Genome-wide CRISPR screens identify novel regulators of wild-type and mutant p53 stability
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Abstract
Tumour suppressor p53 ( TP53 ) is the most frequently mutated gene in cancer. Several hotspot p53 mutants not only lose tumour suppressive capabilities, but also function in a dominant-negative manner, suppressing canonical wild-type p53 function. Furthermore, some hotspot p53 mutants promote oncogenesis by gain-of-function mechanisms. Levels of p53 are regulated predominantly through regulation of protein stability and while wild-type p53 is normally kept at very low levels at steady-state, p53 mutants are often stabilized in tumours, which may be vital for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. We found that most proteins that regulate wild-type p53 also regulate a subset of p53 mutants with the exception of p53 R337H regulators, which are largely private to this mutant. Mechanistically, we identified FBXO42 as a novel positive regulator of a subset of p53 mutants comprising R273H, R248Q and R248W. We show that FBXO42 acts together with CCDC6 to regulate USP28-mediated p53 stabilization. Our work also identifies C16orf72 as a negative regulator of the stability of wild-type p53 and of all p53 mutants tested. C16orf72 is amplified in breast cancer, and we show that C16orf72 regulates p53 levels in mammary epithelium of mice and its overexpression results in accelerated breast cancer with reduced p53 levels. Together, this work provides a network view of the processes that regulate p53 stability, which might provide clues for reinforcing wild-type p53 or targeting mutant p53 in cancer.
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Reply to the reviewers
Reviewer #1.
Reviewer #1 summary:
In this manuscript by Lu et al., the authors describe some CRISPR screens and protein-protein interaction screens to identify novel regulators of wild-type p53 and mutant p53 function and stability. Besides generating a wealth of data, they discover FBXO42-CCDC6 as positive regulators of the some p53 hot-spot mutants, including R273H mutant p53, but not of all p53 mutants tested and also not of wild-type, indicating selectivity. Furthermore, the found C16orf72(TAPR1) as a negative regulator of p53 stability.
Mechanistically, the authors claim a direct interaction between FBXO42 and CCDC6 and p53, but the importance of …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1.
Reviewer #1 summary:
In this manuscript by Lu et al., the authors describe some CRISPR screens and protein-protein interaction screens to identify novel regulators of wild-type p53 and mutant p53 function and stability. Besides generating a wealth of data, they discover FBXO42-CCDC6 as positive regulators of the some p53 hot-spot mutants, including R273H mutant p53, but not of all p53 mutants tested and also not of wild-type, indicating selectivity. Furthermore, the found C16orf72(TAPR1) as a negative regulator of p53 stability.
Mechanistically, the authors claim a direct interaction between FBXO42 and CCDC6 and p53, but the importance of these interactions has not been shown. On the other hand the authors suggest that the FBXO42/CCDC6 regulate p53 via destabilization of USP28, but also the mechanism has not been worked out. For C16orf72, they show that it interacts with USP7, but no relevance of this interaction is shown either.
Response: We sincerely thank the reviewer for the constructive and thorough review. We have incorporated most of the suggestions into our planned revision, with our major focus on the molecular mechanistic follow-up.
Reviewer #1, major points.
One very important point for me is that the authors do not show the levels of expression of p53 in the p53-mClover stable cell lines. It is known that overexpressed p53 is usualy more stable than endogenous levels of wt-p53. Therefore, I think it is necessary that the authors show the levels of the p53-mClover fusion proteins in the stably transduced cell lines compared to endogenous p53 levels in the parental RPE1 cells and also compared to the endogenous levels of R273H mutant in the PANC-1 cells.
Response: We fully agree that the levels of overexpressed p53s are often more than the endogenous ones, due in part to increased expression and stability. In designing the reporter, we first tried to avoid the stabilisation of p53-GFP due to GFP aggregation by using the monomeric mClover-variant. Further, we titrated the WT and R273H clones (similar to our recent work in PMID: 35439056), to select clones with p53 levels closer to endogenous protein, and exhibiting high dynamic response to Nutlin-3a treatment.
In the revised submission, we will include Western blotting comparing the levels of p53-mClover (WT and R273H) expression to the endogenous p53s in RPE1 (WT) and PANC1 (R273H) cell lines, in the presence or absence of Nutlin-3a.
Also the functionality of the wild-type p53-mClover fusion is questionable, at least not shown. One would expect that the overexpression of a functional wt-p53 in p53-KO cells will affect the survival of the RPE1 cells. In Figure 5A the authors show that depletion of MDM2 or C16ORF72 is toxic for the RPE1 cells in a p53-dependent manner, indicating that elevated levels of p53 cannot be handled by these cells. So, experiment(s) showing that the wt-p53/mClover fusion is functional is needed.
Response: We agree that it will be an important point to benchmark the reporter design. The ectopically expressed WTp53 is often observed to have reduced functionality compared to the endogenous WTp53. The WTp53-reporter line behaves similarly to the RPE1 line (p53-proficient), where both chemical (e.g. Nutlin) or genetic perturbation (e.g. depletion of MDM2/C16orf72) would be toxic in a p53-depedent manner. In line with this data, we have observed that the WTp53-reporter line is able to induce a p53 response as demonstrated by induction of p53-target genes such as p21, which is not observed in p53 null RPE cells, albeit the p21 induction is not as dramatic as in RPE1 cells with endogenous WTp53. Together, these data indicate that our WTp53-reporter is functional albeit with a somewhat reduced activity.
In the revised submission, we will better demonstrate the functionality of the WTp53-mClover fusion by probing WTp53 target (e.g. p21), in the presence and absence of Nutlin. This is also performed as a part of the experiment addressing Point #1 above.
A second important point is that the 'verification' of the hits from the screens is only done in one cancer cell line, PANC-1, with mutant p53. I would have like to see at least one other cell line with another p53 mutant endogenously expressed that is also regulated by FBXO42/CCDC6.
Response: we will include validation of the hits (FBXO42, CCDC6) in other 1-2 tumour lines with confirmed R273H endogenous mutation (e.g. MB-MDA-468, etc).
For many of the p53-mutants, a bimodal expression is observed. In the FBXO42- and CCDC6-depleted cells, the equilibrium shifts towards more negative cells but the levels in the two populations itself don’t change (while for example for USP28 depletion also the right peak shifts further up, Fig S4E). Is there any correlation with the cell cycle and p53 expression? And can the authors exclude that FBXO42 and CCDC6 are involved in cell cycle progression and hereby influence p53 indirectly (by combining PI staining with Clover-p53 for example).
Response: we have indeed observed that the “bimodal” levels in the reporters of several mutants, which are also observed in other studies probing the endogenous p53 level (PMID: 29653964); while the population equilibrium shifts, the location of each peak (as a proxy of the level of p53s) are more stable.
Regarding the relation between p53-level and cell cycle stage, indeed, both the authors in the paper above and we have probed this possibility, but were unable to establish a direct connection.
In the revised submission, we will add flow cytometry analysis of the p53-mClover level, and the cell cycle position using Hoechst 33342 (live-cell permeable DNA staining).
The authors claim that the FBXO42-CCDC6 axis regulates stability specifically some p53-mutants, including R273H-mutant, in a manner involving USP28. But USP28 regulates all forms of p53, not just some mutants version. How can the authors reconcile this apparent contradiction?
Response: we thank the reviewer for this critical observation. From our screen (Supplemental Table 1A), we have indeed noticed a pronounced effects (|Z score| >=3) of FBXO42 on R273H and R248Q stability, and a marginal effect on wild-type p53. Similarly, USP28 had pronounced effects on R273H and R248Q and WTp53.
In the discussion of the paper, we noted that USP28 was shown to regulate p53 levels through distinct mechanisms:
‘USP28 was originally implicated as a protective deubiquitinating enzyme counteracting the proteasomal degradation of p53, TP53BP1, CHCK2, and additional proteins68-71. USP28 regulates wild-type p53 via TP53BP1-dependent and -independent mechanisms. Concordantly, our data shows that USP28 and TP53BP1 are strong positive regulators of wild-type p53. However, while USP28 was also a strong hit in the mutant R273H p53 screen, TP53BP1 was not, indicating that the effects we see upon loss of USP28 on R273H p53 are independent of TP53BP1.’
Together, this indicates that the R273H-mutant is regulated by a FBXO42-CCDC6-USP28 axis while wild-type p53 is regulated mainly via a USP28-TP53BP1 axis. We will attempt to address and discuss it in the revision.
On a similar note, the authors show that FBXO42 and CCDC6 interact with p53, but not USP28. Do FBXO42 and CCDC6 interact with each other and with USP28? And is the interaction with p53 specific for the R273H version? This part of the mechanism is very poorly defined and the Co-IPs are not very convincing or relevant for the proposed model.
Response: This comment will be more extensively addressed in the revision. We have indeed observed the interaction between FBXO42 and CCDC6 (via BioID and APMS); however, we failed to recover USP28 as an interactor of either FBXO42 or CCDC6. The interaction between CCDC6/FBXO42 is not specific to R273H; although we were able to IP endogenous R273H with CCDC6 in PANC-1 line, the WTp53 (as in HEK 293 TRex BioID line) was also picked up in the BioID preys of CCDC6/FBXO42. In addition, we have new data to show that FBXO42 directly interacts with WTp53.
In the revised submission, we will improve the molecular underpinning of the FBXO42-CCDC6-USP28-p53 axis we propose. We will specifically address the following.
(1.1.) Biochemically, further support that CCDC6 and FBXO42 regulate p53 via regulating USP28 stability: We will address this by established biochemical assays, e.g. cycloheximide-chase/MG132 experiment. While USP28 is an established WTp53 regulator, little is known about the mechanism, and the “upstream” regulation of USP28; we will attempt to fill this gap:
(1.2.) And to an unbiased systematic approach, how R273H interactome changes upon the loss of CCDC6 or FBXO42.
We will perform R273H-BioID upon loss of CCDC6 and FBXO42 and USP28.
(1.3.) Furthermore, we will specifically exam the interaction of USP28-p53R273H with or without the genetic perturbation of FBXO42/CCDC6.
Through these efforts, we hope to gain further mechanistic insights into this regulatory axis, but hope that the editors and reviewers will agree that a fully annotated mechanistic understanding is probably beyond the scope of this paper.
Reviewer #1, minor points.
The mechanisms of p53 regulation may vary greatly in different cell lines. Can the authors discuss why they choose to do the screen with different mutants, rather than with different cell lines expressing these same mutant endogenously?
Response: While it is certainly very interesting to assess how WT and mutant p53 is regulated in different cell lines, such an approach is confounded by the ‘genetic make-up’ of the respective tested cell lines. For example, TP53BP1 might be a regulator in one cell line but not in another for the simple reason that the later cell line harbors a TP53BP1 deletion or mutation or expression levels. In addition, while working with endogenous p53 mutations certainly has many advantages, comparing different mutants in different cell lines is again very much confounded by the ‘genetic make-up’ of the respective tested cell lines.
Our focus was slightly different, and we wanted to set out and specifically ask what the difference between p53 hotspot mutations are. Are they all the same or are there differences and importantly, are there differences between mutants and WT p53 and this can only be achieved when working in the same cellular background. In designing the screen, we have thus tried to optimise the inclusion of different hotspot mutants in an isogenic screening system. As such, we first depleted the endogenous WTp53 to minimise its interference and built the current isogenic system in the non-transformed RPE1 (“normal”) line.
However, as discussed above, we agree that the screen results will be validated in more cell lines carrying respective endogenous mutants.
Figure 1: Typo in the legends : Nultin ipv Nutlin
Response: We apologise for the typos. This is addressed in the current submission, along with improved figure legends to improve readability.
Figure 1b,1c : Show basal and Nutlin-3 induced MDM2 levels and in the overexpression cell lines; if WT-p53 is functional, MDM2 levels should be higher in WT-transduced cells compared to control or mt-p53 expressing cells.
Response: In the revised submission, we will include Western blotting probing MDM2 levels (antibody permitting); this is a part of the experiment proposed for Points 1 and 2.
Authors should explain which they name USP7 a negative regulator of p53, since it is supposed to de-ubiquitinate p53?!
Response: The effects of USP7 on WTp53 have indeed been difficult to elucidate (by Prof. Vogelstein PMID: 15118411, and PMID: 15058298, and seemingly opposite by Prof. Gu Wei, PMID: 15053880, and PMID: 11923872). However, consistent with Prof. Vogelstein group, the inhibition of USP7 (either by inhibitor or genetically via CRISPR in our studies), has resulted in elevated p53 level.
Figure 2E: the effect of MG132 on p53 seems to be very minimal on this Western blot; it would need quantification to be convincing...Quality of the blot is also not great.The fact that in control cells the levels of p53 R273H are not affected by MG132 treatment fits with Suppl Figure 2E, indicating that the proteasome has no effect on p53 R273H.
Response: We indeed noticed that while the proteasome pathway is largely implicated in the WTp53 screen, it has much reduced effects on R273H. Interestingly, the treatment of MG 132 also has limited effects using PANC-1 line (with endogenous R273H). We will repeat this experiment and provide quantifications and modify the text accordingly.
Suppl figure 3b, 3c, 3d:
Somehow, I have the feeling that the results from the western blots and the FACS do not match fully, although not all the time-points are shown in the various experiments.
For example, the FACS analysis (3b) suggests that in control-transduced cells after 16hr p53 is still increased. However, that is not clear at all in the Western blot (3c)
Is Suppl Figure 3d the quantification of 3c experiment? If so, in the blot also the 24 hrs should be shown.
The blot shown in Suppl Figure 3c suggests that CCDC6 expression increased upon irradiation. Do the authors agree with that? Would that explain why depletion of CCDC6 has more effect upon irradiation?
Suppl Figure S3E: if I am right, this is essentially the same type of experiment as shown in figure 2e, but analysis of p53-expression by Western blot. In that blot no real effect of MG132 on p53 levels could be seen. But here, in the FACS analysis, MG132 clearly increases the p53-Clover fusion levels; for me again that Western blot and FACS data do not neccesarily match.
Response: We apologise for the confusion. In the revised submission, we will improve the figure legends for better readability. Furthermore, in anticipation to the multiple cell lines involved in the revision, we will also clarify the cell lines in the figure.
With regards to the difference between the flow cytometry and WB data, we have generally observed the flow cytometry bimodal shifting to be more sensitive than the WB, e.g. a 50% shift in population (FACS) is reflected by a 15% reduction in WB (which may be partially explained as WB is a measurement across the cell population and FACS determines the p53-GFP levels of every cell and thus the shift of cells between peaks). Similarly, we noticed flow-cytometry based quantification by antibody staining the endogenous p53 yielded similar sensitivity (PMID: 29653964). As such, we will ensure the validation of hits is performed in two modes. For WB experiment, we will do so in two cell lines carrying the endogenous mutants as suggested by Reviewers #1 and 2.
Figure 3B: In the CCDC6 IP a very small amount of p53 can be found. I don't know how much input lysate compared to amount of lysate for IP is used, but the percentage of p53 found interacting with CCDC6 seems so marginal that is difficult to explain the effect of KO of CCDC6 in PANC1 cells.
And, the authors called it a 'reciprocal IP' (Suppl Figure 4a) after transfection of V5-tagged CCDC6 into PANC1 cells, but it actually is the same type of IP. Did the authors try to IP p53 and blot for CCDC6? That would be a reciprocal IP.
Response: We apologise for the confusion. In the revised submission, we will specify the portion of the lysates used for pre-IP (5% lysate) and IP (1 mg). As for the IP, we will also include the true reciprocal IP (IP p53, and blot for CCDC6).
Figure 3H: how can authors explain that basal levels of USP28 in control and CCDC6-KO cells transfected with control plasmid are more or less the same and not reduced in the CCDC6-KO cells?
Response: We will provide a better blot and quantification for this observation. In the current Fig 3H, the CCDC6-KO lane is slightly overladed as seen by the H3 loading control.
Figure 3I: Essentially the whole blot here is of low quality; especially the FBXO42 blot; is deletion of USP28 increasing FBXO42 protein levels, or is it just the quality of the blot? All in all it seems that FBXO42 is very low expressed in the used cell lines.
Response: We apologise for the confusion. In the revised submission, we will repeat and try to include higher quality WB, with more optimised condition for using the FBXO42 antibody.
FBXO42 messenger level is readily detected using qRT.
Figure 4B: I find it a bit surprising that USP7 is also found in the synthetic viability screen, since it has been shown that USP7 has many more essential targets and KO of p53 only partially rescues the development of USP7-KO mouse embryo's.
Response: We thank the reviewer for this critical observation. While the double p53-USP7 knockout line is viable, we acknowledge that it is amongst the top scored hits due to the large differential viabilities between WT and p53-null lines. In the revised submission, we will further clarify the screen analysis and the associated interpretation.
Figure 5: the authors nowhere show the efficacy of the guides targeting c16orf72. A Western blot showing the expression and the reduction upon expressing the guide-RNAs is essential.
Response: We thank the Reviewer for this suggestion. The efficacy of each guide has been verified using ICE (at the genomic level), and in the revised submission, we will include this critical information as part of the Figure S2F.
Figure 5E: First, here probably parental RPE1 cells have been used, but that is not stated. Second, the authors state 'only a slight increase in p53 levels upon siHUWE1'; I would say none compared to scrambled.
I know HUWE1 is a very huge protein, but the blot of HUWE1 is not convincing. I seem to be able to conclude that siMDM2 and siUSP7 reduces HUWE1 levels?
Response: We apologise for the confusion. In the revised submission, we will be specific of the cell line information on the figure, to improve the readability.
We agree with the reviewers that assessment of large protein by WB is often difficult but given that this band almost completely disappears upon HUWE1 knock-down, strongly argues that we are indeed assessing the endogenous HUWE1. We also agree that it is an interesting observation that the levels of HUWE1 seem to be slightly reduced upon knock-down of MDM2 and USP7. We will repeat this experiments and provide quantitative data for HUWE1 and p53. Of note, in the screen, HUWE1 also scored as a negative regulator of wt-p53 and did not quite reach statistical significance for the p53 mutants.
Regarding the relationship between C16orf72 and HUWE1, a newly published work (PMID: 35776542) seems to suggest that siHUWE1 has resulted in an increased C16orf72 level (termed HAPSTR1 in the paper), while siC16orf72 seemed to have no effect on HUWE1 level, although the stability of such a large protein by WB is often difficult to conclude.
Figure 5F, in relation to figure 5D. Here the author overexpress both c16orf72 and USP7, and find an interaction. The implication of that is not clear. If they want to make point of this interaction, they should have looked at endogenous proteins.
Response: We acknowledge the many concerns associated with coIP with ectopically, and especially overexpressed proteins in large quantity. In the revised submission, we will attempt to perform endogenous-based IP experiment (antibody permitting).
It is worrying that USP7 apparently was not one of the hits in the Mass-spec experiment of which results are shown in Figure 5D. Also in that experiment c16orf72 was overexpressed, and USP7 is very highly expressed in essentially all cell lines, so do the authors have an explanation?
Response: We indeed acknowledge this discrepancy. In the revised submission, we will attempt the coIP/IP using endogenous proteins (antibody permitting, or at least using endogenous target for one of the two partners). We also acknowledge that the limitation associated with the APMS for the detection of interactors.
Suppl. figure 5D is missing
Response: We apologise for the confusion. The Figure S5D was inconveniently placed at the top of the figure panel due to space limitation. In the revised submission, we will address this as a part of the overall readability improvement.
Reviewer #1, Significance.
The topic of the paper is of high interest given the relevance of p53 and its gain-of-function mutants in oncology, and the screens are well executed and clearly presented. In terms of novelty, FBXO42 has been linked to p53-degradation before, and c16orf72 was recently shown to be able to destabilize p53. However, the link between CCDC6 and p53 is novel and of interest, since they are both substrates of USP7 and are both regulators of the cell cycle.
We think the manuscript has potential to add something to the field, but would benefit greatly from a better understanding of the molecular underpinnings of their newly described mechanisms, as well as the conditions in which the mechanism is active.
Therefore, it might be advisable to shorten the manuscript, and go more in-depth in finding the mechanisms of regulation.
Response: We sincerely thank the reviewer for all the constructive critiques. We will incorporate them in to our revision.
Reviewer #2.
Reviewer #2 summary:
The paper describes several genome-wide CRISPR screens designed to identify regulators of p53 stability. The authors use a system in which p53 levels are marked by mClover expression, using RFP expression to normalise for gene expression changes.
Reviewer #2, major points.
The bimodal distribution of p53 expression levels in some reporter cell lines (G245S, R248Q, R248W and R273H) hampers the implementation of a robust readout and makes correct interpretation of the results challenging. While it is possible that the bimodal distribution indicates dynamic changes in p53 levels within one population, it also seems possible that a subclone of these cells have acquired additional alterations affecting p53 stability, and that the authors are screening a mixed population of two intrinsically different cell populations. This would make it difficult to interpret the results of the screen in these cell lines and may be a challenge when trying to identify something that has not already been highlighted on depmap.
Response: We thank the reviewer for this critical observation. We strongly believe that this bimodal distribution is actually an inherent property of the p53 mutants in these cells for the following reasons: (1) The observation of the similar bimodal appearance in cell lines harbouring corresponding endogenous mutant p53s (PMID: 29653964) suggest that these two populations are of biological significance. (2) We have established 5-10 clonal lines each from the G245S, R248Q, R248W and R273H p53 reporter line and all of them exhibit a bimodal distribution, making it very unlikely that these populations are all through stochastic outgrowth of sub-populations with spontaneous mutations/alterations. (3) The bimodal distribution is stable over several months to years in culture. If it were a spontaneous mutations giving rise to a clone with higher mutant p53 levels, we would likely expect that over time this clone takes over the population. (4) We observed that such a pool of bimodal cells could be “synchronised” (e.g. by Nutlin, or MDM2 knockout) to one population, and later return to and repopulate the other (e.g. Nutlin washoff, Figure 1B). (5) When we sort out a single cells from the upper or the lower peak, expand them, we obtain again populations of cells with the same bimodal distribution, indicating that this is a dynamic process. Thus, we believe that these two populations were rather intrinsic, such that a cell in the population may assume both states.
We also acknowledge the difficulties of screening using a bimodal population; however, we took advantage of these “bimodal” mutants and using FACS assessed the state of a single cell in relation to a genetic perturbation. Each guide has an equal chance of entering a cell that belongs to one of the two populations. If a gene knock-out really affects p53 levels, the cells with the respective guides enrich in one and deplete in the other population and the analysis comparing the guide abundances from these two peaks ensures the experiment are being perfectly internally controlled.
While many of the top scored hits from the resulting screens are known regulators, it is critical that we validate our hits in an independent system, such as the cell lines harbouring endogenous p53 mutations, echoed by both Reviewers #1 and 2.
The coverage of the sgRNA library (200x) is rather low for a negative selection screen, where a coverage of 500x would be more desirable. The FDR threshold is also rather lenient, a more stringent FDR threshold would seem more appropriate and shorten the list of potential hits.
Response: We thank the reviewer for this constructive suggestion. A higher coverage, along with a more stringent FDR, will ensure an even stronger confidence for the remaining individual hits. The present reporter-based enrichment screen and the synthetical viability drop-out screen used four guides per gene, and with 200x coverage for each guide.
In determining the coverage, we tried to reference recent successful screenings and apply earlier titration result for the 200x coverage (e.g. PMID: 26627737, PMID: 33465779, and reviewed in Nat Rev Methods Primers 2, 8 (2022). https://doi.org/10.1038/s43586-021-00093-4). While the threshold of FDR was often arbitrary, we fully agree that a more stringent FDR, which results in shortened hits list, may further boost the confidence of the hits, though also at the cost of losing potential hits due to collateral effects (e.g. guide efficiency).
We agree with this reviewer that a higher FDR, esp. at the hits that result in p53 stabilization, would make sense as any gene whose loss causes cellular or genotoxic stress, would likely lead at least in part to p53 stabilization. In the revised submission, we will adjust the FDR accordingly.
Although the study is focused on the regulation of p53 stability, there are no experiments to show that any of the manipulations alter the ubiquitination or degradation (half-life) of p53. The rescue of expression by proteasome inhibition is very modest (Figure 2E), suggesting the loss of expression may not be a reflection of degradation. A role for endogenous FBXO42 and C16orf72 in regulating the ubiquitination and half-life of endogenous p53 should be confirmed
Response: We thank the reviewer for this suggestion. In the revised submission, we will monitor the ubiquitination status and also degradation (cycloheximide-chase) experiments for R273H cells, with or without the genetic alteration of CCDC6/FBXO42/C16orf72.
Many p53 mutants are used for the initial screens, but very little validation is carried out to show that the apparent differences in factors regulating their stability persists in cells naturally expressing these mutants. For example, FBXO42 is identified as a protein required to maintain the stability of R273H, 248W and R248Q, but not R175H, G245S and R337H. While the authors show an association of CCDC6 and p53 in PANC1 cells (expressing 273H), it would be important to show a panel of R273H, 248W and R248Q expressing tumor cells and the response of p53 to FBXO42 and CCDC6 depletion, compared to similar experiments in a panel of R175H, G245S and R337H expressing tumor cells. Again, it would be important to show that any changes in protein levels are due to changes in protein stability.
Response: We thank the reviewer for this suggestion. In the revised submission, we will include validations in more cell lines carrying endogenous mutant p53s, with a focus on the R273H mutant. We will also try to involve a line with an endogenous p53 mutation that does not respond to FBXO42/CCDC6 alteration.
The potential hits should also be tested in wild type p53 expressing cells to confirm the specificity to mutant p53s.
Response: In the revised submission, we will include WB for WT lines (e.g. RPE1) upon genetic alteration of CCDC6 and FBXO42. This was already performed for C16orf72 (Figure 6D).
(6A) The role of C16orf72 in restraining p53 activity has been reported previously, as has the interaction with HUWE1 (including a new publication PMID: 35776542). The authors suggest an interaction between C16orf72 and USP7, although this should be shown with endogenous proteins. The relative importance of USP7 and HUWE1 binding is not explored. (6B) The effect of C16orf72 overexpression in promoting mammary tumors is impressive, although maybe the more interesting question is whether inhibition of C16orf72 expression can limit tumor development in this system.
Response to 6A: we are excited about the independent observations by other group(s) confirming similar results! As a part of our improvement for mechanistic work-up, in the revised submission, we will attempt to address, whether C16orf72’ regulation of p53 is dependent on USP7 and/or HUWE1, or other known E3s, such as MDM2.
(1) Whether the interaction of C16orf72 and HUWE1 or USP7 is required for the C16orf72 regulation of p53. Specifically, for example, we will perform epistasis experiments to test USP7’ or HUWE1’ ability to rescue the p53 levels in reporters upon ∆C16orf72. Due to the toxicity/lethality in WTp53 lines induced by the loss of C16orf72, we intend to test using R273H-reporter, or RPE1-line with ∆CDKN1A (p21) that is a synthetic viable rescue for ∆*C16orf72. *
(2) In the revised submission, we will attempt to perform endogenous-based C16orf72-USP7 IP experiment (antibody permitting).
6B. The effect of C16orf72 overexpression in promoting mammary tumors is impressive, although maybe the more interesting question is whether inhibition of C16orf72 expression can limit tumor development in this system.
Response: We are also equally excited about the in vivo result supporting the idea that C16orf72 overexpression in tumour-prone mice (Pik3caH1047R) mice harbouring WTp53 may accelerate tumour formations. In the revised submission, we will further support that this effect is specific to WTp53/C16orf72, by including data of the control cohort with p53-null background (LSL-Pi3kH1047R; p53Flox/Flox).
In regard to the effects of C16orf72-depletion in controlling tumour growth - we agree that this would be a very exciting avenue. Conditional C16orf72 mice are being made at the moment and these mice will allow us to comprehensively address this question. However, it will take several more month to generate and validate this line, and then another 2 breeding rounds to generate homozygous C16orf72fl/fl; Pik3caH1047R mice. In addition, the long time required to form tumours in the control mice with WTp53 (~250 days), it becomes not feasible for us to test whether the inhibition of C16orf72 could limit the tumour development, given the revision timeline. As such we respectfully believe that this would be beyond the scope of this manuscript.
Reviewer #2, Minor comments.
Figure 1b: The nutlin concentration stated in the methods section is wrong. Should be 10 µM instead of 10 nM (correct in figure legend).
Figure 6b: y-axis label is missing.
Figure 1e/f Legend: Should be FDR 0.5.
Response: We apologise for typos. The current submission has incorporated the corrections.
Figure 1c: Include results for a mutant that is not regulated by MDM2, such as R175H. Otherwise, as a standalone experiment, this figure doesn't add much.
Response: We thank the reviewer for this suggestion. In the revised submission, we will include R175H/R337H.
Figure 1h: While an UpSet plot is an elegant way to present unique and overlapping hits between different screens, Venn diagrams might be more 'accessible' to many readers and easier to understand.
Response: We thank the reviewer for this feedback. The choice of UpSet blot was largely motivated by the different categories involved, which made the area representation and the intersection of the conventional Venn diagram no longer feasible.
In the revised submission, we will improve our figure legend for the UpSet blot, to improve the readability.
Might be worth stating that mClover is an eGFP variant and can therefore be targeted by eGFP sgRNAs so that it is easier to understand the following:
o Page 5, paragraph 1: "We used the TKOv3 sgRNA library, which contains [...] 142 control sgRNAs targeting EGFP, LacZ and luciferase"
o Page 5, paragraph 2: "As expected, sgRNAs targeting p53 and mClover were the most depleted sgRNAs, [...]
Response: We thank the reviewer for this suggestion. We believe this will also improve the readability and have incorporated this into our current submission.
Reviewer #2, Significance.
Reviewer #2 (Significance (Required)):
This is an interesting concept and the results could provide a useful resource for groups interested in the regulation of p53. The authors chose to focus on candidate genes that could have been identified by looking for the top 30 p53 co-dependent genes on depmap (C16orf72 is #24 in this list and FBXO42 is #28, most of the other genes ranking above are already known as p53 regulators). While this validates the screen, it would have been interesting if the authors had identified and validated new regulators of p53 that were not apparent from previously published work.
Response: We thank the reviewer for all the thorough and constructive comments! In relation to the DepMap dataset, we are excited that many of the top hits from our screens are indeed top WTp53-correlators/anti-correlators (e.g. MDM2, USP28)!
While the DepMap dataset used cell fitness/viability to construct the genetic relation score, this assay may not effectively rule out the many regulators that could otherwise elicit their regulation of p53 via regulating the general cell response to cell cycle, stress, etc. In our screen systems (i.e. protein stability and synthetic viability screens), we attempted to focus on the regulators of p53-stability (post-translational), and further coupled it with the synthetic viability screens to concentrate on hits that have a more direct role in p53 regulation (e.g. MDM2, C16orf72).
One other difficulty to fully couple our screens to the DepMap dataset is due to the limited cell lines harbouring endogenous mutant p53s, e.g. R337H. This may also contribute to the uniqueness of the identified R337H-reporter specific hits (where cell lines harbouring R337H have not yet been included in the DepMap dataset), e.g. several Aminoacyl tRNA synthetases (SARS, YARS, etc) were identified as R337H unique regulators and subsequently verified using different guides in the reporter line, but could not be obtained via DepMap.
We largely see this paper as a resource for the p53 field and would like to publish it as soon as possible. In fact, when we started working on C16orf72 or CCDC6/FBXO42, these hits were not known for their ability to regulate p53. We will work up several other hits, but this would be beyond the scope of this paper and the first author’s Ph.D. thesis that needs to be completed under a timeline.
Reviewer #3.
Reviewer #3 summary:
The manuscript by Lu and coworkers performed genome wide CRISPR screens to search for genes that when knocked out, lead to p53 accumulation or degradation. Wt p53 and a panel of p53 hotspot mutants were chosen as reporter for the screen. The approach reassuringly identified many previously described regulators of p53 degradation, and also found a large set of new hits that many appear to be indirectly affecting p53 level.
A key step of this approach is the follow up functional and mechanistic study of the hits. To this end, the authors chose FBXO42 as a top hit that blocks mutant p53 degradation, and C16orf72 as a top hit that promotes wt/mutant p53 degradation.
Overall the functional data for FBXO42 is disappointing. FBXO42 knockout has quite modest effect on mutant p53 level (~50% reduction). The knockout also showed some effect on p53 mRNA level (~25% reduction), making the determination of mechanism difficult. It does not appear to be a promising targeting for reducing mutant p53 level and gain of function activity in tumor cells.
We thank the reviewer for this constructive comment! We will address this in the revision, as proposed in Point #3.
The C16orf72 finding unfortunately lost some novelty because it was independently identified as a p53 regulator in a recent study using CRISPR screening (PMID: 33660365). However, the repeated identification is reassuring and the current work provides more convincing functional data, showing C16orf72 knockout increase wt p53 level, inhibits cell proliferation specifically in p53+/+ cells, and overexpression of C16orf72 reduce wt p53 level and accelerates progression of a breast tumor mouse model. Their results suggest C16orf72 is a biologically relevant regulator of p53 in cancer development. In order to provide a reasonable amount of new information and set it further apart from the published study, some biochemical analysis looking into the mechanism of C16orf72 will be helpful.
Reviewer #3 Major and Minor comments:
Specific comments:
There appears to be a mix up in the figure legend for Fig.1A describing line 1 and 2.
Response: We sincerely apologise for the mix up in the figure legend! In the current submission, this has been fixed.
Fig.2. Data for some p53 mutants mentioned in the text cannot be found in the main figure 2D and supplemental figure S3A.
Response: We apologise for having not included the R175H and R337H mutants in Supplemental Figure S3A. In the revised version, we will include these two mutants.
Fig.2 E-F. The effects of FBXO42 and CCDC6 KO on endogenous mutant p53 level is small (~50% decrease). Given that mutant p53 accumulates at high levels, whether a 50% decrease has meaningful effect on its gain of function activities is questionable. The knockouts also caused a ~25% decrease in p53 mRNA (FigS3F) which makes the mechanism quite difficult to investigate further.
Response: We agree with the reviewer that the current data makes it difficult to conclude the mechanism. Given the design of our reporter, we still believe that the regulations could largely be at the post-translational level. In our revised version, we plan to exam the ubiquitination status of p53 upon losses of CCDC6/FBXO42, and also monitor the p53 degradation via cycloheximide chase.
To further address whether this reduced level of mutp53 has biological impacts, we plan to test it in the tumour cell context. Given the difference in migration capability observed between PANC-1 and PANC-1-∆p53 line (e.g. PMID: 35439056), we plan to also evaluate the migration pattern of PANC-1, with the presence and absence of FBXO42/CCDC6 (controlled by similar FBXO42/CCDC6 loss in PANC-1- ∆p53 background). Furthermore, in tissue culture, although there is only marginal to no difference in cell growth rate between many mutant p53 lines (e.g. PANC-1) and their ∆p53 line, we plan to test whether a reduced serum or nutrient level could exacerbate the difference, and hence further be used to monitor the difference resulted from the loss of FBXO42/CCDC6.
Fig.3B. The IP experiment using p53 shRNA and control shRNA should be done by IP of p53 followed by CCDC6 western blot. If CCDC6 IP is used as in the figure, then a CCDC6 shRNA knockdown sample should be compared to control shRNA. The current data does not rule out the possibility that CCDC6 antibody can nonspecifically pull down some p53.
Response: We apologise for the confusion. In the revised version, we will include the proper reciprocal IP, with IP of endogenous p53 (R273H) followed by blotting of CCDC6.
Fig.3D. The in vitro pull down experiment needs specificity controls such as non affected R175H p53 core domain. The data presented would suggest that MBP-FBXO42c captured more than 1:1 molar ratio of R273H core domain, which is unusual for specific binding unless there is aggregation of p53.
Response: We thank the reviewer for this constructive comment! In the revised version, we will incorporate this, by repeating the in vitro pull-down assay including a non-p53 control protein.
To increase the impact of the current study, the authors could provide more mechanism insight on how C16orf72 regulates p53 level, which was also missing in the other published study. For example, addressing whether C16orf72 effect is dependent on MDM2. Does it cooperate with MDM2 to ubiquitinate p53. Does it promote p53 ubiquitination in the absence of MDM2, since it interacts with HUWE1. Does it act by recruiting usp7 to stabilize MDM2.
Response: we thank the reviewer for this very constructive and thorough comment! In our revised version, we will attempt these assays and incorporate them into the submission.
Together with our response to Reviewer #2, Point #6, in the revised submission, we will attempt to address if C16orf72 regulation of p53 is dependent on MDM2 or HUWE1.
(1) Whether the interaction of C16orf72 and HUWE1, or C16orf72 and USP7 is required for the C16orf72 regulation of p53. Specifically, for example, we will perform epistasis experiments to test HUWE1’ or USP7’s ability to rescue the p53 levels in reporters upon the loss of C16orf72 (∆C16orf72). Due to the toxicity/lethality in WTp53 lines induced by the loss of C16orf72, we intend to test using the R273H-reporter, or RPE1-line with ∆CDKN1A (p21) that is a synthetic viable rescue for ∆*C16orf72. *
(2) Whether C16orf72 dependent upon or cooperate with MDM2 in regulating p53.
We will first probe whether C16orf72 overexpression increased the p53 ubiquitination, and then decide whether overexpression of C16orf72 has additive effects to MDM2 overexpression in regulating p53 levels.
We previously observed that overexpressing C16orf72 could not rescue the R273H level resulted from losing MDM2 (using flow-cytometry in R273H-reporter-∆MDM2), and as such, we plan to test the C16orf72-MDM2 relation in the MDM2-proficient context.
The manuscript is in a form extremely unfriendly to review, text, figures and legends are all split up at multiple locations, the pdf figures are very sluggish to scroll.
Response: We sincerely apologise for the inconvenience. In the current submission, we have split the submission into three separate files, (1) main text, (2) main figures, and (3) supplemental figures, along with (4) supplemental tables as individual EXCELs. We will also reduce the resolution of a few images, so the overall higher resolution is retained, while still fitting into the file size limit.
Reviewer #3 (Significance (Required)):
The work is significant in identifying a functionally relevant regulator of p53 stability.
Response: we thank the reviewer again for the very constructive feedback!
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Referee #3
Evidence, reproducibility and clarity
The manuscript by Lu and coworkers performed genome wide CRISPR screens to search for genes that when knocked out, lead to p53 accumulation or degradation. Wt p53 and a panel of p53 hotspot mutants were chosen as reporter for the screen. The approach reassuringly identified many previously described regulators of p53 degradation, and also found a large set of new hits that many appear to be indirectly affecting p53 level.
A key step of this approach is the follow up functional and mechanistic study of the hits. To this end, the authors chose FBXO42 as a top hit that blocks mutant p53 degradation, and C16orf72 as a top hit that …
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 #3
Evidence, reproducibility and clarity
The manuscript by Lu and coworkers performed genome wide CRISPR screens to search for genes that when knocked out, lead to p53 accumulation or degradation. Wt p53 and a panel of p53 hotspot mutants were chosen as reporter for the screen. The approach reassuringly identified many previously described regulators of p53 degradation, and also found a large set of new hits that many appear to be indirectly affecting p53 level.
A key step of this approach is the follow up functional and mechanistic study of the hits. To this end, the authors chose FBXO42 as a top hit that blocks mutant p53 degradation, and C16orf72 as a top hit that promotes wt/mutant p53 degradation.
Overall the functional data for FBXO42 is disappointing. FBXO42 knockout has quite modest effect on mutant p53 level (~50% reduction). The knockout also showed some effect on p53 mRNA level (~25% reduction), making the determination of mechanism difficult. It does not appear to be a promising targeting for reducing mutant p53 level and gain of function activity in tumor cells.
The C16orf72 finding unfortunately lost some novelty because it was independently identified as a p53 regulator in a recent study using CRISPR screening (PMID: 33660365). However, the repeated identification is reassuring and the current work provides more convincing functional data, showing C16orf72 knockout increase wt p53 level, inhibits cell proliferation specifically in p53+/+ cells, and overexpression of C16orf72 reduce wt p53 level and accelerates progression of a breast tumor mouse model. Their results suggest C16orf72 is a biologically relevant regulator of p53 in cancer development. In order to provide a reasonable amount of new information and set it further apart from the published study, some biochemical analysis looking into the mechanism of C16orf72 will be helpful.
Specific comments:
There appears to be a mix up in the figure legend for Fig.1A describing line 1 and 2.
Fig.2. Data for some p53 mutants mentioned in the text cannot be found in the main figure 2D and supplemental figure S3A.
Fig.2 E-F. The effects of FBXO42 and CCDC6 KO on endogenous mutant p53 level is small (~50% decrease). Given that mutant p53 accumulates at high levels, whether a 50% decrease has meaningful effect on its gain of function activities is questionable. The knockouts also caused a ~25% decrease in p53 mRNA (FigS3F) which makes the mechanism quite difficult to investigate further.
Fig.3B. The IP experiment using p53 shRNA and control shRNA should be done by IP of p53 followed by CCDC6 western blot. If CCDC6 IP is used as in the figure, then a CCDC6 shRNA knockdown sample should be compared to control shRNA. The current data does not rule out the possibility that CCDC6 antibody can nonspecifically pull down some p53.
Fig.3D. The in vitro pull down experiment needs specificity controls such as non affected R175H p53 core domain. The data presented would suggest that MBP-FBXO42c captured more than 1:1 molar ratio of R273H core domain, which is unusual for specific binding unless there is aggregation of p53.
To increase the impact of the current study, the authors could provide more mechanism insight on how C16orf72 regulates p53 level, which was also missing in the other published study. For example, addressing whether C16orf72 effect is dependent on MDM2. Does it cooperate with MDM2 to ubiquitinate p53. Does it promote p53 ubiquitination in the absence of MDM2, since it interacts with HUWE1. Does it act by recruiting usp7 to stabilize MDM2.
The manuscript is in a form extremely unfriendly to review, text, figures and legends are all split up at multiple locations, the pdf figures are very sluggish to scroll.
Significance
The work is significant in identifying a functionally relevant regulator of p53 stability.
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Referee #2
Evidence, reproducibility and clarity
The paper describes several genome-wide CRISPR screens designed to identify regulators of p53 stability. The authors use a system in which p53 levels are marked by mClover expression, using RFP expression to normalise for gene expression changes.
- The bimodal distribution of p53 expression levels in some reporter cell lines (G245S, R248Q, R248W and R273H) hampers the implementation of a robust readout and makes correct interpretation of the results challenging. While it is possible that the bimodal distribution indicates dynamic changes in p53 levels within one population, it also seems possible that a subclone of these cells have …
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Referee #2
Evidence, reproducibility and clarity
The paper describes several genome-wide CRISPR screens designed to identify regulators of p53 stability. The authors use a system in which p53 levels are marked by mClover expression, using RFP expression to normalise for gene expression changes.
- The bimodal distribution of p53 expression levels in some reporter cell lines (G245S, R248Q, R248W and R273H) hampers the implementation of a robust readout and makes correct interpretation of the results challenging. While it is possible that the bimodal distribution indicates dynamic changes in p53 levels within one population, it also seems possible that a subclone of these cells have acquired additional alterations affecting p53 stability, and that the authors are screening a mixed population of two intrinsically different cell populations. This would make it difficult to interpret the results of the screen in these cell lines and may be a challenge when trying to identify something that has not already been highlighted on depmap.
- The coverage of the sgRNA library (200x) is rather low for a negative selection screen, where a coverage of 500x would be more desirable. The FDR threshold is also rather lenient, a more stringent FDR threshold would seem more appropriate and shorten the list of potential hits.
- Although the study is focused on the regulation of p53 stability, there are no experiments to show that any of the manipulations alter the ubiquitination or degradation (half-life) of p53. The rescue of expression by proteasome inhibition is very modest (Figure 2E), suggesting the loss of expression may not be a reflection of degradation. A role for endogenous FBXO42 and C16orf72 in regulating the ubiquitination and half-life of endogenous p53 should be confirmed
- Many p53 mutants are used for the initial screens, but very little validation is carried out to show that the apparent differences in factors regulating their stability persists in cells naturally expressing these mutants. For example, FBXO42 is identified as a protein required to maintain the stability of R273H, 248W and R248Q, but not R175H, G245S and R337H. While the authors show an association of CCDC6 and p53 in PANC1 cells (expressing 273H), it would be important to show a panel of R273H, 248W and R248Q expressing tumor cells and the response of p53 to FBXO42 and CCDC6 depletion, compared to similar experiments in a panel of R175H, G245S and R337H expressing tumor cells. Again, it would be important to show that any changes in protein levels are due to changes in protein stability.
- The potential hits should also be tested in wild type p53 expressing cells to confirm the specificity to mutant p53s.
- The role of C16orf72 in restraining p53 activity has been reported previously, as has the interaction with HUWE1 (including a new publication PMID: 35776542). The authors suggest an interaction between C16orf72 and USP7, although this should be shown with endogenous proteins. The relative importance of USP7 and HUWE1 binding is not explored. The effect of C16orf72 overexpression in promoting mammary tumors is impressive, although maybe the more interesting question is whether inhibition of C16orf72 expression can limit tumor development in this system.
Minor comments
- Figure 1b: The nutlin concentration stated in the methods section is wrong. Should be 10 µM instead of 10 nM (correct in figure legend).
- Figure 1c: Include results for a mutant that is not regulated by MDM2, such as R175H. Otherwise, as a standalone experiment, this figure doesn't add much.
- Figure 1e/f Legend: Should be FDR <0.5 not >0.5.
- Figure 1h: While an UpSet plot is an elegant way to present unique and overlapping hits between different screens, Venn diagrams might be more 'accessible' to many readers and easier to understand.
- Might be worth stating that mClover is an eGFP variant and can therefore be targeted by eGFP sgRNAs so that it is easier to understand the following:
- Page 5, paragraph 1: "We used the TKOv3 sgRNA library, which contains [...] 142 control sgRNAs targeting EGFP, LacZ and luciferase"
- Page 5, paragraph 2: "As expected, sgRNAs targeting p53 and mClover were the most depleted sgRNAs, [...]
- Figure 6b: y-axis label is missing
Significance
This is an interesting concept and the results could provide a useful resource for groups interested in the regulation of p53. The authors chose to focus on candidate genes that could have been identified by looking for the top 30 p53 co-dependent genes on depmap (C16orf72 is #24 in this list and FBXO42 is #28, most of the other genes ranking above are already known as p53 regulators). While this validates the screen, it would have been interesting if the authors had identified and validated new regulators of p53 that were not apparent from previously published work.
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Referee #1
Evidence, reproducibility and clarity
Summary:
In this manuscript by Lu et al., the authors describe some CRISPR screens and protein-protein interaction screens to identify novel regulators of wild-type p53 and mutant p53 function and stability. Besides generating a wealth of data, they discover FBXO42-CCDC6 as positive regulators of the some p53 hot-spot mutants, including R273H mutant p53, but not of all p53 mutants tested and also not of wild-type, indicating selectivity. Furthermore, the found C16orf72(TAPR1) as a negative regulator of p53 stability. Mechanistically, the authors claim a direct interaction between FBXO42 and CCDC6 and p53, but the importance of these …
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 #1
Evidence, reproducibility and clarity
Summary:
In this manuscript by Lu et al., the authors describe some CRISPR screens and protein-protein interaction screens to identify novel regulators of wild-type p53 and mutant p53 function and stability. Besides generating a wealth of data, they discover FBXO42-CCDC6 as positive regulators of the some p53 hot-spot mutants, including R273H mutant p53, but not of all p53 mutants tested and also not of wild-type, indicating selectivity. Furthermore, the found C16orf72(TAPR1) as a negative regulator of p53 stability. Mechanistically, the authors claim a direct interaction between FBXO42 and CCDC6 and p53, but the importance of these interactions has not been shown. On the other hand the authors suggest that the FBXO42/CCDC6 regulate p53 via destabilization of USP28, but also the mechanism has not been worked out. For c16orf72, they show that it interacts with USP7, but no relevance of this interaction is shown either.
Major points
One very important point for me is that the authors do not show the levels of expression of p53 in the p53-mClover stable cell lines. It is known that overexpressed p53 is usualy more stable than endogenous levels of wt-p53. Therefore, I think it is necessary that the authors show the levels of the p53-mClover fusion proteins in the stably transduced cell lines compared to endogenous p53 levels in the parental RPE1 cells and also compared to the endogenous levels of R273H mutant in the PANC-1 cells.
Also the functionality of the wild-type p53-mClover fusion is questionable, at least not shown. One would expect that the overexpression of a functional wt-p53 in p53-KO cells will affect the survival of the RPE1 cells. In Figure 5A the authors show that depletion of MDM2 or C16ORF72 is toxic for the RPE1 cells in a p53-dependent manner, indicating that elevated levels of p53 cannot be handled by these cells. So, experiment(s) showing that the wt-p53/mClover fusion is functional is needed.
A second important point is that the 'verification' of the hits from the screens is only done in one cancer cell line, PANC-1, with mutant p53. I would have like to see at least one other cell line with another p53 mutant endogenously expressed that is also regulated by FBXO42/CCDC6.
For many of the p53-mutants, a bimodal expression is observed. In the FBXO42- and CCDC6-depleted cells, the equilibrium shifts towards more negative cells but the levels in the two populations itself don't change (while for example for USP28 depletion also the right peak shifts further up, Fig S4E). Is there any correlation with the cell cycle and p53 expression? And can the authors exclude that FBXO42 and CCDC6 are involved in cell cycle progression and hereby influence p53 indirectly (by combining PI staining with Clover-p53 for example).
- The authors claim that the FBXO42-CCDC6 axis regulates stability specifically some p53-mutants, including R273H-mutant, in a manner involving USP28. But USP28 regulates all forms of p53, not just some mutants version. How can the authors reconcile this apparent contradiction?
On a similar note, the authors show that FBXO42 and CCDC6 interact with p53, but not USP28. Do FBXO42 and CCDC6 interact with each other and with USP28? And is the interaction with p53 specific for the R273H version? This part of the mechanism is very poorly defined and the Co-IPs are not very convincing or relevant for the proposed model.
Minor points
The mechanisms of p53 regulation may vary greatly in different cell lines. Can the authors discuss why they choose to do the screen with different mutants, rather than with different cell lines expressing these same mutant endogenously? .
Figure 1: Typo in the legends : Nultin ipv Nutlin
Figure 1b,1c : Show basal and Nutlin-3 induced MDM2 levels and in the overexpression cell lines; if WT-p53 is functional, MDM2 levels should be higher in WT-transduced cells compared to control or mt-p53 expressing cells. Authors should explain which they name USP7 a negative regulator of p53, since it is supposed to de-ubiquitinate p53?!
Figure 2E: the effect of MG132 on p53 seems to be very minimal on this Western blot; it would need quantification to be convincing...Quality of the blot is also not great. The fact that in control cells the levels of p53 R273H are not affected by MG132 treatment fits with Suppl Figure 2E, indicating that the proteasome has no effect on p53 R273H.
Suppl figure 3b, 3c, 3d:
Somehow, I have the feeling that the results from the western blots and the FACS do not match fully, although not all the time-points are shown in the various experiments. For example, the FACS analysis (3b) suggests that in control-transduced cells after 16 hr p53 is still increased. However, that is not clear at all in theWestern blot (3c) Is Suppl Figure 3d the quantification of 3c experiment? If so, in the blot also the 24 hrs should be shown. The blot shown in Suppl Figure 3c suggests that CCDC6 expression increased upon irradiation. Do the authors agree with that? Would that explain why depletion of CCDC6 has more effect upon irradiation? Suppl Figure S3E: if I am right, this is essentially the same type of experiment as shown in figure 2e, but analysis of p53-expression by Western blot. In that blot no real effect of MG132 on p53 levels could be seen. But here, in the FACS analysis, MG132 clearly increases the p53-Clover fusion levels; for me again that Western blot and FACS data do not neccesarily match.
Figure 3B: In the CCDC6 IP a very small amount of p53 can be found. I don't know how much input lysate compared to amount of lysate for IP is used, but the percentage of p53 found interacting with CCDC6 seems so marginal that is is difficult to explain the effect of KO of CCDC6 in PANC1 cells. And, the authors called it a 'reciprocal IP' (Suppl Figure 4a) after transfection of V5-tagged CCDC6 into PANC1 cells,but it actually is the same type of IP. Did the authors try to IP p53 and blot for CCDC6? That would be a reciprocal IP.
Figure 3H: how can authors explain that basal levels of USP28 in control and CCDC6-KO cells transfected with control plasmid are more or less the same and not reduced in the CCDC6-KO cells?
Figure 3I: Essentially the whole blot here is of low quality; especially the FBXO42 blot; is deletion of USP28 increasing FBXO42 protein levels, or is it just the quality of the blot? All in all it seems that FBXO42 is very low expressed in the used cell lines.
Figure 4B: I find it a bit surprising that USP7 is also found in the synthetic viability screen, since it has been shown that USP7 has many more essential targets and KO of p53 only partially rescues the development of USP7-KO mouse embryo's.
Figure 5: the authors nowhere show the efficacy of the guides targeting c16orf72. A Western blot showing the expression and the reduction upon expressing the guide-RNAs is essential. Figure 5E: First, here probably parental RPE1 cells have been used, but that is not stated. Second, the authors state 'only a slight increase in p53 levels upon siHUWE1'; I would say none compared to scrambled. I know HUWE1 is a very huge protein, but the blot of HUWE1 is not convincing. I seem to be able to conclude that siMDM2 and siUSP7 reduces HUWE1 levels? Figure 5F, in relation to figure 5D. Here the author overexpress both c16orf72 and USP7, and find an interaction. The implication of that is not clear. If they want to make point of this interaction, they should have looked at endogenous proteins. It is worrying that USP7 apparently was not one of the hits in de Mass-spec experiment of which results are shown in Figure 5D. Also in that experiment c16orf72was overexpressed, and USP7 is very highly expressed in essentially all cell lines, so do the authors have an explanation?
Suppl. figure 5D is missing
Referees cross-commenting
I agree essentially with all comments of Reviewer #2. Especially the major points 3 and 4. The use of more cell lines expressing endogenous mutant p53 is very important. In addition, I can agree with almost all comments of Reviewer #3. The effects especially of FBXO42 ablation are rather minimal, so relevance is questionable.
Significance
Nature and Significance
Compare to existing literature
The topic of the paper is of high interest given the relevance of p53 and its gain-of-function mutants in oncology, and the screens are well executed and clearly presented. In terms of novelty, FBXO42 has been linked to p53-degradation before, and c16orf72 was recently shown to be able to destabilize p53. However, the link between CCDC6 and p53 is novel and of interest, since they are both substrates of USP7 and are both regulators of the cell cycle.
We think the manuscript has potential to add something to the field, but would benefit greatly from a better understanding of the molecular underpinnings of their newly described mechanisms, as well as the conditions in which the mechanism is active.
Therefore, it might be advisable to shorten the manuscript, and go more in-depth in finding the mechanisms of regulation.
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