CDK2 activity determines the timing of cell-cycle commitment and sequential DNA replication

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1. 

   eLife

   Aug 21, 2020

   ###Reviewer #3:

   In the manuscript "Kinetics of CDK4/6 inhibition determine different temporal locations of the restriction point" Kim et al., investigate the regulation of the Rb/E2F by CDK4/6 and CDK2 and how mitogen and stress signalling differently regulate kinetics of CDK4/6 inhibition before irreversible cell-cycle entry. Research into restriction point regulation recently experienced a revival due to advanced single cell approaches and the presented study falls into this category as well. Utilizing CDK4, CDK2 and APC/C activity reporters the authors investigate the position of the restriction point in response to external stimuli. Their main conclusions are that i) CDK4/6 activity alone initiates RB hyperphosphorylation and E2F activation, ii) that the CDK2-Rb feedback is the key signalling network controlling the restriction …

   ###Reviewer #3:

   In the manuscript "Kinetics of CDK4/6 inhibition determine different temporal locations of the restriction point" Kim et al., investigate the regulation of the Rb/E2F by CDK4/6 and CDK2 and how mitogen and stress signalling differently regulate kinetics of CDK4/6 inhibition before irreversible cell-cycle entry. Research into restriction point regulation recently experienced a revival due to advanced single cell approaches and the presented study falls into this category as well. Utilizing CDK4, CDK2 and APC/C activity reporters the authors investigate the position of the restriction point in response to external stimuli. Their main conclusions are that i) CDK4/6 activity alone initiates RB hyperphosphorylation and E2F activation, ii) that the CDK2-Rb feedback is the key signalling network controlling the restriction point, iii) that kinetics of CDK4/6 inhibition in response to mitogen removal and stress signalling explain previous observation in asynchronously cycling cells showing different locations of the restriction point and iv), that CDK2 activity alone without other mechanisms in S phase determines the temporal location of the restriction point with respect to CDK4/6 inhibition and S-phase entry.

   I have major concerns with presented work regarding the design of the study in relation to question asked, the one-sided introduction and discussion and imprecise wording of restriction point events, the tendency to overstating/generalize conclusion of their findings, the novelty of their results in relation to old restriction point studies using serum starvation and release regimes and the more recent studies from the Meyer, Spencer and Bakal labs focusing on asynchronously growing cells, and the fact that their results and interpretations are completely at odds with the recent Dowdy and Dyson studies, which are not mentioned at all in either the introduction or discussion. Finally, to my opinion the authors have not yet provided the experimental proof for one of their major claims, namely that CDK4/6 activity alone initiates RB hyperphosphorylation and E2F activation. My detailed criticisms are listed in the major and minor points below.

   Major points:

   1. The authors give the impression in the introduction that they will focus on probing the possibility of different temporal locations of the restriction point depending on the external stimuli (p3, l60ff). However, they only use mitogen withdrawal and NCS-induced DNA damage as "stimuli" but then claim that "we demonstrate that different extracellular environments cause different kinetics of CDK4/6 inhibition (p10, l96ff)". Certainly, these two treatments (in addition to direct CDK4 and CDK2) are not sufficient for such a general statement and in the context of their writing, NCS-induced DNA damage is rather a cell-intrinsic and not an external stimulus/condition as claimed. Similarly, the authors derive from their NCS experiments general and overarching statements about restriction point regulation in response to stress. In fact, CDK4/6 is a target of several integrated stress pathways, e.g. UPR/PERK, which regulate the levels of cyclin D on the translational level (e.g. Brewer at al., PNAS 1999) and are independent on p21. The authors also claim to investigate whether other mechanisms in S phase are required to initiate the restriction point. To me this is another example of unclear wording and unfulfilled expectations as the only factor analysed is the APC/C, which is inactivated at the entry of S phase. From the introduction, discussion and the mentioned literature it is unclear to me why the authors expect that a mechanism in S phase, hence after commitment to proliferation, would feed back on the restriction point during G1 phase of the same cell.

   2. Introduction and discussion are one-sided and completely omit recent findings of the Spencer lab (Min et al., PLOS Biology 2019) in relation to stress and most importantly the Dowdy (Narasimha et al. eLife 2014) and Dyson studies (Sandias et al., Mol Cell 2019), which are both at odds with a major claim of the presented work (see below).

   3. The authors claim throughout the paper that CDK4/6 is sufficient to hyperphosphorylate Rb based on nuclei that can be stained by antibodies specific to 4 Rb phospho sites and in situ extraction experiments that claim to dissociate hyperphopshorylated Rb from the DNA. This claim cannot be made as their results are completely consistent with the alternative, namely that multiple Rb molecules within the same cell (nucleus) are mono-phosphorylated at the analysed sites, or at either of the 14 possible sites. This would be in agreement with the Dowdy and Dyson studies (Fig. 1 & Fig. 2). For the situ extraction experiments investigating nuclear-bound Rb there is no real data shown. Fig. 1J basically shows the segmentation strategy the authors employ and indicate that same cells have less nuclear Rb staining. There are no controls, (e.g. before after extraction) and proof that the assay works in their hands - e.g. treating the cells with CDK4/6 inhibitors and CDK2 inhibitors before the assay. The authors show in Fig 1. that E2F1 is already induced hours before mitosis, yet cells only progress much later into S. However, it is as likely that mono phosphorylation of RB is sufficient to initiate E2F1 transcription, this could be easily tested using the published mutant cell lines expressing Rb variants with only one phosphosite.

   4. The authors claim that "However, previous studies showed that CDK2 inhibitors caused a loss of Rb phosphorylation and induced quiescence (Narasimha et al., 2014; Spencer et al., 2013)" (p5, l60). Reading these papers again it appears to me that this is a wrong statement/interpretation. Narasimha et al, show in Figure 3 that only CDK4 inhibition but not CDK2 inhibition results in a complete loss of Rb phosphorylation. The latter treatment resulted in RB mono phosphorylation (Fig 3i) and did not induce quiescence as the authors claim here. Instead, such cells remained in G1 phase and did not make the transition into G0. Also, the claim the Spencer et al., results are due to off-target effects of CDK2 inhibitors appears flawed, because the authors only detect those after a prolonged time (more than 9 hours), whereas Spencer et al, monitored the effect of such inhibitors on cells immediately after application. Hence, in my opinion this part, the corresponding data (Fig. S3), and interpretations should be removed.

   5. In asynchronously treated cells CDK2 appears to be activated early after mitosis (Spencer et al., 2013), whereas in their experimental setup CDK2 and CDK4 activation are only assessed after mitogen starvation and release. I imagine from the timing that in asynchronously growing cells also CDK2 activity will be tightly coordinated with E2F transcription (Fig 1D) - hence, a main foundation for their study may depend on the experimental setup used and thus this should clearly be discussed. I also wonder how their results on the requirement of CDK4 for RB phosphorylation would be without the synchronization step.

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2. 

   eLife

   Aug 21, 2020

   ###Reviewer #2:

   In this manuscript, Kim et al. investigate the events required for irreversible commitment to division by immortalized mammalian cells in culture. They do so by tracking single, live cells by video-microscopy using an assortment of fluorescent biosensors (augmented by fixed-cell immunofluorescence), and perturbing cell-cycle progression with cyclin-dependent kinase (CDK) inhibitors, DNA-damaging agents, or mitogen withdrawal. This is a complicated problem, which has resisted a comprehensive solution since the initial attempts to define a commitment or "Restriction" point (R point) in mammalian cells over 40 years ago. This study yields some intriguing results, and generally adds significant molecular detail to previous work on this problem by the PI and former colleagues in the Meyer lab. There are serious flaws, …

   ###Reviewer #2:

   In this manuscript, Kim et al. investigate the events required for irreversible commitment to division by immortalized mammalian cells in culture. They do so by tracking single, live cells by video-microscopy using an assortment of fluorescent biosensors (augmented by fixed-cell immunofluorescence), and perturbing cell-cycle progression with cyclin-dependent kinase (CDK) inhibitors, DNA-damaging agents, or mitogen withdrawal. This is a complicated problem, which has resisted a comprehensive solution since the initial attempts to define a commitment or "Restriction" point (R point) in mammalian cells over 40 years ago. This study yields some intriguing results, and generally adds significant molecular detail to previous work on this problem by the PI and former colleagues in the Meyer lab. There are serious flaws, however, both conceptual and technical. Some of them are inherent in the approach, for example, the overreliance on small-molecule inhibitors that are not as selective as one would hope, and on live-cell biosensors that are neither as sensitive nor as specific (for individual CDKs) as they would need to be to justify some of the stronger mechanistic conclusions. Then there is the central take-home message (I think), which is based on the observation that mitogen withdrawal or DNA damaging agents have different windows of sensitivity during G1, such that the former needs to be applied earlier than the latter in order to prevent cell cycle entry. This leads to re-interpretation of the R point as a moving target, occurring at different points in the cell cycle depending on which perturbations cells encounter as they take the necessary steps to commence DNA replication. This makes little biological sense to me. The R point concept seems to lose much or all of its usefulness if it is not understood as a cellular state in which the irreversible commitment to division has been made, irrespective of what might befall an individual cell that has passed it. I think a more reasonable interpretation, of a superficially (at least) similar phenomenon, was put forth by Skotheim and colleagues, who found that the threshold level of CDK1/2 activity that predicted subsequent R-point passage was higher when all mitogens were withdrawn than when a single mitogenic signaling pathway was ablated, e.g. with a MEK inhibitor (Schwarz et al., 2018, ref 22). In this take, the R point per se is not mutable, but the strength of an antimitogenic signal can determine how quickly cells can put on the brakes before reaching it. I would urge the authors to avoid this phrasing, and aim for a bit more clarity in describing an admittedly complicated set of data. Below I Iist my major, specific concerns:

   1. Probably the biggest problem for the current study emerged from a paper by Rubin and colleagues (Guiley et al., 2019, ref. 26), which showed, quite convincingly, that the "CDK4/6 inhibitors" Palbociclib, ribociclib and abemaciclib-used throughout the current study-almost certainly do not work in cells by direct inhibition of CDK4/6, but rather by binding CDK monomers and redistributing CDK inhibitor (CKI) proteins, notably p21, to CDK2. To be fair, this is a very recent paper, which, to their credit, the authors cite and try to address. But they address it only obliquely and, I'm afraid, inadequately; although they show that effects of Palbociclib et al. are partially independent of p21 (Fig. 3B,D), this doesn't rule out contributions by other CKIs such as p27 or p57, all of which could potentially be redistributing to CDK2 complexes if CDK4 complex assembly is impaired (Guiley et al. did not test this possibility and only evaluated CDK2-CKI binding in wild-type cells). Nor do they address the strong implication of Guiley et al., that loss of CDK4/6 activity is not the mechanism by which these compounds act. This is a hugely important point; the entire study (and several previous ones from the Meyer lab) depends on the ability to inhibit CDK4/6 or CDK1/2 with different inhibitors and distinguish the effects on various cellular phenotypes and biosensor signals, which is now in considerable doubt.

   2. More generally, the study relies on small-molecule inhibitors of different CDKs that are at best only modestly selective for their intended targets. The problem with using Palbociclib in this way has been discussed above, and is a recent development, but it should be noted that major "off targets" for the "CDK4/6 inhibitors" include transcriptional CDKs such as CDK9, which are also potently inhibited by "CDK1/2" inhibitors such as roscovitine (and others). One could make the case that these drugs are hitting different targets, because they have different effects on different biosensors, but the specificity of those bioesensors was established in part by using the inhibitors, so the case that their effects occur solely or primarily through their intended targets is in the end circular.

   3. The "CDK4/6 biosensor" has in fact been shown in a previous paper by the PI to detect CDK1/2 activity in addition to CDK4/6; there was residual signal after Palbociclib treatment in cells with high CDK2 activity. Setting aside the aforementioned problem of Palbociclib specificity, if I understand correctly, to "correct" for this lack of specificity, the authors subtract 35% to generate the signal they attribute to CDK4/6. This seems to assume that the relative contributions to this fluorescence by CDK4/6 and CDK1/2 will be in a fixed proportion, or am I missing something?

   4. In previous papers from the Meyer lab, Rb hyperphosphorylation was "inferred" from concurrently increased immunofluorescence signals, in fixed cells, from a panel of phosphoRb-specific antibodies (Chung et al., 2019, ref. 18). I have my problems even with inferring stoichiometry from these types of measurements, but in this manuscript the language is even stronger: IF signals are flatly described (and interpreted) as "markers" of Rb hyperphosphorylation. This too is a major issue; a prevailing model, supported by biochemical data that are by necessity ensemble measurements, holds that CDK4/6 is primarily responsible for Rb monophosphorylation, whereas hyperphosphorylation coincides with and is dependent on activation of CDK2 (Narasimha et al., 2014, ref. 28). Although for the moment the larger concern-that anything the authors have done to inactivate CDK4/6 is likely to be indirectly inhibiting CDK2-renders this more technical point somewhat moot, conclusions-or even inferences-about hyper- versus mono-phosphorylated forms of Rb should be based on actual measurements of stoichiometry.

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3. 

   eLife

   Aug 21, 2020

   ###Reviewer #1:

   This manuscript reports a series of studies probing the relative roles of CDK4/6 and CDK2 in inactivation of the retinoblastoma (Rb) protein and in determining the restriction point, which marks the commitment of a cell to S phase and subsequent cell division. The work builds off the recent development of live-cell reporters for CDK activity, and it primarily uses relationships between those signals to conclude that while CDK4/6 activity is sufficient for Rb inactivation and E2F activation, CDK2 activation determines passage through the restriction point. Though well-studied over the last two decades, the questions addressed here related to the G1-S cell cycle transition are still not sufficiently answered, and they are important to understanding fundamental cell biology and cancer biology. The use of single-cell imaging …

   ###Reviewer #1:

   This manuscript reports a series of studies probing the relative roles of CDK4/6 and CDK2 in inactivation of the retinoblastoma (Rb) protein and in determining the restriction point, which marks the commitment of a cell to S phase and subsequent cell division. The work builds off the recent development of live-cell reporters for CDK activity, and it primarily uses relationships between those signals to conclude that while CDK4/6 activity is sufficient for Rb inactivation and E2F activation, CDK2 activation determines passage through the restriction point. Though well-studied over the last two decades, the questions addressed here related to the G1-S cell cycle transition are still not sufficiently answered, and they are important to understanding fundamental cell biology and cancer biology. The use of single-cell imaging and application of a CDK4/6 sensor is an exciting approach to study Rb inactivation and the restriction point, and many of the experiments here are well designed. In addition, aspects of the authors' approach, including the use of multiple cell lines, make the observations robust. However, there are several significant concerns. While most of the concerns could be addressed through more analysis of experiments already performed and rewriting, more experiments are likely necessary to address the first point.

   Significant concerns:

   1. The study relies on interpretation of the adjusted "CDK4/6 sensor" signal as a specific reporter of CDK4/6 activity. Because this assumption of specificity is so critical, the authors should briefly review the evidence supporting it and better explain the accounting of other activities that may result in sensor phosphorylation. It is problematic that one of the conclusions in the discussion is that the "the CDK4/6 sensor may report other activities which can be targeted by CDK4/6 inhibitors," particularly as these inhibitors were used to validate specificity in ref 19 (Yang et al 2020). It is also important that mounting evidence here (for example Fig. 3A) and elsewhere show that CDK4/6 inhibitors such as palbociclib may also impact CDK2 activity.

   The conclusion that CDK4/6 activity is sufficient for Rb phosphorylation is in large part based on the correlation of the CDK4/6 sensor response with measurements of Rb phosphorylation using phosphospecific antibodies (Fig. 1). However, the sensor was constructed using an Rb-based docking site, which is expected to give the sensor properties of Rb as a substrate. With the perspective that the sensor reports on Rb-like substrate phosphorylation, rather than CDK4/6 activity per se, the reported correlation is inevitable and cannot be used to support the conclusion. The sensor phosphorylation of course correlates with Rb phosphorylation, as it was designed precisely to behave that way. Some other independent measurement of CDK4/6 activity, for example activity toward a different substrate or measurement of the abundance of CDK4/6-CycD complexes is needed to avoid this circular reasoning.

   The plausible interpretation that the sensor merely reports on the threshold of any CDK activity sufficient to phosphorylate Rb would also make other conclusions less novel, for example, that sensor phosphorylation correlates with E2F activation. If one replaces "CDK4/6 activity sensor" with "Rb-phosphorylation sensor," few conclusions from the first two figures are compelling. For this reason, it is critical that the authors further detect and quantify CDK4/6 activity in some independent way. Otherwise, the data as presented are not sufficient to support several of the main conclusions of the paper as stated, and the conclusions that likely could be fairly drawn lack novelty.

   1. Experiments similar to those presented in Fig. S3 were published before in ref 19 (Yang et al 2020). In the previous paper, the effects of the drugs were used to validate the specificity of the CDK sensors. Here, the sensors are invoked to characterize the specificity and effects of the drugs. Again, this circular logic undercuts the validity of the conclusions. It is similarly plausible that either both the sensor and drugs have specificity or both lack specificity; the outcome of the set of experiments would be the same. These experiments are not as critical to the overall study, and the authors may consider removing this part of the manuscript, if further experiments are not possible.

   2. These conclusions following presentation of the data in Fig. 3 are not well substantiated: "the temporal location of the restriction point with respect to stress and CDK4/6 inhibition is closely coupled with engagement of feedback pathways" and "our data demonstrates that inhibition of CDK4/6 activity before threshold-based activation of CDK2-Rb feedback causes cell-cycle exit." The experiments only measure CDK activity and not engagement of CDK2-Rb feedback, so there must be some assumption about the correspondence of a threshold of CDK2 activity to activation of the feedback. How is it known that feedback is engaged? This question persists throughout the study. The authors should more carefully define what CDK2-Rb feedback is and how its initiation is detected experimentally. Is it Rb hyperphosphorylation, mRNA expression of an E2F target gene, or protein levels of CycE? One of these should perhaps be measured in Fig. 3 to state the conclusion in terms of CDK2-Rb feedback rather than a CDK2 activity threshold. Alternatively, if further experimentation is not possible, the conclusions should be carefully stated in terms of CDK2 activity rather than invoking the idea of "CDK2-Rb feedback."

   3. A number of recent studies have similarly used single cell reporter and other analyses to probe the relative roles of CDK4/6, CDK2, and APC-Cdh1 in the restriction point (including Rb inactivation) and S phase entry (e.g. refs 2-4, 16-19, 22, 26, 28). The authors need to better explain how the observations here fit into the paradigms being developed and disputed through this body of work. Several of the conclusions stated here have been reached before. For example, the order that CDK4/6, CDK2, and Apc-CDK1 activity changing en route to S phase, that CDK4/6 is sufficient for Rb hyperphosphorylation, and that CDK2 activity is a threshold for the restriction point have all been described and supported in some of the referenced papers and contradicted in other references. Yet, similar conclusions are stated here as if they are novel. This study still is important in that the use of a CDK4/6 activity reporter may be a powerful approach to investigating these questions. But the subtleties of how this work is distinct and/or confirming needs to be made more clear for the reader to understand its significance.

   A related concern is that the results and conclusions described in Fig. 5 are not particularly surprising or novel. There is extensive literature characterizing high CDK2 activity, including its upregulation through CycE expression, as a mechanism of acquired tumor cell resistance to CDK4/6 inhibitors (see for example references reviewed in PMID: 32289274). Other published studies have examined the effects of ectopic CycE expression on accelerating G1-S, including in the absence of CycD activity or even the absence of Rb (see for example PMID: 8108147, PMID: 7601350, PMID: 1388095, PMID: 14645251, and PMID: 9192874). The authors should place their results in the context of these previous results and emphasize what insights are novel here.

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4. 

   eLife

   Aug 21, 2020

   ##Preprint Review

   This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

   ###Summary:

   Although the reviewers all agreed that you are addressing an important problem, and that a single cell approach is likely to yield important insights, they had serious concerns over the specificity of the probes and reagents you are using, and the degree of advance that your study represents over the current literature. With regard to the latter, the referees strongly suggested that a more comprehensive literature review is needed to put your results in context.

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5. 

   Version published to 10.1101/2020.06.24.169409 on bioRxiv

   Jun 24, 2020