Chloroquine Potentiates the Chemotherapeutic Effect of Carboplatin and ATR/Chk1 Inhibitors by Increasing the Replication Stress

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions changes in the re-submitted files.

Sections "Introduction" and "Material and methods" were improved according to the recommendations.

Comments 1: Have the authors considered the side effects of carboplatin in cancer treatment?

Response 1: Thank you for pointing this out. The following paragraph has been added to the section Introduction (page 2, paragraph 2).

Platinum drugs are common drugs for various malignant tumors treatment including breast cancer, ovarian cancer, colorectal cancer, and others. Despite their central role in cancer treatment long term administration leads to severe adverse effects, including nephrotoxicity (more common for cisplatin), thrombocytopenia caused by myelosuppression (carboplatin), dosage-limiting nausea and vomiting and others. Combination therapy (or multiple-low-dose therapy (MLD)) seems an attractive strategy not only in overcoming drug resistance development but also in reducing the toxicities as the lower therapeutic drugs doses are needed.

Comments 2: As a classic aminoquinoline drug, chloroquine requires the authors to introduce its side effects in the introduction section.

Response 2: Agree. We have, accordingly, added the following paragraph to the section Introduction (page 2, paragraph 5) to emphasize this point.

In general, CQ is a safe drug with low toxicity when used in recommended therapeutic doses. Gastrointestinal toxicities can occur when it is used in short term, including nausea, vomiting, and diarrhea. Long term use and/or overdose can lead to retinopathy, cardiotoxicity and neurotoxicity. But most of these toxicities are reversible if the treatment is stopped. Reducing the CQ dose significantly decreases its possible adverse effects. Note that as CQ has the narrow therapeutic window and does not show good efficiency when used as a single chemotherapeutic agent it is a priority to use it in combination with gentotoxic drugs in order to achieve the maximum chemotherapeutic efficiency along with side effects minimization due to low dose treatment. The reduced concentration of agents in such combination treatment can be used because of the presence of a synergistic effect between the drugs. So it seems important to determine the appropriate drugs which efficiency can be increased when combined with CQ.

Comments 3: Do chloroquine and carboplatin interact with each other, particularly in terms of overlapping toxicities?

Response 3: Thank you for pointing this out. The following paragraph has been added to the section Discussion (page 14, paragraph 3, lines 434-437).

Moreover, as CQ has some overlapping toxicities with carboplatin, such as gastrointestinal, it seems important that low doses combination chemotherapy can be applied to achieve the treatment efficiency thus minimizing the possible side effects.

Comments 4: Did the authors use ATRi or Chk1i as a pharmacological control to confirm that chloroquine-induced Chk1 phosphorylation is ATR-dependent?

Response 4: Thank you for pointing this out. In our study we showed that CQ administration to the combination of carboplatin and RSR inhibitors would be more attractive strategy for cancer treatment as it allowed to reduce the drugs concentrations and thus their possible toxicities without losing the therapeutic effect. But indeed based on our results (western blots in figures 9, 10, 12, 13) we can conclude that CQ induced Chk1 phosphorylation in ATR-independent as its administration together with ATRi significantly enhanced Chk1 phosphorylation compared to the experimental groups without CQ. At the same time when CQ was applied together with Chk1i we could not detect further increase in phospho-Chk1 protein level. The increase in Chk1 phosphorylation in three agents treated groups (carboplatin + Chk1i + CQ) compared to groups without CQ could be explained by the use of low Chk1i concentration. The respective paragraph has been added to the section Discussion (page 16, paragraph 2).

Comments 5: This study uses carboplatin and cisplatin. Do the results extend to oxaliplatin?

Response 5: Thank you for the question. In our study we used only two common drugs from platinum group with similar mechanism of replication stress induction (through replication fork stalling and DDR activation). Despite oxaliplatin can also induce replication stress, the mechanism of this induction is different. So we could not claim that the results would extend to this drug although we did not directly test this combination.

Comments 6: This manuscript focuses on tumor cell sensitization. Did the authors test if chloroquine protects normal cells from platinum toxicity?

Response 6: Thank you for pointing this out. CQ is not typically seen as a normal cells protector. When used in high dosage it can enhance the toxicity of drugs but when used in low concentration CQ, as it is now described in the Introduction, is a safe drug. That’s why we investigated the presence of synergism between CQ and platinum drugs when both were used in low doses in order to minimized side effects.

Comments 1: Clarification of experimental replicates (n):

In several experiments, the number of times each assay was independently replicated (n) is not consistently specified. Providing this information for all experiments is important to ensure transparency and to allow readers to properly assess the robustness of the results.

Response 1: Thank you for pointing this out. The respective information was added:

page 8, figure 4 legend, line 225

page 9, figure 5 legend, lines 236-237

page 14, figure 9 legend, lines 361-362

page 16, figure 10 legend, lines 380-381

page 18, figure 12 legend, line 421

page 19, figure 13 legend, lines 435-436

Comments 2: Figure citation and visual clarity:

Some figures are not cited accurately or consistently within the text. Please revise all figure references for correctness. Additionally, certain graphs would benefit from being slightly enlarged or reformatted to enhance legibility.

Response 2: Thank you for pointing this out. We checked the whole text and accurately cited all the figures (in order of their appearance). Images from figure 13 A were combined with figure 11 (now it is 11 B). The images in some figures were enlarged as recommended. All the figures were improved in their qualities.

Comments 3: Colony formation assays:

While the representative images are useful, including a quantitative plot summarizing colony counts would significantly strengthen the presentation. Such a graph would allow readers to evaluate the results not only visually but also quantitatively, and it would enable the inclusion of statistical significance across experimental conditions.

Response 3: Thank you for pointing this out. This is not a classical colony formation assay (that is why we called it re-proliferation assay) when less than 1000 cells are seeded on the plate. Here we analyzed the ability of the whole cell population to resume its proliferation. So in some cases when, for example, this ability is significantly inhibited (groups with CQ supplementation) we certainly can visualize and count the colonies but in other groups it is impossible as cells form almost monolayer (we also tried some softwares but even they could not identify formed colonies). We believed that images reflect the obtained results well as in most cases this is a qualitative result (CQ completely inhibited cells’ re-proliferation potential with almost complete absence of colonies compared to groups without CQ when significantly greater amount of cells were able to re-growth).

Comments 4: Additional methodological details:

In the re-proliferation assay, the seeding density is reported for the initial plating but not for the second one; please clarify this point.

For the western blot procedures, the percentage of acrylamide used in the gels should be included to ensure full reproducibility.

More generally, please verify that seeding densities and other key experimental parameters are provided consistently for all assays.

Response 4: Agree. We clarified this point (section Materials and Methods, page 22, paragraph 3, lines 571-572).

The percentage of gel was added to the section Materials and Methods, page 22, paragraph 6, line 596.

Seeding densities were verified across the text.