KEAP1 Is Required for Artesunate Anticancer Activity in Non-Small-Cell Lung Cancer

Round 1

Reviewer 1 Report

The revised manuscript has been improved by the authors.

Author Response

Thank you for your previous comments and support for this manuscript

Reviewer 2 Report

The authors have accommodated most of the suggestions and have improved the reading of the manuscript. However, the lack of especially the in vivo experiments is a limitation of the study. Also, the evaluation of DNA damage following KEAP1 knockdown in KEAP1 WT cells and following artesunate and NRF2 inhibition in KEAP1 mutated cells is missing, which in my opinion is not out of the scope of this study as this will prove that DNA damage is indeed a central feature of the treatment effect as proposed by the authors themself. The authors have already used siRNA to knockdown KEAP1, and they also present data on DNA damage, thus this is not a difficult experiment.      

Author Response

We completely agree with your assessment that in in vivo evaluation of artesunate and ML385 are required before the observations of this current study can be applied to the clinical treatment of patients in a clinical trial and this represents a critical future direction.

As requested we evaluated DNA damage following KEAP1 knockdown and NRF2 inhibition (with ML385) and this data has been added to figure 4 (line 180-194) and figure 5 (line 230-240), respectively. Briefly, knocking down KEAP1 in H1299 (KEAP1 WT cells), but not in A549 (KEAP1 mutant) cells resulted in significantly less DNA damage following treatment with artesunate then in cells transfected with non-targeting (siNT) control siRNA (line 164-178). Alternatively, pretreatment of both cell lines with ML385 resulted in a significant increase in DNA damage when cells were also treated with artesunate (line 212-229), this finding agrees with the observation that artesunate and ML385 are synergistic in both A549 and H1299 cells (figure 6).

Reviewer 3 Report

The author's have satisfactorily included additional discussion points that clarified interpretation of data. I have no further comment.

Author Response

Thank you for your previous comments and support for this manuscript

Round 2

Reviewer 2 Report

The authors should perform a one-way ANOVA and not a two-way ANOVA to calculate significance in Figure 5 c and d 

Author Response

We changed the statistical test used for Figure 5c & d to a One-way ANOVA as requested. We have changed Figure 5 (line 227) to reflect the updated statistical results as well as changed the description in the figure legend (line 237-239) and methods section (line 446-448). 

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 1

Reviewer 1 Report

In the manuscript by Hill KS, et al., KEAP1 is required for artesunate anticancer activity in non-small cell lung cancer. The authors suggested that the modulation of KEAP1-NRF2 pathway was able to alter the sensitivity of lung cancer cells to artesunate. In addition, they suggested the combination of artesunate and NRF2 inhibitors to treat NSCLC in patients with mutations of KEPA1-NRF2 pathway. Although the results are of great interest and the manuscript is well written, the lack of validation are major limits for generalizing these results. Specific points are listed below.

1. Authors showed that A549 cells were sensitive to artesunate, while H1299 and H1563 cells were resistance to artesunate. Authors demonstrated that the difference of sensitivity to artesunate was caused by mutation of KEAP1. However, authors should show the changes of KEPA1-NRF2 pathway (for example, translocation and phosphorylation of Nrf2) follow artesunate treatment in the used cells.
2. In the figure 4, authors used the ML385 to inhibit NRF2. Do ML385 treatment affect the proliferation of NSCLC cells?
3. Authors showed that the suppression of KEAP1 did not affect the sensitivity to artesunate in A549. However, the combination of artesunate and NRF2 inhibitor was synergistic in A549 cells. The interaction of KEAP1 and NRF2 is very important in condition of cellular stress. Authors should explain the reasons for these results.
4. Authors described the sensitivity to artesunate in NSCLC. The detailed mechanisms of response to artesunate in molecular levels is necessary to explain the rationale of combined therapy.

Reviewer 2 Report

The authors aim was to demonstrate the importance of KEAP1 for the anticancer activity of artesunate and evaluate NFR2 inhibitors in combination with artesunate in cancer cells with mutations in KEAP1. The authors show that artesunate induce DNA-damage in cancer cells with WT KEAP1 while significantly less DNA damage is observed in KEAP1 mutated cancer cells. The authors show that IC50 value is significantly higher in KEAP1 mutated cell lines and that upon siRNA KEAP1 silencing in KEAP1 WT cells the IC50 value in increased significantly. Finally, the authors show a synergistic effect by combining NRF2 inhibitors and artesunate in KEAP1 mutated cancer cells. The manuscript is still immature, and the authors should address the following issues to increase the impact of the manuscript.

Major points:

• The interaction between KEAP1 and NRF2, the role of NRF2 in the oxidative stress response and how mutations in KEAP1 affect the KEAP1-NRF2 interaction and why the tumor cells benefit from these mutations is very briefly and superficial described. It will improve the understanding of the study if this was more detailed described.
• The discussion seems more to be a summary of the results. The presented results are not discussed in relation to other findings. For instance, a relevant study by Gong et al. 2020 in Cell commun. Signal is not mentioned.    

• The authors should show the level of ROS production following artesunate treatment in the difference cell lines.
• The authors use DNA damage as a measure of oxidative stress following artesunate treatment and that KEAP1 mutations decrease the DNA damage. The authors should show that the DNA damage is decreased in the KEAP1 WT following siRNA silencing of KEAP1 and that DNA damage is increased in KEAP1 mutated cells following combination of artesunate and NRF2 inhibition
• In addition to the DNA damage the authors should more specifically show differences in the activation of the KEAP1/NFR2 between the different cell lines following artesunate treatment.  
• The authors should include more methods to monitor cell viability following artesunate treatment. For instance, it would be interesting to see if artesunate induce apoptosis.
• To really substantiate the effect of combination of KEAP1 and NRF2 inhibitors the authors should include in vivo evaluation of this combination in comparison to the treatment alone.

Minor points.

Line 89: The authors use a two-way ANOVA to compare the pH2AX in cisplatin vs. DMSO treated cells. The two-way ANOVA is used test two different categorical independent variables on one continuous dependent variable, which I do not see this experiment. No information is detailed in Materials and Methods. Can the Authors explain why they have use this test.

91-98. The authors compare directly compare the absolute values of RFU. This is not easy to compare for the reader and I suggest that the authors report the relative values instead.

Line 111: The authors test the expression of NQO-1 following knockdown of KEAP1. It is not clear why the authors test this particular gene.

Reviewer 3 Report

Hill et al nicely showed that KEAP1 is required for artesunate anticancer activity in NSCLC. Artesunateis is an anti malaria drug and shown to have anticancer properties. Kelch13 mutations cause resistance to artesunate, a NSCLC cell line with a KEAP1 inactivating mutation is resistant to artesunate and this resistance to artesunate can be overcome with the addition of a NRF2 inhibitor.

Since KEAP1 mutations are common in NSCLC and are associated with poor prognosis and chemoresistance, the combination of artesunate and an NRF2 inhibitor may be a used as combination therapy for NSCLC. The article is well written and I found this manuscript very interesting and conclusion is fully supported by results.

Major concern to be addressed

Why authors chose to assess cell viability only after 96 hours. Please show these results in lesser time point.

Antimalarial drug promote apoptosis rather than necrosis in most cases although both have been reported. Induction of apoptosis is a major advantage of anti-malignancy action. Does A549 cells were also resistant to apoptosis when treated with artesunate as compared to other cell line?

Similarly when KEAP1 levels were reduced by siRNA still A549 resistant to apoptosis.

Does combining NRF2 inhibitor and artesunate enhances cell apoptosis as compared when use separately show these results. Mitochondria-dependent pathway may be involved in the induction of apoptosis. Combined NRF2 inhibitor and artesunate treatment obviously will be superior to the individual drugs in promoting cell apoptosis.

NRF2 Controls mitochondrial function linking metabolism to redox balance. It will be interesting to see how combined treatment of both artesunate and an NRF2 inhibitor effect mitochondrial function and cancer cell metabolism. Suggestion authors may analyze both OCR and glycolysis. As cancer cell are highly glycolytic.

To prove that both artesunate and an NRF2 inhibitor can be used as combination therapy for NSCLC authors should show in vivo efficacy comparison results of artesunate and NRF2 inhibitor, in human NSCLC xenograft mouse models.

The statement about mycoplasma checking as well as authentication of cells should be provided.

Please provide the catalogue number of all reagent used in the study.