Kinase Inhibitors of DNA-PK, ATM and ATR in Combination with Ionizing Radiation Can Increase Tumor Cell Death in HNSCC Cells While Sparing Normal Tissue Cells

Round 1

Reviewer 1 Report

The study is well done and I have no major comments.

The authors explored whether KI and IR in normal tissue surrounding in the head and neck squamous cancer cell cancer may have a therapeutic effect as in cancer tissue.

The aims of the study should be better defined.

It should also to better explain in a clear manner the cells named SBLF7, BLF9, HSC4, CAL33, UM-SCC-47, UD-SCC-2 and how they were obtained.

The figures are difficult to read and should be reformatted.

Author Response

Dear Reviewer,

thank you for giving us the opportunity to submit a revised draft of our manuscript titled “PARP inhibitors Talazoparib and Niraparib sensitize melanoma cells to ionizing radiation” to the special issue “Mechanism of DNA Damage, Repair and Mutagenesis” published in “Genes – Molecular Genetics and Genomics”. We appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewers for their insightful comments on our paper. We have been able to incorporate changes to reflect the suggestions provided by the reviewers. All changes within the manuscript are reported by the “tracking mode” of MS Word.

Reviewer 1

The study is well done and I have no major comments.

The authors explored whether KI and IR in normal tissue surrounding in the head and neck squamous cancer cell cancer may have a therapeutic effect as in cancer tissue.

The aims of the study should be better defined.

Thank you for your overwhelming feedback. According to your concerns we elaborated the introduction part and defined our aims in more detail. Since HNSCC is still a common malignancy in radiation oncology and sometimes difficult to irradiate, we focused on cell lines representing this malign entity. Since radiation leads to DNA damage (Borrego-Soto et al. 2015 in Genet Mol Biol.) and evidence arises that tumor cells have lost their efficacy of DNA damage repair partly (Srivastava et al. 2015 in Chem Biol), it was of great interest for us to investigate the potential of DNA damage repair pathways using kinase inhibitors to enhance the outcome of the cancer therapy.

We re-worked the introduction section (last paragraph):

These three KI targeting the DDR are the subject of our research, where we combined these KI with IR to investigate the effectiveness of a concomitant therapy in comparison with sole IR in HNSCC tumor cells and normal skin fibroblasts. Combining KI of the DNA damage repair with ionizing radiation, which classically lead to DNA damage, is of great interest for us since HNSCC includes partly radioresistant subtypes. However, due to their radiosensitizing effects [21, 22], KI also pose a risk for the normal tissue surrounding the tumor, that might be affected by combined treatment of KI and IR. It has been observed that increased side effects can occur when IR and KI are combined [23–26]. Nevertheless, it is known that tumor cells harbor mutations in their DDR system in contrast to healthy non-malign cells. It is therefore important to look not only at the tumor cells, but at normal tissue as well. Hence, we included primary skin fibroblasts from healthy donors as a reference in all our experiments. In this study we hypothesize to enhance radiotherapy for HNSCC with a multimodal approach of DDR inhibitors.

It should also to better explain in a clear manner the cells named SBLF7, SBLF9, HSC4, CAL33, UM-SCC-47, UD-SCC-2 and how they were obtained.

Thank you for pointing out your concern here. We described the generating of our cell lines in more detail according to a previous work of us (Hecht et al. 2018 in Oncol Lett.) and the working group of Prof. Dr. Thorsten Rieckmann (https://doi.org/10.1016/j.radonc.2013.03.013), who provided us the HNSCC cell lines generously.

We added to the material and method section (Paragraph: 2.1 Cell culture and inhibitors):

2. Materials and Methods

2.1. Cell culture and inhibitors

Primary human fibroblasts SBLF7 and SBLF9 were derived from the skin of a healthy donor as described previously [27]. In brief, skin biopsies of the cutis and subcutis were taken after local anesthesia. The biopsy was cut in small pieces, placed in tissue culture flasks and each covered with a drop of medium (40 % fetal bovine serum). After the skin pieces had attached to the culture flasks and the first fibroblasts had grown out, they were covered with F-12 Medium (Gibco, Waltham, MA, USA). The tissue samples were obtained after informed consent of the donors. The ethical approval was obtained from the Ethics Committee of the medical faculty of the Friedrich-Alexander-Universität Erlangen-Nürnberg (204_17 BC). The established and commercially available HPV negative HNSCC (head and neck squamous cell carcinoma) cell lines HSC4 and CAL33 as well as the HPV positive cells UM SCC 47 and UD SCC 2 were obtained courtesy of Dr. Thorsten Rieckmann from the University of Medical Centre Hamburg-Eppendorf, Germany.

The figures are difficult to read and should be reformatted.

We agree with your concern and appreciate that you shed light on this important point. We increase the resolution of our images and imported a file type of higher quality. Hopefully, we are now able to satisfy this point in an adequate manner. We changed all graphs included in the manuscript.

Example given: [Figure]

Author Response File: Author Response.docx

Reviewer 2 Report

Faulhaber and co-authors present a work where they compare the effects of ionizing radiation alone or combined with three DNA damage repair inhibitors in four different HNSCC cell lines.

Introduction and discussion are well written and the experimental approach is appropriate and the results are very promising and supporting the conclusions.

I just believe they should improve the English language and eliminate some oversights all over the text and above all, they should add more experimental images. In particular:

1. Results Page 4 and Fig 1: the authors write they have performed experiments on every cell line and with every inhibitor considered in their research, so it is not clear why they show only one cell line in A and B, treated with only one inhibitor, and only two lines in C, and all HPV negative cells. I think it would be better to show the results obtained for every cell line and for every inhibitor (at least, as supporting information).
2. Fig 4: the graphs are not enough to show the results of the colony forming assays. Please show images of the stained colonies for each performed experiment.
3. Fig 5: for the same reason as above, it should be appropriate to show the images of each wound healing experiment.

Author Response

Dear Reviewer,

thank you for giving us the opportunity to submit a revised draft of our manuscript titled “PARP inhibitors Talazoparib and Niraparib sensitize melanoma cells to ionizing radiation” to the special issue “Mechanism of DNA Damage, Repair and Mutagenesis” published in “Genes – Molecular Genetics and Genomics”. We appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewers for their insightful comments on our paper. We have been able to incorporate changes to reflect the suggestions provided by the reviewers. All changes within the manuscript are reported by the “tracking mode” of MS Word.

Reviewer 2

Faulhaber and co-authors present a work where they compare the effects of ionizing radiation alone or combined with three DNA damage repair inhibitors in four different HNSCC cell lines.

Introduction and discussion are well written and the experimental approach is appropriate and the results are very promising and supporting the conclusions.

I just believe they should improve the English language and eliminate some oversights all over the text and above all, they should add more experimental images. In particular:

1. Results Page 4 and Fig 1: the authors write they have performed experiments on every cell line and with every inhibitor considered in their research, so it is not clear why they show only one cell line in A and B, treated with only one inhibitor, and only two lines in C, and all HPV negative cells. I think it would be better to show the results obtained for every cell line and for every inhibitor (at least, as supporting information).

We agree with the reviewer’s feedback here and thank for the support. We included representative images of our gating strategy and data of cell death (dot plots) and cell cycle distribution (histograms). Additionally, we included in the supplementary material the first dose-escalation of all three kinase inhibitors in one of our HPV-pos. cell lines UM SCC 47.

We added to the supp. material section:

Figure S1. Representative dot plots of AnnexinV/7AAD cell death staining of the healthy fibroblasts SBLF7 and SBLF9. Cells were treated with CC-115 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination), AZD0156 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination) or VE-822 (no inhibitor, 0.1 µM, 2 Gy irradiation, 0.1 µM + 2 Gy combination).

Figure S2. Representative dot plots of AnnexinV/7AAD cell death staining of the HPV-positive HSC4 and Cal33. Cells were treated with CC-115 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination), AZD0156 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination) or VE-822 (no inhibitor, 0.1 µM, 2 Gy irradiation, 0.1 µM + 2 Gy combination).

Figure S3. Representative dot plots of AnnexinV/7AAD cell death staining of the HPV-negative UM-SCC-47 and UD-SCC-2. Cells were treated with CC-115 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination), AZD0156 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination) or VE-822 (no inhibitor, 0.1 µM, 2 Gy irradiation, 0.1 µM + 2 Gy combination).

Figure S4. Representative histograms of Hoechst (DNA) cell cycle staining of the healthy fibroblasts SBLF7 and SBLF9. Cells were treated with CC-115 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination), AZD0156 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination) or VE-822 (no inhibitor, 0.1 µM, 2 Gy irradiation, 0.1 µM + 2 Gy combination).

Figure S5. Representative histograms of Hoechst (DNA) cell cycle staining of the HPV-positive HSC4 and Cal33. Cells were treated with CC-115 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination), AZD0156 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination) or VE-822 (no inhibitor, 0.1 µM, 2 Gy irradiation, 0.1 µM + 2 Gy combination).

Figure S6. Representative histograms of Hoechst (DNA) cell cycle staining of the HPV-negative HSC4 and Cal33. Cells were treated with CC-115 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination), AZD0156 (no inhibitor, 1 µM, 2 Gy irradiation, 1 µM + 2 Gy combination) or VE-822 (no inhibitor, 0.1 µM, 2 Gy irradiation, 0.1 µM + 2 Gy combination

Additionally, we included in the supplementary material the first dose-escalation of all three kinase inhibitors in one of our HPV-pos. cell lines UM SCC 47:

Figure S7. Dose escalation study for the induction of apoptosis or necrosis (cell death) by the inhibitors CC 115, AZD0156 and VE 822 conducted on HPV-positive cell line UM-SCC-47. Each value represents mean ± SEM.

1. Fig 4: the graphs are not enough to show the results of the colony forming assays. Please show images of the stained colonies for each performed experiment.

Thank you for your feedback. We agree with it and added a graph to the supplementary material including representative experiments of all tested cell lines and all used kinase inhibitors.

We added to the supp. material section:

Figure S2. Colony forming assay. Representative images of all tested cell lines (healthy: SBLF7, SBLF9; HPV-neg: HSC4, Cal33; HPV-pos: UM-SCC-47, UD-SCC-2) and inhibitor (AZD0156: ATMi; CC-115: DNA-PKi, VE-822: ATRi). Cell were treated with different concentrations of inhibitors (Co: no inhibitor; (1): 5 nM AZD0156, 0.5 µM CC-115, 0.05 nM VE-822; (2): 10 nM AZD0156, 1 µM CC-115, 0.1 nM VE-822). Experiments were seeded in technical replicates (Well 1-3 and well 4-6). In case of healthy fibroblasts, typical morphology of cells leads to slightly stained colonies. Therefore, counting was done microscopical.

1. Fig 5: for the same reason as above, it should be appropriate to show the images of each wound healing experiment.

We thank you and appreciate your feedback. According to the mentioned point above, we re-worked this too and added representative images of our wound healing assays of all tested cell lines and inhibitors. Because of the huge number of images of six different cell lines, treated with three different inhibitors in four different treatment schemes (Co, KI, IR and KI+IR) at in minimum three timepoints (0 h, 24 h and 48 h) we decided to show representative images of timepoint 0 h and 48 h for one candidate of every cell line group: HPV-positive HNSCC cell line (e.g. UM-SCC-47), HPV-negative HNSCC cell line (e.g. Cal33) and healthy fibroblasts (e.g. SBLF7).

We added to the supp. material section:

Figure S9. Representative images of wound healing assay of HPV-positive cell line UM-SCC-47 at time points 0 h and 48 h. Cells were treated either without any treatment, with inhibitor (1 µM CC-115, 1 µM AZD0156 and 0.01 µM VE-822), 2 Gy dose or a combination of inhibitor and 2 Gy.

Figure S10. Representative images of wound healing assay of HPV-negative cell line Cal33 at time points 0 h and 48 h. Cells were treated either without any treatment, with inhibitor (1 µM CC-115, 1 µM AZD0156 and 0.01 µM VE-822), 2 Gy dose or a combination of inhibitor and 2 Gy.

Figure S11. Representative images of wound healing assay of healthy control cell line SBLF7 at time points 0 h and 48 h. Cells were treated either without any treatment, with inhibitor (1 µM CC-115, 1 µM AZD0156 and 0.01 µM VE-822), 2 Gy dose or a combination of inhibitor and 2 Gy.

Author Response File: Author Response.docx