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The EMBO Press journals expect authors to upload the ‘source data’ that were used to generate figures. ‘Source data’ files will be directly linked to specific figures so that the readers can download the associated ‘source data’ for the purpose of detailed inspection, or re-analysis or integration with other data.

Moreover, ‘source data’ files will be automatically submitted to BioStudies and obtain an accession number to make them citable. The respective figures will also be annotated by our SourceData curators to make them searchable. (LINK TO SOURCEDATA)

The source data files are separate from the Expanded View information files and are submitted using the "figure source data" option in the manuscript submission system. We distinguish small scale and large scale source data files. For large scale source data files (>500 MB) please refer to the ‘data availability’ and ‘data deposition’ section as these files have to be submitted to the relevant external databases and referenced in the data availability section. The source data should be submitted at time of resubmission – can also be provided upon initial submission.

For small scale datasets (* provided ethical obligations to the patients and relevant medical and legal issues are respected.

The source data files should be provided as a zipped archive with the collection of minimally processed raw data files supplied in individual folders for each figure and subfolders for each panel – see example below. Individual files should be labelled with the assay and measured biological entities. If necessary, supply a README text file in the subfolder to explain the content of the files:

Starting from here, we could make it a separate 'resources page' to which we link out.

Also "Data Deposition' and 'Data availability' could link out to this resource.

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­Response to Reviewers Comments

We would like to thank all reviewers for carefully considering our manuscript and providing useful suggestions/ideas. The general consensus was that our study provides an important conceptual advance that reveals a new way of thinking about kinetochore phosphatases. However, in light of our surprising findings, it was suggested that additional experiments would be required to fully validate our conclusions. In particular, it was seen as important to test whether PLK1 can activate MPS1 from the BUB complex and to confirm that PP1 and PP2A are effectively inhibited in situations where MELT dephosphorylation can occur normally (Figure 3).

In general, we agree with these and the other points raised by the reviewers, therefore we plan to address all comments as outlined in detail below.

The major new additions to the final paper will be the following:

1) Experiments to test how BUB-bound PLK1 affects MPS1 activity.

2) Experiments to determine the efficiency of phosphatase inhibition in figure 3.

3) Experiments to test whether maintaining PLK1 at the BUB complex causes SAC silencing defects

4) Evolutionary analysis demonstrating that the PLK1 and PP2A-binding modules have co-evolved in the kinetochore BUB complex. This analysis, which has been performed already, strengthens our manuscript because it provides additional independent evidence for a functional relationship between PLK1 and PP2A on the BUB complex.

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Reviewer #1 **Minor comments:** 1) The authors propose that PP1-KNL1 and BUBR1-bound PP2A-B56 continuously antagonise PLK1 association with the BUB complex by dephosphorylating the CDK1 phosphorylation sites on BUBR1 (pT620) and BUB1 (pT609). It is therefore expected that converting these residues to aspartate would increase PLK1 recruitment. It would be interesting to verify if this hypothesis fits with the proposed model.

Response: The general idea to maintain PLK1 at the BUB complex is a good one, but unfortunately polo-box domains do not bind to acidic negatively charged residues. Instead we will attempt to maintain PLK1 at the BUB complex using alternatively approaches (as suggested by reviewer 2).

2) In Figure 1E, are the mean values for BubR1WT+BubWT and BubR1WT+Bub1T609 both normalized to 1? If so, this fails to reveal the contribution of Bub1 T609 for the recruitment of PLK1 when PP2A-B56 is allowed to localize at kinetochores.

Response: The values will be updated and normalised to the BubR1WT+BUB1WT control. We have also performed additional experiments already and overall the results reveal a small reduction in kinetochore PLK1 following BUB1-T609A mutation and a larger reduction upon combined BUBR1-T620A mutation.

3) What underlies the increase in Bub1 levels at unattached kinetochores of siBubR1 cells (Figure S1C?) Is this caused by an increase in Bub1 T609 phosphorylation and consequently unopposed PLK1 recruitment, which consequently increases MELT phosphorylation?

Response: We suspect that PLK1 is not the cause of the increased BUB1 levels because PLK1 kinetochore levels are actually decreased in this situation (Figure S1A).

4) Although the immunoblotting from Figure S1D indicates that BubR1T620A and Bub1T609A are expressed at similar levels as their respective WT counterparts, some degree of single-cell variability is expected to occur. As a complement to Figure 1B,C and Figure S1E,F could the authors plot the kinetochore intensity of BubR1 pT620 and Bub1T609 relative to the YFP-BubR1 and YFP-Bub1 signal, respectively?

Response: There is indeed variability in the level of re-expression of BUBR1/BUB1 on a single cell level, which can at least partially explain the variation on BUBR1-pT620 and BUB1-pT609 observed within in each condition. We can upload these scatter plots at resubmission and include in the supplementary, if required.

5) The authors nicely show that excessive PLK1 levels at the BUB complex are able to maintain MELT phosphorylation and the SAC (independently of MPS1) when KNL1-localised phosphatases are removed (Figures 2A,B). However, it should be noted that PLK1 is able to promote MPS1 activation at kinetochores and so, whether AZ-3146 at 2.5 uM efficiently inhibits MPS1 under conditions of excessive PLK1 recruitment should be confirmed. Can the authors provide a read-out for MPS1 activation status or activity (other than p-MELTs) to exclude a potential contribution of residual MPS1 activity in maintaining the p-MELTs and SAC?

Response: This is a good point because although PLK1 can phosphorylate the MELTs it can also activate MPS1, although it is unknown whether it can do this from the BUB complex. We had left a dotted line in Figure 4B to include this possibility, but we will now test this directly with additional experiments.

6) To examine whether PLK1 removal is the major role of PP1-KNL1 and PP2A-B56 in the SAC or whether they are additionally needed to dephosphorylate the MELTs, the authors monitored MELT dephosphorylation when MPS1 was inhibited immediately after 30-minute of BI2356. This revealed similar dephosphorylation kinetics, irrespective of compromised PP1-KNL1 or PP2A-B56 activity, thus suggesting that these pools of phosphatases are not required to dephosphorylate MELTs. To confirm this and exclude phosphatase redundancy, the authors simultaneously depleted all PP1 and B56 isoforms or treated cells with Calyculin A to inhibit all PP1 and PP2A phosphatases. In both of these situations, the kinetics of MELT dephosphorylation was indistinguishable from wild type cells if MPS1 and PLK1 were inhibited together. These observations led to the conclusion that neither PP1 or PP2A are required to dephosphorylate the MELT motifs. Instead they are needed to remove PLK1 from the BUB complex. This set of experiments is well-designed and the results support the conclusion. However, it would be of value if the authors provide evidence for the efficiency of PP1 and B56 isoforms depletion and for the efficiency of phosphatase inhibition by Calyculin A. An alternative read-out for the activity of PP1 and PP2A-B56 (other than p-MELT dephosphorylation) clearly confirming that both phosphatases are compromised when MPS1 and PLK1 are inhibited together could make a stronger case in excluding the contribution of residual PP1 or PP2A to the observed dephosphorylation of MELT motifs.

Response: This is also a good point. We had attempted many different combinations in Figure 3 to inhibit PP1/PP2A activity as efficiently as possible. This is especially important considering the “negative” results on pMELT are very surprising. However, we will now test how efficiently we have inhibited PP1 and PP2A phosphatase function in these experiments.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Major comments:** 1) In its current state I am not convinced that the key conclusions are fully supported by the experiments and alternative conclusions/interpretations can be drawn. For example the level of MELT phosphorylation will be determined by the balance of kinase and phosphatase activity and if they do not achieve 100% inhibition of Mps1 in their assays then they are not strictly monitoring dephosphorylation kinetics in their assays. If the combination of Mps1 and Plk1 inhibition then more strongly inhibits Mps1 then dephosphorylation kinetics becomes faster. Thus subtle differences in Mps1 activity under their different conditions could lead to misleading conclusions but in its present state a careful analysis of Mps1 activity is not provided. This lack of complete inhibition also applies to the phosphatases and the experiments in Figure 3E indicates that their Calyculin preparation is not really active as at steady state MELT phosphorylation levels are much less affected than in for instance BubR1 del PP2A (Figure 2A as an example). Thus they likely still have phosphatase activity in the experiment in figure 3E making it difficult to draw the conclusions they do. A more careful analysis of kinase and phosphatase activities in their different perturbations would be recommendable and should be possible within a reasonable time frame.

Response: These are good points and we will now more carefully assess MPS1 and PP1/PP2A activities.

2) A more stringent test of their model would also be needed. What happens if Plk1 is artificially maintained in the Bub complex? The prediction would be that SAC silencing should be severely delayed even when Mps1 is inhibited. This is a straightforward experiment to do that should not take too long. If the polobox can bind phosphoSer then one could also make BubR1 T620S to slow down dephosphorylation of this site (PPPs work slowly on Ser while Cdk1 have almost same activity for Ser and Thr).

Response: These are good suggestions and we will try to see if maintaining PLK1 at the BUB complex produces effects on the SAC.

3) Another issue is the relevance of Plk1 removal under normal conditions. As their quantification shows in figure 1D-E (I think there is something wrong with figure 1E - should likely be Bub1) the contribution of BubR1 T620 and Bub1 T609 to Plk1 kinetochore localisation seems minimal. Thus upon SAC satisfaction there is not really a need to remove Plk1 through dephosphorylation as it is already at wild type levels. It is only in their BubR1 and KNL1 mutants that there is this effect so one has to question the impact in a normal setting. This is consistent with the data in Figure S1D showing no phosphorylation of these sites under unperturbed conditions.

Response: The major finding of this study is that kinetochore phosphatases are primarily needed to supress PLK1 activity on the BUB complex and thereby prevent excessive MELT phosphorylation. The relevance of this continued PLK1 removal under normal conditions is clear, because when it cannot occur (i.e. if the phosphatases are removed) then the SAC cannot be silenced unless PLK1 is inhibited. Therefore, whilst it is true that PLK1 localisation to the BUB complex is low under normal conditions, that is because the phosphatases are working to keep it that way. The relevance of that continual removal is an interesting, but in our opinion, separate question that will require a new body of work to resolve. One possibility is that PLK1 recruitment is a continual dynamic process, that is perhaps coupled to a particular stage in MCC assembly. For example, PLK1 could bind the BUB complex to recruit PP2A to BUBR1, before being immediately removed by PP2A. In this sense, PLK1 binding could still be functionally important even if it is only occurs transiently and steady state PLK1 levels are low. We will add a line to the discussion to highlight that it would be interesting to test PLK1 dynamics on the BUB complex in future.

4) They write that in the absence of phosphatase activity Plk1 becomes capable of supporting SAC independently (of Mps1 is implied). They do not show this - only that MELT phosphorylation is maintained. As Mps1 has other targets required for SAC activity I would rephrase this.

Response: Good point, this will be rephrased.

Reviewer #2 (Significance (Required)): The advance is clearly conceptual and provides a new way of thinking about the kinetochore localized phosphatases. These phosphatases and the SAC have been immensely studied but this work brings in a new angle. The discussion would benefit from some evolutionary perspectives as the PP1 and PP2A-B56 binding sites are very conserved but the Plk1 docking sites on Bubs less so. This will be of interest to people in the field of cell division and researchers interested in phospho-mediated signaling.

Response: Since the paper was submitted, we performed evolutionary analysis to examine this point. We discovered that the PLK1 docking sites are surprisingly well conserved and, in fact, they appear to have co-evolved within the same region of MAD/BUB along with the PP2A-B56 binding motif. We believe this new data strengthens our manuscript because it argues strongly for an important functional relationship between PLK1 and PP2A. A new figure containing this evolutionary analysis will be included in the final version.

Reviewer #3 **Major comments:**

1. An important limitation of this study is that KNL1 dephosphorylation at MELT repeats is monitored only by indirect immunofluorescence using phospho-specific antibodies. Thus, reduction of phospho-KNL1 kinetochore signals could be due to protein turnover at kinetochores, rather than to dephosphorylation. This is a serious issue that could be addressed by checking KNL1 dephosphorylation during time course experiments by western blot using phospho-specific antibodies, as previously done (Espert et al., 2014).

Response: This is an important point that we feel is best addressed by examining total KNL1 levels at kinetochores (instead of simply total cellular levels by western blots). The reason is that KNL1 could potentially still be lost from kinetochores even if the total protein is not degraded. In all experiments involving YFP-KNL1 we observe no change in kinetochore KNL1 levels and this data will be included in the final version. We will also perform new experiments to examine total KNL1 levels in the BUBR1-WT/DPP2A situation to test whether KNL1 kinetochore levels are similarly maintained in these cells following MPS1 inhibition.

1. For obvious technical reasons, the shortest time point at which authors compare KNL1 dephosphorylation upon MPS1-PLK1 inhibition is 5 minutes. Based on immunofluorescence data, authors conclude that kinetics of KNL1 dephosphorylation are similar when kinases are inhibited, independent of whether or not kinetochore-bound phosphatases are active. However, in most experiments (e.g. Fig. 3B, 3C, 3E) lower levels of MELT phosphorylation are detected after 5 minutes of kinase inhibition when phosphatases are present than when they are absent, suggesting that phosphatases likely do contribute to KNL1 dephosphorylation. I suspect that differences between the presence and absence of phosphatases might even be more obvious if authors were to look at shorter time points, when phosphatases conceivably accomplish their function. I would therefore suggest that the authors tone down their conclusions, as their data complement but do not disprove the previous model.

Response: We appreciate that small differences can be seen in figure 3B and 3E at the 5-minute timepoint (between the WT and phosphatase inhibited situations). This may reflect a role for the phosphatases in dephosphorylation or in the ability of drugs such as BI-2536 (3B) or Calyculin A (3E) to fully inhibit their targets in the short timeframe. We will perform additional experiments to examine MPS1 and phosphatase activity under these conditions, in response to comments by reviewers 1 and 2. In the final version we will carefully interpret the new and existing data and, if required, modify the conclusions appropriately.

1. In all experiments cells are kept mitotically arrested through nocodazole treatment, which is not quite a physiological condition to study SAC silencing. This could potentially mask the real contribution of phosphatases in MELT dephosphorylation. Indeed, it is possible that higher amounts of phosphatases are recruited to kinetochores during SAC silencing than during SAC signalling (e.g. during SAC signalling Aurora B phosphorylates the RVSF motif of KNL1 to keep PP1 binding at low levels; Liu et al., 2010). What would happen in a nocodazole wash-out? Would phosphatases be dispensable in these conditions for normal kinetics of MELT dephosphorylation and anaphase onset if PLK1 is inhibited?

Response: All SAC silencing assays where performed in nocodazole for 2 main reasons: 1) PP2A-B56, PP1 or PLK1 can all regulate kinetochore-microtubule attachments, and thereby control the SAC indirectly. Therefore, performing our assays in the absence of microtubules allows us to make specific and direct conclusions about SAC regulation; 2) Previous work on pMELT regulation by PP1/PP2A in human cells was also performed following MPS1 inhibition in nocodazole (Espert et al 2014, Nijenhuis et al, 2014). Therefore, we are able to directly compare the contribution of PLK1 to the previously observed phenotypes, which allowed us to conclude that PLK1 has a major influence.

Nevertheless, we appreciate the point that the influence of PLK1 could, in theory, be different during a normal mitosis when microtubule attachment can form. Therefore, we will attempt to address whether PLK1 inhibition can bypass a requirement for PP1/PP2A in SAC silencing during an unperturbed mitosis.

Other data are overinterpreted. For instance, the evidence that CDK1-dependent phosphorylation sites in Bub1 and BubR1 is enhanced when PP1 and PP2A-B56 are absent at kinetochores suggests but does not "demonstrate that PP1-KNL1 and BUBR1-bound PP2A-B56 antagonise PLK1 recruitment to the BUB complex by dephosphorylating key CDK1 phosphorylation sites on BUBR1 (pT620) and BUB1 (pT609)(Figure 1F)". Similarly, the claim "when kinetochore phosphatase recruitment is inhibited, PLK1 becomes capable of supporting the SAC independently" referred to Fig. 2C-D is an overstatement, as residual MPS1 kinase could be still active in the presence of the AZ-3146 inhibitor.

Response: These are good points and the indicated statements will be reworded.

**Minor comments:**

1. In many graphs (Fig. 1A-C, Fig. 2A,C) relative kinetochore intensities are quantified over "CENPC or YFP-KNL1". Authors should clarify when it is one versus the other.

Response: This will be clarified in the axis and in the methods.

1. The drawing in Fig. 1F depicts the action of PP1 and PP2A-B56 in antagonising PLK1 at kinetochores. Thus, the output should be SAC silencing, rather than activation.

Response: The SAC symbol will be removed from the schematic to avoid confusion and because it is not actually the focus of figure 1 anyway.

1. In the Discussion authors speculate that KNL1 dephosphorylation relies on a constitutive phosphatase with unregulated basal activity. Would a phosphatase be needed at all when MPS1 and PLK1 are inhibited? Could phosphorylated KNL1 be actively degraded?

Response: We will insert total KNL1 immunofluorescence quantification so show that KNL1 KT levels are not decreased in this situation. KNL1 remains anchored at kinetochore but the MELTs must be dephosphorylated to remove the BUB complex.

1. What happens to MPS1 when KNL1-bound PP1 and BUBR1-bound PP2A are absent? Do its kinetochore levels increase as observed for PLK1? And what about the kinetochore levels of Bub1 and BubR1?

Response: We have demonstrated previously that BUB1/BUBR1 increase in this situation in line with the pMELTs (Nijenhuis et al 2014;l Smith et al, 2019) – these papers will be referenced in relation to this. We will also address the effect of phosphatase removal on MPS1 activity, in response to comments by reviewers 1 and 2.

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A. The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). B. Though this is not stated in the MS

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<li style="font-weight: 400;">A. The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). <li style="font-weight: 400;">B. Though this is not stated in the MS

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Points of Critique

I would urge them to reposition as a descriptive study rather than making too many grand statements about drug sensitivities.### Other Comments

Van Alphen et al. describe phosphotyrosine proteomic profiling of a panel of AML cell lines and two patient-derived AML samples. Subsequent analyses attempt to identify potentially targetable kinases and pathways that would be considered vulnerabilities for drug treatments. Some hypotheses from these analyses are tested by drug treatment. The patient-derived samples are analyzed as a proof of concept and compared to the cell line profiles.

I think that this study is a potentially valuable resource, but might be served better if positioned more as a catalog of pY signaling in AML and less as a drug-targeting effort. The analysis graphs and charts are quite handy, and perhaps they could be served on a more interactive website that could be expanded in the future as the authors continue similar studies. However, I find that many of the conclusions are overstated and that some of the internal logic is inconsistent. Further, while the bioinformatics analyses are carefully planned and well intentioned, I was confused by the inconsistent quantitative metrics used in different parts of the manuscript and curious why a more modern isobaric labeling technique wasn’t used to compare among this relatively small panel of cell lines. Below I offer several points that could be addressed to help to improve this manuscript.

1. The authors claim that sensitivity to drugs predicted by their inferred kinase activity metrics “validates” their predictions. However, all of the drugs tested have demonstrable polypharmacology. How can they be sure the targets being hit that cause loss in viability are the same ones that they have predicted? Also, it seems curious that they only tested quizartinib in predicted FLT3-GoF lines and ponatanib in inferred FGFR-GoF lines. How do we know that these drugs just don’t kill all lines? It would be more convincing to show some lines where these drugs did not cause loss in viability.

2. Along these same lines, phosphoproteomics seems like a long path to identify vulnerabilities in cancer cell lines. Screening drugs on cell lines is cheaper and easier. Indeed, the CTD2 project has a drug screening arm (as did CCLE), and new Cancer Dependency Map screening is enlarging these screens. These projects also have more comprehensive genetic characterization of the cell lines involved. If the logic is that the drug treatments “validate” the predictions of aberrant kinase activity, couldn’t the drug screening be used to make these predictions, to be later validated by phosphoproteomics? Perhaps screen all TKIs against the AML cell lines and see what common targets emerge?

3. I think that claiming that the patient results match the cell line results is a bit of selective interpretation. The first thing I was drawn to is the whopping amounts of MAPK14 phosphorylation identified by their analysis in these samples. MAPK14 - a.k.a. p38 MAPK alpha - also has drugs that target it. If you were making a therapeutic hypothesis, wouldn’t you start with a p38 inhibitor rather than a FLT3 inhibitor? You already knew that the patients had FLT3-ITD, so you’d probably be starting there anyway. While MAPK14 is found to be phosphorylated in the cell lines as well, the degree to which it rises to the top in the patient samples is striking. This also illustrates an issue with drawing inferences from cell lines to real patient samples.

4. On p.15, you state: “P15: “Kinase activity ranking analysis of the FLT3-ITD mutant cell lines MV4-11, MOLM-13, and Kasumi-6, and the V617F JAK2 cell line HEL showed a lower ranking of FLT3 and JAK phosphorylation than expected based on their mutation status, compared to other kinases (position 6-10). Interestingly, other high-ranking kinases in these cell lines were generally located downstream in the FLT3 and JAK2 cellular signaling hierarchy, thereby still implicating FLT3 and JAK2 as primary suspects of driver activity.”

Perhaps this demonstrates the limitations of the approach? Genetics says FLT3 and/or JAK2 are mutated, proxy measurements say its activated, but the way you are estimating direct activity is not so great? Would a targeted panel on activation loop sites be better?

1. Figure 6 is an interesting analysis but how it was generated is unclear. Again, polypharmacology of the drugs make it hard to interpret. Are the graphs for all potential targets? Just some? Weighted by in vitro IC50 concentrations and/or binding affinities?

Minor points:

IC50 is inappropriate nomenclature here, which describes the concentration at which 50% of an enzyme’s activity is inhibited. The authors should substitute EC50 throughout, as they are referring to the concentration at which viability is decreased by 50%.

Ibrutinib is described as a pan-KI - this is confusing and misleading. It is a pretty specific BTK inhibitor.

Please update sup table 5 to show the exact nature of the mutations. Also, why does Kasumi-6 have two different (presumably) allelic ratios for FLT3 (presumed ITD?)?

Methods - p7 “as described elsewhere” - reference needed?

The statistical rationale is not well explained.

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Referee #1

Other Comments

This manuscript describes phosphotyrosine-focused phosphoproteomics for 16 AML cell lines to obtain molecular profiles of pY towards personal therapy using proper molecular targeting drugs. This is the revised (re-submitted) version and the authors added new data analysis especially on the relationship between kinase-ranking parameters and drug IC50 profiles to kinases. These results indicate the current progress and limitation of phosphoproteomics combined with genomics data and the related computational tools. Overall, the precise descriptions of the experimental procedures as well as the high quality of the experimental datasets are quite useful for researchers in this field. This manuscript should be published after some revisions shown below:

(1) This research group just published a paper on kinase ranking using phosphoproteome datasets, named INKA. Mol Syst Biol. 2019 Apr 12;15(4):e8250. doi: 10.15252/msb.20188250 INKA, an integrative data analysis pipeline for phosphoproteomic inference of active kinases.

INKA seems quite similar to the approaches in this manuscript. The authors should mention about INKA. Especially the parameters in Figure 6 should be described clearly whether these are the same in INKA or not.

(2) Figure 1 as well as Abstract and the first section of RESULTS: the numbers of phosphotyrosine sites and phosphotyrosine peptides should also be described in addition to the current description.

(3) Figure 2: the color for mutation is overlapped with colors for the heatmap. To avoid the misunderstanding, the authors should use the different colors.

they are all wrong.

Reviewer comments

Author rebuttal

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Nice