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. 2021 Jan 20;16(1):e0245715. doi: 10.1371/journal.pone.0245715

PI3k inhibitors (BKM120 and BYL719) as radiosensitizers for head and neck squamous cell carcinoma during radiotherapy

Fu-Cheng Chuang 1,2, Chih-Chun Wang 2,3, Jian-Han Chen 2,4, Tzer-Zen Hwang 2,3, Shyh-An Yeh 1,5, Yu-Chieh Su 2,6,*
Editor: Salvatore V Pizzo7
PMCID: PMC7817006  PMID: 33471836

Abstract

Approximately 500,000 new cases of head and neck squamous cell carcinoma (HNSCC) are reported annually. Radiation therapy is an important treatment for oral squamous cell carcinoma (OSCC). The survival rate of patients with HNSCC remained low (50%) in decades because of radiation therapy failure caused by the radioresistance of HNSCC cells. This study aimed to identify PI3K inhibitors that can enhance radiosensitivity. Results showed that pan-Phosphoinositide 3-kinases (PI3K) inhibitor BKM120 and class I α-specific PI3K inhibitor BYL719 dose-dependently reduced the growth of OSCC cells but not that of radioresistant OML1-R cells. The combination treatment of BKM120 or BYL719 with radiation showed an enhanced inhibitory effect on OSCC cells and radioresistant OML1-R cells. Furthermore, the enhanced inhibitory effect of the combination treatment was confirmed in patient-derived OSCC cells. The triple combination treatment of mTOR inhibitor AZD2014 and BKM120 or AZD2014 and BYL719 with radiation showed a significantly enhanced inhibitory effect on radioresistant OML1-R cells. These results suggest that the PI3K inhibitors are potential therapeutic agents with radiosensitivity for patients with OSCC.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide, remaining a public health concern with an estimated annual incidence of 500,000 new cases [1]. Approximately 90% head and neck cancer are squamous cell carcinoma and appear in upper aerodigestive tract organs, such as oral cavity, larynx, and pharynx [2]. Despite the improvement in HNSCC diagnosis and treatment through radiation, surgery, chemotherapy, concurrent chemoradiation, and monoclonal antibodies, the 5-year survival of patients with HNSS remains low at 40% [3]. Locoregional, distant relapse, and regional recurrences resistant to tumor therapies are the main reason of death in patients with HNSCC [4]. Recurrent tumors arise from radiation-resistant carcinoma cells, leading to re-irradiation treatment failure [5]. However, the combined administration of chemotherapeutics and radiation therapy is accompanied with severe toxic side effects that lead to the limited overall survival of patients [6]. Therefore, effective agents or improving the sensitization of radiation strategies is important for the treatment of these patients.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates the proliferation, survival, and apoptosis of cells [7]. The genetic aberration or dysregulation of the components involved in the PI3K signaling pathway, such as AKT and PTEN, is associated with the development and progression of cancers including HNSCC; it influences the metabolism, tumor growth, and development of metastasis [8]. Furthermore, the PI3K pathway is associated with resistance to anticancer therapies including targeted drugs, radiotherapy, and chemotherapy [9]. However, inhibitors targeting the PI3K/AKT/mTOR signaling pathway reportedly restore sensitivity to inhibit tumor growth under a combination treatment [10]. PI3K inhibitors also increase sensitivity in tumors resistant to chemotherapy, radiation, and hormone therapy [11].

Alpelisib (BYL719) is a class I α-specific PI3K inhibitor with strong inhibitory activity to wild-type and mutant PI3Kα isoforms but with weak inhibition to PI3K β, γ, and δ isoforms [12]. The combination treatment of BYL719 and epidermal growth factor receptor inhibitor synergistically inhibits the growth of HNSCC cells [13]. Buparlisib (BKM120) is a potent pan-PI3K inhibitor that inhibits all class IA PI3K paralogs [14]. Preclinical evidence in HNSCC has shown that BYL719 and BKM120 are under clinical trials. The present study analyzed the radiosensitivity of BYL719 and BKM120 in oral squamous carcinoma cell (OSCC) lines, radioresistant cells, and patient-derived OSCC cells that served as a preclinical model.

Results

Inhibitory effect of BYL719 and BKM120 on OSCC and radioresistant OSCC cell lines

SCC4, SCC25, OML1, and radioresistant OML1-R cells were treated with BYL719 and BKM120 at indicated concentrations to evaluate the inhibitory effect and proper inhibitory concentrations of compounds. The results of cell viability showed that BKM120 significantly dose-dependently reduced the growth of SSC4 and SSC25 cells, and inhibited the growth of OML1 and OML1-R cells at 3 μM. BYL719 significantly dose-dependently inhibited the growth of SSC4 and SSC25 cells but not that of OML1 and OML1-R cells (Fig 1). The results indicated that pan-PI3K inhibitor BKM120 may be a better inhibitor against OSCC cell growth than BYL719.

Fig 1. Reduction effect of BKM120 and BYL719 on the growth of OSCC and radioresistant OML1-R cells.

Fig 1

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SCC4, SCC25, and OML1-R cells were treated with BKM120 or BYL719 at indicated concentrations for 7 days. Cell viability was analyzed with MTS assay. Data are presented as mean ± SD, and the asterisks indicate significant difference. *P < 0.01 compared with the control group.

Enhanced radiosensitization effect of BYL719 and BKM120 on OSCC and radioresistant OSCC cells

OSCC (SCC4 and SCC25), OML1, and OML-R were pretreated with the inhibitors and then irradiated at 0 and 4 Gy to investigate whether BYL719 and BKM120 can sensitize OSCC cells to ionizing radiation (IR) and reduce IR-induced radioresistance. The combination treatment of BYL719 or BKM120 with 4-Gy IR significantly reduced the growth and colony formation of SCC25 cells compared with the single treatment of 4-Gy IR, BYL719, or BKM120 (Fig 2A). However, only BYL719 combined with 4-Gy treatment significantly reduced the growth of SCC4 cells (Fig 2B). In particular, BYL719 and BKM120 combined with 4-Gy treatment significantly inhibited the growth of OML1 and radioresistant OML1-R cells compared with single 4-Gy IR treatment (Fig 2C and 2D). To determine the effect of different radiation dose treated alone or combination treatments with BYL719 or BKM120 in cells growth, the SCC25 were treated with 2, 4, and 6-Gy IR treated alone or combination treatments with PI3K inhibitors. Results indicated that the treatment does-dependent reduced the growth of HNSCC cells (Fig 3).

Fig 2. Enhanced radiosensitivity upon IR exposure combined with BKM120 or BYL719 treatment in OSCC and radioresistant OML1-R cells.

Fig 2

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SCC4, SCC25, and OML1-R cells were pretreated with BKM120 or BYL719 at 2 μM and then irradiated at 0 or 4 Gy. After 14 days, colonies were stained with crystal violet and quantified by an ELISA reader. Data are presented as mean ± SD, and the asterisks indicate significant difference. *P < 0.01 compared with the control group.

Fig 3. IR exposure combined with BKM120 or BYL719 treatment dose-dependent reduced cell growth in OSCC cells.

Fig 3

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SCC25 cells were pretreated with BKM120 or BYL719 at 2 μM and then irradiated at 0, 2, 4 or 6 Gy. After 14 days, colonies were stained with crystal violet and quantified by an ELISA reader. Data are presented as mean ± SD, and the asterisks indicate significant difference. *P < 0.01 compared with the control group.

Whether or not the PI3K signaling inhibitor can increase the sensitivity of OSCC cells to IR treatment was confirmed as follows. Primary tumor cells were isolated from OSCC patients and then pretreated with BYL719 and BKM120 followed by 4-Gy IR treatment. Consistent with the above results, the combination treatment of PI3K inhibitors with IR showed approximately 18% reduction effect on the OSCC cells compared with the single treatment of IR (Fig 4). Therefore, the combination of PI3K inhibitors and IR exposure significantly enhanced the radiosensitivity of OSCC cells by reducing colony formation.

Fig 4. Radiosensitivity enhancement effect of combination treatment of PI3K inhibitors and IR in patient-derived cells.

Fig 4

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Patient-derived cells were derived from patients with OSCC. The cells were pretreated with BKM120 or BYL719 at 2 μM and then irradiated at 0 or 4 Gy. After 14 days, colonies were stained with crystal violet and quantified by an ELISA reader. Typical images of colony growth for the different treatments are shown. *P < 0.01 compared with the control group.

Combination treatment effect of PI3K/mTOR pathway inhibitors with IR on the growth of OSCC cells

The PI3K/AKT/mTOR signaling pathway regulates cell proliferation. Increased radiosensitization can be observed in cells with inhibited PI3k/AKT/mTOR signaling pathway leading to cell cycle arrest [15]. OML-R cells were pretreated with PI3K/mTOR inhibitors combined with or without IR to evaluate the activity of mTOR inhibitor AZD2014 in the combination treatment with PI3K inhibitor or/and IR. As shown in Fig 5, AZD2014 combined with BKM120 or BYL719 treatment and 4-Gy IR showed an enhanced inhibitory effect on the growth of OML-R cells compared with each PI3K/mTOR inhibitor combined with IR. The inhibition percentages of triple combinations were 67% and 60% compared with the IR treatment. However, the combination treatment of AZD2014 with 4-Gy IR showed no increasing reduction effect compared with IR exposure only on OML-R cell growth.

Fig 5. Combination treatment with PI3K/mTOR inhibitors and IR increased radiosensitivity in OML1-R cells.

Fig 5

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The cells were treated with each PI3K/mTOR inhibitor alone or with the combination treatment of PI3K/mTOR inhibitors and IR. After 14 days, colonies were stained with crystal violet and quantified by an ELISA reader. Data are presented as mean ± SD, and the asterisks indicate significant difference. *P < 0.01 compared with the control group.

Discussion

Radiotherapy and surgery are both common treatment for patients with head and neck cancer, and the outcomes for either may is associated with the facility resources and provider expertise. In surgeries, the cancer tissue in local region and surrounding affected lymph node were removed, and the surgery treatment is commonly combined to adjuvant or neoadjuvant radiotherapy, chemotherapy or combined radio chemotherapy [16]. Patients with HNSCC treated with radiation therapy or chemotherapy is accompanied by severe side effects, including mucositis, dysphagia, leukopenia, and thrombocytopenia, which lead to increasing the risk of infection and bleeding resulting in affecting health-related quality of life [17]. Previous study revealed that combination treatment improved the overall survival of patients with advanced HNSCC via induction chemotherapy with cisplatin and 5 FU before radiotherapy or radio-chemotherapy, which not only increase the antitumor activity but also attenuate the administration concentration of treatment [18]. Present study revealed the combination treatment via PI3K inhibitor treatment before radiotherapy increased the antitumor effect in OSCC and patients-derived cells, which may provide information for the development of inhibitors with radio sensitization activity.

Radioresistance decreases the efficiency of radiotherapy, leading to HNSCC treatment failure. Therefore, overcoming the resistance problem has become a serious issue. In recent years, radiation sensitization was suggested to solve the challenges of raidoresistance through enhancing radiation damage to tumor tissue. A previous study showed that PI3k/AKT/mTOR signaling is an important mechanism associated with increasing radiation sensitivity in various cancer types [19, 20]. Our previous studies demonstrated that the mTOR inhibitors RAD001 and AZD2014 as well as the dual PI3K/mTOR inhibitor BEZ235 significantly induce radiation sensitivity by regulating cell cycle arrest, which may serve as potential small-molecular drugs for patients with radioresistance [10]. In the present study, we used radioresistant oral cancer cells and patient-derived OSCC cells to assess the radiosensitivity of PI3K inhibitors. BKM120 and BYL179 enhanced the radiation sensitivity of OSCC cells.

Dysregulation of the PI3K/AKT/mTOR signaling pathway is a common mechanism to initiate the survival, proliferation, and metabolism of abnormal cells [21]. Therefore, drugs targeting PI3K or mTOR in clinical trials can effectively reduce tumor growth, and the combination treatment with radiation can improve anticancer activity [22]. Our previous study showed that the expression level of PI3K/AKT/mTOR-related proteins is higher in radioresistant OML1-R cells than in parental cell lines, suggesting that PI3K/AKT/mTOR signaling plays a critical role in radioresistance [10]. In the present study, the inhibitory effect of the combination treatment of PI3K inhibitor with radiation on radioresistant OML1-R cells was better than that of each single PI3K treatment.

Dual inhibition of mTORC1/mTORC2 activity enhances radiation-induced inhibition for the survival of OSCC cells. However, AZD2014 does not affect the survival of radiation-treated radioresistant OML1-R cells [23, 24]. Results showed that the triple combination treatment of AZD2014 and BKM120 or AZD2014 and BYL719 with radiation exhibited a significantly enhanced inhibitory effect on the growth of radioresistant OML1-R cells. The PI3K/mTOR signaling mechanism plays an important role in cellular mRNA translation and cell cycle progression. Therefore, further studies should investigate the mechanism underlying the inhibitory effect of the triple combination treatment on radioresistant cells. In conclusion, this study confirmed that PI3K/mTOR signaling is the important target for developing anticancer agents against radioresistant oral cancer cells.

Materials and methods

Ethics statement

All patients’ personal information and medical records were collected, and the study was initiated after a formal approval of the institutional review board of the Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taiwan (approved number, B10302008). All experiments involved human samples obtained from patients, and informed consents were obtained from all subjects.

Cell lines and reagents

SCC4 and SCC25, derived from a squamous cell carcinoma of the tongue, were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM/F12 containing 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, and 2 mM l-glutamine. OML1 and OML1-R (radioresistant) cells were established by Hon-Yi Lin and Michael W.Y. Chan et al. and cultured in RPMI1640 containing 10% FBS, 1% penicillin–streptomycin, and 2 mM l-glutamine. All cells were maintained in 37°C under a humidified atmosphere of 5% CO2 with less than 4 months passage and checked for mycoplasma using kit (Lonza, Switzerland). BKM120 and BLY719 were provided by Novartis Pharmaceuticals Corporation (East Hanover, NJ, USA). AZD2014 was obtained from AstraZeneca (London, United Kingdom).

Isolation and maintenance of primary cells

All patient-derived cells were isolated as previously described. In brief, the primary culture was attained from the tumor tissue of the upper lip of patients with OSCC. The cells were maintained in keratinocyte growth media (ScienCell Research Laboratories, Carlsbad, CA) with 15% FBS.

Clonogenic assay

OSCC cells, radioresistant OML1-R cells, and patient-derived cells were pretreated with BKM120, BYL719, or AZD2014 and then irradiated at 0 or 4 Gy. After 14 days, the cells were stained and fixed with a mixed-well solution of 80% crystal violet and 20% methanol. Colonies (defined as group of >50 cells) were photographed with a digital camera. The cells were washed and added with 30% v/v acetic acid to completely dissolve the crystal violate, and the absorbance was detected at 595 nm by a Synergy 3 multimode microplate reader (BioTek, USA).

Cell viability

The OSCC cell lines and radioresistant OML1-R cells were treated with BKM120 or BYL719 at indicated concentrations for 7 days, and the cell viability was determined through a colorimetric MTS assay in accordance with the manufacturer’s instructions. The reagent-added 96 well plates were re-incubated at 37°C for 2 h, and the absorbance was detected at 480 nm by a Synergy 3 multimode microplate reader (BioTek, USA).

Statistical analysis

Data are presented as mean ± standard deviation from at least three independent experiments. Statistically significant differences between the control group and treatment or IR-treated group and combination-treated group were analyzed using Student’s t-test, and p-values less than 0.01 indicated significant differences. Data were analyzed using SPSS 20.0.

Data Availability

All relevant data are within the manuscript.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Salvatore V Pizzo

14 Aug 2020

PONE-D-20-20798

PI3k inhibitors (BKM120 and BYL719) as radiosensitizers for head and neck squamous cell carcinoma during radiotherapy

PLOS ONE

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Reviewer #1: The authors present an interesting analysis of the use of PI3K inhibitors to sensitize OSCC models to radiation therapy that has some novelty in this field. At this point, the manuscript lacks substantial supporting experiments to warrant publication, and some recommendations to help the authors improve the manuscript are listed below.

1) Inclusion of pharmacodynamic analysis of PI3K effector signaling in the models presented, with the doses used (which are often very high).

2) Inclusion of pharmacodynamic analysis of DNA damage responses (e.g. gH2AX, COMET assays) following radiation, as well as more detailed analysis of mechanisms of cell death.

3) In vivo modeling of the various combinations.

4) Supporting genetic approaches to model changes to PI3K isoforms and their regulation of the DNA damage response.

5) Evaluation of alternative modes of DNA damage to determine if the phenotype is unique to radiation and/or DNA double strand break induction.

Reviewer #2: This study describes the effects of the PI3K� inhibitors Alpelisib (BYL719) and Buparlisib (BKM120) on the radiation sensitivity of head and neck cancer cells. Although these studies are of some interest, they are limited in scope and descriptive. There are no mechanistic studies. In addition, the lack of in vivo studies makes it difficult to know if these agents and the combination with the mTOR inhibitor will translate into an actual treatment in patients.

1) Do the authors know if the concentrations used in the in vitro experiments are clinically relevant? What concentrations are found in patients receiving these drugs?

2) The use of a single radiation dose (Figures 2 and 3) give limited information on overall radiation sensitivity. This is usually best assessed by performing a full cell survival curve of control and 3-4 radiation doses.

3) Did the agents actually inhibit PI3K� under the conditions that were investigated for radiosensitization? This would be the best way to verify that the effects.

4) Why did the authors choose combine these inhibitors with an mTOR inhibitor.

5) The MTT like assay is useful for screening large number of conditions rapidly but is not as accurate as a clonogenic assay for detailed assessment of radiosensitization

6) The clinical applicability of these combinations is difficult to assess with in vitro experiments alone.

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Jan 20;16(1):e0245715. doi: 10.1371/journal.pone.0245715.r002

Author response to Decision Letter 0

2 Oct 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors present an interesting analysis of the use of PI3K inhibitors to sensitize OSCC models to radiation therapy that has some novelty in this field. At this point, the manuscript lacks substantial supporting experiments to warrant publication, and some recommendations to help the authors improve the manuscript are listed below.

1) Inclusion of pharmacodynamic analysis of PI3K effector signaling in the models presented, with the doses used (which are often very high).

Our response: As suggested by reviewer, we have considered the in vitro time-course pharmacodynamic analysis of the PI3K inhibitor, but we decided the assay will be performed in vivo study which may provide more information for the clinical trials. However, we still work on the development of the animal model because of the radiotherapy assay being not easy to perform.

2) Inclusion of pharmacodynamic analysis of DNA damage responses (e.g. gH2AX, COMET assays) following radiation, as well as more detailed analysis of mechanisms of cell death.

Our response: Previous study has demonstrated that BYL719 combined with RT induced the tumor γH2AX nuclear formation in HNSCC animal model (1). BKM120 also showed enhanced DNA damage activity in hepatocellular carcinoma cells (2). The detailed mechanisms we will perform in the HNSCC animal model in the further study.

Reference

1. Mizrachi A, Shamay Y, Shah J, Brook S, Soong J, Rajasekhar VK, et al. Tumour-specific PI3K inhibition via nanoparticle-targeted delivery in head and neck squamous cell carcinoma. Nature communications. 2017;8:14292.

2. Liu WL, Gao M, Tzen KY, Tsai CL, Hsu FM, Cheng AL, et al. Targeting Phosphatidylinositide3-Kinase/Akt pathway by BKM120 for radiosensitization in hepatocellular carcinoma. Oncotarget. 2014;5(11):3662-72.

3) In vivo modeling of the various combinations.

Our response: As suggested by reviewer, we understand the importance of animal model for the assessing the clinical applicability. The animal model commonly used the immunodeficiency animal model to assess the activity of the inhibitors, but the model is under the lack of host immunity-tumor cell interaction that may not truly reflect the activity of inhibitors in the in vivo assay. Furthermore, the application of radiotherapy in animal model is not easy to perform. Therefore, we are searching the best solution for the animal model in the further study.

4) Supporting genetic approaches to model changes to PI3K isoforms and their regulation of the DNA damage response.

Our response: As suggested by reviewer, the expression of γH2AX and PARP, which represent the DNA damage response, by western blotting will be checked in the combination treatment of PI3K inhibitor and radiotherapy in HNSCC animal model in the further study.

5) Evaluation of alternative modes of DNA damage to determine if the phenotype is unique to radiation and/or DNA double strand break induction.

Our response: The alternative modes of DNA damage will be identified by performing NGS assay to comprehensively investigate the mechanism in the combination treatment of PI3K inhibitor and radiotherapy in HNSCC animal model in the further study.

Reviewer #2: This study describes the effects of the PI3K� inhibitors Alpelisib (BYL719) and Buparlisib (BKM120) on the radiation sensitivity of head and neck cancer cells. Although these studies are of some interest, they are limited in scope and descriptive. There are no mechanistic studies. In addition, the lack of in vivo studies makes it difficult to know if these agents and the combination with the mTOR inhibitor will translate into an actual treatment in patients.

1) Do the authors know if the concentrations used in the in vitro experiments are clinically relevant? What concentrations are found in patients receiving these drugs?

Our response: Yes, we know the relevant between the in vitro experiments and clinical usage dose. The concentration of alpelisib used in patients is at a dose of 300 mg per day, and the concentration of buparlisib used in patients is 100 mg per day.

2) The use of a single radiation dose (Figures 2 and 3) give limited information on overall radiation sensitivity. This is usually best assessed by performing a full cell survival curve of control and 3-4 radiation doses.

Our response: The suggestion by reviewers was ideal to support more information. Our unshown results demonstrated that different radiation doses dose-dependent reduced the cell growth when treated alone or combination treatment with PI3K inhibitors. We added the result into the manuscript as figure 3.

3) Did the agents actually inhibit PI3K� under the conditions that were investigated for radiosensitization? This would be the best way to verify that the effects.

Our response: The suggestion by reviewers was ideal to confirm the effect of PI3K inhibitor. The pan-PI3K inhibitor buparlisib (BKM120) and the PI3K inhibitor alpelisib (BYL719) are developed by Novartis Oncology for clinical patients, and the specific of the inhibitors against PI3K is definite under the conditions.

4) Why did the authors choose combine these inhibitors with an mTOR inhibitor.

Our response: The PI3K/AKT/mTOR signaling pathway is a common mechanism to initiate the survival and proliferation. Therefore, the combination treatment of PI3K and mTOR inhibitor may boost the anticancer effect in the OSCC cells.

5) The MTT like assay is useful for screening large number of conditions rapidly but is not as accurate as a clonogenic assay for detailed assessment of radiosensitization

Our response: As suggested by reviewer, the MTT assay in present study was the primary experiments to determine the available concentrations of the inhibitors for the further assay. Using clonogenic assay to investigate the appropriate concentrations is more time consuming.

6) The clinical applicability of these combinations is difficult to assess with in vitro experiments alone.

Our response: As suggested by reviewer, we understand the importance of animal model for the assessing the clinical applicability. The animal model commonly used the immunodeficiency animal model to assess the activity of the inhibitors, but the model is under the lack of host immunity-tumor cell interaction that may not truly reflect the activity of inhibitors in the in vivo assay. Furthermore, the application of radiotherapy in animal model is not easy to perform. Therefore, we are searching the best solution for the animal model in the further study.

Attachment

Submitted filename: response to reviewers_BKM_plos one.docx

Click here for additional data file. (24KB, docx)

Decision Letter 1

Salvatore V Pizzo

16 Nov 2020

PONE-D-20-20798R1

PI3k inhibitors (BKM120 and BYL719) as radiosensitizers for head and neck squamous cell carcinoma during radiotherapy

PLOS ONE

Dear Dr. Su,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

One reviewer is concerned that mechanistic studies are lacking and the study is purely descriptive.  Indeed this reviewer recommended rejection.  Nevertheless, we would consider a revised manuscript but these issues should be addressed. 

Please submit your revised manuscript by Dec 31 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Salvatore V Pizzo

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have responded to some comments but not some critical ones.

Comment 1) - the question was about concentrations and the authors have answered in mg

Comments 5 and 6). this reviewer agrees that performing clonogenic assays and animal experiments is time consuming. But they provide important information concerning the clinical applicability of this combination.

Reviewer #3: This study aimed to identify PI3K inhibitors that can enhance radiosensitivity in head and neck cancer cells. The authors showed that a combination of BKM120 or BYL719 with irradiation leads to an increase in radiosensitivity in HNSCC cells.

After a meticulous review of the manuscript, I found that the current study could be pertinent and informative for the readers this journal.

However, I think that there is some room for improvements before a further consideration for publication. At this point there is a need for minor revision before the manuscript could be reconsidered for publication in the journal.

According to my assessments, there are some points to clarify and revise:

In addition to radiotherapy, surgery is a widely used therapeutic option in patients with HNSCC. This should not remain unmentioned in the discussion, since surgery is superior to radiation therapy in early stages. Furthermore, in advanced tumors, radiotherapy is used in combination with chemotherapy and not as the sole modality. The authors should take a more differentiated view on the therapy of HNSCC and side effects of radiotherapy in the discussion section.

I highly recommend investigating the effects of BKM120 and BYL719 in vivo in the near future.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Jan 20;16(1):e0245715. doi: 10.1371/journal.pone.0245715.r004

Author response to Decision Letter 1

23 Dec 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2:

The authors have responded to some comments but not some

critical ones.

Comment 1) - the question was about concentrations and the authors

have answered in mg

Our response: As suggested by reviewer, the concentrations of the inhibitors in patients is associated with how many mg of the inhibitor that patient took, and the inhibitors concentration in the patient plasma appear dynamic which was showed as pharmacokinetics. For example, the alpelisib used in patients at a dose of 350 mg per day in cycle 1 showed the median maximum plasma concentration (Cmax) is 3490 ng/ml. The buparlisib used in patients at a dose of 100 mg per day in cycle 1 showed the Cmax is 1080 ng/ml.

Comments 5 and 6). this reviewer agrees that performing clonogenic

assays and animal experiments is time consuming. But they provide

important information concerning the clinical applicability of this

combination.

Our response: As suggested by reviewer, we understand and agree that the animal model is important information for clinical applicability of the combination. However, previous studies had indicated that the PI3K/Akt/mTOR pathway inhibitors plus radiotherapy were ideal strategy for lung cancer and breast cancer (1, 2). Furthermore, several PI3K/Akt/mTOR inhibitors including alpelisib and buparlisib has been involved in clinical trials, indicating that the use of the inhibitor is available (3, 4). The combination treatment of PI3K/Akt/mTOR inhibitor and radiotherapy has also been identify that the strategy is clinical applicable(1). On the other hand, the use of the hospital facility was limited because of the novel coronavirus disease, which has influenced the progress of the study. In the near future, we will proceed the in vivo analysis to provide the direct evidence.

Reference:

1. Chen K, Shang Z, Dai AL, Dai PL. Novel PI3K/Akt/mTOR pathway inhibitors plus radiotherapy: Strategy for non-small cell lung cancer with mutant RAS gene. Life sciences. 2020;255:117816.

2. DuRoss AN, Neufeld MJ, Landry MR, Rosch JG, Eaton CT, Sahay G, et al. Micellar Formulation of Talazoparib and Buparlisib for Enhanced DNA Damage in Breast Cancer Chemoradiotherapy. ACS applied materials & interfaces. 2019;11(13):12342-56.

3. Rodon J, Brana I, Siu LL, De Jonge MJ, Homji N, Mills D, et al. Phase I dose-escalation and -expansion study of buparlisib (BKM120), an oral pan-Class I PI3K inhibitor, in patients with advanced solid tumors. Investigational new drugs. 2014;32(4):670-81.

4. Beck JT, Ismail A, Tolomeo C. Targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway: an emerging treatment strategy for squamous cell lung carcinoma. Cancer treatment reviews. 2014;40(8):980-9.

Reviewer #3:

This study aimed to identify PI3K inhibitors that can enhance radiosensitivity in head and neck cancer cells. The authors showed that a combination of BKM120 or BYL719 with irradiation leads to an increase in radiosensitivity in HNSCC cells. After a meticulous review of the manuscript, I found that the current study could be pertinent and informative for the readers this journal. However, I think that there is some room for improvements before a further consideration for publication. At this point there is a need for minor revision before the manuscript could be reconsidered for publication in the journal. According to my assessments, there are some points to clarify and revise: In addition to radiotherapy, surgery is a widely used therapeutic option in patients with HNSCC. This should not remain unmentioned in the discussion, since surgery is superior to radiation therapy in early stages. Furthermore, in advanced tumors, radiotherapy is used in combination with hemotherapy and not as the sole modality. Theauthors should take a more differentiated view on the therapy of HNSCC and side effects of radiotherapy in the discussion section. I highly recommend investigating the effects of BKM120 and BYL719 in vivo in the near future.

Our response: As suggested by reviewer, we added more discussion about the importance of surgery and the side effect when patients treated with radiotherapy or chemotherapy in the discussion section. We will proceed the in vivo analysis soon. Because of the novel coronavirus disease, the use of the hospital facility was limited that also influence the progress of the study.

Attachment

Submitted filename: 20201207_Response to reviewer.docx

Click here for additional data file. (26.1KB, docx)

Decision Letter 2

Salvatore V Pizzo

7 Jan 2021

PI3k inhibitors (BKM120 and BYL719) as radiosensitizers for head and neck squamous cell carcinoma during radiotherapy

PONE-D-20-20798R2

Dear Dr. Su,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Salvatore V Pizzo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Acceptance letter

Salvatore V Pizzo

11 Jan 2021

PONE-D-20-20798R2

PI3k inhibitors (BKM120 and BYL719) as radiosensitizers for head and neck squamous cell carcinoma during radiotherapy

Dear Dr. Su:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Salvatore V Pizzo

Academic Editor

PLOS ONE

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    Click here for additional data file. (24KB, docx)
    Attachment

    Submitted filename: 20201207_Response to reviewer.docx

    Click here for additional data file. (26.1KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript.

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