mTOR pathway inhibition stimulates pharmacologically induced nonsense suppression

  1. Amnon Wittenstein
  2. Michal Caspi
  3. Ido Rippin
  4. Orna Elroy-Stein
  5. Hagit Eldar-Finkelmn
  6. Sven Thoms
  7. Rina Rosin-Arbesfeld

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Abstract

A large number of human genetic diseases result from premature termination codons (PTCs) caused by splicing defects, insertions, deletions or point mutations also termed nonsense mutations. Nonsense mutations are the source of various genetic diseases, ranging from rare neuro-metabolic disorders to relatively common inheritable cancer syndromes and muscular dystrophies. Over the years, a wide spectrum of studies has shown that certain antibiotics and other synthetic molecules can act as nonsense mutation suppressors, by inducing readthrough of the stop-codon leading to the expression of a full-length protein. Unfortunately, most readthrough-inducing agents have limited effects and are toxic. Thus, efforts are made to improve the clinical outcome of nonsense mutation suppressors.

Here we show that the mTOR pathway is involved in antibiotic-mediated readthrough of nonsense mutations at the level of protein translation initiation. We demonstrate that inhibition of the mTOR translation-initiation-controlling eIF4E branch induces antibiotic-mediated nonsense mutation readthrough, paving the way to the development of a novel therapeutic strategy for enhancing the restoration of these disease-causing mutated transcripts.

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    Reply to the reviewers

    Reviewer #1:

    We thank the Reviewer for stating that “Overall the article is well structured, the experiments are clearly and logically described. The data is convincing and there does not seem to be a sticking point”, and also for pointing to the fact that “This manuscript will therefore be of interest to people working in the field of readthrough, therapeutic approaches and genetic diseases, but also more generally to people studying gene translation and expression”.

    Specific comments:

    In chapter "Serum starvation increased APC nonsense-mutation readthrough in CRC cell lines", last line please replace "sratvation" by "starvation".

    The mistake has been corrected.

    In chapter "Torin-1 increases antibiotic-mediated nonsense codon readthrough" , 6 lines before the end please replace "Totin-1" by "Torin-1"

    The mistake has now been corrected.

    The following sentence in the discussion has to be rewritten because NMD degrades RNA and not proteins: "In many cases, the cancer cells express a truncated APC protein that is not degraded by the NMD as most of the nonsense mutations occur in a hotspot within the last APC exon, thus they are not recognized by the exon junction complex method of NMD [55] ".

    The sentence has been corrected and rephrased to say: “Mutated APC transcripts are often NMD-resistant as most of the nonsense mutations occur in a hotspot within the last APC exon and therefore not recognized by the exon junction complex that induces NMD”.

    Change "combitation" into "combination" 7 lines from the end of the discussion.

    The mistake has been corrected.

    Figure 5, the authors analyze the effect of an inhibition of the activity of eIF4E using the small molecule 4EGl-1. They are testing for an endogenous nonsense mutation in the APC gene in COLO320 cells. To be consistent with Figure 4, the authors should also show the same effect on SW403 cells.

    The requested missing experiment has been added to Figure 5 (Fig.5D) and the results are discussed.

    Reviewer #2:

    We thank the Reviewer for acknowledging the “nice flow of the paper” and that “The involvement of mTOR pathway in PTC RT is interesting”. We have addressed the Reviewer’s comments and added the requested experiments as follows:

    Major comments -

    1- My major concern is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.

    We thank the Reviewer for raising this point. In the revised manuscript the vast majority of experiments were conducted with a lower dose of antibiotics (500ug/ml). We have used the G418 at 1.5 mg/ml only when comparing it to our previous results showing the effects of serum starvation on readthrough, where this high concentration was used [1] (Fig. 1 & Fig. S1) and when using immunofluorescent experiments on colo320 and SW403 cells (Fig. 4D). In all other experiments 500ug/ml G818 was used. We have now tested cell viability under the different treatments, using the 500ug/ml dose (Fig. S3) and demonstrate that cell survival is between 60%-100% under the different conditions. This point has now been emphasized in the revised manuscript (results section - Torin-1 increases antibiotic-mediated nonsense codon readthrough).

    2- Control cell lines (a CRC cell line without APC mutation to show WT levels of APC, and a CRC cell line with APC mutations other than PTC as negative control) must be included to the experiments. It is much better to report the level of PTC readthrough relative to WT rather than untreated mutant cells. Regarding the low level of PTC RT enhancement in combination treatment it is good to know whether these levels have any biological significance when compared to normal APC levels.

    We have now added the requested missing control cells to the manuscript (Fig. 1C): HCT116

    which harbor an b-catenin mutation (and wt APC) and SW48 expressing an APC gene with a missense mutation. In these cell lines, APC is mostly unaffected by the enhancing readthrough treatment. Please note that the endogenous expression levels of APC in these cells are higher than those achieved by restoring APC levels in Colo320 cells. Importantly, although the induced APC restoration is relatively minor, the effect on reducing active b-catenin levels is significant. The levels of induced readthrough depend on different factors such as the type of the stop codon, the surrounding sequence and the gene itself [2, 3]. As the Reviewer stated, it is important to determine what is the minimal levels of full-length protein induced by the readthrough treatment that has therapeutic effects. It has been shown that in each protein and disease, this level is different. For example, in lysosomal storage disease, even 1 % of normal protein function may restore a near-normal or clinically less severe phenotype [4, 5]. For cystic fibrosis 10 to 35 % of CFTR activity might be needed to significantly alleviate pulmonary morbidity [7] and in DMD – 1-30% of the full-length dystrophin is needed [6]. Similarly, our results indicate that even if we can restore only relatively low amounts of the APC protein [1], these_ levels may _have beneficial therapeutic effects [8]. This important point has now been added to the introduction of the revised manuscript.

    3- In the introduction section the authors mentioned that "there is increasing evidence that APC truncations may exert dominant functions contributing to colon tumorigenesis. These include enhancement of cell migration, interference with spindle formation, and induction of chromosome instability [35-38]." Usually in the course of PTC readthrough the truncated protein is also increased (Baradaran-Heravi et al, 2016, Nucleic Acids Research). In this study, in addition to full length APC authors need to show the truncated form in the CRC cell lines and find out whether this form also increases during mTOR inhibition and G418 treatment. Since the dominant function of APC truncation contributes to colon tumorigenesis, would increase in truncated protein during PTC readthrough be considered as an adverse side effect?

    We have now conducted the missing experiment. In revised Figs. 1B and S1 we show that the increase in full length APC following nonsense mutation-induced readthrough is not observed in the truncated APC protein product. Truncated APC is known to be NMD-resistant [9] and thus accumulates in cancers that originate from APC-premature termination codons. p53, on the other hand, is highly affected by NMD (as discussed in Baradaran-Heravi et al, 2016, Nucleic Acids Research) and thus nonsense mutation readthrough, which leads to prolonged ribosomal protection of the p53 transcripts, could affect the low levels of the truncated p53 protein product.

    4- I am wondering how the authors reconcile diminished translation initiation and increased PTC readthrough? What is the author's proposed model?

    We agree that this is a very important point. Our results show that 4EG1-1 that affects translation initiation, enhanced-PTC readthrough only in the presence of aminoglycosides (Fig. 5). Aminoglycosides exert their PTC readthrough activity by binding at the decoding center of the eukaryotic ribosome and reducing the ability of translation termination factors to accurately recognize the PTC [10, 11]. Similarly to our results, It has been shown that other compounds such as the small molecules CDX5-1 [12] or the drug mefloquine [13], that do not show readthrough activity when used as single agents, potentiate the readthrough activity of aminoglycoside possibly by directly targeting the translation machinery although the exact mechanism is still unclear and should be further studied. Another interesting possibility is that the effect of 4EGI-1 on PTC readthrough arises from its inhibitory effect on mTORC1 signaling which may be independent of its role in cap-dependent translation initiation [14]. This important point has now been discussed in the revised manuscript (discussion paragraph) and, although beyond the scope of the current report, we are currently conducting additional experiments to understand the exact mechanism of enhancing the activity of aminoglycosides on nonsense mutation readthrough.

    5- In figure 2C, can authors induce Gentamicin related PTC RT in TSC-/- cells by treating them with Torin-1 or Rapamycin or 4EGI-1? Please show the results.

    The requested missing data has been added to the Figure (Fig. 2) and corresponding text.

    6- Please show the APC mRNA levels in CRC cell lines and discuss its changes in different treatment combinations.

    We have now measured the APC mRNA levels under the different treated combinations and have added the results to the revised manuscript (Fig. S5). These results have been discussed (in the result section -_ Rapamycin increases antibiotic-mediated nonsense codon readthrough) _as follows: "Interestingly, although mutated APC transcripts are relatively stable, a slight increase in mRNA levels was observed in treated Colo320 cells as opposed to SW403 where mRNA transcripts were unaffected by readthrough or readthrough enhancement".

    7- It would be nice to see the effect of combination treatment on PTC RT response in other CRC cell lines they discussed in Figure 2A.

    We have now added two CRC cell lines, chosen from the group of cells where serum starvation enhances readthrough. These cells respond to the combination treatment on PTC readthrough (SW837 and SW620; Fig. S4).

    Minor comments-

    1- It would be nice to explain in more detail the GFP-BFP cell line when the authors mention it for the first time.

    A detailed explanation on the_ GFP-BFP _reporter plasmin has been added to the revised manuscript (in the results section, under the paragraph - The mTOR pathway may regulate antibiotic-induced nonsense mutation readthrough).

    2- In figure 2A, how many proteins did they end up analyzing? Please mention the number.–

    We tested 214 proteins where we had the data for all 8 cell lines examined. Out of these proteins, 8 were statistically significantly different. Out of these proteins, 4EPB-1 and its three phosphorylated forms had the most statistical significance. This information has now been added to the text.

    3- Authors mentioned that "As can be seen, Totin-1 induced APC restoration in both cell lines, though the re-expression of full-length APC was more complete in COLO320 cells". What do they mean by "complete" when they do not have WT levels of APC to compare with? Do they mean "more efficient" ?

    We apologize for the confusing terminology. We compared the readthrough activity to the null condition and not the wild-type expression. The sentence has been completely rephrased in the discussion paragraph.

    4- Please provide the full image of APC western blots to better visualize to full length and truncated forms in one blot.

    Figures 1C and S1 now show both full-length-APC and truncated APC in untreated and treated cells. Technically, due to the differences in protein sizes (90-160kDa for the truncated APC protein product in the different cell lines and full-length APC-312kDa) and the poor quality of the available antibodies, both APC forms cannot be detected on the same blot and were thus analyzed on separate gels.

    5- In figure 5, please add 4EGI-1 treatment (alone) lane for both panels. Also, please add quantification of active b-catenin for panel B.

    The experiments have been repeated and this missing data has been added to the figure and corresponding text.

    6- In the discussion it is said "As all the CRC cells that responded to mTOR inhibition
    by increased PTC readthrough show high levels of 4E-PB1 we conclude that inhibiting
    cap-dependent protein translation initiation enhances antibiotic mediated PTC
    readthrough". This statement is not accurate. The authors have tested only one cell line, COLO320, which has high 4E-PB1 expression and responds to mTOR inhibition in terms of increased PTC RT.

    This statement has been changed and corrected to state that: "As CRC cells that responded to mTOR inhibition by increased PTC readthrough show high levels of 4EPB-1 (Figs. 3-4 and data not shown)"

    ****Referees cross-commenting****

    I appreciate reviewer #1 and #3 comments and I also agree about the nice flow of the paper. We routinely study G418 effect on PTC readthrough in many different cell lines. My major reservation is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.

    Thank you for this comment. As described above in response to Reviewer #2 comments, the majority of our experiments were now conducted with a lower dose of antibiotics (500ug/ml). Although Reviewer #3 mentioned that “some cell types can tolerate those doses”, we have now tested the survival of the various treatments, using the 500ug/ml dose (Fig. S3) and demonstrated that cell death did not exceed 40% under any condition. Only viable cells were used in our experiments.

    The involvement of mTOR pathway in PTC RT is interesting; however, I am not sure about the biological value of this finding as mTOR inhibition marginally enhances aminoglycoside induced PTC RT (2-2.5-fold in COLO320 cells). Also, the number of cell lines tested in this manuscript is limited to only two CRC cell lines which makes the interpretation of the results more difficult.

    To address this important point additional CRC cell lines have now been used throughout the manuscript. As different studies show that increasing nonsense mutation readthrough levels and inducing some restoration of the full-length protein, even by small amounts could have beneficial value (please see our response to Reviewer # 2, point 2) we suggest that enhancing nonsense mutation readthrough by inhibiting the mTOR pathway may have therapeutic value. We have now emphasized in the manuscript that the different strategies for inducing readthrough (including ours) do not achieve wild-type levels and that this point needs to be considered when evaluating the therapeutic potential of this treatment strategy.

    Reviewer #3 :

    We thank the Reviewer for stating that “ This is an important finding”. We have addressed the specific Reviewer’s comments as follows:

    1. In the first paragraph of the Results Section, you use serum starvation to enhance readthrough. However, I could not find how long you maintained serum starvation, whether it was added before or concurrently to aminoglycoside addition, etc. Please clarify this point.

    We apologize for omitting this point. The treatment conditions of serum starvation have now been added to the results section and to the legend (cells were incubated for 24 h in a medium containing 10% or 1% serum supplemented with 1.5mg/ml G418).

    1. Fig. S1: I can't read the x-axis labels. Please fix this.

    The figure has been corrected (currently Fig. S2).

    1. First paragraph in the torin-1 section: you don't refer the reader to Fig 3B and 3C. I suggest that you revise the text as follows: "Next, the effect of mTOR inhibition on antibiotic-mediated endogenous APC readthrough in the CRC cell lines COLO320 (Fig. 3B) and SW403 (Fig. 3C) was examined where aminoglycosides induced relatively high levels of APC restoration”.

    The text has been revised and corrected.

    1. In figs. 3, 4 and 5, you label the panels using the cell lines COLO320 (panel B) and SW403 (panel C), but not for the APC R1450X line (panel A). The reason for this omission is not clear, but it would help the reader follow your work if you added it.

    The missing panels have now been labeled correctly.

    1. You don't mention Fig. S3 in the text of the manuscript. Please add a sentence to the last paragraph of the Results since it is important to note that 4EGI-1 does not induce readthrough alone.

    The Figure has now been mentioned and the finding that 4EGI-1 does not induce readthrough alone is now shown in Fig. 5 (please see our response to Reviewer #2, point 5, minor points section).

    ****Referees cross-commenting****

    I agree that 1.5 mM G418 sounds high, but some cell types can tolerate those doses. Controls to examine toxicity seem appropriate and won't take too long. In addition, one panel showing that the mTOR inhibition also stimulate readthrough at a lower G418 dose would help to allay this concern.

    Please see our response to this point above (Reviewer #2, point 1). In the current manuscript all experiments except Fig. 1 & Fig.S1 were conducted with 500ug/ml G418.

    References

    [1] A. Wittenstein, M. Caspi, Y. David, Y. Shorer, P.T. Nadar-Ponniah, R. Rosin-Arbesfeld, Serum starvation enhances nonsense mutation readthrough, J Mol Med (Berl), 97 (2019) 1695-1710.

    [2] C. Floquet, I. Hatin, J.P. Rousset, L. Bidou, Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin, PLoS Genet, 8 (2012) e1002608.

    [3] L. Martorell, V. Cortina, R. Parra, J. Barquinero, F. Vidal, Variable readthrough responsiveness of nonsense mutations in hemophilia A, Haematologica, 105 (2020) 508-518.

    [4] I. Maire, Is genotype determination useful in predicting the clinical phenotype in lysosomal storage diseases?, J Inherit Metab Dis, 24 Suppl 2 (2001) 57-61; discussion 45-56.

    [5] I. Nudelman, D. Glikin, B. Smolkin, M. Hainrichson, V. Belakhov, T. Baasov, Repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations, Bioorg Med Chem, 18 (2010) 3735-3746.

    [6] M. Dabrowski, Z. Bukowy-Bieryllo, E. Zietkiewicz, Advances in therapeutic use of a drug-stimulated translational readthrough of premature termination codons, Mol Med, 24 (2018) 25.

    [7] E. Kerem, Pharmacologic therapy for stop mutations: how much CFTR activity is enough?, Curr Opin Pulm Med, 10 (2004) 547-552.

    [8] R. Kariv, M. Caspi, N. Fliss-Isakov, Y. Shorer, Y. Shor, G. Rosner, E. Brazowski, G. Beer, S. Cohen, R. Rosin-Arbesfeld, Resorting the function of the colorectal cancer gatekeeper adenomatous polyposis coli, Int J Cancer, 146 (2020) 1064-1074.

    [9] R.G. Lindeboom, F. Supek, B. Lehner, The rules and impact of nonsense-mediated mRNA decay in human cancers, Nat Genet, 48 (2016) 1112-1118.

    [10] B. Francois, R.J. Russell, J.B. Murray, F. Aboul-ela, B. Masquida, Q. Vicens, E. Westhof, Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding, Nucleic Acids Res, 33 (2005) 5677-5690.

    [11] N. Garreau de Loubresse, I. Prokhorova, W. Holtkamp, M.V. Rodnina, G. Yusupova, M. Yusupov, Structural basis for the inhibition of the eukaryotic ribosome, Nature, 513 (2014) 517-522.

    [12] A. Baradaran-Heravi, A.D. Balgi, C. Zimmerman, K. Choi, F.S. Shidmoossavee, J.S. Tan, C. Bergeaud, A. Krause, S. Flibotte, Y. Shimizu, H.J. Anderson, V. Mouly, E. Jan, T. Pfeifer, J.B. Jaquith, M. Roberge, Novel small molecules potentiate premature termination codon readthrough by aminoglycosides, Nucleic Acids Res, 44 (2016) 6583-6598.

    [13] M.W. Ferguson, C.A.N. Gerak, C.C.T. Chow, E.J. Rastelli, K.E. Elmore, F. Stahl, S. Hosseini-Farahabadi, A. Baradaran-Heravi, D.M. Coltart, M. Roberge, The antimalarial drug mefloquine enhances TP53 premature termination codon readthrough by aminoglycoside G418, PLoS One, 14 (2019) e0216423.

    [14] H. Wang, F. Huang, J. Wang, P. Wang, W. Lv, L. Hong, S. Li, J. Zhou, The synergistic inhibition of breast cancer proliferation by combined treatment with 4EGI-1 and MK2206, Cell Cycle, 14 (2015) 232-242.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study, the authors show that inhibition of the translation initiation-controlled by the cap-dependent (eIF4E) branch of the mTOR pathway enhances antibiotic-mediated nonsense mutation readthrough mediated by aminoglycosides. Interestingly, inhibition of this pathway in the absence of mTOR inhibitors has no effect on readthrough. These studies suggest that inhibition of this pathway may be used to enhance readthrough of disease-causing mutations.

    I suggest that the authors consider the following points to improve the manuscript:

    1. In the first paragraph of the Results Section, you use serum starvation to enhance readthrough. However, I could not find how long you maintained serum starvation, whether it was added before or concurrently to aminoglycoside addition, etc. Please clarify this point.
    2. Fig. S1: I can't read the x-axis labels. Please fix this.
    3. First paragraph in the torin-1 section: you don't refer the reader to Fig 3B and 3C. I suggest that you revise the text as follows: "Next, the effect of mTOR inhibition on antibiotic-mediated endogenous APC readthrough in the CRC cell lines COLO320 (Fig. 3B) and SW403 (Fig. 3C) was examined where aminoglycosides induced relatively high levels of APC restoration. Next, the effect of mTOR inhibition on antibiotic-mediated endogenous APC readthrough in the CRC cell lines COLO320 and SW403 was examined where aminoglycosides induced relatively high levels of APC restoration."
    4. In figs. 3, 4 and 5, you label the panels using the cell lines COLO320 (panel B) and SW403 (panel C), but not for the APC R1450X line (panel A). The reason for this omission is not clear, but it would help the reader follow your work if you added it.
    5. You don't mention Fig. S3 in the text of the manuscript. Please add a sentence to the last paragraph of the Results since it is important to note that 4EGI-1 does not induce readthrough alone.

    Referees cross-commenting

    I agree that 1.5 mM G418 sounds high, but some cell types can tolerate those doses. Controls to examine toxicity seem appropriate and won't take too long. In addition, one panel showing that the mTOR inhibition also stimulate readthrough at a lower G418 dose would help to allay this concern.

    Significance

    Overall, this manuscript demonstrates that inhibition of mTOR-dependent translation initiation by various means (serum starvation, the mTOR inhibitors torin-1 or rapamycin, or 4EGI-1) all stimulate nonsense suppression. This is an important finding.

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    Referee #2

    Evidence, reproducibility and clarity

    This study evaluates the involvement of mTOR pathway in premature termination codon (PTC) readthrough (RT) using cell-based assays. Initially the authors claim that similar to their previous finding serum starvation enhances aminoglycoside induced PTC RT in several cancer cell lines with APC nonsense mutations. They found association between enhanced PTC RT in serum starved cells and increased expression level of 4E-BP using DepMap data and speculated about the role of mTOR in PTC RT. Furthermore, they claim that Torin-1 or Rapamycin treatment of a stable cell line expressing an exogenous PTC construct as well as two colorectal cancer cell lines with APC nonsense mutations increased aminoglycoside induced PTC RT and suppressed active beta-catenin in CRC cells. Finally, they observed enhancement of aminoglycoside induced PTC RT by chemically inhibition of translation initiation factor eIF4E.

    Major comments:

    1. My major concern is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.
    2. Control cell lines (a CRC cell line without APC mutation to show WT levels of APC, and a CRC cell line with APC mutations other than PTC as negative control) must be included to the experiments. It is much better to report the level of PTC readthrough relative to WT rather than untreated mutant cells. Regarding the low level of PTC RT enhancement in combination treatment it is good to know whether these levels have any biological significance when compared to normal APC levels.
    3. In the introduction section the authors mentioned that "there is increasing evidence that APC truncations may exert dominant functions contributing to colon tumorigenesis. These include enhancement of cell migration, interference with spindle formation, and induction of chromosome instability [35-38]."

    Usually in the course of PTC readthrough the truncated protein is also increased (Baradaran-Heravi et al, 2016, Nucleic Acids Research). In this study, in addition to full length APC authors need to show the truncated form in the CRC cell lines and find out whether this form also increases during mTOR inhibition and G418 treatment. Since the dominant function of APC truncation contributes to colon tumorigenesis, would increase in truncated protein during PTC readthrough be considered as an adverse side effect?

    1. I am wondering how the authors reconcile diminished translation initiation and increased PTC readthrough? What is the author's proposed model?
    2. In figure 2C, can authors induce Gentamicin related PTC RT in TSC-/- cells by treating them with Torin-1 or Rapamycin or 4EGI-1? Please show the results.
    3. Please show the APC mRNA levels in CRC cell lines and discuss its changes in different treatment combinations.
    4. It would be nice to see the effect of combination treatment on PTC RT response in other CRC cell lines they discussed in Figure 2A.

    Minor comments:

    1. It would be nice to explain in more detail the GFP-BFP cell line when the authors mention it for the first time.
    2. In figure 2A, how many proteins did they end up analyzing? Please mention the number.
    3. Authors mentioned that "As can be seen, Totin-1 induced APC restoration in both cell lines, though the re-expression of full-length APC was more complete in COLO320 cells". What do they mean by "complete" when they do not have WT levels of APC to compare with? Do they mean "more efficient" ?
    4. Please provide the full image of APC western blots to better visualize to full length and truncated forms in one blot.
    5. In figure 5, please add 4EGI-1 treatment (alone) lane for both panels. Also, please add quantification of active beta-catenin for panel B.
    6. In the discussion it is said "As all the CRC cells that responded to mTOR inhibition
      by increased PTC readthrough show high levels of 4E-PB1 we conclude that inhibiting
      cap-dependent protein translation initiation enhances antibiotic mediated PTC
      readthrough". This statement is not accurate. The authors have tested only one cell line, COLO320, which has high 4E-PB1 expression and responds to mTOR inhibition in terms of increased PTC RT.

    Referees cross-commenting

    I appreciate reviewer #1 and #3 comments and I also agree about the nice flow of the paper. We routinely study G418 effect on PTC readthrough in many different cell lines. My major reservation is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.

    Significance

    The involvement of mTOR pathway in PTC RT is interesting; however, I am not sure about the biological value of this finding as mTOR inhibition marginally enhances aminoglycoside induced PTC RT (2-2.5 fold in COLO320 cells). Also, the number of cell lines tested in this manuscript is limited to only two CRC cell lines which makes the interpretation of the results more difficult.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Wittenstein et al. aims to demonstrate that an optimization of the efficiency of readthrough induced by aminoglycosides can be obtained by inhibiting the mTOR pathway. The results presented in the manuscript show that serum starvation, inhibition of the mTOR pathway using Torin-1 or rapamycin leads to an increase in the efficiency of readthrough induced by G418. These results were shown on mRNA from a transfected construct and on endogenous mRNA coding for APC in cancer cells carrying a nonsense mutation in the APC gene. All results show an increase in readthrough induced by G418 when the mTOR pathway is impacted. Overall the article is well structured, the experiments are clearly and logically described. The data is convincing and there does not seem to be a sticking point. I would only have minor points which would make it possible to improve the reading of the manuscript and a possible additional experience in order to make figures 4 and 5 homogeneous.

    Minor comments:

    In chapter "Serum starvation increased APC nonsense-mutation readthrough in CRC cell lines" , last line please replace "sratvation" by "starvation"

    In chapter "Torin-1 increases antibiotic-mediated nonsense codon readthrough" , 6 lines before the end please replace "Totin-1" by "Torin-1"

    The following sentence in the discussion has to be rewritten because NMD degrades RNA and not proteins: "In many cases, the cancer cells express a truncated APC protein that is not degraded by the NMD as most of the nonsense mutations occur in a hotspot within the last APC exon, thus they are not recognized by the exon junction complex method of NMD [55].".

    Change "combitation" into "combination" 7 lines from the end of the discussion.

    Figure 5, the authors analyze the effect of an inhibition of the activity of eIF4E using the small molecule 4EGl-1. They are testing for an endogenous nonsense mutation in the APC gene in COLO320 cells. To be consistent with Figure 4, the authors should also show the same effect on SW403 cells.

    Significance

    The study described here shows that the efficiency of readthrough by aminoglycosides can be regulated by different parameters (serum concentration; translation efficiency). Few data are available on the regulation of this mechanism whose interest in generating new therapeutic approaches in genetic diseases is increasingly growing. This manuscript will therefore be of interest to people working in the field of readthrough, therapeutic approaches and genetic diseases, but also more generally to people studying gene translation and expression.

  5. Version published to 10.1101/2022.08.31.506003v1 on bioRxiv