Phosphoproteomic analysis reveals the diversity of signaling behind E rb B ‐inhibitor‐induced phenotypes

  1. Katri Vaparanta
  2. Zejia Song
  3. Iman Farahani
  4. Anne Jokilammi
  5. Johannes Merilahti
  6. Johanna Örling
  7. Noora Virtanen
  8. Pekka Haapaniemi
  9. Cecilia Sahlgren
  10. Klaus Elenius
  11. Ilkka Paatero

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Abstract

The impact of kinase inhibitors on the phosphoproteome has been rarely investigated at a whole‐organism level. Here, we performed a phosphoproteomic analysis in embryonic zebrafish to identify the signaling pathways perturbed by ErbB receptor tyrosine‐protein kinase inhibitors gefitinib, lapatinib, and AG1478 at the organism level. The phosphorylation of proteins associated with the phosphoinositide 3‐kinase (PI3K)/protein kinase B (Akt), p38 mitogen‐activated protein kinase (MAPK), Notch, Hippo/Yap, and β‐catenin signaling pathways was differentially regulated by the ErbB inhibitors. Gene set enrichment analyses indicated differential neurological and myocardial phenotypes of different ErbB inhibitors. To assess the neurological and myocardial effects, motility and ventricle growth assays were performed with inhibitor‐treated embryos. The treatment with the inhibitors targeting the PI3K/Akt, p38 MAPK, and Notch signaling pathways, along with the ErbB inhibitors AG1478 and lapatinib, perturbed the overall movement and ventricle wall growth of zebrafish embryos. Taken together, these results indicate that inhibitors with overlapping primary targets can affect different signaling pathways while eliciting similar physiological phenotypes.

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  1. Version published to 10.1111/febs.70197
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    Reply to the reviewers

    The manuscript " Phosphoproteomic* analysis reveals the diversity of signaling behind ErbB inhibitor-induced phenotypes*" authored by Drs. Katri Vaparanta, Anne Jokilammi, Johannes Merilahti, Johanna Örling, Noora Virtanen, Cecilia Sahlgren, Klaus Elenius and Ilkka Paatero was reviewed in Review Commons, and we carried out a full revision based on the received reviewer comments.

    The comments from three reviewers and our point-by-point reply is here below. After each of reviewer´s comment, our reply is formatted in bold.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this study, Vaparanta and co-workers used zebrafish embryos as model to analyze the impact of ErbB tyrosine kinase inhibitors on signaling pathways at the whole organism level. Experimentally, zebrafish embryos were exposed for 1 hour to a single dose of 3 different ErbB tyrosine kinase inhibitors and the global phosphoproteome of the embryos was analyzed by MS/MS. The authors show that the 3 inhibitors differentially modulate the activity of PI3K/Akt, p38 MAPK, Notch, Hippo-YAP/TAZ and β-catenin signaling pathways, associated with different neurological and myocardial phenotypic changes. Using small molecule inhibitors of selective signaling pathways, they show that perturbation of different signaling pathways may induce similar phenotypes in zebrafish embryos.

    Specific comments:

    1. The observation that exposure of zebrafish embryos to lapatinib, gefitinib and AG1478 leads to different global phosphoproteomic changes and to differential modulation of cellular signaling pathways was predictable and supported by an abundant literature. These 3 inhibitors differentially inhibit ErbB homo- and heterodimers and hit many other kinases. This point should be discussed in the paper.

    Indeed, the kinase inhibitors do have different selectivity for the ErbB family kinases as pointed out by the reviewer. We have now discussed this point in the manuscript (new Supplemental Table 1) and added additional data from embryos treated with different ErbB kinase inhibitors with similar selectivity profiles into the manuscript (new Supplemental Figure 3-4). The ErbB family kinase selectivity profile of the inhibitors, however, does not fully explain why treatment with lapatinib (EGFR/ErbB2 inhibitor) induced the most unique phosphoproteomic changes from AG1478 (EGFR/ErbB2/ErbB4 inhibitor) and gefitinib (EGFR inhibitor) treatment in zebrafish embryos. This point is now discussed in the manuscript.

    AG1478 is a first-generation tyrphostin while gefitinib and lapatinib are FDA-approved drugs. These compounds not only have different selectivity profiles, but also different pharmacological properties. Do the authors have any information about the permeability, distribution or concentration of the compounds in zebrafish embryos? Otherwise, how can they compare their effects?

    __The reviewer points out correctly, that not only selectivity but also several other parameters could differ between compounds. The logic of our experimentation was to utilize differences in the properties of inhibitors to get new insights into underlying biological processes. These utilized differences could arise from not only selectivity but also as well from pharmacokinetic and –dynamic properties. Although it can be useful to understand these differences, this information per se is not needed to identify differentially regulated pathways that could affect the studied phenotypes. This is now better clarified in the discussion section. Our data indicates that ErbB inhibition profile explains a significant proportion, but not all, of observed signaling differences (Supplemental Fig. 3C). __

    One major limitation of this study is that phosphoproteomic analysis was performed at a single time point and with a single dose of inhibitor, which compromises the interpretation of the findings. How was the dose of each inhibitor selected?

    __The doses were chosen based on our previous work (Paatero et al, 2019; Vaparanta et al, 2023), where with these inhibitor concentrations we were able to maximize the phenotypic effects without causing significant mortality. This is now mentioned in the results section of the manuscript. Higher dosages were lethal for the embryos, especially of AG1478, which is why a lower concentration of this inhibitor was used. The higher toxicity of AG1478 at lower concentrations compared to other ERBB inhibitors has also been previously noted by another group (Pruvot et al, 2014). Similar concentrations of the inhibitors have also been previously used by other groups with zebrafish embryos (Tran et al, 2007; Gallardo et al, 2015; Zhang et al, 2021; Du et al, 2024). __

    One approach for better exploiting the data would be to correlate changes in phosphopeptides with the kinome selectivity of the inhibitors.

    Indeed, we have now correlated our results from these inhibitors with other ErbB inhibitors of similar ErbB family kinase selectivity. The phosphoproteomic changes induced by inhibitors with similar ErbB family kinase selectivity significantly correlate (P = 0.0002, r:0.80 ,R2:0.65, Supplemental Fig. 3C) indicating that the ErbB selectivity plays a major role in determining the phosphoproteomic changes induced by these inhibitors. We also performed a correlation analysis between dimensionality-reduced phosphoproteomic changes and inhibitor selectivity. There was no significant correlation between the changes in the phosphoproteome and the ERBB selectivity of the inhibitors (P=0.1551, One-tailed Pearson correlation). Taken together, these results indicate that while the phosphoproteomic changes induced by these inhibitors can be reproduced by other inhibitors with similar ERBB selectivity profiles, inhibiting only a subset of the ERBB kinases (especially EGFR and ERBB2, but not ERBB4) produces a unique signaling signature that is not recapitulated with pan-ERBB inhibitor treatment. This information may be of interest since both lapatinib (EGFR/ERBB2 inhibitor) and neratinib (pan-ERBB inhibitor) are both used in the clinic to treat HER2-positive breast cancer. Our data indicates that the administration of these inhibitors to patients will likely have a differential global effect on cell signaling.

    In the same vein, the signaling inhibitors used in Fig. 4 to dissect the phenotypic impact of distinct signaling pathways are non-selective, precluding any rigorous interpretation of the data. This confounding factor should at least be discussed in the manuscript. Again, the choice of the different doses of inhibitors is not justified.

    __Indeed, like all inhibitors, the inhibitors we utilized in Figure 4 can have some off-target effects. We aimed to use the concentration known by previous literature to have a measurable effect on the physiology of the zebrafish embryo (Fujii et al, 2000; Geling et al, 2002; Vasilyev et al, 2012; Jiang et al, 2023). These concentrations for different inhibitors were different in the literature, which is why different concentrations of the different inhibitors were used. We couldn’t find a reference for the concentration for VT-103, so a 30µM concentration was selected. With this concentration, the size of the embryo hearts was significantly reduced (P

    The effect of inhibitors on the motility of embryos appears variable. For example, lapatinib markedly decreases motility in Fig. 4E but has no effect in Fig. 4F. Any explanation?

    Different inhibitor concentrations were used in Figure 4E and Figure 4F. This has been now more clearly indicated in the manuscript in the results section and the figure legend. The lower inhibitor dosages in Figure 4F were to reduce the mortality and allow motility analyses of the embryos treated with a combination of the inhibitors analyses to facilitate observation of potential synergistic actions of inhibitors in co-treated embryos.

    The conclusion that ErbB inhibitors induce similar phenotypes by perturbing different signaling pathways is not justified.

    We have now softened our conclusions in the manuscript in the results section by replacing the sentence:” Taken together, these results suggest that AG1478 and lapatinib induce similar phenotypes by partially perturbing different signaling pathways in zebrafish embryos.” With the sentences: ” Taken together, these results suggest that AG1478 and lapatinib induce similar phenotypes but perturb different signaling pathways. Inhibition of these pathways induce similar phenotypes to lapatinib or AG1478 treatment in zebrafish embryos.”.

    I have a few suggestions which could enhance the study's contribution to the field-

    1. The rationale for this study should be elaborated further. What new information is expected to emerge from these studies, independently of the conceptual and technical limitations outlined above?

    We have now further elaborated the rationale of the study in the introduction section.

    The advantage of studying the whole organism instead of selected tissues is questionable. Analyzing a mixture of organs may mask subtle and physiologically relevant alterations of signaling pathways in specific tissues.

    We agree with the reviewer that if the researcher’s interests reside in a specific tissue then a more targeted approach should be applied to probe the phosphoproteome of this tissue. However, sometimes a more global view of the inhibitor effects is required especially when it is unknown which tissues are affected by the inhibitor treatment. Ideally, the global approach would be followed by a more targeted approach on the tissues that are indicated to be affected by the inhibitor. One must also consider the feasibility, time consumption and costs of probing all tissues separately. If only the targeted approach is applied, the information on what pathway activities are globally most affected in the organism by the inhibitor treatment can be hard to estimate.

    Can the authors correlate neurological and myocardial phenotypes extrapolated from their study with pharmacological effects observed in mice or humans treated with these compounds?

    __We have now correlated our findings in the discussion section with the previous literature on the phenotypes of ErbB inhibitor-treated and ErbB receptor knock-out mice and with the reported adverse effects of ErbB inhibitor treatment in the clinic. __

    Reviewer #1 (Significance (Required)):

    The authors show that the 3 inhibitors differentially modulate the activity of PI3K/Akt, p38 MAPK, Notch, Hippo-YAP/TAZ and β-catenin signaling pathways, associated with different neurological and myocardial phenotypic changes. Using small molecule inhibitors of selective signaling pathways, they show that perturbation of different signaling pathways may induce similar phenotypes in zebrafish embryos.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this study, the authors assess the effects of various ErbB receptor family tyrosine kinase inhibitors on the phosphoproteome of late embryonic and early larval stages of zebrafish. MS, Western blotting, and analysis of a transgenic zebrafish Notch signaling reporter line data suggest differential but overlapping effects of treatment with gefitinib, lapatinib and AG1478. Selected deregulated pathways are further assessed using a range of candidate downstream pathway-targeting inhibitors. Inhibitor treatment followed by quantification of spontaneous larval motility and heart ventricle wall area, which were previously found by the authors to be affected by AG1478 and lapatinib treatment, identifies involved downstream signaling pathways.

    Major comments:

    While I do not question the validity of the presented data showing phosphoproteome perturbations resulting from the performed ErbB inhibitor treatments, the treatment regimens used to assess the differential effects of the compounds may be insuffient to substantiate general statements comparing the phenotypic and phosphorylation effects of lapatinib, gefitinib and AG1478 beyond the effects of the specific doses applied to the embryo media. Unless directly quantified, it is difficult to reliably predict the in vivo dose resulting from drug administered to the embryo medium, and therefore a dose may be too high or too low for drug-to-drug comparison. Rationale for chosen dose of drugs should be provided. If available, inclusion of quantitative data on the drug-induced change in phosphorylation status of the drug target(s) is encouraged, and the discussion of the phosphoproteomic and phenotypical data should include this information.

    The reviewer points out correctly, that not only selectivity but also several other parameters could differ between compounds. The logic of our experimentation was to utilize differences in the properties of inhibitors to get new insights into underlying biological processes. These utilized differences could arise from not only selectivity but also as well from pharmacokinetic and –dynamic properties. Although it can be useful to understand these differences, this information per se is not needed for the identification of differentially regulated pathways that could affect the studied phenotypes. This is now better clarified in the manuscript.

    The rationale for the chosen drug doses has now been added to the manuscript in the results section. We used drug concentrations that were known to produce a phenotypic effect without causing significant mortality in the zebrafish embryos.

    The ErbB receptors themselves are expressed at low levels, and unfortunately, we couldn´t reliably observe phosphopeptides of ErbB tyrosine autophosphorylation sites. To address this issue from a different angle, we treated embryos with other ErbB inhibitors exhibiting similar ErbB inhibition profiles as AG1478, lapatinib, and gefitinib (Supplemental Figure 3-4). This data indicates that the ErbB inhibition profile correlates quite well with the observed changes in the downstream signaling pathways p38, pAkt, pErk and Notch (Figure 3C and 4C).

    Husbandry: The statement that "Zebrafish were maintained (...) following standard procedures." is insufficient without a specific reference. Please provide details on water quality parameters, temperature, light/darkness cycle and feeding regimen.

    The requested information has now been added to the manuscript.

    Western analysis: How many embryos were pooled in each sample? Please specify standard protocol or provide reference.

    We have now amended the western analysis chapter in the materials and methods section as suggested by the reviewer. Five embryos were pooled for each sample.

    Ventricle growth assay: The method of ventricle wall quantification is insufficiently described and might result in unnecessarily high variation. At which stage of the cardiac contraction-relaxation cycle were ventricle wall thickness and ventricle area measured? The confounding effect of contraction could be avoided altogether by stopping the heartbeat pharmacologically e.g. by administration of blebbistatin or verapamil. Subtracting ventricle lumen area from total ventricle area seems a much more direct measure of ventricle wall area than the estimation obtained by multiplying ventricle wall thickness with ventricle area.

    We apologize for the mistake in the materials methods section, where we had written area instead of perimeter. We have now amended the ventricle growth assay chapter in the materials and methods sections and added more details on the ventricle wall area estimation. The ventricle wall area was measured from high-speed movies in diastole and systole, and the average perimeter over these states was reported. The ventricle wall thickness was only measured in systole. We chose this quantification method since the lumen area is difficult to estimate in the systole.

    Phosphopeptide enrichment: How many embryos per sample? Final DMSO concentration is not stated.

    __Twenty embryos per sample and 1% DMSO was used. This information is now included in the materials and methods section. __

    P-values are presented for comparison of select groups only and a statement that e.g. only P-values We have added the recommended statement and the mean/median value with deviation values for the data indicated by the reviewer in the figure legends.

    Minor comments:

    Overall, the manuscript is well written and data and methods are well presented.

    The relevant targets within the ErbB family of receptors should be introduced including information on well-established functions and downstream signaling pathways to enable the non-specialist reader to place the presented data in the context of known gene and protein function. Furthermore, conservation of target proteins in zebrafish should be touched upon.

    We have now rewritten the introduction and results sections to include information on the ErbB family kinase selectivity of these inhibitors, the well-established functions and the target downstream pathways of ErbB receptors. We have now performed a multiple sequence alignment on the kinase domain of the ErbB receptors in human and zebrafish to estimate the conservation of the inhibitor targets in the zebrafish model. Human ErbB kinase domains had a high 86+/-9% sequence identity with zebrafish counterparts (Supplemental Figure 2) compared to 67+/-14% identity with the other ErbB kinase domain sequences in zebrafish (P=0.012).

    Given different target profiles of the tested drugs among receptors of the ErbB family, differences in protein phosphorylation perturbations and in treatment-induced phenotypes may not be unexpected. Statements such as: "An unexpectedly large cluster of phosphopeptides that were increased in lapatinib-treated embryos but reduced in AG1478 and gefitinib treated embryos was detected" and "AG1478 and lapatinib may induce similar phenotypes by partially perturbing different signaling pathways in zebrafish embryos" should be discussed in the context of known drug target(s) and their functions.

    We have now rewritten these statements as suggested by the reviewer and the target profiles are now discussed in the manuscript.

    **Referee Cross-commenting**

    I agree with the other reviewers on almost all points.

    1. While the sensitivity to smaller or highly local effects is most likely reduced using the whole organism approach compared to e.g. single tissue analysis, I do believe that it is highly relevant due to its ability to identify potential effects beyond a single tissue or organ.
    1. I maintain that while the presented data nicely show the effects of each administered dose of the individual compounds, the data does not allow for meaningful drug-to-drug comparisons without quantitative information on in vivo dose or direct target effect. If such information cannot be included, cross-drug conclusions and discussion should be done very carefully.

    Reviewer #2 (Significance (Required)):

    The evaluation of systemic molecular and phenotypic consequences of anti-cancer drugs in a vertebrate model system represents a relevant advancement. Although drug effects are likely to differ somewhat between embryonic and larval zebrafish and human cancer patients, the authors' comparison of obtained zebrafish data with human data supports translatability of the presented phosphoproteomics data. Also, the presented data pose a relevant advancement facilitating the informed use of the tested inhibitors as tools in basic science.

    Expertise: Molecular biology, signaling, zebrafish. Limited expertise in omics data analysis and pharmacology.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The authors evaluated selected EGFR inhibitors developed as targeted cancer therapeutics, using zebrafish embryos and larvae as an in vivo model system. They performed mass spectrometry to analyze phosphorylation levels in target proteins, in combination with western blotting and gene set enrichment analyses; using this data, they assessed overlap between the inhibitors and overlap with known human data. They also performed imaging and locomotion analyses to assess alterations in phenotypes and phosphorylation-dependent signaling due to the inhibitor(s). The study generates novel information that is potentially relevant to the toxicity and efficacy of clinically used kinase inhibitors.

    • The statistical analyses are appropriate to the data and the experimental design.
    • The claims made by the authors are consistent with the data. In my opinion, the following revisions are needed for the manuscript to be accepted for publication:
    1. There is no mention of Gefitinib in the Abstract; please include it.

    Gefitinib is now included in the Abstract.

    Please state the target selectivity profiles (from known preclinical and/or clinical data) of the three inhibitors used.

    __These are now presented in supplemental data (Supplemental Table 1), and analysed in relation to the observed signaling changes (Supplemental Figures 3 and 4). __

    Please clarify whether the residues mentioned in the phospho-specific antibody data refer to zebrafish or human proteins.

    Residues refer to human proteins as they are more widely used. This is now more clearly indicated in the materials and methods section.

    Please state whether the pan-antibodies corresponding to the phospho-specific antibody targets were used, and mention any problems associated with their use. This will help readers not familiar with antibody use in zebrafish experiments. It will also help emphasize the value of mass spectrometric analysis in zebrafish protein work.

    __As pointed out, the target specificity of antibodies is not often defined in zebrafish models on residue level, and phospho-specific antibodies may bind several closely related targets. The availability of robustly validated antibodies for zebrafish work, especially for phosphospecific epitopes, is quite limiting and therefore other, non-antibody-based techniques would be highly useful. This is now discussed in the manuscript. The phosphorylation site-specific antibodies used in this study indeed recognize the phosphorylated residue in several protein family members which further complicates the result interpretation. This is less of a limitation in the DIA-MS based phosphoproteomics approach which is now additionally discussed in the manuscript. __

    Please attempt to describe the clinically documented cardiovascular and neurological effects of the inhibitors and any correlation(s) with your data. This will enhance the impact of the study.

    See our reply for reviewer#1, comment 3.

    **Referee Cross-commenting**

    The common points raised in all the Reviews are the following:

    1. The rationale of the study should be described in more detail, especially the utility of zebrafish as an in vivo model, addressing its advantages and limitations.

    This is now discussed more extensively in the manuscript.

    The findings need to be described in the context of the target selectivity profiles and clinical effects of the inhibitors, especially the approved inhibitors (Gefitinib and Lapatinib).

    We have added data on target selectivity profiles (Supplemental Table 1), target conservation (Supplemental Figure 2) and also compared our observations to zebrafish embryos treated with other ErbB inhibitors with similar ErbB selectivity profiles (Supplemental Figure 3 and 4).

    1. In my opinion, while the comments regarding target site drug concentration (within the embryos/larvae) and dose-response are relevant, I consider these experiments to be appropriate in a more detailed follow-up study.

    We agree with the reviewer that the comprehensive pharmacokinetic studies fall outside the scope of this manuscript. As discussed before, in this manuscript we utilize differential inhibitor properties to gain new insight into phenotypes and underlying biological processes. This logic works even if the differences arise from properties other than the target selectivity.

    One of the main value additions of the study is that it highlights a useful alternative to conventional strategies used in preclinical cellular and mammalian model studies of kinase inhibitors. I would urge the authors to discuss specific future directions, giving due importance to all the reviewers' comments.

    This is now more extensively elaborated in the discussion section.

    Reviewer #3 (Significance (Required)):

    The experiments are well-described and provide sufficient information and detail for readers to understand and reproduce.

    The study is highly relevant to the use of zebrafish as a whole-organism model for in vivo evaluation of drugs, specifically kinase inhibitors.

    References

    Du K, Liu Y, Zhang L, Peng L, Dong W, Jiang Y, Niu M, Sun Y, Wu C, Niu Y et al (2024) Lapatinib combined with doxorubicin causes dose-dependent cardiotoxicity partially through activating the p38MAPK signaling pathway in zebrafish embryos. Biomed Pharmacother 175. doi:10.1016/J.BIOPHA.2024.116637.

    Fujii R, Yamashita S, Hibi M, Hirano T (2000) Asymmetric p38 activation in zebrafish: Its possible role in symmetric and synchronous cleavage. Journal of Cell Biology 150. doi:10.1083/jcb.150.6.1335.

    Gallardo VE, Varshney GK, Lee M, Bupp S, Xu L, Shinn P, Crawford NP, Inglese J, Burgess SM (2015) Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion. DMM Disease Models and Mechanisms 8. doi:10.1242/dmm.018689.

    Geling A, Steiner H, Willem M, Bally-Cuif L, Haass C (2002) A γ-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep 3. doi:10.1093/embo-reports/kvf124.

    Jiang Y, Zhao X, Chen J, Aniagu S, Chen T (2023) PM2.5 induces cardiac malformations via PI3K/akt2/mTORC1 signaling pathway in zebrafish larvae. Environmental Pollution 323. doi:10.1016/j.envpol.2023.121306.

    Paatero I, Veikkolainen V, Mäenpää M, Schmelzer E, Belting HG, Pelliniemi LJ, Elenius K (2019) ErbB4 tyrosine kinase inhibition impairs neuromuscular development in zebrafish embryos. Mol Biol Cell 30. doi:10.1091/mbc.E18-07-0460.

    Pruvot B, Curé Y, Djiotsa J, Voncken A, Muller M (2014) Developmental defects in zebrafish for classification of EGF pathway inhibitors. Toxicol Appl Pharmacol 274. doi:10.1016/j.taap.2013.11.006.

    Tran TC, Sneed B, Haider J, Blavo D, White A, Aiyejorun T, Baranowski TC, Rubinstein AL, Doan TN, Dingledine R et al (2007) Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish. Cancer Res 67. doi:10.1158/0008-5472.CAN-07-3126.

    Vaparanta K, Jokilammi A, Paatero I, Merilahti JA, Heliste J, Hemanthakumar KA, Kivelä R, Alitalo K, Taimen P, Elenius K (2023) STAT5b is a key effector of NRG ‐1/ ERBB4 ‐mediated myocardial growth . EMBO Rep 24. doi:10.15252/embr.202256689.

    Vasilyev A, Liu Y, Hellman N, Pathak N, Drummond IA (2012) Mechanical stretch and PI3K signaling link cell migration and proliferation to coordinate epithelial tubule morphogenesis in the zebrafish pronephros. PLoS One 7. doi:10.1371/journal.pone.0039992.

    Xin M, Kim Y, Sutherland LB, Qi X, McAnally J, Schwartz RJ, Richardson JA, Bassel-Duby R, Olson EN (2011) Development: Regulation of insulin-like growth factor signaling by Yap governs cardiomyocyte proliferation and embryonic heart size. Sci Signal 4. doi:10.1126/scisignal.2002278.

    Zhang Y, Cai Y, Zhang SR, Li CY, Jiang LL, Wei P, He MF (2021) Mechanism of hepatotoxicity of first-line tyrosine kinase inhibitors: Gefitinib and afatinib. Toxicol Lett 343. doi:10.1016/j.toxlet.2021.02.003.

  3. 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

    The authors evaluated selected EGFR inhibitors developed as targeted cancer therapeutics, using zebrafish embryos and larvae as an in vivo model system. They performed mass spectrometry to analyze phosphorylation levels in target proteins, in combination with western blotting and gene set enrichment analyses; using this data, they assessed overlap between the inhibitors and overlap with known human data. They also performed imaging and locomotion analyses to assess alterations in phenotypes and phosphorylation-dependent signaling due to the inhibitor(s). The study generates novel information that is potentially relevant to the toxicity and efficacy of clinically used kinase inhibitors.

    • The statistical analyses are appropriate to the data and the experimental design.
    • The claims made by the authors are consistent with the data.

    In my opinion, the following revisions are needed for the manuscript to be accepted for publication:

    1. There is no mention of Gefitinib in the Abstract; please include it.
    2. Please state the target selectivity profiles (from known preclinical and/or clinical data) of the three inhibitors used.
    3. Please clarify whether the residues mentioned in the phospho-specific antibody data refer to zebrafish or human proteins.
    4. Please state whether the pan-antibodies corresponding to the phospho-specific antibody targets were used, and mention any problems associated with their use. This will help readers not familiar with antibody use in zebrafish experiments. It will also help emphasize the value of mass spectrometric analysis in zebrafish protein work.
    5. Please attempt to describe the clinically documented cardiovascular and neurological effects of the inhibitors and any correlation(s) with your data. This will enhance the impact of the study.

    Referee Cross-commenting

    The common points raised in all the Reviews are the following:

    1. The rationale of the study should be described in more detail, especially the utility of zebrafish as an in vivo model, addressing its advantages and limitations.
    2. The findings need to be described in the context of the target selectivity profiles and clinical effects of the inhibitors, especially the approved inhibitors (Gefitinib and Lapatinib).
    3. In my opinion, while the comments regarding target site drug concentration (within the embryos/larvae) and dose-response are relevant, I consider these experiments to be appropriate in a more detailed follow-up study.
    4. One of the main value additions of the study is that it highlights a useful alternative to conventional strategies used in preclinical cellular and mammalian model studies of kinase inhibitors. I would urge the authors to discuss specific future directions, giving due importance to all the reviewers' comments.

    Significance

    The experiments are well-described and provide sufficient information and detail for readers to understand and reproduce.

    The study is highly relevant to the use of zebrafish as a whole-organism model for in vivo evaluation of drugs, specifically kinase inhibitors.

  4. Review Commons

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

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this study, the authors assess the effects of various ErbB receptor family tyrosine kinase inhibitors on the phosphoproteome of late embryonic and early larval stages of zebrafish. MS, Western blotting, and analysis of a transgenic zebrafish Notch signaling reporter line data suggest differential but overlapping effects of treatment with gefitinib, lapatinib and AG1478. Selected deregulated pathways are further assessed using a range of candidate downstream pathway-targeting inhibitors. Inhibitor treatment followed by quantification of spontaneous larval motility and heart ventricle wall area, which were previously found by the authors to be affected by AG1478 and lapatinib treatment, identifies involved downstream signaling pathways.

    Major comments:

    While I do not question the validity of the presented data showing phosphoproteome perturbations resulting from the performed ErbB inhibitor treatments, the treatment regimens used to assess the differential effects of the compounds may be insuffient to substantiate general statements comparing the phenotypic and phosphorylation effects of lapatinib, gefitinib and AG1478 beyond the effects of the specific doses applied to the embryo media. Unless directly quantified, it is difficult to reliably predict the in vivo dose resulting from drug administered to the embryo medium, and therefore a dose may be too high or too low for drug-to-drug comparison. Rationale for chosen dose of drugs should be provided. If available, inclusion of quantitative data on the drug-induced change in phosphorylation status of the drug target(s) is encouraged, and the discussion of the phosphoproteomic and phenotypical data should include this information.

    Husbandry: The statement that "Zebrafish were maintained (...) following standard procedures." is insufficient without a specific reference. Please provide details on water quality parameters, temperature, light/darkness cycle and feeding regimen.

    Western analysis: How many embryos were pooled in each sample? Please specify standard protocol or provide reference. Ventricle growth assay: The method of ventricle wall quantification is insufficiently described and might result in unnecessarily high variation. At which stage of the cardiac contraction-relaxation cycle were ventricle wall thickness and ventricle area measured? The confounding effect of contraction could be avoided altogether by stopping the heartbeat pharmacologically e.g. by administration of blebbistatin or verapamil. Subtracting ventricle lumen area from total ventricle area seems a much more direct measure of ventricle wall area than the estimation obtained by multiplying ventricle wall thickness with ventricle area.

    Phosphopeptide enrichment: How many embryos per sample? Final DMSO concentration is not stated. P-values are presented for comparison of select groups only and a statement that e.g. only P-values < 0.05 are plotted would be helpful if applicable. Also, please provide mean +/- standard deviation for data presented in figures 3A, 3B, 4C, 4E, and 4F.

    Minor comments:

    Overall, the manuscript is well written and data and methods are well presented. The relevant targets within the ErbB family of receptors should be introduced including information on well-established functions and downstream signaling pathways to enable the non-specialist reader to place the presented data in the context of known gene and protein function. Furthermore, conservation of target proteins in zebrafish should be touched upon.

    Given different target profiles of the tested drugs among receptors of the ErbB family, differences in protein phosphorylation perturbations and in treatment-induced phenotypes may not be unexpected. Statements such as: "An unexpectedly large cluster of phosphopeptides that were increased in lapatinib-treated embryos but reduced in AG1478 and gefitinib treated embryos was detected" and "AG1478 and lapatinib may induce similar phenotypes by partially perturbing different signaling pathways in zebrafish embryos" should be discussed in the context of known drug target(s) and their functions.

    Referee Cross-commenting

    I agree with the other reviewers on almost all points.

    1. While the sensitivity to smaller or highly local effects is most likely reduced using the whole organism approach compared to e.g. single tissue analysis, I do believe that it is highly relevant due to its ability to identify potential effects beyond a single tissue or organ.
    2. I maintain that while the presented data nicely show the effects of each administered dose of the individual compounds, the data does not allow for meaningful drug-to-drug comparisons without quantitative information on in vivo dose or direct target effect. If such information cannot be included, cross-drug conclusions and discussion should be done very carefully.

    Significance

    The evaluation of systemic molecular and phenotypic consequences of anti-cancer drugs in a vertebrate model system represents a relevant advancement. Although drug effects are likely to differ somewhat between embryonic and larval zebrafish and human cancer patients, the authors' comparison of obtained zebrafish data with human data supports translatability of the presented phosphoproteomics data. Also, the presented data pose a relevant advancement facilitating the informed use of the tested inhibitors as tools in basic science.

    Expertise: Molecular biology, signaling, zebrafish. Limited expertise in omics data analysis and pharmacology.

  5. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this study, Vaparanta and co-workers used zebrafish embryos as model to analyze the impact of ErbB tyrosine kinase inhibitors on signaling pathways at the whole organism level. Experimentally, zebrafish embryos were exposed for 1 hour to a single dose of 3 different ErbB tyrosine kinase inhibitors and the global phosphoproteome of the embryos was analyzed by MS/MS. The authors show that the 3 inhibitors differentially modulate the activity of PI3K/Akt, p38 MAPK, Notch, Hippo-YAP/TAZ and β-catenin signaling pathways, associated with different neurological and myocardial phenotypic changes. Using small molecule inhibitors of selective signaling pathways, they show that perturbation of different signaling pathways may induce similar phenotypes in zebrafish embryos.

    Specific comments:

    1. The observation that exposure of zebrafish embryos to lapatinib, gefitinib and AG1478 leads to different global phosphoproteomic changes and to differential modulation of cellular signaling pathways was predictable and supported by an abundant literature. These 3 inhibitors differentially inhibit ErbB homo- and heterodimers and hit many other kinases. This point should be discussed in the paper.
    2. AG1478 is a first-generation tyrphostin while gefitinib and lapatinib are FDA-approved drugs. These compounds not only have different selectivity profiles, but also different pharmacological properties. Do the authors have any information about the permeability, distribution or concentration of the compounds in zebrafish embryos? Otherwise, how can they compare their effects?
    3. One major limitation of this study is that phosphoproteomic analysis was performed at a single time point and with a single dose of inhibitor, which compromises the interpretation of the findings. How was the dose of each inhibitor selected?
    4. One approach for better exploiting the data would be to correlate changes in phosphopeptides with the kinome selectivity of the inhibitors.
    5. In the same vein, the signaling inhibitors used in Fig. 4 to dissect the phenotypic impact of distinct signaling pathways are non-selective, precluding any rigorous interpretation of the data. This confounding factor should at least be discussed in the manuscript. Again, the choice of the different doses of inhibitors is not justified.
    6. The effect of inhibitors on the motility of embryos appears variable. For example, lapatinib markedly decreases motility in Fig. 4E but has no effect in Fig. 4F. Any explanation?
    7. The conclusion that ErbB inhibitors induce similar phenotypes by perturbing different signaling pathways is not justified.

    I have a few suggestions which could enhance the study's contribution to the field-

    1. The rationale for this study should be elaborated further. What new information is expected to emerge from these studies, independently of the conceptual and technical limitations outlined above?
    2. The advantage of studying the whole organism instead of selected tissues is questionable. Analyzing a mixture of organs may mask subtle and physiologically relevant alterations of signaling pathways in specific tissues.
    3. Can the authors correlate neurological and myocardial phenotypes extrapolated from their study with pharmacological effects observed in mice or humans treated with these compounds?

    Significance

    The authors show that the 3 inhibitors differentially modulate the activity of PI3K/Akt, p38 MAPK, Notch, Hippo-YAP/TAZ and β-catenin signaling pathways, associated with different neurological and myocardial phenotypic changes. Using small molecule inhibitors of selective signaling pathways, they show that perturbation of different signaling pathways may induce similar phenotypes in zebrafish embryos.

  6. Version published to 10.1101/2024.03.12.584564 on bioRxiv