Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction

Round 1

Reviewer 1 Report

Giocosa S. et al. shows that CK2/ATM inhibitors drug could be an interesting possibility to fight metastatic clear cell renal cell carcinoma.

I think that not only this manuscript is well done from a basic scientific point of view but it can be a valid indicator for any clinical trials. 

For this reason I suggest to improve your point of view with in vivo experiment in a next paper.

Only a minor revision and suggestion: while your work is fascinating and besides being interesting it stimulates reading, on the contrary, the abstract is not. It should be improved especially when you talk about 786-O-VHL^-. I mean, it should also be explained in the abstract why you have choiced these cells and why it is so important that your results were found precisely on VHL ^- cells.

Thanks a lot

Author Response

Dear Reviewer,

Thank you for your very kind comments. Please, find the implemented abstract bellow.

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL^- cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2,880 drug-gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rational for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL^- ccRCC.

Thank you again for your comments.

Best regards

Odile Filhol

Reviewer 2 Report

Giacosa and colleagues demonstrate their systematic screening approach novel therapeutic approach for renal cell carcinoma. Combinatorial treatment with both candidate molecules facilitates inhibition of the CK2 and ATM kinases which proved especially effective against VHL tumors.

Major comments

• do both compounds also act synergistically with regards to migration? 
• are other independent studies using this kind of 3D migration assay, please provide references if possible

Minor comments

• please check spaces (l. 35, 198-200, 289f, 323, 389, 400, 402), spaces before units (l. 119, 205, 234f, 294)
• rephrase sentence 197-200, see usage of plural
• formatting with regard to justification also lacks some diligence
• introduction and consequent use of abbreviations, see ds (l. 254)
• check pb (l. 260)
• please rephrase first sentence of section 2.11

Author Response

Dear Reviewer,

Thank you for your kind comments. Please, find bellow our answers.

Major comments:

• do both compounds also act synergistically with regards to migration? 

According to data presented in the Figure 2F, there was a clear synergistic effect of the drug combination on cell spreading at the time point 5 days. After 7 days, both inhibitors alone induce a significant inhibition that consequently attenuate the synergistic effect of their combination.

This comment is added in the text lane 430.

• are other independent studies using this kind of 3D migration assay, please provide references if possible

The 3D migration assay used in our study was previously described on glioma cell spheroids (reference 49). This reference is now added in the text lane 426.

Minor comments were all addressed , spaces, abbreviations and the sentence lane 281: “The first read contains 16 bases that must have a quality score higher than 10. The first 6 bp correspond to a unique sample specific barcode and the following 10 bp to a unique molecular identifier (UMI)”.

Thank you again for your comments.

Best regards

Odile Filhol