Selective killing of p53-deficient cancer cells by SP600125

The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53+/+ and TP53−/− human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53−/− (but not TP53+/+) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53−/− cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53−/− cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.

Jemaa investigate the anticancer properties of SP600125, a small molecule inhibitor of a large number of kinases. Using a large portfolio of experimental approaches, the authors demonstrate that SP600125 preferentially kills p53-deficient tumor cells, as opposed to their wild type counterparts, and implicate as the underlying mechanism for this selectivity the activation of mitochondrial apoptosis following rounds of abortive mitoses and tetraploidization in the absence of p53.
The paper continues on an already rich line of investigation that has extensively characterized the SP600125 compound as a potential anticancer agent, and provides additional lines of evidence in support of a p53-regulated tetraploidization checkpoint, a topic that has generated conflicting results in the literature. Overall, the work is well done and appropriate for the readership of the journal. From an experimental standpoint, the manuscript is extensive and supported by an impressive array of well-controlled approaches. On the other hand, the authors are dealing with a compound that has virtually no specificity, so that dissecting the precise molecular mechanism(s) of its anticancer activity in p53-deficient cells remains a remarkable challenge. Strong evidence is presented in the paper that inhibition of the spindle assembly checkpoint kinase Mps1 contributes, at least in part, to SP600125-mediated killing. However, as multiple other mitotic kinases are clearly inhibited at the concentrations of the agent used, the interpretation of these results is not straightforward. In this context, further characterization of the mitotic events induced by SP600125 and the demonstration that cells treated with SP600125 truly die from mitosis would further support the authors' conclusions and improve on the overall priority of the paper.

Referee #1 (Other Remarks):
Jemaa investigate the anticancer properties of SP600125, an extensively characterized small molecule inhibitor of a large number of kinases, including mitotic kinases. Using a large portfolio of experimental approaches, the authors demonstrate that SP600125 preferentially kills p53-deficient tumor cells, as opposed to their wild type counterparts, and implicate as the underlying mechanism for this selectivity the activation of mitochondrial apoptosis following rounds of abortive mitoses and tetraploidization occurring in cells lacking p53.
Critique A potential molecular approach to selectively kill p53-deficient tumor cells, as addressed in the present paper, would hold considerable promise for anticancer strategies in patients and is clearly of great pathophysiological relevance. From an experimental standpoint, the manuscript is very extensive, supported by all the necessary controls, and provides an impressive array of complementary experimental strategies. On the other hand, the authors are dealing with a compound that has virtually no specificity, so that unambiguously dissecting the precise molecular mechanism(s) of anticancer activity in p53-deficient cells remains a remarkable challenge. Strong evidence is presented that inhibition of the spindle assembly checkpoint kinase Mps1 contributes, at least in part, to SP600125-mediated tumor cell killing. However, as multiple other mitotic kinases are clearly inhibited at the same concentrations of the agonist used, the interpretation of these results is not straightforward. In order to clarify the molecular requirements of the proposed pathway, the authors should revisit some of the critical mitotic events associated with SP600125 treatment, and provide unambiguous evidence that treated cells actually die from mitosis, as opposed to a pseudo-G1 state observed for other anticancer agents (taxanes).
Specific points 1. Data in Figure 2B suggest that p53-deficient cells treated with SP600125 do not arrest at mitosis, but continue through the cell cycle and undergo at least three rounds of aberrant mitosis ( Figure 5B). In contrast, treated wild type p53 cells exhibit a stable 4N mitotic arrest. This finding is a cornerstone of the authors' proposed model, as mitotic catastrophe is predicted to ensue selectively from polyploidization of p53-deficient cells. However, it is confusing that cyclin B levels are progressively lost over time in the wild type population, but are stable in the p53-/-cells, and that phospho-H3 is very modestly maintained in the p53-deficient cells, but not at all in the wild type population. Given that SP600125 also inhibits Cdk1, is this truly a mitotic arrest or is it instead a G2 block? And, secondly, without Cdk1 activity, how do p53-/-cells actually enter metaphase? 2. The experiments in Figure 4A are designed to establish a role of the Mps1 kinase as a target of SP600125 in the proposed pathway. However, the interpretation of the data is confused by the fact that transfection of the empty plasmid alone generates a large population of cells with intermediate polyploidization (>4N -<8N) after treatment with SP600125. Similar considerations apply to the experiments of siRNA silencing of Mps1 ( Figure 4F), which seem to produce a large S-phase arrest, a phenotype not observed under any condition described in the paper. These results should be explained.
3. As indicated above, the authors should demonstrate that p53-/-cells killed by SP600125 die from mitosis, thus validating the model of mitotic catastrophe proposed in the paper.
4. The xenograft data presented in Figure 7 are not convincing. From the description of the experiment it appears that mice were started on the drug one week after injection, which at this time point does not show any measurable tumor growth. In order to validate the proposed conclusions in vivo, drug treatment should start when tumors are established, typically with a volume of 150 mm3.

Referee #2 (Comments on Novelty/Model System):
The experiments are carefully performed, but non-immortalized wt cells are required as controls and early passages must be similarly used.

Referee #2 (Other Remarks):
In this manuscript the authors present a systematic analysis of the effects of the Ser/Thr inhibitor SP600125 on cells impaired in their p53 function, identifying its biological effect and proposing a mechanism of action. After screening a library of compounds (480 molecules from ICCB) using a videomicroscopy-based technology, they have identified SP600125 as a powerful molecule able to preferentially kill p53 -/-HCT 116 cells. These data were followed by a more stringent validation of the effects of the inhibitor on p53 deficient cells measuring mitochondrial transmembrane potential, Propidium Iodine labeling and proliferation. p53 deficient cells acquire more rapidly than WT controls a 4n DNA content, in response to the drug. This effect was accompanied by unimpaired DNA synthesis and significant increment of mitotic cells. In addition, , p53 null cells treated with SP600125 undergo abortive mitoses, increasing their DNA content but failing karyokinesis and cytokinesis. SP600125 has a broad target spectrum, ranging from JNKs to MPS1 to Aurora kinase. Authors have focused on the effect on MPS1 Kinase. Using ectopic expression of mutant forms of the protein insensitive to the inhibitor and siRNA studies they could show that MPS1 inhibition is sufficient to trigger polyploidization and mitotic entry of p53 deficient cells. Nevertheless they have failed to demonstrate that such effect is sufficient to induce cell death of p53 -/-cells. Using detailed videomicroscopy analysis, they demonstrate that although both WT and p53 deficient cells show abortive mitosis when treated with SP600125, only p53 cells escape quiescence. This effect is suggested as the cause of their selective killing by the Ser/Thr inhibitor, via the activation of the mitochondrial apoptotic pathway. Interestingly, SP600125 can significantly reduce the growth of subcutaneously injected p53-/-HCT 116 cells.
The identification of compounds that can specifically target p53 deficient cells is instrumental for the development of new cancer therapies, considering the high percentage of cancers that display a direct or indirect impairment in the p53 pathway. Nevertheless, there are several issues that must be addressed by the authors in order render this work suitable for publication I. The finding that p53 deficient cells are specifically sensitive to SP600125 inhibitors is not completely novel. In particular: ï Mingo-Sion AM et al. (Oncogene 2004) reported that MDA MB-231 cells but not MCF7 that have a wt p53 are sensitive to SP600125 induced apoptosis. These effects are reproduced also when p53 is targeted by shRNA and are JNKs dependent. • Potapova O et al. (MCB 2000) show that loss of JNKs expression by antisense treatment leads to apoptosis preferentially in HCT 116 p53 -/-as compared to their wt counterparts II. It has already been shown that SP600125 induces per se endoreplication from G2 and/or mitotic failure: • Kim et al. (Oncogene 2010) • Schmidt et al. (EMBO reports 2005) III. p53 deficient cells have been described in several different context to become spontaneously tetraploid. The authors should discuss these points and these previous publications more clearly and emphasize the novelty of their work when compared with the above-mentioned ones.
2. Although it has been extensively clarified that the endoreplicative effect in WT cells is JNK1 and JNK2 independent, there is no evidence that the MAP Kinase pathway is not involved in the additional apoptosis measured in p53 -/-cells following SP600125 treatment. This must be addressed 3. The authors identify MPS1 as an important piece of the kinase network that is disrupted by SP600125, however the rationale to concentrate on this particular kinase seems unclear. It would be therefore useful to identify and test additional kinases that can mediate the effect (JNK1-3, Aurora A, etc.) and to unravel how such a network would lead to the activation of the apoptotic pathway specifically in p53 deficient cells.

What is the level of apoptosis in cells expressing the MPS1 mutant (MPS1-M602Q) insensitive to SP600125?
5. p53 deficient MEFs are immortal and present a much higher proliferation rate in culture. In addition, regularly after few passages they become tetraploid and may acquire additional mutations due to their intrinsic genetic instability. Nevertheless, MEFs are a very useful tool compared to other tumor cell lines. It is anyway necessary to work with early passages MEFs, especially in a p53 -/background and to avoid SV40-immortalized MEFs as controls, where the p53 response is impaired. The experiments should be repeated with WT and p53 deficient non-immortalized early passage MEFs.

Referee #3:
This article presents a series of robust, inter-correlating experiments that support the conclusions described within, namely: (i) SP600125 inhibits the viability of TP53-cells, in relation to TP53+ cells (ii) It leads to polyploidization through abortive mitosis, in the absence of metaphase chromosome alignment, anaphase or attempted cytokinesis (iii) It increases mitochondrial-induced apoptosis (iv) These effects are transduced, at least in part, through Mps1 (v) Injection of SP600125 into mice with tumors originating from TP53-HCT xenografts, showed increase in tumor size in relation to TP53+, suggesting SP600125 has therapeutic potential.
Each conclusion (apart from the last) is backed-up through multiple assays, with almost all corroborating the other, and together they stand as a substantial body of work that should be published.
However, I have two essential queries/recommendations that need addressing: (i) The data concerning the presence or absence of Cyclins under different experimental conditions appears to be at odds (or at least is not well presented and I have mis-interpreted it). In Figure 2F, 24 hrs after treatment with SP600125, ~20% of TP53+ (WT) cells stain with Cyclin B (this decreases to <5% after 48 hrs), and we are told in the text that there is a "marked decline in Cyclin B" (p7), and 40% of cells stain for Cyclin E. However, in Figure 4, where the authors are looking at effect of introducing Mps1 variants into cells, WT cells 24 hr post-treatment (with or without vectors) have 30% 50% Cyclin B and 30% Cyclin E staining. This discrepancy needs explaining.
(ii) Throughout the article, the authors talk about ""quantitative results". What they mean is "bar charts", or "histograms". With only 2 exceptions, none of these figures come with any statistical analysis. Therefore, although I agree with their interpretations of the experiments, it is difficult to conclude, for example, "transfection-enforced expression of WT MPS1 and more so of an SP600125-refractory MPS1 variant (MPS1-M602Q) (Schmidt et al, 2005), reduced the capacity of SP600125 to trigger polyploidization in TP53-/-" (Figure 4; p8). This needs to be shown with a statistical significance test that is described and represented in the Figure. All histograms in this study, where differences between control and treated samples are seen and commented upon should have such a statistical analysis performed, and p values associated.
I also have one recommended experiment that would be nice to undertake, and could significantly assist in our understanding of how SP600125 triggers mitotic catastrophe which, I admit, may lie outside of the scope of this particular study: (iii) The authors should/could undertake live imaging of WT/TP53-cells expressing GFP-Cyclin B in the presence/absence of SP600125, quantitatively analysing the intensity of GFP-CyclinB over time. This would show whether the accumulation and degradation of CyclinB occurs with normal dynamics in TP53-SP-treated cells, potentially defining the mitotic catastrophe event with greater clarity. Our response: The reviewer requests further characterization of the mitotic events induced by SP600125, in particular with regard to the question as to whether cells treated with SP600125 truly die from mitosis. We have performed an extensive cell fate profiling of p53-positive or p53-negative cells (which all express GFP-histone H2B to monitor chromatin morphology) by videomicroscopy, and we found that p53-deficient cells, which are susceptible to SP600125-mediated killing, did not die during mitosis but approximately 10±2 hours after an aberrant mitosis (featuring transient rapid chromatin condensation with abortive karyokinesis and no cytokinesis) and did not manifest a mitotic arrest as they continued to duplicate their chromosomal material. Thus, in TP53 -/cells, the mitotic abortion triggered by SP600125 activates a delayed program of cell death, a process that overall represents one particular type of mitotic catastrophe (Vitale I. et al., Nat Rev Mol Cell Biol. 2011;Galluzzi et al., Cell Death Differ. 2012). These data are shown in Figure 5 and in Supplementary Videos 1-3 (for the SP600125-induced polyploidization and for the DNA rereplication of newly generated polyploids please also refer to Figure 2). To further investigate the link between abortive mitosis and cell death, we arrested the proliferation of p53-negative cells by culturing them in the presence of low serum concentrations (which induces a blockade in the G1phase) or at high confluence, and then exposed them to SP600125. Non-proliferating cells were killed less efficiently by SP600125, and the differential susceptibility of p53-deficient cells to SP600125-mediated killing was lost. Of note, upon the re-establishment of normal culture conditions, the susceptibility of TP53 -/cells to SP600125-mediated cell killing was also restored. These new results, which suggest a causal link between proliferation and cell death induced by SP600125 are shown in Figure 6A. Figure 2B suggest that p53-deficient cells treated with SP600125 do not arrest at mitosis, but continue through the cell cycle and undergo at least three rounds of aberrant mitosis ( Figure 5B)

. In contrast, treated wild type p53 cells exhibit a stable 4N mitotic arrest. This finding is a cornerstone of the authors' proposed model, as mitotic catastrophe is predicted to ensue selectively from polyploidization of p53-deficient cells. However, it
is confusing that cyclin B levels are progressively lost over time in the wild type population, but are stable in the p53-/-cells, and that phospho-H3 is very modestly maintained in the p53-deficient cells, but not at all in the wild type population. Given that SP600125 also inhibits Cdk1, is this truly a mitotic arrest or is it instead a G2 block? And, secondly, without Cdk1 activity, how do p53-/cells actually enter metaphase?
Our response: We thank the reviewer for his/her constructive criticism. In p53-negative cells, we did not detect CDK1 inhibition. Rather, in a substantial fraction of p53-negative cells exposed to SP600125, we did detect the expression of cyclin B1 (the obligatory cofactor of Cdk1) (Fig. 2E,F and new Supplementary Figure S2) as well as the phosphorylation of the bona fide Cdk1 substrate histone H3 and a positive staining for the mitotic marker MPM2 (an antibody that recognizes several phosphoproteins containing similar phosphoepitopes, which are phosphorylated during the M phase by Cdk1-Cyclin B1 complex) (new Fig. 2D and new Supplementary Figure S1). These result suggest that Cdk1 is not inhibited by SP600125, at least in our experimental model, in accord with the fact that HCT 116 cells treated with SP600125 entered successive rounds of mitosis, although these mitoses were abortive and did not lead to nuclear and cellular division (please refer to videomicroscopy data in Fig. 5 and to the functional and morphological characterization of mitosis and interphase FISH in Fig 3). As a possibility, SP600125 inhibits Cdk1 in p53-positive but not in p53 negative cells, perhaps through an indirect mechanism (please see also Kim et al. Oncogene 2010). Finally, the dynamics of the accumulation and degradation of cyclin B in p53-proficient and p53-deficient cells is different. Indeed, SP600125 provoked a complete degradation of cyclin B only in p53-proficient cells, suggesting these cells are stably arrested in a G 1 tetraploid phase rather that in a G 2 and/or M diploid phase (Fig. 2E,F and new Supplementary Figure 2; see also our response to the minor point n° 3 raised by reviewer n° 3). Figure 4A are designed to establish a role of the Mps1 kinase as a target of SP600125 in the proposed pathway. However, the interpretation of the data is confused by the fact that transfection of the empty plasmid alone generates a large population of cells with intermediate polyploidization (>4N -<8N) after treatment with SP600125. Similar considerations apply to the experiments of siRNA silencing of Mps1 ( Figure 4F), which seem to produce a large S-phase arrest, a phenotype not observed under any condition described in the paper. These results should be explained.

Our response:
The reviewer correctly points out that in Fig. 4A peaks are relatively broad, suggesting intermediate polyploidization levels. However, the data in Fig. 4A have been obtained by staining intact cells with Hoechst 33342, followed by cytofluorometric analysis. The resolution of this method is somehow lower than that resulting from ethanol fixation, RNA digestion with RNAse and staining with propidium iodide, resulting in broader G 1 and G 2 /M peaks, which however do not represent intermediate levels of polyploidization. As to Fig. 4F, we performed this experiment seven times, and we always failed to detect a consistent S phase arrest. Thus, we chose to show more representative panels.

Specific point 3 raised by reviewer 1: As indicated above, the authors should demonstrate that p53-/-cells killed by SP600125 die from mitosis, thus validating the model of mitotic catastrophe proposed in the paper.
Our response: See our response to the general critique raised by the reviewer.

Specific point 4 raised by reviewer 1:
The xenograft data presented in Figure 7 are not convincing. The authors should discuss these points and these previous publications more clearly and emphasize the novelty of their work when compared with the above-mentioned ones.
Our response: Driven by the reviewer's indications, we have discussed, compared, cited and mentioned all these works, while giving more emphasis to the novelty of our results, either in the introduction (page 3, lines 19-20), in the results and discussions (page 6, lines 18-19; page 7, lines 4-5) or in the conclusion (page 12, lines 6-11; page 13, lines 1-11; page 13, lines 11-16) of the revised version of the paper

Specific point 3 raised by reviewer 2:
. The authors identify MPS1 as an important piece of the kinase network that is disrupted by SP600125, however the rationale to concentrate on this particular kinase seems unclear. It would be therefore useful to identify and test additional kinases that can mediate the effect (JNK1-3, Aurora A, etc.) and to unravel how such a network would lead to the activation of the apoptotic pathway specifically in p53 deficient cells.
Our response to points 2 and 3: The reviewer requests information on other kinases that might be inhibited by MPS1. As a result, we knocked down with specific siRNAs c-jun N-terminal kinase 1 (JNK1), JNK2, Aurora kinase A (AURKA), Aurora kinase B (AURKB), MEK1, MPS1, FLT3, CHEK1 and CHEK2, alone or in combination. As the reviewer will appreciate, none of these single or combined knockdown experiments led to the preferential killing of p53-deficient over p53proficient cells. These results have been added as supplemental Figures S6-S8. We provided the following hypothetical explanation for these results: SP600125 induces preferential killing of p53deficient cells through its inhibitory action on several kinases (and perhaps off-target effects). At this point, we only know that one of the kinases that must be inhibited by SP600125 to observe these effects is MPS1. However, inhibition of MPS1 is not sufficient to kill p53-deficient cells in a preferential fashion. At present, we do not know which other kinases, beyond MPS1, must be inhibited to selectively ablate p53-negative cells.

Specific point 4 raised by reviewer 2:. What is the level of apoptosis in cells expressing the MPS1 mutant (MPS1-M602Q) insensitive to SP600125?
Our response: MPS1 overexpression is reported to overactivate the spindle assembly checkpoint (SAC) in cells that have normal spindles and, consequently, to induce a prolonged metaphase arrest (Hardwick et al., Science 1996;Liang H et al, EMBO J 2011). In addition, the upregulation of MPS1 may lead (at least in some cell lines) to mitotic alteration such as centrosomes amplification and multipolar mitotic spindles (Fisk and Winey, Cell, 2001). Extendedly arrested, aberrant and/or abortive mitoses all can trigger mitotic catastrophe, an oncosuppressive mechanism that eliminates mitosis-incompetent cells, which -among other outcomes -can lead to cell death (Vitale I. et al., Nat Rev Mol Cell Biol. 2011;Galluzzi et al., Cell Death Differ. 2012). Consistent with this, we found that the overexpression of MPS1 (be it wild type or the SP600125-refractory mutant M602Q) was partially toxic and killed a significant fraction of cells. As a result, the background levels of apoptosis induced by the overexpression of MPS1 alone are too high to assess the effects of SP600125. To perform the cell cycle analysis, debris, shrunken and subdiploid cells were excluded. This allowed us to retrieve information on the effects of MPS1 on cell cycle, yet precluded the analysis of MPS1 effects on apoptosis. Our response: To meet the referee's desiderata, we obtained adult fibroblasts from the ears of young (8 week old) Tp53 -/-C57Bl/6 mice and their isogenic wild type counterparts (3 mice for each genotype). After two (data shown) or three (data not shown) passages, these cells were exposed to SP600125 for 48, 72 or 96h. Clearly, SP600125 killed more Tp53 -/than Tp53 +/+ fibroblasts, supporting the contention that SP600125 also kills non-immortalized p53-deficient cells more efficiently than their p53-sufficient counterparts (Fig. 7H). Of note, both the p53-deficient and p53proficient cells used in these experiments were diploid with a percentage of spontaneous tetraploid (cell displaying a >4n DNA content) lower than 8% (as assessed by cytofluorimetric analysis upon Hoechst 33342 or propidium iodide staining). Figure 2F,  Our response: We thank the reviewer for raising this point. Indeed, the description of our results was not sufficiently clear. In Fig. 4E, all the cells included in the experiment were p53-deficient, and this has now been clearly indicated in the figure, as well in the revised version of the corresponding legend. As a result, there is no internal contradiction, and in both experiment the percentage of cyclin B-positive p53 -/cells, 24 hours after the addition of SP600125, amounts to 40-50%.

Specific point 2 raised by reviewer 3:
Throughout the article, the authors talk about ""quantitative results". What they mean is "bar charts", or "histograms". With only 2 exceptions, none of these figures come with any statistical analysis. Therefore, although I agree with their interpretations of the experiments, it is difficult to conclude, for example, "transfection-enforced expression of WT MPS1 and more so of an SP600125-refractory MPS1 variant (MPS1-M602Q) (Schmidt et al, 2005), reduced the capacity of SP600125 to trigger polyploidization in TP53-/-" (Figure 4; p8). This needs to be shown with a statistical significance test that is described and represented in the Figure. All histograms in this study, where differences between control and treated samples are seen and commented upon should have such a statistical analysis performed, and p values associated.
Our response: We apologize for the missing statistical studies. Statistical comparisons have now been introduced into most figures, and the methods used to perform statistical calculations have been precisely described in the Materials & Methods section, as well as in the figure legends, of the revised paper.
Optional point 3 raised by reviewer 3: I also have one recommended experiment that would be nice to undertake, and could significantly assist in our understanding of how SP600125 triggers mitotic catastrophe which, I admit, may lie outside of the scope of this particular study: The authors should/could undertake live imaging of WT/TP53-cells expressing GFP-Cyclin B in the presence/absence of SP600125, quantitatively analysing the intensity of GFP-CyclinB over time. This would show whether the accumulation and degradation of CyclinB occurs with normal dynamics in TP53-SP-treated cells, potentially defining the mitotic catastrophe event with greater clarity.
Our response: We have attempted to do these experiments (with adenovirus-mediated transfer of GFP-cyclin B) without success. However, we do have data suggesting that cyclin B accumulates and degrades with a normal dynamics in p53-deficient cells. These results, as well as data suggesting that cyclin B1/Cdk1 is active in such cells (and hence phosphorylates histone H3 and stain positively for MPM2) are shown in Figures 2D-F and in new Supplementary Figures S1 and S2. Only in wild type cells, cyclin B degrades completely after treatment with SP600125, presumably as a consequence of their polyploidization followed by a prolonged cell cycle arrest in a G1 tetraploid phase (in this context, please see also the cell fate profiling by videomicroscopic observation of WT cells reported in Figure 5A and 5B).
2nd Editorial Decision 02 February 2012 Please find enclosed the final reports on your manuscript. We are pleased to inform you that your manuscript is accepted for publication and will be sent to our publisher to be included in the next available issue of EMBO Molecular Medicine if or once we have received your licenses (see below).
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Yours sincerely, Editor EMBO Molecular Medicine ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): The model system and experimental approaches are appropriate for the investigation of a p53dependent cell killing pathway mediated by the investigational agent SP600125.
Referee #1 (Other Remarks): I have re-reviewed the manuscript by Jemaa et al. The authors have satisfactorily and thoroughly addressed the concerns raised in my previous review, and added new experimental evidence to further clarify a potential mechanism of tumor cell killing by the SP600125 compound. A detailed evaluation of the effect of the agent on mitotic transitions is also now provided. I have no further concerns.
Referee #2: Authors have satisfactorily addressed all my previous concerns. The manuscript is now acceptable for publication.