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    Reply to the reviewers

    Response to all reviewers

    We thank all the reviewers for carefully considering our manuscript and providing useful comments and suggestions. We agree with the general comment that testing our key findings in breast cancer cells is important. We will therefore carry out this work over the coming months and include this data in the revision. The other specific comments we address individually in the point-by-point responses below, which provides an outline of the other new experiments we plan to carry out prior to revision.

    *In addition to this, we would like to just highlight one general point that we only picked up when considering these responses. It is important to highlight this to all reviewers now, since we believe it adds clinical weight to our conclusions. This relates to the issue of P53, which our manuscript shows drives resistance to CDK4/6 inhibition in cells by inhibiting long-term cell cycle withdrawal following genotoxic damage. *

    *P53 loss has been implicated in abemaciclib resistance in breast cancer patients (P53 mutation was detected in 2/18 responsive patients and 10/13 non-responsive patents (Patnaik et al., 2016)). This was recently corroborated in a larger scale study in breast cancer: the first whole exome sequencing study aimed at **characterising intrinsic and acquired resistance to CDK4/6 inhibitors (Wander et al., 2020). In this recent study, P53 loss/mutation was identified in 0/18 sensitive tumours, 14/28 intrinsically resistant tumours, and 9/13 tumour with acquired resistance. This was the most frequent single genetic change associated with resistance (58.5%), although 8 other genetic changes were also associated with resistance to differing degrees (7-27%). *

    Most of these other resistance events occurred in pathways known previously to help drive G1/S progression following CDK4/6 inhibition: i.e. fully predictable resistance mechanism (RB loss, CCNE2 amplification, ER loss, RAS/AKT1 activation, FGFR2/ERB22 mutation/amplification).* Importantly, when the authors attempted to recapitulate these resistance event in breast cancer cell lines, they could demonstrate the expected increase in proliferation following CDK4/6 inhibition in all situation tested, except for P53 loss. This caused the authors to conclude that “loss of P53 function is not sufficient to drive CDK4/6i resistance”. This would appear to us to be an unsatisfactory explanation given the clinical data. However, the authors speculated further that: “*Enrichment of TP53 mutation in resistant specimens may result from heavier pre-treatment (including chemotherapies), may be permissive for the development of other resistance-promoting alterations, or may cooperate with secondary alterations to drive CDK4/6i resistance in vivo.”

    *We believe that our data provide a crucial alternative explanation for these clinical findings. P53 does not affect the efficiency of a G1 arrest (fig.2), but rather it prevents the resulting genotoxic damage from inducing long-term cell cycle withdrawal (figs.2,3). Therefore, this could explain why it drives resistance in clinical disease but not in the in vitro cell growth assays employed by Wander et al. This highlights a crucial general point of our paper – important effects like this can be missed or misinterpreted until the true nature of long-term cell cycle withdrawal is appreciated. *

    As part of our breast cancer work at revision we will analyse this closely by comparing the effect of p53 loss on long-term cell cycle withdrawal. If the current RPE1 data holds true in breast cancer, then we believe that out study would provide a crucial explanation for these clinical findings, and in turn, these clinical data would throw weight behind our conclusion that genotoxic damage and p53 loss is a clinically important consequence of CDK4/6 inhibition in patients.


    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

    Comments on 'CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal'

    The rationale for this work is to understand the mechanism by which Cdk4/6 inhibitors inhibit tumour cell growth, specifically via senescence which seems to be a frequent outcome of Cdk4/6 inhibition. Although several mechanisms by which Cdk4/6 inhibition induce senescence have been proposed these have varied with the cancer cell model studied. To examine the mechanism for the cytostatic effect of cdk4/6i in therapy without potential confounding effects of different cancer cell line backgrounds, Crozier et al tackle this question in the non-transformed, immortalised diploid human cell line, RPE1. They use live cell imaging and colony formation to track the impact of G1 arrests of different lengths induced by a range of clinically relevant cdk4/6 inhibitors. They also use CRISPR-mediated removal of p53 to examine the role of p53 in the observed cell cycle responses. After noting that G1 arrest of over 2 days leads to a pronounced failure in continued cell cycle and proliferation that is associated with features of replication stress, they perform a proteomics analysis to determine the factors responsible for this. They discover that MCM complex components and some other replicative proteins are downregulated and overall suggest a mechanism whereby downregulation of these essential replication components during a prolonged G1 induce replication stress and ultimate failure of proliferation. They show the impact of cdk4/6 inhibition can be increased by combining with either aneuploidy induction (to indirectly elevate replication stress), aphidicolin (to directly elevate replication stress) or chemotherapy agents that damage DNA.

    Overall this is a well written and presented manuscript. Data are extremely clearly presented and described clearly within the text. Most appropriate controls were included and the work is performed to a high standard. I have a few comments about the proteomic analysis, and the link between MCM component deregulation and the induction of replication stress:

    - We thank the reviewer for this careful, detailed review, and for their kind comments about our work.

    **Major points:**

    1. Relevance to cancer. I appreciate that examining the mechanism in a diploid line is a sensible place to start. However it remains a bit unclear precisely which aspects of this mechanism might be conserved in cancer. It could be helpful to provide evidence (if it exists) of the impact of cdk4/6 inhibition in tumour cells. For example, are catastrophic mitosis, senescence, etc observed? And is there anything further known about the relationship between tumour mutations such as p53 and clinical response to Cdk4/6i?

    - It is important to point out that senescence is a common outcome of CDK4/6 inhibition in tumour cells, but exactly why tumour cells become senescent is still unclear. There have been many possible explanations proposed (see introduction), but so far, none of these implicate DNA damage. This is surprising for us, considering that DNA damage remains the best-known inducer of senescence and this is how most other broad-spectrum anti-cancer drugs induce permanent cell cycle exit. P53 loss has been associated with CDK4/6i resistance in the clinic, but this has also not previously been linked to genotoxic stress or senescence following CDK4/6 inhibition (see detailed description of this in comment to all reviewers above).* Therefore, our data could help to explain both of these key findings. However, we appreciate the importance of testing these results in breast cancer cells, therefore we will perform these experiments and include the data after revision.*

    Also - many of the phenotypes followed in this manuscript vary considerably with the length of G1 and the length of release. Which of these scenarios might mimic in vivo conditions?

    *- We see that a prolonged arrest (> 2 days) is necessary to see genotoxic effects in RPE cells. Clinically, palbociclib is administered in 3-week on/1-week off cycles, therefore this is consistent with the possibility that replication stress is induced during the off periods to cause genotoxic damage and cell cycle withdrawal. *

    Relating to the downregulation of MCM complex members, and the potential impact on origin licensing, how would this mechanism be manifest in cancer cells that have already deregulated gene transcription programs, and are already experiencing replication stress?

    - We hypothesise that cancer cells with ongoing replication stress maybe more sensitive to the MCM downregulation caused by CDK4/6 inhibition. The rationale is that a reduction in licenced origins would impair the ability of dormant origins to fire in response to replication problems, therefore making elevated levels of replication stress less tolerable. This is consistent with the enhanced effect of CDK4/6 inhibition seen when replication stress is elevated in RPE cells. Moreover, others have shown that experimentally reducing MCM protein levels induces hypersensitivity to replication stress in transformed cell lines such as U2OS and HeLa (Ge et al., 2007; Ibarra et al., 2008). Thus, low MCM levels and reduced origin licensing can contribute to replication failure in cancer cells.

    1. MCM protein levels and proposed impact on chromatin loading and origin licensing. Several MCM components are clearly reduced at the protein level. A chromatin assay (assaying fluorescence of signal remaining after pre-extraction of cytosolic proteins) suggests that MCM loading on chromatin is reduced, and this is taken to suggest a reduction in origin licensing. This is quite an indirect method - and it is difficult to conclude that the reduced chromatin bound fraction really represents a meaningful reduction in origin licensing. It would be more convincing if either positive and negative controls for this assay were included. Moreover it is not clear if this MCM reduction and proposed reduction in licensed origins would actually impact replication in an otherwise unperturbed state? Many more origins are licensed than actually fire during a normal S-phase, so it is not entirely clear that MCM levels could lead directly to replication stress here.

    *- Quantifying the non-extractable MCM proteins is in truth the most direct assay for origin licensing (not origin firing) available in human cells. To our knowledge, there are no reports of MCM loading by this or similar assays that are not strongly correlated with origin licensing per se. The reviewer is correct that modest reductions in MCM loading are well-tolerated in the absence of other perturbations. Specifically, Ge et al found no proliferation effects after 50% MCM loading reduction, but any further reduction introduced a proliferation delay (Ge et al., 2007). Of note, the U2OS cells used in that study also have a functional p53 response. *

    - Another important point that is worth emphasizing, is that many of the differentially downregulated proteins only function at replication forks (fig.4c). Therefore, we believe that the replication stress is a combined result of poor licencing and reduced levels of replication fork proteins that are needed after the origins fire. We will clarify this point in the revised manuscript.

    1. Loss of MCM protein levels and chromatin loading occurs after 1 day, not 4 days, of Cdk4/6 inhibition. The current proposal (based on evidence from the live cell imaging, and the induction of hallmarks of replication stress in figures 1-3) seems to be that something occurs between 2 and 7 days of cdk4/6i to prevent cells from resuming a normal cell cycle. Thus the proteomics was performed between 2 and 7 days, and MCM proteins identified as major changed proteins between those times. However, according to Western blots and FACS profiles in Figure 4, the major reduction in MCM protein levels, and chromatin loading occurs already at 1 day of of cdk4/6i (Figure 4d,e,f). However, replication stress is not observed after this timepoint (Figure 3) - so this seems to decouple the timings of MCM reduction from induction of replication stress. How can this be reconciled?

    - We agree that some of the observed changes to replisome components are quite considerable after just 1 day of arrest (some of these downregulations such as Cdc6 or phospho-Rb can be attributed to the cell cycle arrest itself - Cdc6 is unstable in G1 - but others, such MCM proteins, are not typically lost during G1). We were initially surprised by this too, considering that the phenotype clearly appears later than 1 day of arrest. It is important to state though, that the levels of almost all replisome components continue to decline as the duration of arrest is extended, eventually falling to considerably lower levels than seen after just 1 day. This is observed for MCM2, MCM3 and PCNA by western (fig.4e,e) and a large number of other replisome components by proteomics (fig.4c, 2 vs 7 days). Even MCM loading, which is 58% reduced after just 1-day arrest, is still reduced even further to just 20% of controls after 7 days (p- Our interpretation of the phenotypic data in light of this, is that replication problems become apparent when the number of licensed origins and the function of the replisome is compromised below a certain threshold; which most likely depends on cell type and, in particular, the levels of endogenous replication stress. So, in RPE cells, 1-day treatment is clearly tolerable, perhaps because there are still enough origins to complete DNA replication successfully. But, importantly, if replication stress is enhanced in these cells then 1-day of palbociclib arrest now starts to cause observable defects. This is evident in Figure 5h, where 1-day palbociclib treatment causes minimal effect on long-term growth on its own, but growth is reduced considerably when replication stress is elevated with genotoxic drugs. We interpret this to mean that the reduction in licenced origins and replisome components observed after 1 day of arrest, starts to become problematic in situations when replication stress is elevated.*

    *- This is actually an important point that we will highlight this at revision, because one prediction is that other cells with elevated replication stress (e.g. tumour cells with oncogene-induced replication stress) may begin to see defects after as little as 1-day palbociclib arrest. *

    **Minor points:**

    1. All the live cell tracking figures would be even more informative if a quantification of key features (such as a cumulative frequency of S-phase entry, or a mean+SD of time in G1, S and G2) were also presented.

    - We agree this will be useful, and we will include this information after revision.

    1. In Figure 2D the cells released from palbociclib seem to delay longer in G1 until they start to enter S phase, compared to cells co-treated with STLC (Figure 2B). Why would this be? It is difficult to tell if other subtle effects might be present in between the +STCL and -STLC conditions, so additional graphs such as those suggested above might be informative here in particular.

    *- Fig.2d shows a representative experiment (50 cells) because it is difficult to interpret these individual cell cycle profiles when more than 50 cells are presented. However, we have all the data from 3 experiments (150 cells), therefore we will also calculate timings as suggested and present this information after revision. *

    1. Figure 4f It would be helpful to see the FACS plot for at least one of the conditions quantified in the graph as a comparison.

    - These plots will be included after revision

    1. MCM2 protein is not down in p53 wt, but is reduced in p53 KO cells - why is this? And why is MCM2 not impacted when the other MCM complex members are?

    - We think perhaps there has been a mistake in interpreting these graphs. MCM2 is actually slightly lower in WT than KO cells at 1 days, and similar at 4 and 7 days (Fig.4d,e). MCM2 is also reduced slightly more than MCM3 (fig.4d,e) and MCM2, 3, 4, and 5 are all reduced by similar extents between 2 and 7 days palbociclib arrest (30-40% reductions; Fig.4c).

    Inducing aneuploidy with reversine to elevate replication stress may result in additional aneuploidy-related stresses that confound this interpretation. For example, aneuploidy per se is known to elevate p21 and p53 levels, and chromosome mis-segregation could elevate DNA damage. For these reasons these experiments are not as compelling as the direct elevation of replication stress using aphidicolin.

    - We agree that the aneuploidy experiment could have many different interpretations, and only one of these relates specifically to replication stress. This was also commented on by reviewer 3, so we feel it is best to remove this data and just keep the data on drugs that affect replication stress or DNA damage directly. We will address the effects of aneuploidy more extensively in a separate study.

    **Interesting points to follow up/add more mechanism**

    1. What is mechanism of protein downregulation of MCM etc? Was gene transcription impacted, or is this a question of protein stability? Depletion of one subunit can destabilise the complex leading to protein loss of the other MCM subunits, so perhaps this effect could be due to downregulation of a single MCM complex member.
    2. Are these findings specific to Cdk4/6 inhibitors, or would another means or arresting cells in G1 have the same impact?

    Both of these points are interesting questions and they are actually the focus of an entirely separate study that is ongoing. In particular, we are working on the mechanism(s) of MCM and replisome downregulation.

    Reviewer #1 (Significance (Required)):

    The central question of the paper is an important one so this work would be of interest to many in the clinical and preclinical fields, and also to the cell cycle and replication stress fields.

    - We thank the reviewer for this, and we agree that linking CDK4/6 inhibitors to genotoxic stress is important both for our understanding of cell cycle control and for cancer treatment. We are actually amazed that these drugs have not previously been linked to genotoxic stress, given that they appear to have broad pan-cancer activity and all other broad-spectrum anti-cancer drug work by causing genotoxic stress.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    In this paper, Saurin and colleagues investigate the effects of CDK4/6 inhibitors on cell cycle arrest and re-entry. The authors report that long-term G1 arrest induced by CDK4/6i interferes with DNA replication during the next cell cycle, leading to DNA damage and mitotic catastrophe. Additionally, this compromised replication state sensitizes cells to chemotherapeutics that enhance replication stress.

    The major claims advanced in this paper are well-supported by the presented evidence. Well I have several questions regarding the significance (see below), I have only a few minor points regarding the methodology.

    1. Regarding the down-regulation of MCM components induced by long-term palbo treatment shown in Figure 4: MCM levels are tightly regulated by cell cycle phase. I could imagine that this gene expression change may be a consequence of, for instance, 2 days CDK4/6i treatment arresting 95% of cells in G1 while 7 days of CDK4/6i treatment causes a 99.9% G1 arrest. The data in Figure 1B seems to argue against this hypothesis, but how was that data generated? Can the authors rule out a subtle change in S-phase % over 7 days in palbo?

    Alternately, is the down-regulation of MCM genes a consequence of cells entering senescence?

    *- We have performed extensive long-term movies with these cells, and we never see cells dividing or exiting G1 after the first day of palbociclib treatment. This is illustrated in fig.1b which demonstrates that 100% of FUCCI cells are in G1 (Red) at each of the timepoints. This will be clarified in the legend. In addition, MCM protein levels do not actually oscillate with cell cycle phase (Matson et al., 2017; Méndez and Stillman, 2000), although their mRNA levels certainly do (Leone et al., 1998; Whitfield et al., 2002). Furthermore, RPE and mammalian fibroblasts retain MCM proteins after 2 days of growth factor withdrawal despite transcriptional repression of their respective genes *(Cook et al., 2002; Matson et al., 2019)

    - We see significant changes in MCM levels at a time when cells are still permissive to enter the cell cycle following drug release. Therefore, MCM reduction is not a consequence of senescence. Rather, we believe that it is one of the causes of cell cycle withdrawal following the subsequent S-phase.

    1. For the drug studies presented in figure 5, it is important that the authors perform the appropriate statistical comparisons and analyses to demonstrate true synergy. The authors show that combining palbo and certain chemotherapies causes a greater decrease in clonogenicity than palbo alone. This may or may not be surprising (see below) - but this by itself is insufficient to support the claim that palbo "sensitizes" cells to genotoxins. If you treat cells with two poisons, in 9 out of 10 cases, you'll kill more cells than if you treat cells with one poison alone. But that could be due to totally independent effects - see, for instance, Palmer and Sorger Cell 2017. There are several well-established statistical methods for investigating drug synergy - like Loewe Additivity or Bliss Independence - and one of these methods should be used to analyze the drug-combination studies presented in Figure 5.

    - This analysis will be performed at revision

    Reviewer #2 (Significance (Required)):

    While this study is a comprehensive analysis of the effects of CDK4/6i in RPE1 cells in 2d culture, I am not convinced of its broader significance.

    1. So far as I can tell, the authors do not cite any studies establishing that CDK4/6i results in a significant increase in G1-arrested cells in treated patients. What evidence is there for this claim? I am aware that this has been demonstrated in xenografts and in mouse models, but I could not find evidence for this from actual clinical studies. Here, I am reminded of the very interesting work from Beth Weaver's group on paclitaxel - Zasadil STM 2014. While it had been widely assumed that paclitaxel causes a mitotic arrest, they actually show that this drug kills tumor cells by promoting mitotic catastrophe without inducing a complete mitotic arrest. Similarly, in the absence of existing clinical data, the underlying assumption regarding the effects of CDK4/6i that motivates this paper may not be accurate. For instance, if CDK4/6i acts through the immune system (as suggested by Jean Zhao and others), then this G1 arrest phenotype could be entirely secondary to the drug's actual mechanism-of-action.

    - We are very surprised by the suggestion that CDK4/6 inhibitors may not need to cause a G1 arrest in patient tumours. We appreciate that that these inhibitors effect the immune system in many different ways to combat tumourigenesis, but there is also an overwhelming amount of evidence that a G1 arrest in patient tumours is critical for the overall response. Perhaps the most striking evidence is the fact that RB loss in tumours is one of the best-characterised mechanism of resistance in breast cancer patients (Condorelli et al., 2018; Costa et al., 2020; Li et al., 2018; O'Leary et al., 2018; Wander et al., 2020). In addition, tumours types that typically achieve a poor CDK4/6i-induced G1 arrest in preclinical models, such as TNBCs, also exhibit a poor response to CDK4/6i therapy in patients. Recently a luminal androgen receptor subtype of TNBCs has been identified that responds to CDK4/6 inhibition, due to low CDK2 activity which can otherwise drive G1 progression independently of CDK4/6 in basal-like TNBCs (Asghar et al., 2017; Liu et al., 2017). This rationalises combination therapies that converge to inhibit G1 more effectively in this subtype (e.g. AR antagonist + CDK4/6 inhibition (Christenson et al., 2021)), which is akin to the oestrogen receptor and CDK4/6 combinations that have proven so successful at treating HR+ breast cancer. Many other combinations are also currently in trials based on the same premise that inhibiting upstream G1/S regulators can enhancing the response by inducing a more efficient G1 arrest (MEK, PI3K, AKT, mTOR) (Klein et al., 2018).

    *- In response to the specific question about clinical G1 arrest in patients, tumour samples from breast cancer patients shows a decrease in S-phase specific markers pRB and TopoIIa following abemaciclib treatment (Patnaik et al., 2016) and there is extensive evidence of a profound cell cycle arrest following CDK4/6 inhibition as judged by staining with the mitotic marker Ki67 (Hurvitz et al., 2020; Johnston et al., 2019; Ma et al., 2017; Prat et al., 2020). Whilst this does not formally prove a G1-arrest is specifical responsible for this overall cell cycle arrest, that is the implicit assumption given the known mechanism of action of CDK4/6 inhibitors in cells. *

    1. How relevant are RPE1 cells? Clinically, CDK4/6 inhibitors are combined with fulvestrant (which would not have an effect in RPE1), and the activity that they exhibit in breast cancer has not been matched in any other cancer types. The underlying biology of HR+ breast cancer (particularly regarding the regulation of CCND1 expression and the G1/S transition by estrogen) may not be recapitulated by other cell types. Moreover, the artificial media used in cell culture experiments may alter the regulation of the G1/S transition. I do not believe that these experiments conducted in RPE1 cells in 2d cell culture are generalizable.

    - Fulvestrant/tamoxifen are effective because they enhance the efficiency of a CDK4/6i arrest by reducing Cyclin D expression to enhance Cyclin D-CDK4/6 inhibition. That convergence onto the G1/S transition is why ER antagonists enhance the CDK4/6 response. i.e. CDK activity is inhibited and CycD transcription is reduced, therefore this double hit allows breast cancer cells to arrest in G1 more efficiently than healthy tissue which is not oestrogen-responsive (this provides yet more evidence the G1 arrest in tumours is crucial for the clinical response). It is true that RPE1 cells do not respond to the oestrogen treatment, but that is not really relevant here in our opinion. We are not testing the efficiency of a G1 arrest beyond the initial characterisation in figure 1. We are mainly examining how cells respond to that G1 arrest afterwards. It could be that components of the cell culture media affect that downstream response in unanticipated ways, but we feel that is very unlikely.

    - Having said that, we agree that the general point on the relevance of RPE cells is a valid one, and we will repeat key experiment in breast cancer cells. We suspect that the reason replisome components become widely downregulated during a G1 arrest will not be a specific phenomenon that is characteristic of one particular cell type. Nevertheless, it is important to validate that assumption.

    1. I am confused about the effects of CDK4/6i on genotoxin sensitivity. Replogle and Amon PNAS 2020 and several citations contained therein report that CDK4/6i protects cells from DNA damage. Moreover, trilaciclib has recently received FDA approval for its ability to protect the bone marrow from cytotoxic chemotherapy! Is this a question of dose timing/intensity? The FDA approval of trilaciclib for this indication should certainly be discussed. This underscores my concern that certain findings in this paper are RPE1/tissue culture artifacts, with limited generalizability.

    - The studies the reviewer refers to demonstrate that halting cell cycle progression can protect cells from genotoxic drugs that cause DNA damage during S-phase. However, we can only think that the reviewer must have missed the critical point here: The genotoxic agents in figure 5 were added after washout from CDK4/6 inhibition (we will highlight this more clearly in the revised manuscript). After drug removal, cells enter S-phase with replication competence problems (as a result of the CDK4/6 arrest) and they then experience additional problems during S-phase (as a result of the genotoxic agents included following washout). These effects synergise to enhance replication stress, a key conclusion of figure 5.

    - This does is in no way support that notion that “findings in this paper are RPE1/tissue culture artefacts with limited generalizability”. Experiments in 2D tissue culture have furnished some of the most important fundamental discoveries in cancer research. It remains to be seen whether our study will cause a paradigm shift in our thinking about how CDK4/6 inhibitors work, but we believe that it may do. We appreciate that this will not become clear until our findings are followed up and validated in preclinical models and human disease, but that does not, in our opinion, make them any less valid at this stage. As stated earlier, we will confirm this is not a RPE1 cell phenomenon, but if this holds up in breast cancer cells then we believe our data will have an important impact on future preclinical and clinical work in this area.

    **Referees cross-commenting**

    I think that we largely agree that RPE1 is not a great model for this study, and repeating certain key experiments in an ER+ BC line like MCF7 may be warranted.

    *- We agree that it would add value to examine our findings in BC cells, therefore we will address this point at revision by repeating key experiments in BC cells. *

    Additionally, I wanted to draw attention to the fact that, to my knowledge, the evidence for palbociclib inducing a G1 arrest in patients is incredibly spotty. For early-stage breast tumors where palbo is most effective, nearly all tumor cells are in G1 anyway. I think that it makes the most sense that palbo is actually working through immune modulation or through some secondary mechanism, rather than enforcing a G1 arrest. So I'm not sure about the premise of this study.

    - As discussed above, there is extensive evidence that proliferation is reduced in response to CDK4/6 inhibition in patients (Hurvitz et al., 2020; Johnston et al., 2019; Ma et al., 2017; Patnaik et al., 2016; Prat et al., 2020). We agree that proliferation in patient tumours can be slower than observed in preclinical models, and there can be many reasons for this, especially within solid tumour where hypoxia is a major factor that limits proliferation. However, we do not agree that this implies that drugs that target these tumours do not act on proliferating cells. In fact, most other broad-spectrum non-targeted chemotherapies used to treat cancer also work by targeting dividing cells, and many of these are also more effective in early stage breast cancer. In addition, and as discussed extensively above, there are many studies supporting the interpretation that a G1 arrest is critical for CDK4/6i response in breast cancer patients. Considering all of these points, we strongly believe that the premise of our study – to characterise why a G1 arrest becomes irreversible – is valid and important. This point Is also made in numerous recent reviews which also highlight that this key mechanistic information is currently lacking (Goel et al., 2018; Klein et al., 2018; Knudsen and Witkiewicz, 2017; Wagner and Gil, 2020).

    *- We do not disagree that the immune effects are important in patients – indeed, we cited and discussed these studies in our manuscript. However, we would argue that this works together with a G1 arrest in tumour cells. The G1 arrest most likely induces a senescent response that stimulates immune engagement and tumour clearance. These multifactorial effect of CDK4/6 inhibition, on both the tumour and the immune system, are discussed at length in these reviews: (Goel et al., 2018; Klein et al., 2018; Wagner and Gil, 2020). *

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

    The authors clearly demonstrate, with appropriate techniques, that cells treated with clinically relevant CDK4/6 inhibitors lead to a cell cycle arrest, that is only partly reversible.

    The authors also demonstrate clearly that release from a cdk4/6i arrest leads to two phenomena: the inability to initiate S-phase, and a cell cycle exit in G2.

    The inability to initiate S-phase is partly dependent on p53, the cell cycle exit is fully dependent on p53.

    In the absence of p53, cells that are released from a CDK4/6i block frequently enter mitosis with unrepaired DNA lesions.

    The authors clearly demonstrate that cdk4/6 inhibition leads to down regulation of key replication genes.

    Combined treatment with genotoxic agents further exaggerates the phenotype of cell cycle exit upon cdk4/6 inhibition.

    **Specific comments:**

    Figure 1B: the loss of reversibility remains at approximately 50%. Does the phenotype of replication protein depletion not happen in the 50% of cells that do restart the cell cycle? it would be good if the authors could experimentally address the heterogeneity that is observed.

    *- This is actually a result of the fixed analysis use in fig.1B. The irreversibility is much higher than 50% after long durations of arrest, but at the 24h timepoint used in this fixed assay many cells have exited G1 but not yet had a chance to revert back into G1 from S/G2 phase. We will reinforce this point in the legend. This highlights the value of our extensive live cell assays that can fully capture cell cycle profiles, and accurately determine when cell do/don’t enter or withdraw from different stages of the cell cycle. We believe that an overreliance of fixed endpoints in previous studies may have contributed to the genotoxic effects in S-phase being missed previously: many studies show senescence after drug washout, but the cause of that senescence only becomes apparent when you observe that cells withdraw with defects after the first S-phase. *

    Figure 1C: the G1 state after S-phase. The read-out here is loss of the Fucci reporter geminin. Does observation reflect p53-dependent activation of the APC/C-Cdh1 prematerely? this is a known effect of persistent DNA damage in G2 cells.

    - Yes, we expect that APC/C-Cdh1 activation causes geminin and cyclin degradation when cells permanently withdraw from the cell cycle from G2. This is likely caused by p53-dependent p21 activation in response to DNA replication defects, as has been shown previously in direct response to DNA damage.

    Figure 2: there seem to be two distinct phenotypes when comparing p53-wt and p53-KO: the ability to initiate S-phase after CDK4/6i removal (which is largely gone in p53 KO, only slight number after 7d treatment). And cell cycle-drop-out after S-phase (this seems to be fully p53 dependent). I am not sure if a single mechanisms explains both.

    - We agree that there are p53-dependent effects on speed/extent of S-phase entry and on the resulting withdrawal from G2. It may not be a single mechanism that connected these effects, although they may be related. Our manuscript mainly focusses on the DNA replication defects and cell cycle withdrawal, but in the future, it will be important to also characterise what causes the delay in cell cycle re-entry following CDK4/6 inhibition. We suspect that this could reflect differing depths of quiescence, potentially caused by p21, which would explain the p53-dependence.

    Figure 3a: related to the proviso point. it is unclear if the p21 up regulation happens in G1 or G2 cells, and related to the inability of cells to initiate S-phase, or the cell cycle exit in G2.

    *- This is a good point, and as discussed above, we suspect both maybe related to p21. We will examine p21 levels during a G1 arrest to compare to the levels seen following release, and we will include this data after revision. *

    It is stated that a combined action of the p53 pathways and ATR signaling prevent mitotic entry in RPE-wt cells. However, ATR should also be able to do this in p53-KO cells. Does cdk4/6i inhibiton also down-regulation of ATR pathway components?

    - We do not detect downregulation of any ATRi components in the mass spec data comparing 2 and 7 day palbociclib arrest.

    Following the observation that CDK4/6i leads to replication stress, I would hypothesise that these cells would be very sensitive to agents that inhibit the response to replication stress (inhibitors of Wee1, ATR or Chk1). Yet, these agents work preferentially in p53-deficient cells, and require cell cycle progression. Sequential treatment with CDK4/6 inhibition followed by cell cycle checkpoint inhibition may help in uncovering the phenotype.

    - This is a good point and we will perform experiments with ATR inhibitors after release from CDK4/6 inhibition to examine if this enhances the phenotype.

    The authors increase the amount of replication stress using chemotherapeutic approaches or MPS1 inhibitors. The chemotherapeutic approaches are relevant clinically, but mechanistically it don't understand this beyond adding up treatments that lead to replication defects.

    - We agree that the main value of these experiments is not to provide mechanistic insight, but rather to demonstrate that CDK4/6 inhibition can enhance the effect of current genotoxic drugs. Considering CDK4/6 inhibitors are well-tolerated, this could represent an effective way to enhance the tumour-selectivity of current genotoxic therapeutics. This has been suggested previously in a pancreatic cancer study (Salvador-Barbero et al., 2020), but the reasons given for synergy were different (DNA damage repair) and the order of drugs exposure was reversed (genotoxic before CDK4/6i). This underscores the potential importance of our new data.

    - From a mechanistic point of view, these data do still suggest that CDK4/6i and genotoxic drugs converge onto the same replication stress phenotype, thereby supporting our overall conclusions. One interpretation is that a reduction in replisome levels and licenced replication origins impairs the ability of cells to overcome replication problems induced by chemotherapy drugs. Conceptualising how these drugs may synergize in this way will be important in designing new studies and trials to address this synergy more broadly.

    The aneuploidy treatment is a bit weird, because it may trigger a p53 response, before the cells are released from a cdk4.6i arrest. besides, mps1 inhibition does more than just cause replication stress and is not very clinically relevant in this context.

    - We agree that the aneuploidy experiment could have many different interpretations, and only one of these relates specifically to replication stress. This was also commented on by reviewer 1, so we feel it is best to remove this data and just keep the data on drugs that affect replication stress or DNA damage directly. We will address the effects of aneuploidy more extensively in a separate study.

    Reviewer #3 (Significance (Required)):

    In their manuscript entitled: Crozier and co-workers studied the effects of CDK4/6 inhibition on cell growth. CDK4/6 inhibitors are currently used in the treatment for hormone-positive breast cancers, but their cell biological effects on tumor cells remain incompletely clear, which may hamper the further clinical development of these drugs for breast cancer or other cancers.

    Inhibition of CDK4/6 is known to trigger a cell cycle arrest, and it is currently unclear how this could lead to long-term tumor control. This manuscript addresses the question why cdk4/6 inhibitors cause long-term cell cycle exit.

    *- We thank the reviewer for this simple description of our work, which we think pitches the significance very clearly. There are currently 15 different CDK4/6 inhibitors in clinical trials, and more than 100 further trials using the 3 currently licenced inhibitors in a wide variety of tumour types and drug combinations. Although the clinical work on these drugs is huge, it is unclear how they cause long-term cell cycle arrest and we now link this to genotoxic stress for the first time. This explains clearly why this work is potentially very significant. We agree, however, that the main caveat is the need to demonstrate our findings are also applicable to breast cancer cells. But, if this is the case, we believe this would represent a paradigm shift in our understanding of how these drugs work, especially considering that genomic damage is an universal route to prolonged cell cycle exit in response to almost all other broad-spectrum anti-cancer drugs. *

    There are two issues that affect the significance of the findings:

    the authors start their manuscript with a strong translational/clinical issue, but solely use RPE1 cell lines to address this issue2. it remains unclear if their observations hold true in breast cancer models. it would be advised to repeat key findings in a hormone receptor-positive breast cancer model.

    - We will examine the applicability of our findings in breast cancer cells and include this work at revision.

    the effects of CDK4/6 inhibitors are observed in clinically relevant doses. however, the effects are observed upon switch-like wash out. this does not per se reflect the pharmacodynamics of more gradual increase and decrease of drug concentrations in tuner cells. by washing out the CDK4/6 inhibitors. the significant of this work would be greater if cell cycle exit with replication stress would be observed either in clinical samples or in vivo treated cancer cells.

    - We agree that the significance of this work will ultimately only become fully apparent if replication stress is confirmed in clinical samples or in vivo. We envisage that our study will stimulate exactly this type of analysis in future. However, we would also add that the gradual increase/decrease in drug concentrations seen in patients is still likely to lead to switch like cell cycle re-entry given the switch-like nature of cell cycle controls at the G1/S transition. So, the timing may be different, but we would not predict that the downstream response in S-phase would be. However, whether replication stress is seen during drug-free washout periods in patients is clearly a critical future question, as we highlight in the discussion.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors clearly demonstrate, with appropriate techniques, that cells treated with clinically relevant CDK4/6 inhibitors lead to a cell cycle arrest, that is only partly reversible.

    The authors also demonstrate clearly that release from a cdk4/6i arrest leads to two phenomena: the inability to initiate S-phase, and a cell cycle exit in G2.

    The inability to initiate S-phase is partly dependent on p53, the cell cycle exit is fully dependent on p53.

    In the absence of p53, cells that are released from a CDK4/6i block frequently enter mitosis with unrepaired DNA lesions.

    The authors clearly demonstrate that cdk4/6 inhibition leads to down regulation of key replication genes.

    Combined treatment with genotoxic agents further exaggerates the phenotype of cell cycle exit upon cdk4/6 inhibition.

    Specific comments:

    Figure 1B: the loss of reversibility remains at approximately 50%. Does the phenotype of replication protein depletion not happen in the 50% of cells that do restart the cell cycle? it would be good if the authors could experimentally address the heterogeneity that is observed.

    Figure 1C: the G1 state after S-phase. The read-out here is loss of the Fucci reporter geminin. Does observation reflect p53-dependent activation of the APC/C-Cdh1 prematerely? this is a known effect of persistent DNA damage in G2 cells.

    Figure 2: there seem to be two distinct phenotypes when comparing p53-wt and p53-KO: the ability to initiate S-phase after CDK4/6i removal (which is largely gone in p53 KO, only slight number after 7d treatment). And cell cycle-drop-out after S-phase (this seems to be fully p53 dependent). I am not sure if a single mechanisms explains both.

    Figure 3a: related to the proviso point. it is unclear if the p21 up regulation happens in G1 or G2 cells, and related to the inability of cells to initiate S-phase, or the cell cycle exit in G2.

    It is stated that a combined action of the p53 pathways and ATR signaling prevent mitotic entry in RPE-wt cells. However, ATR should also be able to do this in p53-KO cells. Does cdk4/6i inhibiton also down-regulation of ATR pathway components?

    Following the observation that CDK4/6i leads to replication stress, I would hypothesise that these cells would be very sensitive to agents that inhibit the response to replication stress (inhibitors of Wee1, ATR or Chk1). Yet, these agents work preferentially in p53-deficient cells, and require cell cycle progression. Sequential treatment with CDK4/6 inhibition followed by cell cycle checkpoint inhibition may help in uncovering the phenotype.

    The authors increase the amount of replication stress using chemotherapeutic approaches or MPS1 inhibitors. The chemotherapeutic approaches are relevant clinically, but mechanistically it don't understand this beyond adding up treatments that lead to replication defects.

    The aneuploidy treatment is a bit weird, because it may trigger a p53 response, before the cells are released from a cdk4.6i arrest. besides, mps1 inhibition does more than just cause replication stress and is not very clinically relevant in this context.

    Significance

    In their manuscript entitled: Crozier and co-workers studied the effects of CDK4/6 inhibition on cell growth. CDK4/6 inhibitors are currently used in the treatment for hormone-positive breast cancers, but their cell biological effects on tumor cells remain incompletely clear, which may hamper the further clinical development of these drugs for breast cancer or other cancers.

    Inhibition of CDK4/6 is known to trigger a cell cycle arrest, and it is currently unclear how this could lead to long-term tumor control. This manuscript addresses the question why cdk4/6 inhibitors cause long-term cell cycle exit.

    There are two issues that affect the significance of the findings:

    -the authors start their manuscript with a strong translational/clinical issue, but solely use RPE1 cell lines to address this issue2. it remains unclear if their observations hold true in breast cancer models. it would be advised to repeat key findings in a hormone receptor-positive breast cancer model.

    -the effects of CDK4/6 inhibitors are observed in clinically relevant doses. however, the effects are observed upon switch-like wash out. this does not per se reflect the pharmacodynamics of more gradual increase and decrease of drug concentrations in tuner cells. by washing out the CDK4/6 inhibitors. the significant of this work would be greater if cell cycle exit with replication stress would be observed either in clinical samples or in vivo treated cancer cells.

    -the effects of CDK4/6 inhibitors are observed in clinically relevant doses. however, the effects are observed upon switch-like wash out. this does not per se reflect the pharmacodynamics of more gradual increase and decrease of drug concentrations in tuner cells. by washing out the CDK4/6 inhibitors. the significant of this work would be greater if cell cycle exit with replication stress would be observed either in clinical samples or in vivo treated cancer cells.

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper, Saurin and colleagues investigate the effects of CDK4/6 inhibitors on cell cycle arrest and re-entry. The authors report that long-term G1 arrest induced by CDK4/6i interferes with DNA replication during the next cell cycle, leading to DNA damage and mitotic catastrophe. Additionally, this compromised replication state sensitizes cells to chemotherapeutics that enhance replication stress.

    The major claims advanced in this paper are well-supported by the presented evidence. Well I have several questions regarding the significance (see below), I have only a few minor points regarding the methodology.

    1. Regarding the down-regulation of MCM components induced by long-term palbo treatment shown in Figure 4: MCM levels are tightly regulated by cell cycle phase. I could imagine that this gene expression change may be a consequence of, for instance, 2 days CDK4/6i treatment arresting 95% of cells in G1 while 7 days of CDK4/6i treatment causes a 99.9% G1 arrest. The data in Figure 1B seems to argue against this hypothesis, but how was that data generated? Can the authors rule out a subtle change in S-phase % over 7 days in palbo?

    Alternately, is the down-regulation of MCM genes a consequence of cells entering senescence?

    1. For the drug studies presented in figure 5, it is important that the authors perform the appropriate statistical comparisons and analyses to demonstrate true synergy. The authors show that combining palbo and certain chemotherapies causes a greater decrease in clonogenicity than palbo alone. This may or may not be surprising (see below) - but this by itself is insufficient to support the claim that palbo "sensitizes" cells to genotoxins. If you treat cells with two poisons, in 9 out of 10 cases, you'll kill more cells than if you treat cells with one poison alone. But that could be due to totally independent effects - see, for instance, Palmer and Sorger Cell 2017. There are several well-established statistical methods for investigating drug synergy - like Loewe Additivity or Bliss Independence - and one of these methods should be used to analyze the drug-combination studies presented in Figure 5.

    Significance

    While this study is a comprehensive analysis of the effects of CDK4/6i in RPE1 cells in 2d culture, I am not convinced of its broader significance.

    1. So far as I can tell, the authors do not cite any studies establishing that CDK4/6i results in a significant increase in G1-arrested cells in treated patients. What evidence is there for this claim? I am aware that this has been demonstrated in xenografts and in mouse models, but I could not find evidence for this from actual clinical studies. Here, I am reminded of the very interesting work from Beth Weaver's group on paclitaxel - Zasadil STM 2014. While it had been widely assumed that paclitaxel causes a mitotic arrest, they actually show that this drug kills tumor cells by promoting mitotic catastrophe without inducing a complete mitotic arrest. Similarly, in the absence of existing clinical data, the underlying assumption regarding the effects of CDK4/6i that motivates this paper may not be accurate. For instance, if CDK4/6i acts through the immune system (as suggested by Jean Zhao and others), then this G1 arrest phenotype could be entirely secondary to the drug's actual mechanism-of-action.

    2. How relevant are RPE1 cells? Clinically, CDK4/6 inhibitors are combined with fulvestrant (which would not have an effect in RPE1), and the activity that they exhibit in breast cancer has not been matched in any other cancer types. The underlying biology of HR+ breast cancer (particularly regarding the regulation of CCND1 expression and the G1/S transition by estrogen) may not be recapitulated by other cell types. Moreover, the artificial media used in cell culture experiments may alter the regulation of the G1/S transition. I do not believe that these experiments conducted in RPE1 cells in 2d cell culture are generalizable.

    3. I am confused about the effects of CDK4/6i on genotoxin sensitivity. Replogle and Amon PNAS 2020 and several citations contained therein report that CDK4/6i protects cells from DNA damage. Moreover, trilaciclib has recently received FDA approval for its ability to protect the bone marrow from cytotoxic chemotherapy! Is this a question of dose timing/intensity? The FDA approval of trilaciclib for this indication should certainly be discussed. This underscores my concern that certain findings in this paper are RPE1/tissue culture artifacts, with limited generalizability.

    Referees cross-commenting

    I think that we largely agree that RPE1 is not a great model for this study, and repeating certain key experiments in an ER+ BC line like MCF7 may be warranted.

    Additionally, I wanted to draw attention to the fact that, to my knowledge, the evidence for palbociclib inducing a G1 arrest in patients is incredibly spotty. For early-stage breast tumors where palbo is most effective, nearly all tumor cells are in G1 anyway. I think that it makes the most sense that palbo is actually working through immune modulation or through some secondary mechanism, rather than enforcing a G1 arrest. So I'm not sure about the premise of this study.

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    Referee #1

    Evidence, reproducibility and clarity

    Comments on 'CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal'

    The rationale for this work is to understand the mechanism by which Cdk4/6 inhibitors inhibit tumour cell growth, specifically via senescence which seems to be a frequent outcome of Cdk4/6 inhibition. Although several mechanisms by which Cdk4/6 inhibition induce senescence have been proposed these have varied with the cancer cell model studied. To examine the mechanism for the cytostatic effect of cdk4/6i in therapy without potential confounding effects of different cancer cell line backgrounds, Crozier et al tackle this question in the non-transformed, immortalised diploid human cell line, RPE1. They use live cell imaging and colony formation to track the impact of G1 arrests of different lengths induced by a range of clinically relevant cdk4/6 inhibitors. They also use CRISPR-mediated removal of p53 to examine the role of p53 in the observed cell cycle responses. After noting that G1 arrest of over 2 days leads to a pronounced failure in continued cell cycle and proliferation that is associated with features of replication stress, they perform a proteomics analysis to determine the factors responsible for this. They discover that MCM complex components and some other replicative proteins are downregulated and overall suggest a mechanism whereby downregulation of these essential replication components during a prolonged G1 induce replication stress and ultimate failure of proliferation. They show the impact of cdk4/6 inhibition can be increased by combining with either aneuploidy induction (to indirectly elevate replication stress), aphidicolin (to directly elevate replication stress) or chemotherapy agents that damage DNA.

    Overall this is a well written and presented manuscript. Data are extremely clearly presented and described clearly within the text. Most appropriate controls were included and the work is performed to a high standard. I have a few comments about the proteomic analysis, and the link between MCM component deregulation and the induction of replication stress:

    Major points:

    1. Relevance to cancer. I appreciate that examining the mechanism in a diploid line is a sensible place to start. However it remains a bit unclear precisely which aspects of this mechanism might be conserved in cancer. It could be helpful to provide evidence (if it exists) of the impact of cdk4/6 inhibition in tumour cells. For example, are catastrophic mitosis, senescence, etc observed? And is there anything further known about the relationship between tumour mutations such as p53 and clinical response to Cdk4/6i? Also - many of the phenotypes followed in this manuscript vary considerably with the length of G1 and the length of release. Which of these scenarios might mimic in vivo conditions? Relating to the downregulation of MCM complex members, and the potential impact on origin licensing, how would this mechanism be manifest in cancer cells that have already deregulated gene transcription programs, and are already experiencing replication stress?
    2. MCM protein levels and proposed impact on chromatin loading and origin licensing. Several MCM components are clearly reduced at the protein level. A chromatin assay (assaying fluorescence of signal remaining after pre-extraction of cytosolic proteins) suggests that MCM loading on chromatin is reduced, and this is taken to suggest a reduction in origin licensing. This is quite an indirect method - and it is difficult to conclude that the reduced chromatin bound fraction really represents a meaningful reduction in origin licensing. It would be more convincing if either positive and negative controls for this assay were included. Moreover it is not clear if this MCM reduction and proposed reduction in licensed origins would actually impact replication in an otherwise unperturbed state? Many more origins are licensed than actually fire during a normal S-phase, so it is not entirely clear that MCM levels could lead directly to replication stress here.
    3. Loss of MCM protein levels and chromatin loading occurs after 1 day, not 4 days, of Cdk4/6 inhibition. The current proposal (based on evidence from the live cell imaging, and the induction of hallmarks of replication stress in figures 1-3) seems to be that something occurs between 2 and 7 days of cdk4/6i to prevent cells from resuming a normal cell cycle. Thus the proteomics was performed between 2 and 7 days, and MCM proteins identified as major changed proteins between those times. However, according to Western blots and FACS profiles in Figure 4, the major reduction in MCM protein levels, and chromatin loading occurs already at 1 day of of cdk4/6i (Figure 4d,e,f). However, replication stress is not observed after this timepoint (Figure 3) - so this seems to decouple the timings of MCM reduction from induction of replication stress. How can this be reconciled?

    Minor points:

    1. All the live cell tracking figures would be even more informative if a quantification of key features (such as a cumulative frequency of S-phase entry, or a mean+SD of time in G1, S and G2) were also presented.
    2. In Figure 2D the cells released from palbociclib seem to delay longer in G1 until they start to enter S phase, compared to cells co-treated with STLC (Figure 2B). Why would this be? It is difficult to tell if other subtle effects might be present in between the +STCL and -STLC conditions, so additional graphs such as those suggested above might be informative here in particular.
    3. Figure 4f It would be helpful to see the FACS plot for at least one of the conditions quantified in the graph as a comparison.
    4. MCM2 protein is not down in p53 wt, but is reduced in p53 KO cells - why is this? And why is MCM2 not impacted when the other MCM complex members are?
    5. Inducing aneuploidy with reversine to elevate replication stress may result in additional aneuploidy-related stresses that confound this interpretation. For example, aneuploidy per se is known to elevate p21 and p53 levels, and chromosome mis-segregation could elevate DNA damage. For these reasons these experiments are not as compelling as the direct elevation of replication stress using aphidicolin.

    Interesting points to follow up/add more mechanism

    1. What is mechanism of protein downregulation of MCM etc? Was gene transcription impacted, or is this a question of protein stability? Depletion of one subunit can destabilise the complex leading to protein loss of the other MCM subunits, so perhaps this effect could be due to downregulation of a single MCM complex member.
    2. Are these findings specific to Cdk4/6 inhibitors, or would another means or arresting cells in G1 have the same impact?

    Significance

    The central question of the paper is an important one so this work would be of interest to many in the clinical and preclinical fields, and also to the cell cycle and replication stress fields.

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