Conformational change of adenine nucleotide translocase‐1 mediates cisplatin resistance induced by EBV‐LMP1

Abstract Adenine nucleotide translocase‐1 (ANT1) is an ADP/ATP transporter protein located in the inner mitochondrial membrane. ANT1 is involved not only in the processes of ADP/ATP exchange but also in the composition of the mitochondrial membrane permeability transition pore (mPTP); and the function of ANT1 is closely related to its own conformational changes. Notably, various viral proteins can interact directly with ANT1 to influence mitochondrial membrane potential by regulating the opening of mPTP, thereby affecting tumor cell fate. The Epstein–Barr virus (EBV) encodes the key tumorigenic protein, latent membrane protein 1 (LMP1), which plays a pivotal role in promoting therapeutic resistance in related tumors. In our study, we identified a novel mechanism for EBV‐LMP1‐induced alteration of ANT1 conformation in cisplatin resistance in nasopharyngeal carcinoma. Here, we found that EBV‐LMP1 localizes to the inner mitochondrial membrane and inhibits the opening of mPTP by binding to ANT1, thereby favoring tumor cell survival and drug resistance. The ANT1 conformational inhibitor carboxyatractyloside (CATR) in combination with cisplatin improved the chemosensitivity of EBV‐LMP1‐positive cells. This finding confirms that ANT1 is a novel therapeutic target for overcoming cisplatin resistance in the future.

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Lise Roth
Lise Roth, PhD Editor EMBO Molecular Medicine ***** Reviewer's comments ***** Referee #1 (Remarks for Author): The paper by Zhao et al. deals with the role of adenine nucleotide translocase-1 (ANT1), a death-stimulatory isoform of ANT, in the response of nasopharygeal carcinomal cell lines expressing the Epstein Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) to cisplatin-based chemotherapy in vitro. The authors show that LMP1 physically interacts with ANT1, forcing into a confirmation (the m-state) that is incompatible with the opening of the mitochondrial permeability transition pore (mPTP), hence rendering the cell resistant to cisplatin-induced cell death. However, carboxyatractyloside (CATR), which induces a pro-mPTP conformation of ANT1 (the c-state) can reestablish the response of lMP1-expressing cells to cisplatin. This is shown both in vitro and in vivo, in mice bearing xengrafted human cancer cell lines. Altogether, this a convincing story supported by an orthogonal approach, careful experimental design and compelling data. The authors should consider to improve their paper in the following points: Results: • Explain the two sets of NPC cells (CNE1/CNE1-LMP1 and HK1/HK1-LMP1) the first time that you mention them. Define what JC-1 stands for "the mPTP allows free passage of substances less than or equal to 1.5 KD". Replace "substances" by "solutes" Define what means "significantly" in the Results (p value and statistical test) It is not common language to say that mice were inoculated into the "armpit". It is pressing to elucidate the mechanism of drug resistance for new cancer treatment approaches and strategies. In this regard, the authors of this study identified a novel role for EBV-LMP1-induced ANT1 conformation changes in cisplatin resistance of nasopharyngeal carcinoma. They found that EBV-LMP1 localizes to the inner mitochondrial membrane and inhibits the opening of mPTP by binding to ANT1 fixed to m-state, thereby favoring tumor cell survival and drug resistance. On the whole, the major findings of this research are novel and interesting. But the manuscript may need some revisions before publication. 1. The titles of the figures are kind of different from the subheads of the manuscript. The authors should check them carefully to make sure of the consistency between the conclusions and the data provided in the figures, especially the titles of Figure 4 and Figure 6. 2. In Figure 2, the authors proved that ANT1 binds to LMP1 and identified the specific binding regions, but they didn't provide evidence to show that the binding of these two proteins is necessary for the conformation change of ANT1.The authors may need further experiments or an interpretation in this regard. 3. In Figure 3A, the results indicate that BKA, an inhibitor keeping ANT1 in m-state, inhibits the binding of ANT1 and VDAC1, while CATR, an inhibitor stabilizing ANT1 in c-state, promotes the binding. But the authors drew a totally opposite conclusion in the manuscript (line 211-214), which should be corrected. 4. In Figure 3F, this reviewer would expect that the cell viability in BKA treatment groups is comparable with that in LMP1overexprssed cells without BKA treatment because ANT1 is at m-state in these three groups, but it was not the case as shown in the results. Besides, it's better to use CATR to attenuate ANT1 at c-state to prove LMP1 modulate mitochondrial membrane potential and cell viability by keeping ANT1 at m-state. 5. The authors should check through the manuscript carefully to correct typo errors and other mistakes.  Figure 6 showed that the xenograft experiment used up to 5×107 cells, which was far beyond common sense. Please check through the texts to correct these mistakes and make sure that the figure legends and methods contain enough information to allow interpretation and replication of the results.

Dear Prof. Cao,
We have now received the report from reviewer #3, that you will find attached to this email. As mentioned in my decision letter, we would like you to address the issues raised by this referee, but do not ask for further reaching experiments. Specifically, we would like you to address the points 1, 3, 6, 7, 8, and 9 (CDDP) in writing. Please also improve the quality of the figures and their description throughout the manuscript, as mentioned in point 4. Regarding point 2, we would be happy for you to include additional experiments, or at least discuss the limitations of the method as mentioned by the referee. Please address experimentally points 5 and 9 (IP).
Do not hesitate to contact me should you have any question.
Looking forward to receiving your revised manuscript, Sincerely,

Lise Roth
Lin Zhao and colleagues aimed to identify mechanism by which a latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus (EBV), induces alteration of ANT1 conformation in cisplatin (CDDP)-resistant nasopharyngeal carcinoma cells (NPC). The authors described that EBV-LMP1 inhibits the mPTP opening by binding to ANT1, leading to the increased tumour cell survival and drug resistance. ANT1 inhibitor CATR and especially its combination with CDDP improved a chemosensitivity of EBV-LMP1-positive cells.
In my opinion several obtained results not necessarily support the made conclusion, the interpretation of several experiments is not convincing and important controls are lacking. The manuscript itself reminds me of the first draft. Major comments 1. The introduction describes the current research field very narrowly and subjectively. The fact that the ANT1 is a part of mPTP is highly controversial. Zhao et al. didn´t mention the existing hot debates and other hypotheses, excluding ANT1. ANT1 was shown to be not only ATP/ADP transporter, but obviously also participates in the proton transport in the presence of fatty acids. As recently shown, the proton transport is also affected by CATR and BA. 2. Several very important conclusions are based on the measurements of mitochondrial membrane potential (MMP) using fluorescent dyes TMRM and JC1. However, only measurements which investigate the immediate MMP changes after direct addition of substances potential are easy to interpret. The comparison of two different cell cultures (e.g. wt and KO or cells before and after 24 h incubation ( Fig. 5 B/C)) is very challenging because of the different dye/cell concentration, change in mitochondrial surface/volume ratio and loading time. 3. It is not clear how the authors calculated the fluorescent intensity ratio F/F0 (e.g. Fig. 1, A)? How they have taken in account a different amount of cells in each snapshot? Please explain, how you perform statistics in the experiment shown in Fig. 1, D? My concern is that the fluorescence will depend on the cell amount. 4. The scientific quality of the figures and their descriptions is below the usual requirements for a bachelor thesis. Sometimes I get the impression that the authors do not know exactly what they are measuring. This applies to all Figures. I will give only some examples below. Fig. 1, B: y-axis is entitled scientifically incorrect! Fig. 1, C: x-axis is entitled scientifically incorrect! Line 141 I don´t see how the conclusion "suggesting that EBV-LMP1 ..... increases the mitochondrial membrane potential" can be made from Fig. 1C. The experiment shown in Fig. 1D should be explain better. The use of ionomycin is controversial (PMID: 28655872), the authors should take better A23187, to induce a decrease in calcein fluorescence. Suppl. Fig. 1 Actin is not appropriately shown in Western Blots and cannot be used as a control. Please explain what we see in the Western Blot (?) at 35 kD. There is no appropriate description of the Fig. 3D. Suppl. Fig. 2C. The line description: "19F-NMR assay of ANT1-WT" doesn´t help to understand what was measured. 5. In several experiments the antibodies (AB) against ANT1 and ANT2 are used. Due to relatively high homology between mitochondrial transporters AB against ANT1 are known to be highly unspecific and cannot be used in immunohistochemical staining. Authors should perform (i) full size WB for ANT1 expression in HK1 cell to validate the use of the antibody for staining and (ii) to show the specificity of this AB using ANT1 KO cells/tissues as negative control. Also appropriate positive control (e.g. recombinant ANT1) is required. 6. Which absolute values for ATP/ADP exchange were measured in the experiments shown in Fig. 4b? How this exchange was measured? 7. Please explain how the Seahorse experiment justifies the conclusion "the process is closely related to ANT1 conformation-based functional restrictions." (Fig. 4, C). 8. What is the hypothesis for the usage of two sets of NPC cells? 9. A representative IP for LPM1 (Fig. 3 B) is not convincing. Authors should show at least two other experiments. 9. CDDP (cis-diamminedichloroplatinum? this abbreviation is not explained in the text) and cisplatin are obviously the same substances. However, authors describe their experiment like this (Line 291): "... cells were divided into a control group, CDDP-treatment only group, cisplatin-treatment only group, and a combination of CDDP and cisplatin-treated group, respectively." This description should be corrected.    Figure 3A in the original figure is Figure 3B in the revised manuscript, and Figure 3B in the original figure is Figure 3D in the revised manuscript. • It should be noted that the results in vivo (on tumor growth) appear additve, not synergistic. This should be mentioned as a caveat in the discussion. (2)Minor: Avoid unexplained abbreviations in the abstract (CATR). Define what JC-1 stands for "the mPTP allows free passage of substances less than or equal to 1.5 KD". Replace "substances" by "solutes" Author Response: Under normal physiological conditions, the mPTP allows free passage of solutes armpit less than or equal to 1.5 KD (line 133).
Define what means "significantly" in the Results (p value and statistical test) Author Response: The statistical significance of the data was analyzed by using a standard Student's t-test. A p-value of < 0.05 was deemed statistically significant and p< 0.01 was considered statistically significant (line 644).
It is not common language to say that mice were inoculated into the "armpit".
Author Response: The revised manuscript has been modified to inoculate subcutaneously into mice (line 630-631).

Reviewer #2:
It is pressing to elucidate the mechanism of drug resistance for new cancer treatment approaches and strategies. In this regard, the authors of this study identified a novel  Figure 2, the authors proved that ANT1 binds to LMP1 and identified the specific binding regions, but they didn't provide evidence to show that the binding of these two proteins is necessary for the conformation change of ANT1.The authors may need further experiments or an interpretation in this regard. 4. In Figure 3F, this reviewer would expect that the cell viability in BKA treatment groups is comparable with that in LMP1-overexprssed cells without BKA treatment because ANT1 is at m-state in these three groups, but it was not the case as shown in the results. Besides, it's better to use CATR to attenuate ANT1 at c-state to prove LMP1 modulate mitochondrial membrane potential and cell viability by keeping ANT1 at m-state. 2) The antibodies used for IP in Fig 2F and  5) The results and methods of Figure 6 showed that the xenograft experiment used up to 5×10 7 cells, which was far beyond common sense. Please check through the texts to correct these mistakes and make sure that the figure legends and methods contain enough information to allow interpretation and replication of the results.

Reviewer #3:
Lin Zhao and colleagues aimed to identify mechanism by which a latent membrane 3. It is not clear how the authors calculated the fluorescent intensity ratio F/F0 (e.g. Fig. 1, A)? How they have taken in account a different amount of cells in each snapshot? Please explain, how you perform statistics in the experiment shown in Fig.   1, D? My concern is that the fluorescence will depend on the cell amount.  Line 141 I don´t see how the conclusion "suggesting that EBV-LMP1 ..... increases the mitochondrial membrane potential" can be made from Fig. 1C.
Author Response: Mitochondria underwent swelling mainly caused by mPTP opening. The swelling rate of HK1-LMP1 (6%) shown in Figure 1C was significantly lower than that of HK1 (23%), and similar conclusions were obtained in CNE1 (27%) /CNE1-LMP1 (7%). Combining the results in Figure 1A/B, we confirmed that LMP1 can increase the mitochondrial membrane potential. Supplementary Table 1: 7. Please explain how the Seahorse experiment justifies the conclusion "the process is closely related to ANT1 conformation-based functional restrictions." (Fig. 4, C).
Author Response: ANT1 enables mitochondrial ADP/ATP exchange through uninterrupted switching of its c-and m-state conformations. BKA and CATR are conformational inhibitors of ANT1 (BKA puts ANT1 in m-stae; CATR puts ANT1 in c-stae). In Figure 4C/D the maximal respiration of BKA and CATR-treated HK1 cells was significantly lower than that with the control group, with similar findings in other 9. A representative IP for LPM1 (Fig. 3 B) is not convincing. Authors should show at least two other experiments.
Author Response: First, we confirmed by IP that LMP1 functions similarly to BKA to maintain ANT1 in the m-state (Fig. 3 A/C), and next we directly confirmed by 19-NMR that LMP1 induces conformational changes in ANT1 and that the binding of LMP1 to ANT1 is necessary for its occurrence (Fig. 3 G/H). Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have received the enclosed report from referees #1 and #2, and as you will see, they are now supportive of publication. I am therefore pleased to inform you that we will be able to accept your manuscript once the following minor editorial points will be addressed: 1/ Please address the minor comment from referee #1.
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20th Sep 2021 2nd Authors' Response to Reviewers
The authors performed the requested editorial changes. We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.
Please note that I have replaced the TPE in the manuscript as discussed previously:

Problem
The Epstein-Barr virus (EBV) encodes the key oncogenic protein LMP1, which plays an important role in promoting resistance to therapy. In this study, we explored novel targets and potential mechanisms by which EBV-LMP1 regulates resistance to cisplatin in nasopharyngeal carcinoma (NPC).

Results
We found that EBV-LMP1 can localize to the mitochondria to bind directly to adenine nucleotide translocase-1 (ANT1), fixing the ANT1 conformation in the m-state, thereby increasing the mitochondrial membrane potential and promoting the viability of NPC cells. Carboxyatractyloside (CATR), a conformational inhibitor of ANT1, which contributes to mPTP opening and cell death, enhanced the sensitivity of tumor cells to cisplatin. Impact Our study links for the first time ANT1 conformational changes to cisplatin chemosensitivity, highlighting the importance of protein conformational changes in tumor chemotherapy.
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We numbered the mice sequentially and grouped them using a randomized method.

Manuscript Number: EMM -2021-14072
Appropriate statistical tests are used according to the experimental data and purpose.The experimental data are presented as the mean values ± S.E.M. The statistical significance of the data was analyzed by using a standard student's t-test. A p-value of < 0.05 was deemed statistically significant and p< 0.01 was considered statistically significant.

NA
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A double-blind method was used in this study.
A double-blind method was used in this study.

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