Loss of CDK4/6 activity in S/G2 phase leads to cell cycle reversal

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle1,2, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein3–5. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell’s progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle 1,2 , when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein [3][4][5] . Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell's progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.
The restriction point (R point) marks the point in the cell cycle when mammalian cells become independent of mitogen signalling and are irreversibly committed to proliferation 1,2 . Molecularly, this irreversible cell fate decision is thought to arise because cells convert extracellular mitogen signals into a self-sustaining positive feedback loop (Fig. 1a). Mitogens activate cyclin-dependent kinases 4 and 6 (CDK4/6), which phosphorylate the tumour suppressor retinoblastoma protein (Rb), leading to activation of the transcription factors E2F1-3. These E2Fs promote transcription of cyclins E and A, which form complexes with CDK2 that also phosphorylate Rb and further drive E2F-mediated transcription of cyclins E and A. Thus, once activated, CDK2 is thought to form a positive feedback loop with Rb that can maintain continuous CDK2 activity even in the absence of upstream mitogen signalling 6 , exhibiting properties of bistability and irreversible hysteresis with respect to mitogen concentration (Fig. 1b) 7,8 . Therefore, the textbook model of the R point is that CDK2 activation and Rb phosphorylation determine the transition from a pre-R to post-R state [3][4][5] . However, recent studies have also found that CDK2 activity and Rb phosphorylation status cannot determine whether all cells have crossed the R point, observing outlier cells that appear to contradict the textbook model 3,4,[9][10][11] . This discrepancy calls into question both the long-standing R-point model and our basic understanding of mammalian cell cycle control. Thus, new single-cell studies are needed to uncover a universal model of cell cycle control.

Mitogen signalling maintains CDK2 activity in S/G2
The principal tenet of the R-point model is that pre-R cells are sensitive to loss of mitogen signalling and will exit the cell cycle to G0 if mitogens are removed, whereas post-R cells are insensitive and will complete mitosis, after which their daughter cells will arrest in G0 (refs. 9,12) (Fig. 1c). To test this fundamental prediction of the R-point model, we serum starved MCF-10A cells or treated them with a mitogen-activated protein kinase kinase inhibitor (MEKi) or a cyclin-dependent kinases 4 and 6 inhibitor (CDK4/6i) for 48 h and then, measured DNA content and Rb phosphorylation 13 . Contrary to the R-point model, as many as 15% of MCF-10A cells had 4N DNA content (4 copies of each chromosome, for example diploid), indicating that these cells did not complete mitosis and arrest in G0 but instead, exited the cell cycle from G2 phase (Fig. 1d, histograms). However, unlike a typical G2 arrested cell with hyperphosphorylated Rb, these cells had hypophosphorylated Rb (Fig. 1d, scatterplots). Thus, rather than all cells arresting in G0 with 2N DNA content following loss of mitogen signalling, we found that some cells entered a 'G0-like' state with 4N DNA content and hypophosphorylated Rb, suggesting that the proposed feedback loop between CDK2 and Rb was not indefinitely maintained in these cells. Indicative of a general phenomenon, we recapitulated this cell cycle state in primary cells, non-transformed cells, transformed cells and cells lacking the stress-induced CDKis p21, p27 and p16 (Extended Data Fig. 1).
To assess how loss of mitogen signalling affected CDK2 activity and Rb phosphorylation, we transduced MCF-10A cells with a CDK2 activity sensor (Extended Data Fig. 2a), the activation of which corresponds with Rb phosphorylation 4 . Since it has been shown that the R point precedes S phase 14 and inactivation of the anaphase-promoting complex/ cyclosome (APC/C) marks entry into S phase 15 , we utilized an APC/C activity sensor 16 to identify pre-R (APC/C on) or post-R (APC/C off) cells (Extended Data Fig. 2a,b). We combined these activity sensors Article with live-cell imaging and automated single-cell tracking to measure CDK2 activity after mitogen removal, MEK inhibition or CDK4/6 inhibition in post-R cells.
Post-R cells treated with dimethylsulfoxide (DMSO) divided repeatedly (Fig. 1e,g and Supplementary Video 1), while treatments perturbing the mitogen signalling pathway resulted in two distinct cell cycle trajectories (Fig. 1f,g and Extended Data Fig. 2b,c). Most post-R cells built up CDK2 activity until they divided into two daughter cells, which arrested in G0 with low CDK2 activity and high APC/C activity (Fig. 1f, upper panel and Supplementary Video 2). However, up to 15% of post-R cells did not enter mitosis but instead, gradually lost CDK2 activity and prematurely reactivated the APC/C (Fig. 1f, lower panel, Fig. 1h   Rb and 4N DNA content) (Fig. 1i). Notably, entry into this G0-like state explained the appearance of a persistent G2 population when looking at DNA content alone (Fig. 1d, pink boxes). Therefore, in contrast to the textbook model of the R point (Fig. 1c), we find that not all post-R cells are irreversibly committed to proliferation since some cells exit the cell cycle to a G0-like state after loss of mitogen signalling (Fig. 1j). Furthermore, these G0-like cells eventually exhibited senescence-associated β-galactosidase activity (Extended Data Fig. 2d-f), consistent with previous reports that premature APC/C reactivation in S/G2 phase is a precursor to cellular senescence 17,18 .

Competition between mitosis and exit determines cell fate
To understand why these outlier post-R cells failed to enter mitosis after loss of mitogen signalling, we sorted all cells by time of treatment with respect to S-phase entry (that is, APC/C inactivation) and found that cells were more likely to exit the cell cycle when closer to the start of S phase than mitosis when treated ( Fig. 2a and Extended Data Fig. 3). Since mitosis and cell cycle exit are mutually exclusive fates that preclude the observation of each other 19 , we hypothesized that a cell closer to mitosis when mitogen signalling was lost may not have had enough time to respond and would enter mitosis instead of exiting the cell cycle, while a cell closer to S phase would exit the cell cycle instead of entering mitosis. This temporal competition between opposing fate outcomes can be conceptualized as a competition between two molecular clocks, representing the time taken to progress from S phase to mitosis (the mitosis clock) and the time required to lose CDK2 activity after loss of mitogen signalling (the cell cycle exit clock) (Fig. 2b). If, for a given cell, its cell cycle exit clock is longer than its mitosis clock, then mitosis wins the competition and cell cycle exit would not be observed and vice versa. The competing clock model makes two main predictions. The first prediction is that since all cells contain both a mitosis clock and a cell cycle exit clock that operate independently, blocking either clock would allow the opposing fate to win the competition (Fig. 2c). To test this first prediction, we tipped the balance in favour of mitosis by growing cells in full-growth media and observed that nearly all cells reached mitosis, revealing an underlying 'pre-competition' distribution of mitosis times (Fig. 2d,e and Extended Data Fig. 4d-f). Conversely, we tipped the balance in favour of cell cycle exit by disabling the mitosis clock using a CDK1i (Extended Data Fig. 4a,b) at the same time as blocking mitogen signalling ( Fig. 2d and Extended Data Fig. 4a-c). With the mitosis clock disabled, nearly all post-R cells lost CDK2 activity and exited the cell cycle after loss of mitogen signalling, revealing an underlying 'pre-competition' distribution of cell cycle exit times that could only be revealed by blocking mitosis (Fig. 2e and Extended Data Fig. 4c). Disabling the mitosis clock by arresting cells in G2 phase with a small molecule inhibitor of Polo-like kinase 1, a key regulator of the G2/M transition, or triggering the G2/M DNA damage checkpoint using the radiomimetic drug neocarzinostatin yielded similar results (Extended Data Fig. 4g,h). Thus, by disabling the mitosis clock, we found that all post-R cells and not just the 10-15% of outlier cells contain a cell cycle exit clock. This means that all post-R cells are unable to sustain CDK2 activity in the absence of CDK4/6 signalling, in stark contrast to the textbook model in which CDK2 activity is self-sustaining.
The second prediction made by the competing clock model is that that knowledge of the 'pre-competition' mitosis and cell cycle clock distributions alone should be sufficient to explain whether a cell will decide to enter mitosis or exit the cell cycle after loss of mitogen signalling. To test this second prediction, we first measured the 'pre-and post-competition' distributions of mitosis and cell cycle exit times (Fig. 2e,f and Extended Data Fig. 5a,b). We then used a Monte Carlo algorithm to randomly sample from the 'pre-competition' distributions for cell cycle exit and mitosis and simulated a competition (Extended Data Fig. 5c). For the competing clock model to be correct, the simulated competition should match the experimentally measured 'post-competition' distributions of cell cycle exit and mitosis times. Cell cycle exit and mitosis were modelled as independent competing processes because once the mitosis clock is disabled, the probability of cell cycle exit was independent of a cell's proximity to mitosis upon treatment (Extended Data Fig. 5d). Results from our simulation agreed with our experimental observations for CDK4/6i-treated cells, including the frequency of cell cycle exit (Fig. 2g,h and Extended Data Fig. 5e), 'post-competition' cell cycle exit clock times ( Fig. 2h and Extended Data Fig. 5f) and the relationship between proximity to the start of S phase and the probability of cell cycle exit (Extended Data Fig. 5g). Notably, despite absolute differences in the cell cycle exit and mitosis clocks between cell lines (Extended Data Fig. 5h-j), we found that the simulations agreed with experimental observations from multiple cell lines (Extended Data Fig. 5k-m). These simulations indicate that the reason some cells are observed to exit the cell cycle in G2 phase is because these cells had a shorter cell cycle exit clock than their mitosis clock and that the relative difference in the timing of each clock combined with cell-to-cell variability at the population level can account for the frequency of cells that exit the cell cycle upon loss of mitogen signalling that we observed experimentally (Fig. 2i). Thus, our data support an alternative model for cell cycle commitment that can account for the behaviour of all cells after loss of mitogen signalling.
An important implication of this revised model of cell cycle commitment is that cells have limited time to complete the cell cycle given that the median 'pre-competition' cell cycle exit and mitosis clock times differed on average by only 4 h (for example, 15 versus 11 h, respectively, in MCF-10A cells) ( Fig. 2e and Extended Data Fig. 5h-j). To test this, we sought to extend the mitosis clock rather than blocking it completely by treating cells transiently with either hydroxyurea or thymidine, which stalls DNA replication in S phase without causing DNA damage (Extended Data Fig. 6a-d), to extend the time from S phase to mitosis of post-R cells by 2-h increments (Extended Data Fig. 6e,f). Imposing this delay on the mitosis clock in combination with CDK4/6i treatment increased the frequency of cell cycle exit as a function of the increase in the mitosis clock ( Fig. 2j and Extended Data Fig. 6g-i), revealing that in the absence of mitogen signalling, cells have limited time to complete the cell cycle (Fig. 2k). While we observed a drop in CDK2 activity following hydroxyurea or thymidine treatment, we could rescue this drop by inhibiting Wee1, a kinase that negatively regulates CDKs (Extended Data Fig. 6j-l). Again, we observed cells exiting the cell cycle into the G0-like state when treated with the CDK4/6i and as little as a 4-h pulse of thymidine with or without the Wee1 inhibitor. Thus, we extended S/G2 length without profoundly interfering with CDK2 activity, and we still observe cells exiting into the G0-like state after CDK4/6 inhibition.

CDK4/6 promotes cyclin A2 synthesis in S/G2
Our data demonstrating that CDK2 activation and Rb phosphorylation are reversible in all post-R cells after loss of mitogen signalling raise the question of whether CDK2 and Rb comprise a bona fide self-sustaining feedback loop. To address this, we measured each component of the proposed CDK2-Rb feedback loop in post-R cells after CDK4/6i treatment (Fig. 3a). While cyclin E activates CDK2 in G1 phase, it is degraded [20][21][22] and does not contribute to CDK2 activity in S/G2 phase; therefore, it was not included in this analysis (Extended Data Fig. 7a,b). Cyclin A2 protein levels, CDK2 activity, Rb phosphorylation and E2F1 mRNA remained high for 6-8 h before declining concomitantly, while cyclin A2 mRNA levels fell within 2 h (Fig. 3b and Extended Data Fig. 7c-e). Real-time quantitative reverse-transcriptase PCR (qRT-PCR) analysis confirmed that a 2-h CDK4/6i treatment reduced cyclin A2 mRNA levels in post-R cells by 50%, while mRNA levels of canonical E2F target genes (CCNE1 and E2F1) remained unchanged (Extended Data Fig. 7f). Expression of cyclin A2 from an unregulated promoter prevented post-R cells from losing CDK2 activity after CDK4/6i treatment, establishing that repression of CDK4/6-mediated cyclin A2 transcription is the cause of cell cycle exit (Fig. 3c-e).

Article
To identify proteins regulated by CDK4/6 that may mediate the transcription of cyclin A2, we tested whether small-interfering RNA (siRNA) knockdown of various known CDK4/6 substrates that control cell cycle gene transcription could rescue the loss of CDK2 activity    (Fig. 3f). Knockdown of the CDK4/6-regulated transcription factors FOXO3 and FOXM1 did not rescue the loss of CDK2 activity (Extended Data Fig. 7g-i), nor did knockdown of Rb (Fig. 3g,h and Extended Data Fig. 8a,b). However, knockdown of p107 and p130, two members of the same protein family as Rb, did rescue loss of CDK2 activity (Fig. 3g,h and Extended Data Fig. 8a,b). Likewise, knockdown of p107 and p130, but not Rb, prevented loss of cyclin A2 mRNA after CDK4/6i treatment (Extended Data Fig. 8c), indicating that CDK4/6 represses cyclin A2 transcription primarily through p107/p130 and not Rb. Consistent with this mode of regulation, we found that p107 and p130 were dephosphorylated within 1-2 h of CDK4/6i treatment, while Rb remained hyperphosphorylated, indicating that p107 and p130, but not Rb, were activated by CDK4/6i treatment (Fig. 3i and Extended Data Fig. 8d-f). Activated forms of p107 and p130 repress transcription through complex formation with repressor E2Fs E2F4 and E2F5 [23][24][25][26] . Consistent with this, CDK4/6 inhibition induced complex formation between E2F4 and p107 (Extended Data Fig. 8g), and combined knockdown of E2F4 and E2F5 also prevented the loss of CDK2 activity (Extended Data Fig. 8h-j). Therefore, we find that CDK4/6-p107/p130 regulate cyclin A2 transcription and CDK2 activity in post-R cells, implying a critical role for CDK4/6 activity in maintaining CDK2 activity in S/G2 phase.

Article
Given that CDK2 activity is driven by cyclin A2 in post-R cells 4 and that the cyclin A2 protein has an approximately 12-h half-life in post-R cells (Extended Data Fig. 9a,b), this suggests that cyclin A2 protein levels are the primary contributor to the timing of the cell cycle exit clock, which had a median time of approximately 15 h. To test this, we expressed the CDK2 biosensor in U2OS cells with cyclin A2 endogenously tagged with yellow fluorescent protein (YFP), incubated them with cyclin A2 siRNA (Extended Data Fig. 9c,d) and then, measured endogenous cyclin A2 levels and CDK2 activity over time after CDK1i + CDK4/6i treatment. In keeping with our prediction that cyclin A2 protein levels set the timing of the cell cycle exit clock, we found that cells with lower starting levels of cyclin A2 took less time to lose CDK2 activity and exit the cell cycle after loss of mitogen signalling (Fig. 3j,k), that the time to lose CDK2 activity and exit the cell cycle was strongly correlated with the time to lose cyclin A2 protein (Extended Data Fig. 9e) and that the cyclin A2 levels at the time mitogen signalling was lost were predictive of whether cells reached mitosis or exited the cell cycle (Extended Data Fig. 9f,g). Furthermore, in support of the mitosis and cell cycle exit clocks being driven by independent molecular processes, we noted that only a small reduction in cyclin A2 levels was able to shorten the timing of the cell cycle exit clock, while the duration of the mitosis clock remained unchanged (Extended Data Fig. 9h).
To further test whether cyclin A2 protein stability is the primary contributor to the timing of the cell cycle exit clock, we directly manipulated cyclin A2 protein stability using a CRISPR-engineered RPE-1 cell line where the endogenous cyclin A2 protein was fused to two inducible degrons: an auxin-inducible degron and a small molecule-assisted shutoff (SMASh) tag 27,28 (RPE-1 CCNA2 dd cells) (Extended Data Fig. 10a). The addition of the inducible degron already reduced the stability of cyclin A2 from 10 h in normal RPE-1 cells to 5 h in the engineered line (Fig. 3l). The cyclin A2 stability could be further reduced to 2 h by the addition of the DIA cocktail (doxycycline, indole-3-acetic acid and asunaprevir) (Supplementary Methods). Shortening the half-life of cyclin A2 and treating cells with a CDK4/6i led to more rapid cell cycle exit (Extended Data Fig. 10b,c), allowed a greater proportion of cells to exit the cell cycle in S/G2 phase (Extended Data Fig. 10d) and extended the period when cells were sensitive to CDK4/6 inhibition up to 3 h before they entered mitosis (Extended Data Fig. 10d-f).
Finally, we took advantage of natural variation between cell lines and plotted the cyclin A2 protein half-life as well as the cell cycle exit time for five different cell lines. We found a strong correlation between the cyclin A2 half-life and the cell cycle exit times (Fig. 3l). Notably, the best-fit line has a y intercept of less than 2 h, which is strikingly similar to the time it takes for cyclin A2 mRNA to be lost after CDK4/6 inhibition, and a slope of approximately one, indicating that cyclin A2 stability is the primary contributor to the cell cycle exit clock.

A feed-forward loop underlies CDK2 reversibility
Our data demonstrating that after loss of mitogen signalling, cyclin A2 transcription is repressed by p107/p130 several hours before any detectable change in CDK2 activity or Rb phosphorylation are at odds with the textbook model, which predicts that cyclin A2-CDK2 activity is self-sustaining due to a positive feedback loop (Fig. 4a), although is consistent with the presence of a dominant feed-forward loop (Fig. 4b).
However, how this signalling architecture, as opposed to one with only a feedback loop, could generate the apparent irreversible cell cycle commitment commonly associated with the R-point phenomenon remains unclear. To address this, we used a mathematical model developed by Yao et al. 8 , which demonstrates irreversibility in the proposed CDK2-Rb loop after mitogen removal (Fig. 4c) and adapted it to model the feed-forward pathway after mitogen removal (Fig. 4d). The steady-state levels of CDK2 generated by the feedback loop model remained consistently high over time after loss of mitogen signalling, inconsistent with our experimental observations (Fig. 4c,e). However, CDK2 activity generated by the feed-forward pathway remained high at the time when a cell would normally enter mitosis (Fig. 4d,e), but at steady state, corresponding to the median time to exit the cell cycle, CDK2 activity was low (Fig. 4d,e). Thus, the feed-forward signalling pathway matches our experimental observations that CDK2 activity is reversible in post-R cells. This lack of true irreversibility in CDK2 activation challenges the textbook model, which states that hysteresis in CDK2 activity with respect to mitogen signalling underlies irreversible cell cycle commitment at the R point. To test for hysteresis, we treated pre-and post-R cells with varying doses of MEKi and measured the fraction of cells with high CDK2 activity at different times after treatment. Demonstrating a lack of hysteresis in CDK2 activity with respect to mitogen signalling at the R point, we observed that both pre-R and post-R cells lost CDK2 activity at the same dose of MEKi if given enough time to reach steady state (Fig. 4e). These results agreed with the competing clock model, arguing that irreversible commitment to the cell cycle after loss of mitogen signalling has only been observed previously because a cell enters mitosis before the molecular signalling pathway driving CDK2 activity reaches a steady state that forces cell cycle exit. In other words, the R-point phenomenon is observed because in most cells, the half-life of cyclin A2 protein allows CDK2 activity to persist after loss of mitogen signalling for a longer time than is required for a cell to reach mitosis. This revised model of cell cycle commitment implies that there is no single point when cells irreversibly commit to proliferation that can be defined by a single molecular event, but it is rather determined by the cell's proximity to mitosis as well as the levels of cyclin A2 protein when mitogen signalling is lost (Fig. 4f).

Discussion
Here, we used high-throughput single-cell imaging and tracking to study how the loss of mitogen signalling affected cell cycle commitment in post-R cells. We detected outlier cells whose behaviour contradicted the main prediction of the R-point model that all post-R cells will make it to mitosis and divide even in the absence of mitogen signalling (Fig. 1d). Notably, these outlier cells are found in numerous previous studies 3,4,10 , but their contradictory behaviour has been left largely unexplained. In our attempt to understand why these post-R cells exited the cell cycle instead of dividing, we found that the temporal distance of any post-R cell from mitosis at the time of mitogen removal was predictive of whether it would exit the cell cycle or make it to mitosis and divide. Subsequently, by blocking mitosis in cells deprived of mitogen signalling, we found that CDK2 activity and Rb phosphorylation are reversible in all cells. We could observe this reversibility in CDK2 activity and cell cycle commitment because by preventing cells from undergoing mitosis, the molecular signalling pathway driving CDK2 activity was allowed sufficient time to reach steady state. Previous studies failed to observe this reversibility because cells were allowed to enter mitosis before CDK2 activity reached steady state, precluding the observation of cell cycle exit in post-R cells. Our study also uncovered an unexpected role for CDK4/6 in S and G2 phases by maintaining cyclin A2 transcription throughout the cell cycle, and it implies that small molecule CDK4/6 is may be effective in phases of the cell cycle beyond G1 phase, particularly if combined with traditional chemotherapies that extend the mitosis clock by inducing DNA damage and triggering cell cycle checkpoints.
Using genetic, biochemical and pharmacological approaches as well as mathematical modelling, we find that the signalling pathway connecting mitogen signalling with CDK2 activity is predominantly regulated by a feed-forward loop rather than containing a dominant positive feedback loop. This signalling architecture pathway contains ultrasensitive signalling nodes, which make the system bistable and switch like, but the lack of a dominant positive feedback loop means the system is still reversible. We also cannot exclude the existence of additional positive feedback loops being involved in this signalling pathway since similar signalling systems, such as the one controlling the G2/M transition, have also been shown to contain positive feedback loops yet are still reversible 29,30 . Nevertheless, our data demonstrate that the dominant signalling architecture contains a feed-forward loop, resulting in a lack of hysteresis. Given that the two competing fates (mitosis versus cell cycle exit) are mutually exclusive, there is likely a double-negative feedback loop at the level of these cell fate decisions that ensures that once a cell exits, it makes it impossible to undergo mitosis and vice versa. Our findings provide a simple explanation for why the R-point phenomenon has been previously observed and led us   The observed (bottom panel) dose response relationship between CDK2 activity and MEKi concentration for pre-and post-R cells is shown. CDK2 activity was evaluated after MEKi treatment at the times indicated. Mitosis was blocked using a CDK1i. Error bars are s.e.m. from n = 2 replicates. f, For MCF-10A cells, in the absence of mitogens, competition between mitosis and cell cycle exit determines the fate of the cell due to a feed-forward loop regulating cyclin A2. A cell that is greater than approximately 15 h away from mitosis at the time mitogen signalling is blocked (cell 1) will lose cyclin A2 and exit, while a cell further along the cell cycle (cell 2) will reach mitosis before it will lose cyclin A2 and divide into two daughter cells.
Article to a new model of cell cycle commitment that does not contain a single decision point. This model of temporal competition between mitosis and cell cycle exit can explain the behaviour of all cells, including the apparent contradictory behaviour of outlier cells observed here and in previous studies 3,4,10 .
Our results show that mutually exclusive competing cell fates (mitosis and cell cycle exit) coupled with changes in cell state (either dividing or exiting the cell cycle) can function as irreversible fate transitions. More generally, temporal competition between any two mutually exclusive fates could be utilized by cells as a means of cellular decision-making in other contexts [31][32][33] : for example, deciding between proliferation or differentiation or deciding between mitosis and apoptosis. Thus, our findings may have implications for cellular decision-making outside of cell cycle regulation and may be a more general theme that underpins other cell decision-making.

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