Potent synthetic lethality between PLK1 and EYA-family inhibitors in tumours of the central and peripheral nervous system
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (Review Commons)
Abstract
The Eyes Absent family of protein phosphatases (EYA1-4) are aberrantly expressed and tumour-promoting across many devastating cancers of neurological origin affecting both children and adults. It has recently been demonstrated that EYA1 and EYA4 promote tumour cell survival by increasing the active pool of Polo-like kinase 1 (PLK1) molecules. This discovery provides a rationale for the therapeutic combination of EYA inhibitors with direct, ATP-competitive, PLK1 inhibitors. Here, we demonstrate potent and synergistic effects of EYA and PLK1 inhibition in cancer cell lines that overexpress EYA1 and/or EYA4, including in neuroblastoma and glioblastoma models. We identify decreases in PLK1 activity and RAD51 foci formation, and increases in mitotic arrest and cell death, as mechanistic contributors to combination sensitivity. Combined EYA and PLK1 inhibition is also effective in glioblastoma stem cell models that overexpress EYA1/EYA4 and specifically targets the cancer stem cell state. Finally, through multi-omic correlational analysis, we identify high levels of the NuRD complex and SOX9 as contributors to combination treatment sensitivity. Overall, this work identifies a novel synthetic lethal combination therapy with potential utility across a wide range of neurological cancers.
Article activity feed
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This manuscript addresses the question of whether inhibitors of the phosphatases Eya1-4 and of the kinase PLK1 provide an effective therapeutic approach to a range of cancers. Both Eyas and PLK1 have well documented roles in development, and have been implicated in a subset of tumors. Moreover, the authors have previously shown that PLK1 is a substrate of Eya phosphatase activity. Building on these previous findings, the authors assess the possibility of combining an Eya inhibitor, benzarone, with a PLK1 inhibitor, BI2536.
There are several concerns with the study:
- The authors suggest that …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This manuscript addresses the question of whether inhibitors of the phosphatases Eya1-4 and of the kinase PLK1 provide an effective therapeutic approach to a range of cancers. Both Eyas and PLK1 have well documented roles in development, and have been implicated in a subset of tumors. Moreover, the authors have previously shown that PLK1 is a substrate of Eya phosphatase activity. Building on these previous findings, the authors assess the possibility of combining an Eya inhibitor, benzarone, with a PLK1 inhibitor, BI2536.
There are several concerns with the study:
- The authors suggest that these two drugs are synergistic. Synergy is usually taken as indicative of a greater than additive effect of the two drugs. The ZIP synergy score tested here indicates that the combination of the two drugs has a synergy score between 0 and 10 (figure1, and figure 5). According to "Synergy Finder", "A ZIP synergy score of greater than 10 often indicates a strong synergistic effect, while a score less than -10 suggests a strong antagonistic effect. Scores between -10 and 10 are typically considered additive or near-additive." The data in figure 2 on mitotic cell fraction and on cell death also seems to be more of an additive effect of the two drugs than synergy. The data in figure 3 are also additive effects on RAD51. Therefore a conclusion that "These data indicate that the drug combination was broadly synergistic" seems unwarranted.
There is a general lack of nomenclature standardisation for defining synergy. Furthermore, multiple synergy models exist, with discrepancies between them. However, as the reviewer states, the prevailing view is that synergy is a combination effect that is stronger than the additive effect of the two drugs. Synergy scores derived from dose-response matrices using different synergy scoring models with scores that fall above 5 are considered truly synergistic (Malyutina A et al., 2019). To strengthen our conclusion of synergy between PLK1 and EYA inhibitors, we have calculated synergy scores using additional synergy models for both benzarone + BI2536 and benzarone + volasertib in H4 and T98G cell lines. Specifically, we find robust synergy (>5) using ZIP, HSA and Bliss calculations with the Benzarone + BI2536 drug combination in H4 cells and with Benzarone + Volasertib in H4 and T98G cells. Synergy scores for Benzarone + BI2536 fell just below 5 in T98G cells. These data are now included in Supplemental Fig S1G of the revised manuscript.
The discovery of synergistic drug combinations can be further strengthened by evaluating synergy across multiple cellular models. In this study, we have tested a total of 27 different cancer models that universally support synergy.
Regarding the phenotypic outcomes (mitotic cell fraction, cell death, RAD51 foci), we agree that the observed effects are additive. This is consistent with overall synergistic effects on viability being caused by a combination of additive mechanistic effects. We have amended the text in the revised manuscript to clarify this point.
There was no statistical difference in the synergy scores of the "high expressing" versus "low expressing cells". So the conclusion that the drug combination "t was effective at lower doses in cell lines with high levels of EYA1 and/or EYA4" seems unwarranted based on the data. Moreover, since there was no statistical difference in synergy between high and low expressing cells, stating that "the potential utility of the combination treatment depends on the specific overexpression of EYA1 and/or EYA4 in cancer cells," seems unwarranted by the data.
Synergy scores quantify the interaction between drugs, but do not capture absolute treatment effectiveness or dose sensitivity, both of which are crucial for therapeutic considerations. We have included the following sentence in the revised manuscript to clarify this distinction: “While synergy scores did not significantly differ between high and low EYA expressors, high EYA1/4 expression was associated with increased sensitivity to the combination treatment at lower doses, as evidenced by decreased cell viability.” We have also amended the conclusions in the Abstract and Discussion to reflect that the potential utility of the combination therapy in EYA1/4-high cancers is supported by potency rather than synergy scores alone.
Benzarone and benzbromarone and their derivatives have been shown to bind and inhibit Eya phosphatases, albeit at fairly high doses. However, these two compounds also have a number of other, unrelated targets. The only demonstration that Eyas are a target of benzarone in this study are the CETSA data in supplemental figure 1. The data here seem to represent an n of 1, with no error bars shown. Even more importantly, there is no control. Looking at the blot of actin, it seems as if there may be a benzarone- temperature effect on this protein as well. It would be very helpful to show some evidence that knockdown of Eya similarly synergizes with the PLK1 inhibitor, show data that benzarone is in fact inhibiting Eya activity in these cells by looking at known targets (ie the carboxyterminal tyrosine of H2AX), and other evidence of specificity.
The specificity of benzarone to the EYA proteins has been demonstrated previously using both in vitro phosphatase assays and the assessment of EYA-mediated pathways (Tadjuidje et al., 2012; Wang et al., 2021; Nelson et al., 2024). These publications have been cited in the manuscript. In addition, benzarone produces phenotypes consistent with the known functions of the EYAs (ie, reduction of PLK1 activity, reduction in RAD51 foci, G2/M arrest, and apoptosis). To further validate EYA target specificity, we have performed viability assays on control and EYA4-depleted HeLa, H4 and T98G cells in response to BI2536 treatment, demonstrating EYA4 depletion-mediated sensitization to BI2536. These data are now included in Fig 1H of the revised manuscript.
To strengthen our CETSA data, we have now included: (i) densitometry of actin, demonstrating a lack of benzarone-temperature effect, (ii) CETSA analysis for an additional cell line (T98G), demonstrating enhanced thermal stability of the EYAs in the presence of benzarone, and (iii) CETSA analysis of an additional protein (BUB1) to demonstrate target specificity. These data are now included in Supplemental Fig S1E and F of the revised manuscript.
The proteomic and transcriptomic data of cell lines that were vulnerable to the combination of BI2536 and benzarone implicate overall changes in chromatin with sensitivity. These findings call into question the idea that these two compounds are acting selectively on PLK1 and Eyas. The authors don't really provide any model for explaining this correlation of Nurd complex components with targeting Eyas and PLK1.
The proteomic and transcriptomic data demonstrate that sensitivity to the combination treatment is associated with higher expression of NuRD complex members and other chromatin regulators. This suggests that cell lines with certain chromatin configurations might be more susceptible to the combined inhibition of PLK1 and EYA. This does not undermine the demonstrated on-target effects of the two compounds, but rather suggests a potential contextual dependence of drug efficacy on chromatin state. Our data thereby implicate NuRD complex expression as a predictive biomarker for tumours that are likely to respond to EYA and PLK1 combination therapy. This has now been clarified in the discussion section of the revised manuscript.
Specificity of antibodies: I would like to see validation of the Eya antibodies, given the difficulty with such reagents in the field.
All EYA antibodies have now been validated by western blot analysis following siRNA-mediated depletion. These data are presented in Supplemental Fig S1A of the revised manuscript.
Reviewer #1 (Significance (Required)):
New therapies targeting glioblastoma would be welcome. It is not clear that the combination tested here is an effective approach to therapy. It would be necessary to know the targets of the combination and understand the mechanism so that the approach could be pursued further,
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This study explores the sensitivity of cancer cell lines, particularly GBM cells, to dual inhibition of EYA and PLK1, aiming to uncover the connection between these pathways and the cancer stem cell state. Additionally, it investigates whether the NuRD complex modulates GBM cell responses to EYA and PLK1 inhibition. While the findings are interesting, further clarification is needed to establish the mechanistic links between EYA, PLK1, and NuRD, as well as a stronger rationale for their targeted inhibition in GBM therapy- this can be better clarified.
Some key comments and recommendations: The findings demonstrate that the combination of Benzarone (EYAi) and volasertib (PLKi) significantly reduced cell proliferation in H4 and T98G GBM cell lines, both of which show high expression of EYA. In contrast, the low EYA-expressing A172 cells exhibited limited response. A possible explanation is the inherently slower proliferation rate of A172 cells, which may reduce their dependence on G2/M arrest, thereby diminishing the impact of PLK1i. Does A172 line show a similar growth or cell division rate to H4 and T98G lines.
A172 cells have a slower proliferation rate than H4 or T98G cells, which may diminish their response to EYA/PLK1 inhibitors. However, in this study we have tested a total of 15 cancer cell lines and 12 GBM stem cell line models. No clear correlation between cell growth rate and sensitivity was observed. As a specific example, the low EYA expressing SJSA-1 cell line has a high proliferation rate but is a low responder to EYA1/PLK1 inhibitors.
Additionally, although protein expression levels of EYA were assessed across these cell lines, the activity and expression levels of PLK1 were not fully characterized. Since PLK1 is a crucial regulator of mitotic entry and DNA damage repair, its activity across cell lines may contribute to the observed variations in drug sensitivity. Could the authors investigate levels of PLK in these cell lines?
To address this point, we compared PLK1 expression levels across the panel of cancer cell lines used in our study. These data are now included in Supplemental Fig S1D of the revised manuscript, and show that PLK1 levels are comparable across the cell lines, indicating that baseline PLK1 abundance does not fully explain the observed differential sensitivity.
The study describes the combination treatment as synergistic in H4 and T98G cells, however this synergy is unclear in Fig 2A and Supplemental Fig S2A. The data suggest that H4 and T98G cells exhibit sensitivity to either EYA or PLK1 inhibition alone, with combined treatment showing enhanced effects rather than synergy. This distinction is evident as BI2536 alone induces robust G2/M arrest with decreased G1 and S phase cells. To validate these findings, combination treatment should be tested in additional GBM cell lines. Additionally, repeating FUCCI cell cycle assays in A172 and H4 cells, particularly in H4, where increased γH2AX and phospho-H3 were detected in response to individual inhibitors, would provide more definitive insights into treatment-induced cell cycle dynamics.
We agree that several of the phenotypic outcomes, for example G2/M arrest (Fig 2A) and micronuclei formation (Supplemental Fig S2A), produce additive rather than synergistic effects in the combination treated cells. The major claim of the study is that the combination treatment results in potent loss of cell viability in EYA1/EYA4 overexpressing cancer cell models. This is consistent with a combination of additive mechanistic effects causing overall synergistic effects on cancer cell viability. We have clarified this point in the revised manuscript.
We have previously struggled to get adequate FUCCI sensor expression in H4 cells. However, to address this point, we have quantified cell cycle phase distribution in H4 cells treated with benzarone, BI2536, and the drug combination, using our quantitative image-based cytometry data (Fig 3A, B). These data demonstrate an accumulation of H4 cells in G2/M following combination treatment, consistent with the FUCCI data from T98G cells. Cell cycle dynamics of H4 cells are now included in Supplemental Fig S2A of the revised manuscript.
A notable inconsistency: Figure 1 utilizes volasertib, whereas Figure 2 employs BI2536. Given that both inhibitors target PLK1 why these specific inhibitors were chosen for each experiment.
This is not the case. To clarify, BI2536 is used in both Fig 1 and 2. Volasertib is used in Supplemental Fig S1 to reproduce the synergy matrix, thereby demonstrating consistent results with a second PLK1 inhibitor.
The observation of increased Rad52 foci and sister chromatid exchange (SCE) upon EYA and PLK1 inhibition (Figure 3) is interesting. These findings suggest that dual inhibition impairs homologous recombination (HR), reinforcing the role of EYA and PLK1 in maintaining genomic stability.
We agree.
Figure 4 suggests that SJH1 cells, with low EYA expression, exhibit increased sensitivity to EYA inhibition - does this cell line show high expression of PLK or NuRD?
To clarify, Fig 4 shows that SJH1 cells, which display moderate levels of EYA expression, are highly sensitive to EYA/PLK1 inhibition. Consistent with the observed positive correlation between NuRD protein expression and EYA/PLK1 inhibitor sensitivity, SJH1 cells exhibit the highest levels of NuRD components relative to the other GBM stem cell lines. Expression levels of NuRD components across the slightly sensitive, moderately sensitive, and highly sensitive GBM stem cell lines from publicly available proteomic data and western blot analysis have now been included in Supplemental Fig S5A and B of the revised manuscript, further demonstrating this positive correlation.
It seems like EYA1 (HW1) and EY4 (SB2B and PB1) expression levels are better predictors of sensitivity to treatment, but not EYA2 and 3 (which is high in H4)- can the authors comment on this?
Overall, EYA1 and EYA4 expression levels are the major predictors of EYA/PLK1 inhibitor sensitivity in both the cancer cell lines (Fig 1) and the GBM stem cell models (Fig 4). EYA3 levels are also positively associated with sensitivity in the GBM stem cell models, but not in the cancer cell lines. Despite being consistently high, EYA2 expression levels were not associated with sensitivity in either model. These intricacies are likely to reflect functional differences between the proteins, and their ability to form different sub-complexes with each other. We have now clarified these points in the discussion of the revised manuscript.
Reviewer #2 (Significance (Required)):
It remains unclear whether NuRD complex involvement is independent of EYA expression levels. Since EYA and PLK1 regulate cell cycle progression and DNA repair, further investigation is needed to delineate their connection to NuRD-mediated chromatin remodeling and differentiation programs. Overall, this study provides some interesting evidence for targeting transcriptional and mitotic vulnerabilities in GBM but requires further validation of synergistic mechanisms, differential inhibitor effects, and NuRD complex involvement in regulating the EYA-PLK1 axis.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
This manuscript extends the findings of the interactions between EYA family members and PLK1. The idea to combine EYA inhibitors and PLK1 inhibitors is a thoughtful approach. The effects on proliferation and DNA damage are useful. This effort is a combination of preclinical efforts and some mechanistic efforts and will require additional efforts to support the conclusions drawn.
Major concerns:
- The preclinical studies will absolutely require in vivo studies. All brain tumor treatments are limited by delivery across the blood-brain barrier. It is critical to have intracranial survival studies to support the significance of the findings.
In this study, we have focused on in vitro models including cancer cell lines, GBM stem cell models and 3D tumor spheroids, to establish proof-of-principle as well as mechanistic insight for combined EYA/PLK1 inhibition. We recognize that blood-brain-barrier penetration and therapeutic efficacy in vivo are key translational steps; however, we feel that benzarone is a suboptimal drug candidate for in vivo evaluation. Future development of second-generation EYA inhibitors with higher potency, improved selectivity, and better blood-brain-barrier permeability, is currently underway by ourselves and other groups. These compounds are likely to be more suitable for future in vivo studies, including pharmacokinetic profiling, blood-brain-barrier penetration assays, and orthotopic intracranial tumour models to assess their therapeutic potential more rigorously.
Likewise, cancer stem cell studies require in vivo studies.
As outlined above, we feel that in vivo studies fall beyond the scope of this study.
The proper studies of sphere formation would include in vitro limiting dilution assays. I would suggest greater depth in stem cell and differentiation marker studies to understand what the connection to stemness is.
The limiting dilution assay is used to measure the self-renewal potential of cancer stem cells, and would be used in this context to determine whether the treatments impact cellular differentiation. This is not the focus of this study. Rather, we are interested in comparing drug sensitivity in cancer stem cells versus differentiated cancer cells. Nevertheless, this is a great suggestion for future investigation as part of a more detailed evaluation of stemness and how these drugs and drug combinations impact self-renewal.
DNA damage responses differ between cancer stem cells and differentiated tumor cells. I would suggest comparison of effects between matched cells with different cell states.
We agree that cancer stem cells and their differentiated counterparts often display distinct DNA damage responses. We have tried to mimimise the impact of these differences on the overall conclusions by using multiple cancer cell lines and GBM stem models. To address this comment, we performed western blot analysis of DNA damage response proteins in matched PB1 stem cells and differentiated cells, demonstrating comparable expression of DNA damage response proteins. These data have now been included in Supplemental Fig S5C of the revised manuscript.
While the inhibitors used may have general specificity for the molecular targets, I would suggest that the authors use genetic loss-of-function and gain-of-function studies to validate the findings. It is particularly important because the primary targets do not predict treatment responses. I would suggest that rescues with PLK1 phosphorylation mutants would be helpful.
Our data demonstrate that EYA expression levels are predictive of treatment response in both cancer cell lines and GBM stem cell models. To further validate EYA target specificity, we have used a genetic loss-of-function approach. Specifically, we performed viability assays on control and EYA4-depleted HeLa, H4 and T98G cells in response to BI2536 treatment, demonstrating EYA4 depletion-mediated sensitization to BI2536. These data are now included in Fig 1H of the revised manuscript.
We have previously performed comprehensive rescue experiments with PLK1 phosphorylation mutants (Fig 5C–K; Nelson et al., Nat. Commun. 2024). These experiments demonstrated that cell death in response to EYA depletion or inhibition is attributable to the phosphorylation status of pY445 on PLK1, with an accumulation of Y445 phosphorylation reducing PLK1 activity and functionality, culminating in the potent induction of mitotic cell death.
Figure 5 should be performed with several lines across different response groups.
Our study currently includes cell viability and proliferation data from multiple models including 15 cancer cell lines and 12 GBM stem cell line models, spanning different EYA expression levels, and displaying varying sensitivities to both single agents and the EYA/PLK1 combination treatment. We then narrowed the number of models significantly for follow-up analysis. In Fig 5, we selected the highly sensitive PB1 GBM stem cell line based on its ability to form and grow as spheroids. While we appreciate the suggestion to expand these analyses to additional lines, we would like to respectfully decline growing additional spheroids at this time due to limitations inherent in the expansion of these models. We believe that the current dataset adequately demonstrates the reproducibility and relevance of our findings across different response groups.
The molecular associations are currently just associations. I would suggest greater analysis using genetic manipulation to test causation.
To address this concern, we have performed additional experiments using siRNA-mediated knockdown of EYA4 in HeLa, H4 and T98G cells. These experiments demonstrate that depletion of EYA4 sensitizes cells to PLK1 inhibition, mimicking the effects observed with pharmacological EYA inhibition. These data have been included in Fig 1H of the revised manuscript, and provide additional functional evidence supporting a causal relationship between EYA activity and sensitivity to PLK1 inhibition.
Figure 6 should be better developed to include protein testing and validation.
To address this point, expression levels of NuRD components have been compared using publicly available proteomic datasets and western blot analysis across the slightly sensitive, moderately sensitive and highly sensitive GBM stem cell lines, supporting a positive correlation with sensitivity. These data have been included in Supplemental Fig S5A and B of the revised manuscript.
Reviewer #3 (Significance (Required)):
This is a modest advance in understanding how EYA family members may function with PLK1.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
This manuscript extends the findings of the interactions between EYA family members and PLK1. The idea to combine EYA inhibitors and PLK1 inhibitors is a thoughtful approach. The effects on proliferation and DNA damage are useful. This effort is a combination of preclinical efforts and some mechanistic efforts and will require additional efforts to support the conclusions drawn.
Major concerns:
The preclinical studies will absolutely require in vivo studies. All brain tumor treatments are limited by delivery across the blood-brain barrier. It is critical to have intracranial survival studies to support the significance of the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
This manuscript extends the findings of the interactions between EYA family members and PLK1. The idea to combine EYA inhibitors and PLK1 inhibitors is a thoughtful approach. The effects on proliferation and DNA damage are useful. This effort is a combination of preclinical efforts and some mechanistic efforts and will require additional efforts to support the conclusions drawn.
Major concerns:
The preclinical studies will absolutely require in vivo studies. All brain tumor treatments are limited by delivery across the blood-brain barrier. It is critical to have intracranial survival studies to support the significance of the findings.
Likewise, cancer stem cell studies require in vivo studies.
The proper studies of sphere formation would include in vitro limiting dilution assays. I would suggest greater depth in stem cell and differentiation marker studies to understand what the connection to stemness is.
DNA damage responses differ between cancer stem cells and differentiated tumor cells. I would suggest comparison of effects between matched cells with different cell states.
While the inhibitors used may have general specificity for the molecular targets, I would suggest that the authors use genetic loss-of-function and gain-of-function studies to validate the findings. It is particularly important because the primary targets do not predict treatment responses. I would suggest that rescues with PLK1 phosphorylation mutants would be helpful.
Figure 5 should be performed with several lines across different response groups.
The molecular associations are currently just associations. I would suggest greater analysis using genetic manipulation to test causation.
Figure 6 should be better developed to include protein testing and validation.
Significance
This is a modest advance in understanding how EYA family members may function with PLK1.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This study explores the sensitivity of cancer cell lines, particularly GBM cells, to dual inhibition of EYA and PLK1, aiming to uncover the connection between these pathways and the cancer stem cell state. Additionally, it investigates whether the NuRD complex modulates GBM cell responses to EYA and PLK1 inhibition. While the findings are interesting, further clarification is needed to establish the mechanistic links between EYA, PLK1, and NuRD, as well as a stronger rationale for their targeted inhibition in GBM therapy- this can be better clarified.
Some key comments and recommendations:
The findings demonstrate that the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This study explores the sensitivity of cancer cell lines, particularly GBM cells, to dual inhibition of EYA and PLK1, aiming to uncover the connection between these pathways and the cancer stem cell state. Additionally, it investigates whether the NuRD complex modulates GBM cell responses to EYA and PLK1 inhibition. While the findings are interesting, further clarification is needed to establish the mechanistic links between EYA, PLK1, and NuRD, as well as a stronger rationale for their targeted inhibition in GBM therapy- this can be better clarified.
Some key comments and recommendations:
The findings demonstrate that the combination of Benzarone (EYAi) and volasertib (PLKi) significantly reduced cell proliferation in H4 and T98G GBM cell lines, both of which show high expression of EYA. In contrast, the low EYA-expressing A172 cells exhibited limited response. A possible explanation is the inherently slower proliferation rate of A172 cells, which may reduce their dependence on G2/M arrest, thereby diminishing the impact of PLK1i. Does A172 line show a similar growth or cell division rate to H4 and T98G lines.
Additionally, although protein expression levels of EYA were assessed across these cell lines, the activity and expression levels of PLK1 were not fully characterized. Since PLK1 is a crucial regulator of mitotic entry and DNA damage repair, its activity across cell lines may contribute to the observed variations in drug sensitivity. Could the authors investigate levels of PLK in these cell lines?
The study describes the combination treatment as synergistic in H4 and T98G cells, however this synergy is unclear in Figure 2A and EV 2A. The data suggest that H4 and T98G cells exhibit sensitivity to either EYA or PLK1 inhibition alone, with combined treatment showing enhanced effects rather than synergy. This distinction is evident as BI2536 alone induces robust G2/M arrest with decreased G1 and S phase cells. To validate these findings, combination treatment should be tested in additional GBM cell lines. Additionally, repeating FUCCI cell cycle assays in A172 and H4 cells, particularly in H4, where increased γH2AX and phospho-H3 were detected in response to individual inhibitors, would provide more definitive insights into treatment-induced cell cycle dynamics.
A notable inconsistency: Figure 1 utilizes volasertib, whereas Figure 2 employs BI2536. Given that both inhibitors target PLK1 why these specific inhibitors were chosen for each experiment.
The observation of increased Rad52 foci and sister chromatid exchange (SCE) upon EYA and PLK1 inhibition (Figure 3) is interesting. These findings suggest that dual inhibition impairs homologous recombination (HR), reinforcing the role of EYA and PLK1 in maintaining genomic stability.
Figure 4 suggests that SJH1 cells, with low EYA expression, exhibit increased sensitivity to EYA inhibition - does this cell line show high expression of PLK or NuRD?
It seems like EYA1 (HW1) and EY4 (SB2B and PB1) expression levels are better predictors of sensitivity to treatment, but not EYA2 and 3 (which is high in H4)- can the authors comment on this?
Significance
It remains unclear whether NuRD complex involvement is independent of EYA expression levels. Since EYA and PLK1 regulate cell cycle progression and DNA repair, further investigation is needed to delineate their connection to NuRD-mediated chromatin remodeling and differentiation programs.
Overall, this study provides some interesting evidence for targeting transcriptional and mitotic vulnerabilities in GBM but requires further validation of synergistic mechanisms, differential inhibitor effects, and NuRD complex involvement in regulating the EYA-PLK1 axis.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This manuscript addresses the question of whether inhibitors of the phosphatases Eya1-4 and of the kinase PLK1 provide an effective therapeutic approach to a range of cancers. Both Eyas and PLK1 have well documented roles in development, and have been implicated in a subset of tumors. Moreover, the authors have previously shown that PLK1 is a substrate of Eya phosphatase activity. Building on these previous findings, the authors assess the possibility of combining an Eya inhibitor,benzarone, with a PLK1 inhibitor, BI2536.
There are several concerns with the study:
The authors suggest that these two drugs are synergistic. Synergy …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This manuscript addresses the question of whether inhibitors of the phosphatases Eya1-4 and of the kinase PLK1 provide an effective therapeutic approach to a range of cancers. Both Eyas and PLK1 have well documented roles in development, and have been implicated in a subset of tumors. Moreover, the authors have previously shown that PLK1 is a substrate of Eya phosphatase activity. Building on these previous findings, the authors assess the possibility of combining an Eya inhibitor,benzarone, with a PLK1 inhibitor, BI2536.
There are several concerns with the study:
The authors suggest that these two drugs are synergistic. Synergy is usually taken as indicative of a greater than additive effect of the two drugs. The ZIP synergy score tested here indicates that the combination of the two drugs has a synergy score between 0 and 10 (figure1, and figure 5) . According to "Synergy Finder" , "A ZIP synergy score of greater than 10 often indicates a strong synergistic effect, while a score less than -10 suggests a strong antagonistic effect. Scores between -10 and 10 are typically considered additive or near-additive." The data in figure 2 on mitotic cell fraction and on cell death also seems to be more of an additive effect of the two drugs than synergy. The data in figure 3 are also additive effects on RAD51. Therefore a conclusion that "These data indicate that the drug combination was broadly synergistic" seems unwarranted. Indeed, the data form
There was no statistical difference in the synergy scores of the "high expressing" versus "low expressing cells". So the conclusion that the drug combination "t was effective at lower doses in cell lines with high levels of EYA1 and/or EYA4" seems unwarranted based on the data. Moreover, since there was no statistical difference in synergy between high and low expressing cells, stating that "the potential utility of the combination treatment depends on the specific overexpression of EYA1 and/or EYA4 in cancer cells," seems unwarranted by the data.
Benzarone and benzbromarone and their derivatives have been shown to bind and inhibit Eya phosphatases, albeit at fairly high doses. However, these two compounds also have a number of other, unrelated targets. The only demonstration that Eyas are a target of benzarone in this study are the CETSA data in supplemental figure 1. The data here seem to represent an n of 1, with no error bars shown. Even more importantly, there is no control. Looking at the blot of actin, it seems as if there may be a benzarone- temperature effect on this protein as well. It would be very helpful to show some evidence that knockdown of Eya similarly synergizes with the PLK1 inhibitor, show data that benzarone is in fact inhibiting Eya activity in these cells by looking at known targets (ie the carboxyterminal tyrosine of H2AX), and other evidence of specificity.
The proteomic and transcriptomic data of cell lines that were vulnerable to the combination of BI2536 and benzarone implicate overall changes in chromatin with sensitivity. These findings call into question the idea that these two compounds are acting selectively on PLK1 and Eyas. The authors don't really provide any model for explaining this correlation of Nurd complex components with targeting Eyas and PLK1.
Specificity of antibodies: I would like to see validation of the Eya antibodies, given the difficulty with such reagents in the field.
Significance
New therapies targeting glioblastoma would be welcome. It is not clear that the combination tested here is an effective approach to therapy. It would be necessary to know the targets of the combination and understand the mechanism so that the approach could be pursued further,
-
