Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells

Round 3

Reviewer 1 (original reviewer 2) Report

The authors have addressed my concerns.  I don't have any further comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 2

Reviewer 1 (original reviewer 2) Report

In the study presented in the manuscript, Dr. Ròdland and colleagues reported the effect of combining ATR and WEE1 inhibitors in different cancer cell lines. In the study different in vitro studies show the effect of VE-822, MK-1775 as single agents, as combination or as radio-sensitizer against osteosarcoma and lung cancer cell lines. The effect of the combination was stronger in the osteosarcoma cells U2OS in comparison with the effect on the four lung cancer cell lines. Although the study is well designed, there is a lack of novelty. Indeed, the findings are similar to that founds in other tumor entities.

Before considering for publication, some improvements are needed to follow the conclusions of the authors:

Major comment:

• In the Introduction, the authors should insert and comment some recent studies regarding the effect of CHK1 and WEE1 inhibitors in combinations (DOI: 10.1158/0008-5472.CAN-17-3932 and https://doi.org/10.3390/cancers11111654). In a similar way, the authors should also insert and comment a recent study that evaluate the effect of the combination between ATR and WEE1 inhibitors in combination and as chemo-sensitizer agent (gemcitabine) against diffuse large B-cell lymphoma (DLBCL) cells ( DOI: 10.1158/0008-5472.CAN-18-2480).

• In the Result section 2.4, the authors evaluated the efficacy of the ATR/WEE1 inhibitors combination as radio-sensitizer agent in U2OS and A549 cells. Based on cell viability and clonogenic assays, they concluded that the combination was not able to enhance the efficacy of radiotherapy in the two cell models. In addition, they found antagonism in A549 cells combining IR with VE-822 in combination with MK-1775. The results reported in this section are however very superficial. No functional studies have been presented in order to explain the results of the experiments. Cell cycle analysis and/or immunoblotting analysis should be performed in order to explain the effect of the combination.

Minor comments:

• In Figure 1A, the authors compare the expression of different G2/M cell cycle checkpoint markers in U2OS cells treated with the ATR and WEE1 inhibitors alone or in combinations. Although there is evidences of protein expression changes between single agent and combinations during the time points, controls cells (mock) are reported only once. When did the control cells have been harvested?

• In Figure 1C, the authors showed the effect on cell cycle progression of U2OS cells treated with the two inhibitors in single agent or in combination. The authors concluded that the combination increased the number of cells in S phase. No statistical test is reported in the figure.
• In Figure 1D, statistical test are missing.

Author Response

Point-by-point response to reviewers comments:

REVIEWER 1

Open Review

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( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style

Comments and Suggestions for Authors

In the study presented in the manuscript, Dr. Ròdland and colleagues reported the effect of combining ATR and WEE1 inhibitors in different cancer cell lines. In the study different in vitro studies show the effect of VE-822, MK-1775 as single agents, as combination or as radio-sensitizer against osteosarcoma and lung cancer cell lines. The effect of the combination was stronger in the osteosarcoma cells U2OS in comparison with the effect on the four lung cancer cell lines. Although the study is well designed, there is a lack of novelty. Indeed, the findings are similar to that founds in other tumor entities.

Before considering for publication, some improvements are needed to follow the conclusions of the authors:

Major comment:

• In the Introduction, the authors should insert and comment some recent studies regarding the effect of CHK1 and WEE1 inhibitors in combinations (DOI: 10.1158/0008-5472.CAN-17-3932 and https://doi.org/10.3390/cancers11111654). In a similar way, the authors should also insert and comment a recent study that evaluate the effect of the combination between ATR and WEE1 inhibitors in combination and as chemo-sensitizer agent (gemcitabine) against diffuse large B-cell lymphoma (DLBCL) cells ( DOI: 10.1158/0008-5472.CAN-18-2480).

** Authors reply:

We have now included these references in the introduction and inserted a comment (p.2: lines 63-65, line 60 and line 66). Of note, we cannot see that ATR and WEE1 inhibitors were tested in combination with gemcitabine in the reference DOI: 10.1158/0008-5472.CAN-18-2480. However, gemcitabine was included in one of the articles with CHK1 and WEE1 inhibitors, reference DOI: 10.1158/0008-5472.CAN-17-3932.

• In the Result section 2.4, the authors evaluated the efficacy of the ATR/WEE1 inhibitors combination as radio-sensitizer agent in U2OS and A549 cells. Based on cell viability and clonogenic assays, they concluded that the combination was not able to enhance the efficacy of radiotherapy in the two cell models. In addition, they found antagonism in A549 cells combining IR with VE-822 in combination with MK-1775. The results reported in this section are however very superficial. No functional studies have been presented in order to explain the results of the experiments. Cell cycle analysis and/or immunoblotting analysis should be performed in order to explain the effect of the combination.

**Authors reply:

-Based on both clonogenic assays and cell viability assays performed in two different cell lines, we have found that the synergy between ATR and WEE1 inhibitors is weakened when combined with radiation. Based on these results we concluded that for combination treatment with radiaton, the combined ATR/WEE1 inhibition would not likely be better than ATR inhibitor alone, (the latter which appeared to give the biggest radiosensitizing effects in our cell lines).

-To further strengthen these results, we have now performed additional Bliss analysis evaluating synergistic effects between IR + VE822, IR + MK1775, and IR+ VE822/MK1775 (new Figure S6D). The new analysis confirms our previous conclusion that the ATR inhibitor alone gives better effects on radiosensitization than the combined ATR/WEE1 inhibition. (See new Figure S6D and main text p. 14,  lines 321-322.)

-We have also performed additional flow cytometry experiments investigating the cell cycle distribution of cells treated with the inhibitors in the presence and absence of IR (new Figure S6B). U2OS showed a transient accumulation in S and G2 phase after IR, as expected due to checkpoint activation and as measured at 6 hours after treatment. Interestingly, no further increase in the S-phase population was observed in inhibitor treated irradiated cells at this time point, which might contribute to explain that effects are different in irradiated versus non-irradiated cells. On the other hand, the highest inhibitor concentrations caused a strong S-phase accumulation regardless of IR at 24 hours after treatment. In A549 cells, in which the synergy between MK1775 and VE822 was completely abrogated in response to IR, there were very few cells in S-phase at 24h after IR (new Figure S6B). Although the cell cycle analysis cannot fully explain the mechanisms, these results are important to give a more thorough picture of the effects. The cell cycle effects after IR and inhibitors are complex, likely because the inhibitors cause checkpoint abrogation and inhibition of DNA repair in addition to inducing S-phase damage. (See new Figure S6B and main text on p. 14, lines 302-312.)

Minor comments:

• In Figure 1A, the authors compare the expression of different G2/M cell cycle checkpoint markers in U2OS cells treated with the ATR and WEE1 inhibitors alone or in combinations. Although there is evidences of protein expression changes between single agent and combinations during the time points, controls cells (mock) are reported only once. When did the control cells have been harvested?

**Authors reply:

The control (mock) cells were harvested at the same time point as the cells that had been treated with inhibitors for 3h. The protein expression in control (mock) is not expected to change over time as the sample contains cells in unperturbed growth. We have previously confirmed this in other experiments where we harvested control (mock) samples at multiple time points.

• In Figure 1C, the authors showed the effect on cell cycle progression of U2OS cells treated with the two inhibitors in single agent or in combination. The authors concluded that the combination increased the number of cells in S phase. No statistical test is reported in the figure.

**Authors reply:

Student t-test analysis has now been performed, and p-values < 0.05 are indicated with an asterisk in the figure.  The figure legend is updated accordingly.

• In Figure 1D, statistical test are missing.

**Authors reply:

Student t-test analysis has now been performed, and p-values < 0.05 are indicated with an asterisk in the figure.  The figure legend is updated accordingly.

REVIEWER 2

Open Review

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English language and style

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(x) English language and style are fine/minor spell check required
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Comments and Suggestions for Authors

The authors have addressed my concerns.  I don't have any further comments.

**Authors reply:

Thank you for the comment.

Submission Date

16 April 2021

Date of this review

06 May 2021 06:37:21

REVIEWER 3

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

Comments and Suggestions for Authors

I reviewed the revised manuscript and the responses from the authors.
I had no additional concerns.

**Authors reply:

Thank you for the comment.

Submission Date

16 April 2021

Date of this review

05 May 2021 11:01:32

Author Response File: Author Response.docx

Reviewer 2 (original reviewer 3) Report

The authors have addressed my concerns.  I don't have any further comments.

Author Response

Thank you for the comment.

Author Response File: Author Response.docx

Reviewer 3  (original reviewer 4) Report

I reviewed the revised manuscript and the responses from the authors.
I had no additional concerns.

Author Response

Thank you for the comment.

Author Response File: Author Response.docx

Round 1

Author Response for the reviewers' comments

Academic Editor Notes

Dear Dr. Syljuåsen,
the four reviewers that’ve read your manuscript have raised several, important concerns about it. Among all, I have to point out that the reviewers agree that the main weakness of the manuscript is the lack of any  explanation for the variation that you observe in cell sensitivity to the diverse treatments. I strongly suggest you to design and perform more experiments to determine the mechanisms of resistance/sensitivity to the treatments you used.  All things considered, the manuscript, to be published in Cancers, needs many experiments to be done.  Please, resubmit the paper only if you can properly address all the reviewers’ comments.

Reviewer 1

( ) I would not like to sign my review report 
(x) I would like to sign my review report 

English language and style

( ) Extensive editing of English language and style required 
( ) Moderate English changes required 
(x) English language and style are fine/minor spell check required 
( ) I don't feel qualified to judge about the English language and style 

Comments and Suggestions for Authors

The authors build on previous data combining a WEE1i and a CHK1i in U2OS cells.  They hypothesized that if the above combination worked, then perhaps by combining a WEE1i with an ATRi would be beneficial.  They demonstrated that this is indeed true for U2OS cell line and in some lung cancer cell lines.  Overall the data supports their hypothesis, however there are some specifics that need addressing prior to acceptance.

1. In Figure 2A it appears that the cell lines were treated for different days and it is not explained in the text why this is.  I think it would be more descriptive if a single time point was chosen to assess sensitivity/resistance.

**Authors reply:

-Note that all cell lines were treated with the Wee1 inhibitor for 24 hours in Figure 2A. The only difference is that cell viability was measured at 4 days after drug removal, instead of at 5 days after drug removal, in one of the cell lines (H1975). We wanted to make sure that the non-treated cells were in sub-confluent conditions throughout the assay. Since H1975 appeared slightly denser than the other cell lines at 4 days (based on visual inspection by phase contrast microscopy), we did the CellTiterGlo assay at 4 days in this cell line. We have previously measured cell viability at both 4, 5 and 6 days after drug removal in analogous experiments, and got very similar results at 4-6 days ¹. We have altered the text of the figure legend (p7, line 162-163) and materials and methods (p19-20, line 483-486) to clarify this issue.

2. Figure 3D and F only show 2 of the 5 cell lines and it is not explained why the other cell lines were not shown.

**Authors reply:

-We have performed new experiments measuring CDC45 loading in all four lung cancer cell lines (new figure 5B, replacing old fig 3D).

-In addition, we have now measured CDK activity in all four lung cancer cell lines by examining the phosphorylation of three different CDK1 and CDK2 substrates: phospho-MPM2, phospho-B-Myb and phospho-BRCA2. These results are included in new figure 3C and new figure S3. Together, these results indicate that MK1775 affects replication initiation factors similarly in all cell lines. Note that our flow cytometry method measures CDK activity in individual S-phase cells in a highly accurate manner (see raw data and barcoding strategy in fig S3A and S3B).

-The DNA fiber assay results in old Fig 3F have been moved to new fig S2B-D. In this assay we used H460 cells because they were most resistant to the Wee1 inhibitor, and we wanted to explore if replication was affected differently, by increased CDC45 loading and CDK activity, than it was in WEE1 inhibitor sensitive U2OS cells. Furthermore, U2OS cells were included for comparison because they are very sensitive to the Wee1 inhibitor and these cells were used in fiber assays in our previous study ². We have included a comment on this on p6, line 155-159.

3. Figure 4A shows the combination data and different doses were used and not explained why.  It also states strong synergy was observed in 2 of the cell lines but there are no stats or Bliss data to prove this.  The only Bliss data was for the radiation experiments.  In Figure 4A, there appears to be no effect of the single agent VE compound but want to confirm if the authors normalized to ND.  Also in Figure 4A the Y axis is not consistent.  In Figure 4B, there is no mention at why the concentrations of MK1775 were chosen.  

**Authors reply:

-In figure 4A, a different range of doses of MK1775 were used for the most sensitive and resistant cell lines, similarly as in figure 2A (for H460 cells the results with the highest concentration of MK1775, 2000nM, are only shown in 4A). The effects of single agent VE822 are stated in the figure legend, and the results are normalized. We have altered the figure legend text to clarify these issues (p11, line 233-237).

-Bliss independence analysis is now shown in Figure 4B, 4C, S4A and S4C (Figure 4C and S4C show results from new experiments testing a range of different concentrations of both MK1775 and VE822 in three of the lung cancer cell lines.) The results support our previous conclusion that H460 and H1975 cells show better synergy for the combined treatment than A549 and SW900 cells.

-The Y-axis has been corrected in Figure 4A.

-In the clonogenic assays in Figure 4B, we aimed at using concentrations of the inhibitors for which synergy was expected based on viability assays in H1975 and H460. For A549 we used similar concentrations as for H460 and we also tested additional concentrations in A549 in Figure 6B and S6B. This has now been clarified in the text on p9, line 210-213 and 216.

4. Line 246 has A549 as A459.

**Authors reply:

-The error has been corrected

5. Although the data does appear to support their statements of strong synergy in the combination, without stats to support this it is an overstatement.

**Authors reply:

-We have now performed Student t test analysis of Bliss independence from 3 independent experiments, and have found p-values <0.05 for all cell lines in at least one of the three different survival assays performed* (Figure S4A, S4C and 4B). Note that the magnitude of the “excess over Bliss” score is larger in some cell lines than others, indicating stronger synergy. Comments on this have been included on p 9, line 216-219, and p 9-10, line 224-227.

(*For H460 cells the large effects in Fig S4A were non-significant, likely due to different stocks of the inhibitors being used in the three experiments. However, the effect of the combined treatment in H460 was more than additive in all replicates of the experiments performed. Furthermore, significant synergistic results (p<0.05) were obtained in the new set of experiments performed with multiple inhibitor concentrations in Fig S4C.)

6. It would be nice to see some in vivo data to support the findings.

**Authors reply:

-Although we agree supporting in vivo data would always be nice, measuring in vivo responses with the two inhibitors alone and in combination in two to four lung cancer cell models would require unreasonable effort and is out of scope for the present study.

Reviewer 2

(x) I would not like to sign my review report 
( ) I would like to sign my review report 

English language and style

( ) Extensive editing of English language and style required 
( ) Moderate English changes required 
(x) English language and style are fine/minor spell check required 
( ) I don't feel qualified to judge about the English language and style 

Comments and Suggestions for Authors

The work analyze the effect of targeting WEE1 and ATR kinase in different cancer cell lines. The paper is well written, however some improvements are needed to follow the conclusions of the authors:

1) In Results 2.1, the authors showed that the combination of the two inhibitors synergize in the induction of DNA-damages in S phase cells. However, in Figure 1A several markers are missing WEE1, DNA-PK, ATM and ATR (as well as the auto-phosphorylation site ATR Thr1989). Moreover, to enforce the data on the effect of the combination in cell cycle progression, the authors could evaluate the protein level of Cyclin A, Cyclin E and Cyclin B.

**Authors reply:

-To enforce the data on the effect of the combination on cell cycle progression, we have included quantification of our cell cycle analysis in new bar charts in Fig 1C, Fig 5A and Fig S2A. Note also that the percentage of mitotic cells has been measured in Fig S1C and new Fig S5B and C, and see also revised text on p 11, line 258-261.

(Cyclin A, E and B would not be reliable markers to assess cell cycle changes in this case, as we have previously observed that they are degraded upon MK1775-induced S-phase DNA damage (³, and unpublished results).)

-According to the reviewers suggestion, immunoblots for WEE1, DNA-PK, ATM and ATR are now included in Figure 1A.

-For assessment of ATR activity, we have used the downstream substrate phospho-Chk1-Ser317 instead of phospho-ATR Thr1989. Our group has previously made considerable efforts trying to find a reduction in phospho-ATR Thr1989 upon addition of ATR inhibitors. However, ATR Thr1989 was often not reduced at all, or only very minorly affected, by addition of ATR inhibitors, indicating that this residue on ATR is also phosphorylated by other kinase(s) (unpublished results). We therefore think the downstream substrate is a better marker for ATR activity.

2) In Results 2.2, the authors tested the potential efficacy of the combination in a panel of lung cancer cell lines. They highlighted heterogeneity in term of sensitivity to MK1775. The difference in term of response was related to the induction of S-phase DNA damages. However, several studies (e.i. doi: 10.18632/oncotarget.18728) have reported that the mutational status of TP53 significantly affects MK1775 efficacy in different cancer models. Authors should evaluate the mutational status of TP35. Again, in Figure 2C several markers of the DNA damage response pathway are missing. Moreover, authors did not reported any cell cycle analysis on lung cancer cell lines treated in combination with MK1775 and VE822. Is the effect consistent with the one reported for

**Authors reply:

-In our study the two most resistant lung cancer cell lines H460 and A549 cells have wt p53, and the two most sensitive lung cancer cell lines SW900 and H1975 cells have mut p53. However, U2OS cells have wt p53 and are also very sensitive to MK1775. It is therefore not a strict correlation with p53 status and sensitivity to MK1775 across our cell lines. Furthermore, a previous study examining a larger panel of ~70 lung cancer cell lines did not find a correlation between p53-status and sensitivity to MK1775 as single agent ⁴. We therefore conclude that p53-status does not appear to predict sensitivity to MK1775 as single agent. This issue has been discussed on p 17, line 396-403 (discussion).

-Immunoblots for WEE1, DNA-PK and ATM are now included in Figure 2C.

-Cell cycle analysis has now been performed after treatment of lung cancer cell lines with MK1775 and VE822 (Bar charts in Fig 5A, and main text p11, line 249-254).  The results support our previous conclusion that combined treatment of ATR and Wee1 inhibition does not cause major S-phase effects in A549, H460 and H1975 cells.

3) In results 2.4 , the authors combined X-ray radiation with MK-1775 and VE822 in combination and in single agent. Overall, this result section is not clear and the authors should perform additional experiments to highlight the value of adding radiotherapy to MK-1775/VE822 combination. Firstly, the biological reason of combining ATR inhibitors instead of ATM inhibitors, which are frequently combined with ionizing radiation, is not well presented. Finally, why did the authors combine radiotherapy with the combination treatment? Are the tested cell lines arrested in S phase after radi otherapy?

The authors concluded that the synergy of the two inhibitors was weakened when combined with radiation, however they tested only a drug schedule (inhibitors and radiation simultaneously). Thus, the authors should performed additional drug schedules such as pre-treatment with MK-1775/VE822 and the radiation or vice versa to address the efficacy of the combination. Why did the authors performed viability assays instead of clonogenic assays to evaluate the long term efficacy of the combination?

**Authors reply:

-Many previous preclinical studies have reported radiosensitizing effects of ATR inhibitors (see e.g. ^{5, 6}), and several clinical trials are actually ongoing to test radiotherapy in combination with ATR inhibitors. ATR inhibitors are therefore highly relevant for combining with radiotherapy. Furthermore, the WEE1 inhibitor Adavorsetib (MK1775/ AZD1775) also has radiosensitizing capabilities and is currently being tested in clinical trial in combination with radiotherapy. As our main goal was to address whether combined treatment with ATR and WEE1 inhibitors might be advantageous, we therefore asked whether combined ATR/WEE1 inhibition might potentially be even more promising together with radiotherapy than single inhibition of ATR or WEE1. Given the strong interest in combining such inhibitors with radiotherapy, we think this is a highly important issue to address.

-Mechanistically, the radiosensitizing effects of ATR or WEE1 inhibitors are mainly thought to be caused by their roles in  checkpoint abrogation and inhibition of HR repair, as well as induction of DNA damage in S-phase (reviewed in ⁷). As these responses take place in the first 48 hours after radiation, the inhibitors are typically added shortly prior to the radiation treatment and kept in the cell medium for 24-48 hours after irradiation, such as done in our experiments.

- According to the reviewer’s suggestion, we have now performed clonogenic assays to address the efficacy of combined ATR/WEE1 inhibition with radiation (see new Fig 6B and S6B). The results support our previous conclusions obtained with the viability assay.

-The advantage of the viability assay is that many different concentrations of the inhibitors can be tested. In fact, for evaluation of the radiosensitizing effects of drugs, a robust correlation has been reported between results from cell growth assays performed at 4-6 days after irradiation and results from clonogenic assays ⁸. We therefore performed the CellTiterGlo assay at 5 days after irradiation in our experiments. However, we agree that it was important to verify the main findings by the clonogenic assay.

-To better explain the reasoning behind combining ATR/WEE1 inhibition with radiation and the treatment schedule, we have altered the text on p13, line 282-287 and 290-293. We have also revised the text regarding the CellTiterGlo assay in materials and methods p20, line 495-499.

 Reviewer 3

Open Review

(x) I would not like to sign my review report 
( ) I would like to sign my review report 

English language and style

( ) Extensive editing of English language and style required 
( ) Moderate English changes required 
(x) English language and style are fine/minor spell check required 
( ) I don't feel qualified to judge about the English language and style 

Comments and Suggestions for Authors

Rodland et al. manuscript investigated the effect of combining the inhibition of the ATR and Wee1 kinases with small molecule inhibitors on lung cancer cell lines.  They nicely showed that lung cancer cells, like other cancer cells such as breast, AML and biliary tract, exhibited synthetic lethality with the combination of ATR and Wee1 inhibitors. The authors also found that less synergy was found when the combination of inhibiting ATR and Wee1 with radiation actually decreased synergy in killing U2OS and A549 cells. The authors concluded that, depending on the specific cell lines, benefit might not result from combining ATR/Wee1 inhibition with radiation treatment. This study nicely confirmed a number of other published works and showed the efficacy of ATR/Wee1 combination treatment in lung cancer cells.  There is no novel mechanistic finding in this manuscript.  The experiments showing a lack of synergy between the combined ATR/Wee1 inhibition and radiation was not properly done.  I recommend rejection.

 **Authors reply:

-Our results highlight that the effects of ATR and WEE1 inhibitors and their combined effects vary between different cell lines. This is likely because both inhibitors affect multiple processes in the cells, resulting in different effects depending on many different intrinsic signaling events in each cell line.  We have improved the mechanistic aspects of the paper first by showing chromatin loading of CDC45 and CDK activity in all cell lines (new data in Fig. 5B and Fig. 3C). These experiments showed us that increased activity of replication initiation factors does not correlate with induction of DNA damage in S-phase in all cell lines. Furthermore, we also show that synergistic reduction in cell survival can occur upon the combined treatment, without any apparent induction of DNA damage in S-phase or premature mitotic entry. These results are discussed p11, the whole paragraph, and briefly on p 7, line 172-175. Second, we have compared the protein levels of proposed biomarkers for WEE1 and ATR inhibitor sensitivity in all cell lines, but found no clear correlation between levels of such single biomarkers and sensitivity/resistance to the inhibitors alone or in combination. See also our reply to reviewer 4. Clonogenic assays have now been performed confirming the results for the inhibitors in combination with radiation.

Major concerns:

The effect of radiation treatment on cells is normally assayed by colony formation assay. Figure 6, while convincingly showed the decrease in synergy between radiation and ATR/Wee1 inhibition, was based on viability assays 3 days after drug washout.  I cannot actually see if there is any effect from radiation in this timeline. Based on the literature, radiation treatment usually causes delayed cell death and the viability assay might not capture this effect. Of course, the major concern here is that the conclusion is based on negative data.  This experiment should be done with the colony formation assay.

 **Authors reply:

-We have now performed clonogenic assays (new Figure 6B and S6B). The results support our previous conclusions. Furthermore, when comparing the effect of radiation alone in the two assays, we found that the survival fractions were the same. Note that the effects of radiation alone are stated in the figure legends (Figure 6A, 6B and S6). See also our reply to reviewer 2, last point.

The authors claimed that lung cancer cell lines have a variable response to combined Wee1 and ATR inhibition, but they only checked this at a fixed concentration of the ATR inhibitor that yielded about 10-20% reduction in cell viability. If their cell lines exhibit variable response, the approach would be to evaluate the response at increasing concentrations of both the drugs to see if there truly is any synergy or not (this is done only for U2OS and A549 and not the other three cell lines).

**Authors reply:

 -According to the reviewer’s suggestion, we have now performed viability assays at multiple concentrations of both inhibitors also in H460 and SW900 cells* (new Figure 4C). We have also included statistical analysis of Bliss Independence scores to better evaluate the synergistic effects (Fig S4A and Fig S4C and Fig 4B). The results support our previous conclusions. Note also that the differences in response to a fixed concentration of the ATR inhibitor are highly relevant to show that the cell lines have a variable response to the inhibitors. When the inhibitors are used in the clinic, a fixed concentration will most likely be used for all tumors. See also response to reviewer 1, point 5.

(*H1975 cells clearly showed significant synergistic effects based on the results in Fig S4A and 4B.)

Minor concerns:

Figure 1: pATM should be normalized to total ATM level.

**Authors reply:

 -Total ATM level is now included in Figure 1. See also our reply to Reviewer 2, point 1.

Fig 2C: needs total DNA-PK, RPA and ATM as controls

**Authors reply:

  -Total DNA-PK and ATM level is now included in Figure 2. See also our reply to Reviewer 2, point 2. Total RPA shows multiple bands when it is phosphorylated and is therefore not a good loading control.

Fig 3C: Were Myt1 levels normalized to total protein? No description in the method section.

 **Authors reply:

  -Yes. We have now included a description of this in legend to Figure 3D on p9, line 190-192.

Fig 3D – how does CDC45 loading of the resistant cell lines compared to the sensitive cell lines? What is IC50 of MK-1775 in U2OS cells?

  **Authors reply:

  -We have now measured CDC45 loading in all cell lines after treatment with the inhibitors alone and in combination (new Figure 5B). The pattern of CDC45 loading looks very similar for all cell lines, as it increases more with ATR than WEE1 inhibition and is highest upon the combined treatment. The most resistant cell line H460 shows a slightly less increase in CDC45 loading upon treatment with these inhibitors than the other cell lines. However, we do not find a correlation between the extent of CDC45 loading and sensitivity to the ATR or WEE1 inhibitors or the combined ATR/WEE1 inhibitor treatment across our cell panel. A discussion of this has been included on p11, line 261-267.

-The IC50 of MK1775 for U2OS cells is 150-200 nM (Fig S6A, and data not shown).

Table 1 and Figure 4 – requires the IC50 for ATRi(VE822) alone

   **Authors reply:

-Cell viability assays and IC50 values for ATRi alone are now included in new figure S4B and Supplementary Table 1. Interestingly, there is no correlation between sensitivity to the WEE1 inhibitor and sensitivity to the ATR inhibitor. A comment on this is included on p15, line 331-333.

What is the p53 status on the cell lines used? This information should be included.

**Authors reply:

  -This is now included on p 19, line 446-448 (methods).  See also our reply to Reviewer 2, point 2.

Sup fig 1 – Y15 CDK1 status was not shown after Wee1 siRNA.

**Authors reply:

  -This is now included in Fig S1B

All experiments were done in a mixed cell population using flow cytometry. Without actual microscopic examinations, the flow data is superficial at best.

 **Authors reply:

  -We strongly disagree that our flow cytometry data are superficial. With flow cytometry, fluorescence signals from many thousands of individual cells are accurately measured. We do multiparameter analysis to also assess the cell cycle position of each cell, in addition to e.g. measurement of CDC45 loading or CDK activity. We also include barcoding dyes, to obtain highly accurate quantification of results. The bar-coding strategy eliminates sample-to-sample variation due to differences in antibody staining or flow cytometry processing. See our experimental setup and analysis strategy in figure S3.  

No raw data were shown for the DNA fiber analysis.  Are there heterogeneities in replication fork rate in the other cell lines?

 **Authors reply:

  -The distribution of fork rates has now been included in Figure S2C to show the heterogeneity in replication fork rate. DNA fiber analysis was performed in the cell line most resistant to the WEE1 inhibitor (H460) in order to confirm that the lack of S-phase accumulation observed by flow cytometry (Figure 2B) was not due to the cells not cycling, but that the replication was unperturbed. U2OS cells were included for comparison, as they show a big accumulation in S-phase after the treatment with MK1775 and we had previously used U2OS in the DNA fiber assay ². The other cell lines were not examined by this assay. A revised text explaining these issues has been included on p6, line 155-159.

Reviewer 4

( ) I would not like to sign my review report 
(x) I would like to sign my review report 

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Comments and Suggestions for Authors

This is a well-written and performed analysis of the impact of blocking both WEE1 and ATR kinases using one very sensitive osteosarcoma cell line, U2OS, and four lung cancer cell lines with varying sensitivities to WEE1 inhibition. Ultimately, it was found that combining AZD1775 and VE822 resulted in synergy in lines that were initially sensitive to WEE1 inhibition alone, but the combination did not markedly enhance sensitivity using cell lines that were relatively resistant to AZD1775 alone. Radiation plus kinase inhibitors did not enhance the therapeutic effects of radiation to the extent predicted by the authors, and they indicate that this strategy may be less effective than expected. Ultimately, no firm explanation for the variation in cell sensitivity was identified. While the findings of the paper are interesting and may be worthy of publication after major additions, such as testing the hypotheses for why certain cells are resistant that the authors bring up briefly in the discussion, it is unclear how the present results should be used in future clinical trials. Unfortunately, no biomarker of response versus resistance was identified nor tested. The reader is left unsatisfied with more questions than answers.

**Authors reply:

  -We have now compared the levels of possible resistance/sensitivity markers in our cell lines, including levels of cyclin E, cdc25A, ATM, pATR, p21, RRM2  and γH2AX (see results in new Figure 7) and Myt 1 (was already included in previous Fig 3C, now 3D). In addition, we have evaluated the impact of p53 mutational status (p 17, line 396-403 (discussion)). The most striking findings from this analysis were that that Cyclin E levels are very high in U2OS cells and γH2AX levels are very high in SW900 cells. This could indicate elevated endogenous replication stress in these cell lines, which might be consistent with their high sensitivity to the WEE1 inhibitor. However, in U2OS cells the high Cyclin E levels were not accompanied by high γH2AX levels which would have been expected if they were suffering from replication stress. Moreover, SW900 cells were relatively resistant to the ATR inhibitor for which replication stress is expected to predict sensitivity. When examining all our results, we have not found one universal biomarker that can predict the responses to the WEE1 or ATR inhibitor or to the combined effects. See a more extensive description of these results on p15, line 325-349.

-We think that an important issue to note regarding biomarkers is that both inhibitors affect multiple mechanisms in the cells. They can therefore cause cell death by affecting different mechanisms in the different cell lines (e.g. mechanisms affecting S-phase, G2/M transition, mitosis or DNA repair). Our results suggest that e.g. S-phase DNA damage is more important in U2OS than in the other cell lines. As the mechanisms in S, G2/M transition, mitosis and DNA repair involve different factors, it might not be possible to find one universal biomarker for the treatment response to these inhibitors. We think this view would be interesting to many scientists working on Wee1 and ATR inhibitors. We also think it would fit with the scope of Cancers to publish negative as well as positive results.

-We have improved our discussion of biomarkers on p 17, paragraph on lines 375-405.

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