TopBP1 biomolecular condensates: a new therapeutic target in advanced-stage colorectal cancer

Reviewer #1 (Public review):

Summary:

Laura Morano and colleagues have performed a screen to identify compounds that interfere with the formation of TopBP1 condensates. TopBP1 plays a crucial role in the DNA damage response, and specifically the activation of ATR. They found that the GSK-3b inhibitor AZD2858 reduced the formation of TopBP1 condensates and activation of ATR and its downstream target CHK1 in colorectal cancer cell lines treated with the clinically relevant irinotecan active metabolite SN-38. This inhibition of TopBP1 condensates by AZD2858 was independent from its effect on GSK-3b enzymatic activity. Mechanistically, they show that AZD2858 thus can interfere with intra-S-phase checkpoint signaling, resulting in enhanced cytostatic and cytotoxic effects of SN-38 (or SN-38+Fluoracil aka FOLFIRI) in vitro in colorectal carcinoma cell lines.

Major comments from the first round of peer review:

Overall the work is rigorous and the main conclusions are convincing. However, they only show the effects of their combination treatments on colorectal cancer cell lines. I'm worried that blocking the formation of TopB1 condensates will also be detrimental in non-transformed cells. Furthermore it is somewhat disappointing that it remains unclear how AZD2858 blocks self-assembly of TopBP1 condensates, although I understand that unraveling this would be complex and somewhat out-of-reach for now. Here are some specific points for improvement:

1. The authors conclude that "These data supports [sic] the feasibility of targeting condensates formed in response to DNA damage to improve chemotherapy-based cancer treatments". To support this conclusion the authors need to show that proliferating non-transformed cells (e.g. primary cell cultures or organoids) can tolerate the combination of AZD2858 + SN-38 (or FOLFIRI) better than colorectal cancer cells.

2. Page 19 "This suggests that the combination... arrests the cell cycle before mitosis in a DNA-PKsc-dependent manner." I find the remark that this arrest would be DNA-PKcs-dependent too speculative. I suppose that the authors base this claim on reference 55 but if they want to support this claim they need to prove this by adding DNA-PKcs inhibitors to their treated cells.

3. When discussing Figure S5B the authors claim that SN-38 + AZD2858 progressively increases the fractions of BrdU positive cells, but this is not supported by statistical analysis. The fractions are still very small, so I would like to see statistics on these data. Alternatively, the authors could take out this conclusion.

Comments on revised version:

I have reviewed the revised manuscript and read the rebuttal. The authors have carefully addressed my concerns. There is however one point that needs further work:

This follows up on my major point #1 in my initial review. I had I asked the authors to demonstrate that FOLFIRI + AZD are less toxic to untransformed colorectal cells than colorectal cancer cell lines.

It is good to see that the authors took my advice and show effects of the drug treatments on the untransformed colorectal cell line CCD841. It seems to be less sensitive to AZD and FOLFIRI in the figure in the rebuttal. What surprises me is that I cannot find these new figures anywhere in the revised manuscript. Also, the data seem preliminary, because I do not see any standard errors in the graphs, and I cannot find a description of the time of drug incubation. I ask the authors to make sure that the CCD841 data are reproducible, and make sure they incorporate the data in the revised manuscript.

Reviewer #2 (Public review):

Summary:

In 2021 (PMID: 33503405) and 2024 (PMID: 38578830) Constantinou and colleagues published two elegant papers in which they demonstrated that the Topbp1 checkpoint adaptor protein could assemble into mesoscale phase-separated condensates that were essential to amplify activation of the PIKK, ATR, and its downstream effector kinase, Chk1, during DNA damage signalling. A key tool that made these studies possible was the use of a chimeric Topbp1 protein bearing a cryptochrome domain, Cry2, which triggered condensation of the chimeric Topbp1 protein, and thus activation of ATR and Chk1, in response to irradiation with blue light without the myriad complications associated with actually exposing cells to DNA damage.

In this current report Morano and co-workers utilise the same optogenetic Topbp1 system to investigate a different question, namely whether Topbp1 phase-condensation can be inhibited pharmacologically to manipulate downstream ATR-Chk1 signalling. This is of interest, as the therapeutic potential of the ATR-Chk1 pathway is an area of active investigation, albeit generally using more conventional kinase inhibitor approaches.

The starting point is a high throughput screen of 4730 existing or candidate small molecule anti-cancer drugs for compounds capable of inhibiting the condensation of the Topbp1-Cry2-mCherry reporter molecule in vivo. A surprisingly large number of putative hits (>300) were recorded, from which 131 of the most potent were selected for secondary screening using activation of Chk1 in response to DNA damage induced by SN-38, a topoisomerase inhibitor, as a surrogate marker for Topbp1 condensation. From this the 10 most potent compounds were tested for interactions with a clinically used combination of SN-38 and 5-FU (FOLFIRI) in terms of cytotoxicity in HCT116 cells. The compound that synergised most potently with FOLFIRI, the GSK3-beta inhibitor drug AZD2858, was selected for all subsequent experiments.

AZD2858 is shown to suppress the formation of Topbp1 (endogenous) condensates in cells exposed to SN-38, and to inhibit activation of Chk1 without interfering with activation of ATM or other endpoints of damage signalling such as formation of gamma-H2AX or activation of Chk2 (generally considered to be downstream of ATM). AZD2858 therefore seems to selectively inhibit the Topbp1-ATR-Chk1 pathway without interfering with parallel branches of the DNA damage signalling system, consistent with Topbp1 condensation being the primary target. Importantly, neither siRNA depletion of GSK3-beta, or other GSK3-beta inhibitors were able to recapitulate this effect, suggesting it was a specific non-canonical effect of AZD2858 and not a consequence of GSK3-beta inhibition per se.

To understand the basis for synergism between AZD2858 and SN-38 in terms of cell killing, the effect of AZD2858 on the replication checkpoint was assessed. This is a response, mediated via ATR-Chk1, that modulates replication origin firing and fork progression in S-phase cell under conditions of DNA damage or when replication is impeded. SN-38 treatment of HCT116 cells markedly suppresses DNA replication, however this was partially reversed by co-treatment with AZD2858, consistent with the failure to activate ATR-Chk1 conferring a defect in replication checkpoint function.

Figures 4 and 5 demonstrate that AZD2858 can markedly enhance the cytotoxic and cytostatic effects of SN-38 and FOLFIRI through a combination of increased apoptosis and growth arrest according to dosage and treatment conditions. Figure 6 extends this analysis to cells cultured as spheroids, sometimes considered to better represent tumor responses compared to single cell cultures.

Significance:

Liquid phase separation of protein complexes is increasingly recognised as a fundamental mechanism in signal transduction and other cellular processes. One recent and important example was that of Topbp1, whose condensation in response to DNA damage is required for efficient activation of the ATR-Chk1 pathway. The current study asks a related but distinct question; can protein condensation be targeted by drugs to manipulate signalling pathways which in the main rely on protein kinase cascades?

Here, the authors identify an inhibitor of GSK3-beta as a novel inhibitor of DNA damage-induced Topbp1 condensation and thus of ATR-Chk1 signalling.

This work will be of interest to researchers in the fields of DNA damage signalling, biophysics of protein condensation, and cancer chemotherapy.

Comments on latest version:

Morano et al. have revised their manuscript in response to the points raised by reviewer #3 as follows.

1. Fig. 2E: Correcting the previously erroneous labelling of this Fig. makes it match the textual description.

2. Figs 3A and B: The revised textual description of the flow cytometry BrdU incorporation is now precise.

3. Fig. 3E: Removing the suspect WB images is a pragmatic decision that does not significantly affect the overall conclusions of the paper.

4. Fig. 3D: Despite its puzzling appearance this data is now described accurately in the text as "DSBs remained elevated after the combined treatment" rather than "increased after the combined treatment. A more convincing increase in the presumed damaged DNA band is evident in Fig. 4D when AZD2858 is combined with a much lower concentration of SN38 (1.5nM) which could mean that the concentration used in Fig. 3D (300nM) induced maximal damage that could not be further enhanced.

Reviewer #3 (Public review):

Summary:

The authors have extended their previous research to develop TOPBP1 as a potential drug target for colorectal cancer by inhibiting its condensation. Utilizing an optogenetic approach, they identified the small molecule AZD2858, which inhibits TOPBP1 condensation and works synergistically with first-line chemotherapy to suppress colorectal cancer cell growth. The authors investigated the mechanism and discovered that disrupting TOPBP1 assembly inhibits the ATR/Chk1 signaling pathway, leading to increased DNA damage and apoptosis, even in drug-resistant colorectal cancer cell lines.

Comments on latest version:

The authors have addressed most of the concerns that I raised in the first round of revision and I have no further questions. I appreciate the authors's efforts in carrying out an preliminary in vivo work, although as the authors pointed out the compound seems to be not effective in vivo. Future work is desired to address this to clarify the significance of the work.

Author response:

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary:

Laura Morano and colleagues have performed a screen to identify compounds that interfere with the formation of TopBP1 condensates. TopBP1 plays a crucial role in the DNA damage response, and specifically the activation of ATR. They found that the GSK-3b inhibitor AZD2858 reduced the formation of TopBP1 condensates and activation of ATR and its downstream target CHK1 in colorectal cancer cell lines treated with the clinically relevant irinotecan active metabolite SN-38. This inhibition of TopBP1 condensates by AZD2858 was independent from its effect on GSK-3b enzymatic activity. Mechanistically, they show that AZD2858 thus can interfere with intra-S-phase checkpoint signaling, resulting in enhanced cytostatic and cytotoxic effects of SN-38 (or SN-38+Fluoracil aka FOLFIRI) in vitro in colorectal carcinoma cell lines.

Major comments:

Overall the work is rigorous and the main conclusions are convincing. However, they only show the effects of their combination treatments on colorectal cancer cell lines. I'm worried that blocking the formation of TopB1 condensates will also be detrimental in non-transformed cells. Furthermore it is somewhat disappointing that it remains unclear how AZD2858 blocks selfassembly of TopBP1 condensates, although I understand that unraveling this would be complex and somewhat out-of-reach for now.

We appreciate your feedback and fully recognize the importance of understanding how AZD2858 blocks the assembly of TopBP1 condensates. While we understand your disappointment, addressing this question remains a key focus for us. Keeping in mind that unravelling such a mechanism in vitro or in vivo is rather challenging, we have consulted an expert who has made efforts to predict the potential docking sites of AZD2858 on TopBP1, which may provide valuable insights for future experimental investigations. Using an AlphaFold model (no crystal or cryo-EM structure available) and looking for suitable pockets or cavities in which AZD2858 could bind, the analyses, though requiring cautious interpretation, suggested that AZD2858 may target the BRCT1 and BRCT8 domains (as shown below, two pockets n°1 and 7 with sufficient volume and surrounded by b-sheets structures like other GSK3 inhibitor) of TopBP1.

However, these are preliminary results that require further exploration and experimental validation to confirm their significance and mechanistic implications.

Author response image 1.

Here are some specific points for improvement:

(1) The authors conclude that "These data supports [sic] the feasibility of targeting condensates formed in response to DNA damage to improve chemotherapy-based cancer treatments". To support this conclusion the authors need to show that proliferating non-transformed cells (e.g. primary cell cultures or organoids) can tolerate the combination of AZD2858 + SN-38 (or FOLFIRI) better than colorectal cancer cells.

We would like to thank the reviewer for this vital suggestion to prove that this combination is effective on tumor cells and not very toxic on healthy cells. We therefore used a healthy colon cell line (CCD841) and tested the efficacy of each treatment alone (FOLFIRI and AZD2858) as well as the combination FOLFIRI+AZD2858. We compared the results obtained in the CCD841 cell line with those obtained in the HCT116 colorectal cancer cell line. The results presented below show not only that each treatment alone is much less effective on CCD841 lines, but also that the combination is not synergistic.

Author response image 2.

Page 19 "This suggests that the combination... arrests the cell cycle before mitosis in a DNAPKsc-dependent manner." I find the remark that this arrest would be DNA-PKcs-dependent too speculative. I suppose that the authors base this claim on reference 55 but if they want to support this claim they need to prove this by adding DNA-PKcs inhibitors to their treated cells.

Thank you for your thoughtful comment. We agree with the reviewer that claiming the G2/M arrest is DNA-PKcs-dependent without direct experimental evidence is speculative. While we initially based this hypothesis on reference 55, we acknowledge that further experiments, such as the use of DNA-PKcs inhibitors, would be necessary to robustly support this claim.

Given that this observation was intended as a potential explanation for the G2/M arrest observed at 6 and 12 hours of treatment with AZD2858 + SN-38 (compared to SN-38 alone), and considering that exploring this pathway is not the primary focus of our study, we have decided to remove this hypothesis from both the figure and the text to avoid any ambiguity.

We appreciate the reviewer’s input and will consider investigating this pathway in future studies.

(2) When discussing Figure S5B the authors claim that SN-38 + AZD2858 progressively increases the fractions of BrdU positive cells, but this is not supported by statistical analysis.

The fractions are still very small, so I would like to see statistics on these data. Alternatively, the authors could take out this conclusion.

Thank you for your valuable comment. In response, we have conducted a statistical analysis (Mann-Whitney test) on the data, and the results have been added to Figure S5C for the 6-hour time point and Figure S5D for the 12-hour time point, based on three independent biological replicates. We hope this provides the necessary clarification.

Minor comments:

- Page 5 Materials and methods - Cell culture. Last sentence "Add in what medium you cultured them" looks like an internal review remark and should probably be removed?

We apologize for this oversight. The medium has now been specified, and the sentence has been removed.

- The numbers in all the synergy matrices (in white font) are extremely small and virtually unreadable, and visually distracting. I recommend taking these out altogether.

We believe that the reduction in figure quality may be due to the PDF compression, which affected the resolution of the figures. We are happy to provide high-resolution versions of the figures separately for clarity. If the issue persists even with the higher resolution, we will consider removing the numbers, as suggested.

- The legends of the synergy matrices (for example Fig 1D, 4E, 5, 6) are often extremely small, making it difficult to understand them intuitively. Please enlarge them and label them more clearly, and use larger fonts. In the legend of Figure 5D,E a green matrix indicating % live cells is mentioned but I don't see it. Do they mean the grey matrix?

We have enlarged the figure legends and will provide high-resolution versions of the figures to ensure all details are clearly readable. Regarding Figure 5D,E: we acknowledge that the color may appear differently (more green or gray) depending on the display or printer settings. To avoid any confusion, we have corrected the legend to specify that the color in question is khaki, rather than green. Moreover, following suggestions of the reviewer #2, these figures have been respectively moved to Figure S6B and S6C.

- Figure S2. Perhaps I misunderstand the PML body experiment but the authors seem to use PML body formation to support their idea that AZD2858 blocks TopBP1 condensate formation and not just any condensate formation. However, if this is the case they would need a proper positive control, i.e. an additional experimental condition in which they do see PLM bodies.

Arsenic is a well-known positive control for experiments involving PML bodies due to its ability to induce specific responses in PML proteins and modify PML nuclear bodies (NBs) structure and function (Jaffray et al., 2023, JCB ; Zhu et al., 1997, PNAS). Thus, we used Arsenic as a positive control and observed a significant increase in PML NBs vs the other conditions (Kruskal-Wallis test) as indicated below. We thus implemented the results in the corresponding figure S2B and text.

Author response image 3.

PML condensates were tested after 2 h of incubation. AZD2858 : 100nM ; SN-38 : 300nM ; Arsenic : 6µM. ****: p<0.0001 (Kruskal-Wallis test).

- The quantification of the flow cytometry data needs to be clarified. I find it strange that in the figures (for example Figure 3A and 3C) representative examples are shown of apparently 3 replicates, and that the percentages shown in these examples are then the given in the text as the overall numbers; for example on page 18 "...BrdU incorporation increased from 16.11% (SN38 alone) to 41.83% (combination)...". This type of description is done in multiple places in the Results section and is confusing. It would be clearer if the authors show proper quantifications (mean +/- sem) of the percentages of (the relevant) gated populations. Besides, I don't think it make a lot of sense to mention in the text the percentages with 2 decimals behind the comma. This suggests a level of precision that does not seem justified in flow cytometry data. Finally, all flow cytometry plots look visually very busy and all the text is crammed in with really small fonts. Cleaning them up and enlarging the fonts of the remaining text/numbers would really improve the readability of the figures.

Thank you for your helpful comments. We understand your concern regarding the flow cytometry quantification. Indeed, the percentages presented in the figures are derived from representative replicates, and we acknowledge that this presentation could be confusing. To address this, we have included a table summarizing the data from all replicates to improve readability [Table S2 and S3 in the new version]. Second, we specified in the text that the data are representative biological replicates when needed. Third, we have performed statistical analyses on the three replicates when necessary, as shown in Supplementary Figure S5C-F in the new version. The text has been revised to reflect the correct statistical interpretation.

Regarding the use of two decimal, we are unable to remove them due to limitations in the software (Kaluza) used for flow cytometry analysis. However, we agree that this level of precision may not be warranted, and we have revised the text where appropriate to reduce confusion.

- In Figure 5G the authors show that FOLFIRI + AZD2858 are synergistic in two SN-38-resistant cell lines. They conclude that this combination may overcome drug resistance. But tried to figure out the used FOLFIRI concentrations used in these cell lines and they still seem far higher than the SN-38-sensitive HCT116 cell lines, so I would like to see a bit more nuance in their interpretation. I think overcoming drug resistance is an overstatement, and perhaps alleviating would be a better term

Thank you for highlighting this important point; we have adjusted the text accordingly.

- The legend in Table S2 refers to Figure 5A-B; this should be Figure 4A-B.

Thank you, this has been corrected and Table S2 is now moved to Table S4 .

Reviewer #1 (Significance (Required)):

The finding that AZD2858 block TOPbp1 condensate formation via a pleiotropic effect of this compound is interesting and convincing. To my best knowledge it's a novel finding which is interesting to the potential target audience mentioned below. Their findings that inhibition of TOPbp1 condensation and ATR signaling via AZD2858 may synergize with FOLFIRI therapy in colorectal cancer cells are still very preliminary, because the effects on non-cancerous cells are not tested.

Researchers involved in early cancer drug discovery and cell biologists studying DNA damage responses in cancer cells seem to me typical audience interested and influenced by this paper.

I'm a cell biologist studying cell cycle fate decisions, and adaptation of cancer cells & stem cells to (drug-induced) stress. My expertise aligns well with the work presented throughout this paper.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The authors have extended their previous research to develop TOPBP1 as a potential drug target for colorectal cancer by inhibiting its condensation. Utilizing an optogenetic approach, they identified the small molecule AZD2858, which inhibits TOPBP1 condensation and works synergistically with first-line chemotherapy to suppress colorectal cancer cell growth. The authors investigated the mechanism and discovered that disrupting TOPBP1 assembly inhibits the ATR/Chk1 signaling pathway, leading to increased DNA damage and apoptosis, even in drug-resistant colorectal cancer cell lines. Addressing the following concerns would enhance clarity and further in vivo work may improve significance:

(1) How does the optogenetic method for inducing condensates compare to the DNA damage induction mechanism?

Optogenetics provides a versatile and precise approach for controlling the condensation of scaffold proteins in both space and time. This method enables us to study the role of biomolecular condensates with minute-scale resolution, separating their formation from potentially confounding upstream events, such as DNA damage, and providing valuable insights into their specific function. Importantly, based on our previous publications on TopBP1 or SLX4 optogenetic condensates, we have substantial evidence indicating that light-induced condensates closely mimic those formed in response to DNA damage:

- Functional similarity: Optogenetic condensates recapitulate endogenous condensates formed upon exposure of the cells of DNA damaging agents, and include most known partner proteins involved in the DNA damage response. It was shown for light induced-TopBP1 and SLX4 condensates (1-3).

- Dynamic reversibility: Optogenetic condensates and DNA damage induced condensates are both dynamic and reversible. They dissolve within 15 minutes of light deactivation or after removal of the damaging agent (1,3).

- Chromatin association: Both optogenetic and DNA damage-induced condensates are bound to chromatin or localized at sites of DNA damage (3).

- Regulation: Both types of condensates are regulated similarly, with their formation triggered by the same signaling pathways. ATR basal activity drives the nucleation of opto-TopBP1 condensates and endogenous TopBP1 structures upon light exposure (1). Likewise, sumoylation modifications regulate the formation of opto-SLX4 condensates and endogenous SLX4 condensates (3).

- Structurally: Using super-resolution imaging by stimulation-emission-depletion (STED) microscopy, we observed that endogenous SLX4 nanocondensates formed globular clusters that were indistinguishable from recombinant light induced SLX4 condensates (1,3).

(1) Frattini C, Promonet A, Alghoul E, Vidal-Eychenie S, Lamarque M, Blanchard MP, et al. TopBP1 assembles nuclear condensates to switch on ATR signaling. Molecular Cell. 18 mars 2021;81(6):1231-1245.e8.

(2) Alghoul E, Basbous J, Constantinou A. An optogenetic proximity labeling approach to probe the composition of inducible biomolecular condensates in cultured cells. STAR Protocols. 2021;2(3):100677.

(3) Alghoul E, Basbous J, Constantinou A. Compartmentalization of the DNA damage response: Mechanisms and functions. DNA Repair. août 2023;128:103524.

(2) Why wasn't the initial screen conducted on the HCT116-SN50 resistant cell line?

Thank you for raising this important question, which we also considered at the outset of the project. After careful consideration, we decided to use the HCT116 WT cells in order to obtain initial data from an unmodified cell line. It is worth mentioning that HCT116-SN50 cells exhibit slower proliferation compared to WT cells, and they also express an efflux pump capable of pumping out SN38. We were concerned that these factors might interfere with the optogenetic assay, which is why we chose to perform the screen using the WT HCT116 cells.

(3) The labels in Fig. 1D are difficult to recognize.

This issue was also raised by Reviewer #1. We suspect that the PDF conversion may have reduced the resolution of the figures, so we will provide them separately in high resolution. In addition, we have increased the size of some labels to improve their clarity.

The selected cell image in Fig. 2A for SN-38 seems over-representative; unselected cells appear similar to other groups. Why does AZD2858 itself induce TopBP1 condensates in the plot, yet this is not evident in the images?

Thank you for your comment; we have updated the figure with a more representative image. We indeed observe that AZD2858 alone induces a slight increase in TopBP1 condensates. However, this increase did not lead to the activation of the ATR/Chk1 signaling pathway, as shown by the Western blot data presented in Fig. 2B. In addition, AZD2858 specifically prevents the formation of TopBP1 condensates induced by SN38 treatment, and the level of TopBP1 condensates does not return to the basal levels observed in untreated cells, but rather to those observed with AZD2858 treatment. During the 2-hour AZD2858 treatment, the progression of replication forks was unaffected (Fig. 3A and 3B). However, when AZD2858 was added alone to the Xenopus egg extracts, there was increased recruitment of TopBP1 to the chromatin (Fig. 2E). This result suggests that AZD2858 alone can induce the assembly of TopBP1 on chromatin to initiate DNA replication (a well-established role of TopBP1), but the number and concentration of TopBP1 molecules did not reach levels sufficient to activate the ATR/Chk1 pathway.

(4) In Fig. 3A, despite the drastic change in the FACS plot shape, the quantifications appear quite similar.

Thank you for this insightful observation. The gates for the S phase were intentionally set wider to avoid biasing the results and inadvertently excluding the population that incorporates BrdU weakly (but still incorporates it) in the SN-38 only condition. As a result, the percentage of cells within this gate remains similar, even though the overall shape of the FACS plot changes, reflecting a shift in the distribution of BrdU incorporation. This point has now been clarified in the legend of the Figure 3A.

This effect can also be attributed to the relatively short treatment time (2 hours), which captures early changes in DNA synthesis. The effect becomes more pronounced at later time points, as shown in Figure 3C. For example, after 6 hours of treatment, the percentage of BrdU-positive cells increases from 15% with SN-38 alone to 41% with the AZD2858 combination, demonstrating a clearer impact on DNA synthesis. A graph summarizing the statistical analysis has been added to Figure S5C for the 6-hour time point and Figure S5D for the 12-hour time point, based on data from three independent biological replicates.

(5) The results section is imbalanced; Figs. 5 and 6 could be combined into one figure.

We have combined Figures 5 and 6 into a single figure to optimize the presentation of results. To avoid overloading the new figure, some of the data have been moved to supplementary figures, ensuring the main figure remains clear and focused.

(6) An in vivo study is anticipated to assess the drug's efficacy.

Although AZD2858 was developed a few years ago, there is a limited amount of in vivo data available, which led us to consider potential issues related to the drug's biodistribution or its pharmacokinetics (PK). Despite these concerns, we proceeded with preliminary in vivo studies, testing various diluents and injection routes for AZD2858. However, we observed that the compound was not effective in vivo. Given the strong synergistic effects observed in vitro, we concluded that AZD2858 was likely not being distributed properly in the mice. As a result, we have decided to conduct a more detailed investigation into the pharmacokinetics (PK), pharmacodynamics (PD), and absorption, distribution, metabolism, and excretion (ADME) of AZD2858 to better understand its in vivo behavior and efficacy. Therefore, the in vivo evaluation of AZD2858 will be addressed in a separate study specifically focused on this aspect.

Reviewer #2 (Significance (Required)):

Addressing the stated concerns would enhance clarity and further in vivo work may improve significance.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary

In 2021 (PMID: 33503405) and 2024 (PMID: 38578830) Constantinou and colleagues published two elegant papers in which they demonstrated that the Topbp1 checkpoint adaptor protein could assemble into mesoscale phase-separated condensates that were essential to amplify activation of the PIKK, ATR, and its downstream effector kinase, Chk1, during DNA damage signalling. A key tool that made these studies possible was the use of a chimeric Topbp1 protein bearing a cryptochrome domain, Cry2, which triggered condensation of the chimeric Topbp1 protein, and thus activation of ATR and Chk1, in response to irradiation with blue light without the myriad complications associated with actually exposing cells to DNA damage.

In this current report Morano and co-workers utilise the same optogenetic Topbp1 system to investigate a different question, namely whether Topbp1 phase-condensation can be inhibited pharmacologically to manipulate downstream ATR-Chk1 signalling. This is of interest, as the therapeutic potential of the ATR-Chk1 pathway is an area of active investigation, albeit generally using more conventional kinase inhibitor approaches.

The starting point is a high throughput screen of 4730 existing or candidate small molecule anticancer drugs for compounds capable of inhibiting the condensation of the Topbp1-Cry2mCherry reporter molecule in vivo. A surprisingly large number of putative hits (>300) were recorded, from which 131 of the most potent were selected for secondary screening using activation of Chk1 in response to DNA damage induced by SN-38, a topoisomerase inhibitor, as a surrogate marker for Topbp1 condensation. From this the 10 most potent compounds were tested for interactions with a clinically used combination of SN-38 and 5-FU (FOLFIRI) in terms of cytotoxicity in HCT116 cells. The compound that synergised most potently with FOLFIRI, the GSK3-beta inhibitor drug AZD2858, was selected for all subsequent experiments.

AZD2858 is shown to suppress the formation of Topbp1 (endogenous) condensates in cells exposed to SN-38, and to inhibit activation of Chk1 without interfering with activation of ATM or other endpoints of damage signalling such as formation of gamma-H2AX or activation of Chk2 (generally considered to be downstream of ATM). AZD2858 therefore seems to selectively inhibit the Topbp1-ATR-Chk1 pathway without interfering with parallel branches of the DNA damage signalling system, consistent with Topbp1 condensation being the primary target. Importantly, neither siRNA depletion of GSK3-beta, or other GSK3-beta inhibitors were able to recapitulate this effect, suggesting it was a specific non-canonical effect of AZD2858 and not a consequence of GSK3-beta inhibition per se.

To understand the basis for synergism between AZD2858 and SN-38 in terms of cell killing, the effect of AZD2858 on the replication checkpoint was assessed. This is a response, mediated via ATR-Chk1, that modulates replication origin firing and fork progression in S-phase cell under conditions of DNA damage or when replication is impeded. SN-38 treatment of HCT116 cells markedly suppresses DNA replication, however this was partially reversed by co-treatment with AZD2858, consistent with the failure to activate ATR-Chk1 conferring a defect in replication checkpoint function.

Figures 4 and 5 demonstrate that AZD2858 can markedly enhance the cytotoxic and cytostatic effects of SN-38 and FOLFIRI through a combination of increased apoptosis and growth arrest according to dosage and treatment conditions. Figure 6 extends this analysis to cells cultured as spheroids, sometimes considered to better represent tumor responses compared to single cell cultures.

Major comments

Most of the data presented is of good technical quality and supports the conclusions drawn. There are however a small number of instances where this is not true; ie where the data are of insufficient technical quality, or where the description or interpretation of the results is at variance with the data which is presented. Some examples:

(1) Fig.2E - the claim that "we observed an increase in RPA, Topb1 and Pol-epsilon levels when CPT and AZD2858 were added together" do not seem to be justified by the data provided. It is also unclear what the purpose/ significance of this experiment is.

Thank you for pointing out the contradiction in Figure 2E. Upon review, we identified an error in the labeling of conditions (CPT and AZD2858 were inadvertently swapped). The corrected figure now clearly shows that, at the 60-minute timepoint after starting replication, the combination of

CPT and AZD2858 results in a greater accumulation of TopBP1, Pol ε, and RPA on chromatin compared to CPT alone. We have revised the sentence to: "Our data demonstrate that combining CPT and AZD2858 earlier enhances the accumulation of replication-related factors (RPA, TopBP1, and Pol ε) on chromatin compared to CPT treatment alone, particularly visible at the 60minute after starting replication."

The significance of this experiment lies in its connection to the earlier observation that AZD2858 restores BrdU incorporation when combined with SN-38, as shown in flow cytometry data (Figure 3A). At a molecular level, this was further supported by DNA fiber assays, which revealed that replication tracks (CldU tracts) were longer in the combination treatment compared to SN-38 alone (Figure 3B).

To strengthen and validate these findings, we chose to employ the Xenopus egg extract system for several reasons. This model provides a highly controlled environment where DNA replication occurs without confounding effects from transcription or translation. Moreover, replication is limited to a single round, offering a unique opportunity to specifically interrogate replication mechanisms. These attributes make the Xenopus model an ideal system to confirm that AZD2858 facilitates replication recovery in the presence of replication stress induced by agents like CPT. This will lead, in longer treatment, to accumulation of DNA damage and apoptosis (Figure 3D-E and Figure 4A-D)

(2) Figs. 3 A and C certainly show that the SN-38-mediated suppression of DNA synthesis is modified and partially alleviated by co-treatment with AZD2858. The statement however that "prolonged co-incubation with AZD2858 for 6 and 12 hours effectively abolished the SN-38 induced S-phase checkpoint" is clearly misleading. If this were true, then the BrdU incorporation profiles of the respective samples would be similar or identical to control, which clearly they are not. Clearly AZD2858 is affecting the imposition of the S-phase checkpoint in some way, but not "abolishing" it.

We appreciate the reviewer’s detailed observations regarding Figures 3A and 3C and the phrasing in our manuscript. We agree that the term "abolished" is not precise in describing the effects of AZD2858 on the SN-38-induced S-phase checkpoint.

To clarify: our data indicate that co-treatment with AZD2858 modifies and partially alleviates the SN-38-induced suppression of DNA synthesis, as demonstrated by increased BrdU incorporation relative to SN-38 treatment alone. However, as the reviewer correctly points out, the BrdU incorporation profiles of the co-treated samples do not fully return to control non treated cells levels. This suggests that while AZD2858 significantly mitigates the S-phase checkpoint, it does not completely abolish it.

We have revised the statement in the manuscript to better reflect these findings, as follows: "Prolonged co-incubation with AZD2858 for 6 and 12 hours significantly alleviated the SN-38induced S-phase checkpoint, as evidenced by the partially increased BrdU incorporation. However, the population of co-treated cells is heterogeneous: some cells exhibit BrdU incorporation levels similar to those of untreated control cells, while others incorporate BrdU at levels comparable to cells treated with SN-38 alone. This indicates that AZD2858 does not fully restore DNA synthesis to control levels across the entire cell population."

This revised phrasing aligns with the data presented and acknowledges the partial recovery of DNA synthesis observed. Thank you for bringing this to our attention and helping us improve the accuracy of our conclusions.

(3) Fig. 3 E. The western blots of pDNA-PKcs (S2056) and total DNA-PKcs are really not interpretable. It is possible to sympathise that these reagents are probably extremely difficult to work with and obtain clear results, however uninterpretable results are not acceptable.

We agree that the data presented in the Fig3E are difficult to interpret. As noted by Reviewer 1, we recognize the challenge of obtaining clear and reliable results with these specific reagents. Based on this feedback, and to ensure the robustness of our conclusions, we have decided to exclude these specifics blots from the revised manuscript.

We believe that this adjustment will enhance the clarity and reliability of the manuscript while focusing on the other, more interpretable data presented. Thank you for pointing this out, and we appreciate your understanding.

(4) Fig. 3D. This is a puzzling image. Described as a PFGE assay, it presumably depicts an agarose gel, with intact genomic DNA at the top and a discrete band below representing fragmented genomic DNA. This is a little surprising, as fragmented genomic DNA does not usually appear as a specific band but as a heterogenous population or "smear". Nevertheless, even if one accepts this premise, it is unclear what is meant by "DSBs remained elevated after the combined treatment" when the intensity of this band is equivalent for both SN-38 and SN-38 + AZD2858 treatments.

We thank the reviewer for his insightful comments regarding the PFGE results in Figure 3D. We agree that the appearance of a discrete band, rather than a heterogeneous smear, is atypical for fragmented genomic DNA in this assay. However, by enhancing the signal intensity (as shown below), the expected smear becomes more appreciable.

Author response image 4.

Regarding the interpretation of the band intensities, we agree that the signals for SN-38 and SN38 + AZD2858 appear similar under these specific conditions. At the relatively high concentration of SN-38 used in this experiment (300 nM), it is indeed challenging to observe a more pronounced effect on DNA breaks. This is why we proposed the "DSBs remained elevated after the combined treatment" because the band intensity of SN-38 single agent treated cells or combined with AZD2858 is comparable. However, we note a slightly more intense γH2AX signal over time when AZD2858 is combined with SN-38 compared to SN-38 alone (Figure 3E). Furthermore, under lower, sub-optimal doses of SN-38 and over extended incubation treatment (48h), we observe a clearer increase in fragmented DNA bands, as demonstrated in Figure 4D.

Minor comments

(1) Fig. 1. A surprisingly large number of compounds scored positive in the primary screen for inhibition of Topbp1 condensation (>300). Of the 131 of these selected for secondary screening using Chk1 activation (S345 phosphorylation) as a readout approximately 2/3 were negative, implying that a majority of the tested compounds inhibited Topbp1 condensation but not Chk1 activation. What could explain that?

Thank you for this thoughtful comment. The discrepancy between the large number of compounds scoring positive for TopBP1 condensation inhibition and the smaller number inhibiting Chk1 activation (S345 phosphorylation) could be attributed to several factors:

• Different cell lines and induction methods: The initial screen was conducted in HEK293 TrexFlpin cells overexpressing optoTopBP1, while the secondary screen used HCT116 cells. In addition, the methods used to induce the respective pathways were distinct: in the primary screen, we employed a blue light induction of opto-TopBP1 condensates, whereas in the secondary screen, we used an SN-38 treatment to induce DNA replication stress and activate the Chk1 pathway. These differences could account for the varying responses observed in the two screens.

• The compounds that inhibited TopBP1 condensation might not fully block Chk1 activation. While they disrupt TopBP1 condensation, they may still allow for partial activation of Chk1 or Chk1 activation through alternative mechanisms. For instance, Chk1 activation could be mediated by other signaling pathways or molecules, such as ETAA1, a known Chk1 activator (1). Thus, TopBP1 condensation inhibition does not necessarily translate to complete inhibition of Chk1 activation, especially if ETAA1 is employed by cells as a rescue activator.

• Some compounds may affect chromosome dynamics, potentially generating mechanical forces or torsional stress that could activate the ATR/Chk1 pathway independently of TopBP1

(2).

These factors suggest that while the compounds effectively disrupt TopBP1 condensation, they may not always fully inhibit the downstream Chk1 activation, pointing to the complexity of the DNA damage response pathways.

(1) Bass, T. E. et al. ETAA1 acts at stalled replication forks to maintain genome integrity. Nat Cell Biol 18, 1185–1195 (2016).

(2) Kumar, A. et al. ATR Mediates a Checkpoint at the Nuclear Envelope in Response to Mechanical Stress. Cell 158, 633–646 (2014).

(2) Fig. 2D. The protein-protein interaction assay shown demonstrates that AZD2858 ablates the light-induced auto-interaction between exogenous opto-Topbp1 molecules and ATR plus or minus SN-38, but clearly endogenous Topbp1 molecules do not participate. Why is this?

The biotin proximity labeling assay was conducted without exposing cells to light, using a TurboID module fused to TopBP1-mCherry-CRY2. Stable cell lines were then generated in HEK293 TrexFlpIn cells, where endogenous TopBP1 is still expressed. Upon adding doxycycline, the recombinant TurboID-TopBP1-mCherry-Cry2 (opto-TopBP1) is induced at levels comparable to endogenous TopBP1 (Fig 2D).

Since the opto-TopBP1 construct exhibits behavior similar to that of endogenous TopBP1 (1), we used it to investigate whether TopBP1 self-assembly and its interaction with ATR are influenced by AZD2858 alone or in combination with SN38. Our results show that treatment with SN38 increases the proximity between opto-TopBP1 and the endogenous TopBP1 (not fused to TurboID). However, AZD2858, either alone or in combination with SN38, disrupts the selfassembly of recombinant TopBP1 with itself as well as its interaction with endogenous TopBP1.

(1) Frattini C, Promonet A, Alghoul E, Vidal-Eychenie S, Lamarque M, Blanchard MP, et al. TopBP1 assembles nuclear condensates to switch on ATR signaling. Molecular Cell. 18 mars 2021;81(6):1231-1245.e8.

Reviewer #3 (Significance (Required)):

Significance

Liquid phase separation of protein complexes is increasingly recognised as a fundamental mechanism in signal transduction and other cellular processes. One recent and important example was that of Topbp1, whose condensation in response to DNA damage is required for efficient activation of the ATR-Chk1 pathway. The current study asks a related but distinct question; can protein condensation be targeted by drugs to manipulate signalling pathways which in the main rely on protein kinase cascades?

Here, the authors identify an inhibitor of GSK3-beta as a novel inhibitor of DNA damage-induced Topbp1 condensation and thus of ATR-Chk1 signalling.

This work will be of interest to researchers in the fields of DNA damage signalling, biophysics of protein condensation, and cancer chemotherapy.