraterone sensitivity remains speculative.
5. In the in vivo CDX experiments (Figure 5), treatment efficacy is reported exclusively as endpoint tumor weight. Although the authors state that irregular tumor geometry precluded reliable volume measurements, omission of longitudinal growth data limits assessment of treatment kinetics, variability, and early versus late effects. Presentation of individual tumor trajectories or waterfall plots would strengthen confidence in the in vivo conclusions.
6. The Discussion emphasizes clinical trial outcomes from PROpel and related studies at length, yet the experimental systems used here do not model the AR-positive or BRCA-altered patient populations that benefited most in those trials. The authors should more explicitly reconcile this discrepancy and clarify how AR-negative, BRCA-wildtype CDX data inform interpretation of these clinical results.
7. The claim that the combination therapy “overcomes abiraterone resistance” is reiterated in the Abstract, Results, and Discussion; however, no model of acquired resistance (e.g., long-term abiraterone adaptation or resistant derivatives) is presented. The language should be revised to reflect the specific biological context tested rather than generalized resistance reversal.
8. Figure legends for Figures 2–5 use multiple statistical symbols (≠, #, $, *) within the same panels, requiring frequent cross-referencing to the legend text. This substantially reduces readability and could be simplified without loss of statistical rigor.
9. While the study convincingly demonstrates that combination treatment increases tumor cell death relative to single agents, the absence of a unifying mechanistic schematic or model makes it difficult to integrate the PC3 versus DU145 differences (niraparib-sensitive vs olaparib-sensitive) into a coherent biological framework.
10. Overall, the data are technically sound and internally consistent, but the conclusions would benefit from more precise alignment between the biological models used, the claims made about resistance and synergy, and the clinical context emphasized in the Discussion.

 

Comments on the Quality of English Language

Can be improved. 

Author Response

Comments and responses to Comments of Reviewer 3:

As for Reviewers 1 and 2, we greatly thank Reviewer 3 for his/her appreciation of our work and for the comments raised. We responded to them the best we can. As for Reviewer 1 and 2, these comments have a very favorable impact on the results depicted in the present study, now in the new manuscript.

We made changes according to the comments in the revised manuscript. We have highlighted all the changes in yellow in the text of the revised manuscript.

Major Comment:

The manuscript presents technically sound preclinical data supporting enhanced antitumor effects of PARP inhibitors combined with abiraterone; however, the central claims require refinement. The study frames its findings as overcoming abiraterone resistance and demonstrating synergistic activity, yet the experimental models used are intrinsically AR-negative and no formal synergy analysis is provided. In addition, mechanistic insight remains limited, and the translational relevance to clinically studied AR-positive or BRCA-altered populations is not fully reconciled. Addressing these conceptual, mechanistic, and interpretive issues is necessary to strengthen the manuscript.

Comment 1: The manuscript positions the study as addressing abiraterone resistance; however, resistance in this work is defined by lack of response at ≤10 µM abiraterone in PC3 and DU145 cells, both of which are intrinsically AR-negative. As shown in Figures 1A–D and Figure 6, these models lack detectable AR expression at baseline. The authors should explicitly clarify that the study interrogates intrinsic hormone insensitivity rather than acquired abiraterone resistance, as this distinction materially affects interpretation of the conclusions.

Response: We thank the Reviewer for this important clarification and fully agree that the distinction between intrinsic hormone insensitivity and acquired abiraterone resistance is critical for accurate interpretation of our findings. To address this concern, we have revised the manuscript to explicitly clarify that the PC3 and DU145 models used in this study represent intrinsic hormone-insensitive, AR-negative prostate cancer, rather than models of acquired resistance following abiraterone exposure. Specifically, we have added a dedicated paragraph at the end of the Introduction section that describes baseline AR expression in PC3 and DU145 cells, supported by the data shown in Figures 1A–D and Figure 6, which confirm the absence of detectable AR expression in these models.

In addition, we have refined the language throughout the manuscript—particularly in the Introduction and Discussion to replace references to “acquired abiraterone resistance” with more precise terminology, such as intrinsic hormone insensitivity or AR-independent disease, where appropriate. These revisions ensure that the scope and implications of our study are accurately framed and aligned with the experimental models employed.

Comment 2: In Figures 2A–D, the combination of niraparib or olaparib with abiraterone is described as synergistic; however, the dose–response matrices shown do not include combination index calculations or reference models for additivity. In several conditions (e.g., niraparib ≥10 µM in PC3 cells), the observed loss of viability is comparable between monotherapy and combination treatment, suggesting dominant PARP inhibitor cytotoxicity rather than combination-specific effects.

Response: We thank the Reviewer for this important point and agree that the use of the term “synergistic” requires formal quantitative assessment using established additivity reference models (e.g., Bliss, Loewe, or combination index calculations), which were not performed in this study.

Treatment with PARP inhibitors in combination with abiraterone resulted in greater reductions in cell viability compared with abiraterone alone in selected conditions. However, at higher PARP inhibitor concentrations, particularly in PC3 cells treated with niraparib, the observed effects were predominantly driven by PARP inhibitor monotherapy, as we indicated in 3.2 ‘suggesting that the observed effects are likely due to Niraparib monotherapy rather than a synergistic interaction’.

Comment 3: In the 3D organoid assays (Figures 3 and 4), combination effects are only apparent within a narrow intermediate dose range. At higher PARP inhibitor concentrations (20–50 µM), organoid viability approaches baseline-independent toxicity, making it difficult to distinguish combination effects from monotherapy-driven cell death. The authors should explicitly delineate this effective window and avoid extrapolating synergy across the full concentration range tested.

Response: We thank the Reviewer for this comment and appreciate the opportunity to clarify the rationale for the concentration range used in the 3D organoid experiments. The higher concentrations of PARP inhibitors employed in the organoid assays reflect well-established differences between 2D monolayer cultures and 3D organoid systems. In particular, 3D organoids exhibit increased drug tolerance due to limited compound penetration, altered diffusion kinetics, cell–cell interactions, and extracellular matrix components, which frequently necessitate the use of higher drug concentrations to achieve biologically meaningful effects compared with 2D cultures.

Accordingly, the 20–50 µM range was included to ensure adequate exposure and to capture treatment responses across the full dynamic range of the 3D model. While we agree that at the highest concentrations organoid viability is substantially reduced and effects are likely driven predominantly by PARP inhibitor cytotoxicity, these doses were necessary to define the upper boundary of response in this system. We hope that the Reviewer will agree with these explanations.

Comment 4: The mechanistic interpretation relies heavily on TUNEL staining (Figure 8) to infer apoptosis as the primary driver of tumor suppression. However, Ki67 staining (Figure 7) shows minimal differences between treatment groups, and no DNA damage or repair markers are assessed. Without evidence of increased DNA damage or impaired homologous recombination, the mechanistic link between PARP inhibition and enhanced abiraterone sensitivity remains speculative.

Response: We thank the Reviewer for this thoughtful comment and agree that the mechanistic interpretation of PARP inhibitor–mediated effects would be strengthened by the inclusion of direct DNA damage or homologous recombination repair markers. In the present study, TUNEL and Ki67 staining were used primarily as phenotypic readouts of treatment efficacy, reflecting changes in cell death and proliferation rather than serving as a comprehensive mechanistic dissection of PARP inhibitor activity.

The primary objective of this work was to address a clinically relevant question: whether the addition of PARP inhibition can enhance therapeutic response in intrinsically hormone-insensitive, abiraterone-resistant prostate cancer models, rather than to fully delineate the molecular pathways underlying this effect. As such, analyses of DNA damage signaling or homologous recombination status were beyond the scope of the current study. To avoid overinterpretation, we have indicated at various places throughout the former manuscript that the intimate mechanism of the observed effects was not the purpose of the present study. We agree that future studies incorporating markers of DNA damage response and repair will be important to further elucidate the mechanistic basis of the observed combination effects. We hope that the Reviewer will agree with these explanations.

Comment 5: In the in vivo CDX experiments (Figure 5), treatment efficacy is reported exclusively as endpoint tumor weight. Although the authors state that irregular tumor geometry precluded reliable volume measurements, omission of longitudinal growth data limits assessment of treatment kinetics, variability, and early versus late effects. Presentation of individual tumor trajectories or waterfall plots would strengthen confidence in the in vivo conclusions.

Response: We thank the Reviewer for this thoughtful comment. In our study, we monitored tumor size throughout the experiment, but we chose to report end point tumor weights as the main measure of treatment efficacy. The reason is that many of the tumors had irregular shapes, which made volume estimates using calipers less reliable and sometimes misleading. Tumor weight, in contrast, provides a straightforward and accurate measure of the actual tumor burden at the end point, making comparisons between groups more dependable. We agree that longitudinal growth curves or waterfall plots can provide extra insight into treatment dynamics. However, in this model, the irregular tumor geometry limited the usefulness of volume-based data. We have added a brief explanation in the Results section to clarify why we focused on tumor weight, so readers understand our reasoning. Specifically, we modified the sentence in the corresponding Results section in the revised manuscript by indicating: ‘Since the growth of tumors was irregular in shape, we decided to show the effects of the treatments on tumor weights at the end of the experimental period. Indeed, many of the tumors had irregular shapes, which made volume estimates using calipers less reliable and sometimes misleading. Tumor weight, in contrast, provides a straightforward and accurate measure of the actual tumor burden at the end point, making comparisons between groups more dependable.’

Comment 6: The Discussion emphasizes clinical trial outcomes from PROpel and related studies at length, yet the experimental systems used here do not model the AR-positive or BRCA-altered patient populations that benefited most in those trials. The authors should more explicitly reconcile this discrepancy and clarify how AR-negative, BRCA-wildtype CDX data inform interpretation of these clinical results.

Response: We thank the Reviewer for highlighting this important point. It is true that recent clinical trials, such as PROPEL (Abiraterone + Olaparib) and MAGNITUDE (Abiraterone + Niraparib), showed the most benefit in AR-positive and BRCA-altered mCRPC patients, whereas our experimental models are AR-negative and BRCA-wildtype.

This is why we have clarified that in the Discussion section in the former manuscript indicating that these clinical studies primarily evaluated treatment-naïve patients or those without prior Abiraterone exposure and therefore do not directly address resistance mechanisms in patients who have already progressed on Abiraterone therapy. In contrast, our study focuses on intrinsically hormone-insensitive, abiraterone-resistant models to ask whether PARP inhibition can enhance treatment efficacy in a clinically distinct, high-need population that is not represented in those trials.

While our models do not replicate the exact genetic and AR context of the patients who benefited most in PROPEL or MAGNITUDE, they provide complementary insights into how PARP inhibitors may overcome resistance in AR-independent disease, highlighting potential avenues for patient populations that remain largely underserved.

Comment 7: The claim that the combination therapy “overcomes abiraterone resistance” is reiterated in the Abstract, Results, and Discussion; however, no model of acquired resistance (e.g., long-term abiraterone adaptation or resistant derivatives) is presented. The language should be revised to reflect the specific biological context tested rather than generalized resistance reversal.

Response: Thank you. We have modified the terms in the different sections.

Comment 8: Figure legends for Figures 2–5 use multiple statistical symbols (≠, #, $, *) within the same panels, requiring frequent cross-referencing to the legend text. This substantially reduces readability and could be simplified without loss of statistical rigor.

Response: We thank the Reviewer for this comment. We used multiple statistical symbols in Figures 2–5 to clearly distinguish between different comparisons within each panel—for example, comparing each treatment to control versus comparing treatments to one another. Using a single symbol for all comparisons would make it difficult for readers to follow which differences are being highlighted. To help with readability, all symbols are explicitly defined in the Figure legends so that each comparison is clearly explained. We believe this approach strikes a balance between clarity and statistical rigor, ensuring that readers can accurately interpret all the relevant comparisons while maintaining transparency about the tests performed. Both additional Reviewers did not make specific comments about that. We hope that the Reviewer will agree with these explanations.

Comment 9: While the study convincingly demonstrates that combination treatment increases tumor cell death relative to single agents, the absence of a unifying mechanistic schematic or model makes it difficult to integrate the PC3 versus DU145 differences (niraparib-sensitive vs olaparib-sensitive) into a coherent biological framework.

Response: We thank the Reviewer for this insightful comment. To provide a clearer framework, we have added a paragraph in the Introduction highlighting the distinct HRR-related genetic backgrounds of PC3 and DU145 cells, which likely underlie their differential sensitivity to PARP inhibitors. Specifically, PC3 cells harbor mutations associated with HRR deficiency, making them more sensitive to niraparib, while DU145 cells carry BRCA-related alterations, favoring olaparib sensitivity. In this way, the two models can be seen as preclinical analogs to patient populations in clinical trials: PC3 reflecting the MAGNITUDE trial (patients with HRR mutations) and DU145 reflecting the PROPEL trial (patients with BRCA mutations). This addition provides a unifying context to interpret the cell line–specific responses and helps readers connect the preclinical findings to relevant clinical observations.

Comment 10: Overall, the data are technically sound and internally consistent, but the conclusions would benefit from more precise alignment between the biological models used, the claims made about resistance and synergy, and the clinical context emphasized in the Discussion.

Response: Thank you very much. As explicated in the responses to previous comments, and responses to other Reviewers, we think that the we have precise that in a much better and accurate manner.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed my queries, and I congratulate them on this study.