Release from cell cycle arrest with Cdk4/6 inhibitors generates highly synchronised cell cycle progression in human cell culture

Each approach used to synchronise cell cycle progression of human cell lines presents a unique set of challenges. Induction synchrony with agents that transiently block progression through key cell cycle stages are popular, but change stoichiometries of cell cycle regulators, invoke compensatory changes in growth rate and, for DNA replication inhibitors, damage DNA. The production, replacement, or manipulation of a target molecule must be exceptionally rapid if the interpretation of phenotypes in the cycle under study are to remain independent of impacts upon progression through the preceding cycle. We show how these challenges are avoided by exploiting the ability of the Cdk4/6 inhibitors, palbociclib, ribociclib and abemaciclib to arrest cell cycle progression at the natural control point for cell cycle commitment: the restriction point. After previous work found no change in the coupling of growth and division during recovery from CDK4/6 inhibition, we find high degrees of synchrony in cell cycle progression. Although we validate CDK4/6 induction synchronisation with hTERT-RPE-1, THP1 and H1299, it is effective in other lines and avoids the DNA damage that accompanies synchronisation by thymidine block/release. Competence to return to cycle after 72 hours arrest enables out of cycle target induction/manipulation, without impacting upon preceding cycles.

Synchronised progression through the cell division cycle throughout a population supports 41 the ability to extrapolate the biochemical and functional attributes of the synchronised bulk 42 population back to infer behaviour in an individual cell [1,2]. Many approaches are popular. 43 Bulk levels of DNA or cell cycle markers support fractionation of live, or fixed, cell 44 populations into pools enriched for discrete cell cycle stages [3,4]. Although yields are low, 45 selection synchronisation based upon size, or mitotic shake off, are highly effective 46 approaches to isolate cells in one cycle phase from a large population of asynchronous cells 47 [5,6]. However, the ease of induction synchrony makes it the most widely applied approach. 48 49 Induction synchrony exploits the ability of transient exposure to a particular context to 50 accumulate cells at a discrete cell cycle stage, before removal of the context simultaneously 51 releases all cells in the population, to progress synchronously through subsequent phases of 52 the cell division cycle [1]. In yeasts transient ablation of cell cycle regulators through 53 reversible conditional mutations and the addition of mating pheromones predominate [7,8].

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Although the advent of analogue sensitive versions of cell cycle kinases has introduced 55 analogous chemical genetic approaches into human tissue culture studies [9][10][11][12], induction many lines, the stalled forks are prone to collapse over the extended arrest and subsequent 66 attempts at repair introduce damage and chromosomal rearrangements [19][20][21] . There are 67 also reports of understandable impacts upon RNA biology during the long block [22,23].

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Thus, this popular approach can be of limited utility in the study of S phase, some 69 transcriptional and chromatin associated events.

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When early cell cycle events are to be analysed, induction synchrony via release from a 72 mitotic arrest in the previous cell cycle, provides an attractive alternative. However, like 73 other forms of induction synchrony that rely upon an arrest within the cell division cycle, 74 prolonged cell cycle arrest will generate an imbalance in the many regulators, whose levels 75 fluctuate with cell cycle progression as a consequence of stage dependent transcription 76 and/or destruction [24]. Consequently, the next cycle may well be altered by excessive 77 regulatory activities, or substrates, inherited from the preceding, arrested, cycle. Incisive 78 studies by Ginzberg and colleagues revealed how counter-measures to accommodate some 79 imbalances promote adjustments in growth rates at two points in the cycle [25]. Prolonged 80 mitotic arrest can also initiate apoptotic pathways [26] and/or leave a memory of the mitotic 81 arrest that modifies cell cycle progression in the next cycle and beyond [27][28][29][30]. 82 83 Thus, while highly informative for some questions, data obtained through traditional 84 induction synchrony approaches, that rely upon arrest within the cycle, have to be 85 interpreted with caution. They must be consolidated with complementary data from 86 alternative approaches, to reveal the commonalities that exclude the artefacts incurred in 87 one given approach to synchronisation. 88 89 A further challenge in synchronising cell cycle progression throughout a population arises 90 when there is a need to assess the impact of protein depletion, induction or replacement. It is critical to ensure that the destruction, induction, or activation of a mutant variant starts 92 after the synchronising procedure is complete. If not, then the phenotype can be a legacy 93 arising from perturbation of progression through the previous cycle, rather than a direct 94 impact upon the cycle being studied. Advances in degron and PROTAC technologies may 95 overcome many of these challenges [31][32][33]. However, even with many induction 96 synchronisation approaches, the switch from one version of a protein to another must be 97 exceptionally rapid and complete if perturbation of the preceding cycle is to be avoided.

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For adherent cell lines (RPE1, A549 and H1299), cells were released from the substrate by 171 treatment with trypsin (15400054, Gibco), pelleted by centrifugation at 300 g for 5 minutes 172 before resuspension in growth media. Cells were counted using a TC20 automated cell 173 counter (BioRad) and 2.5 x 10 5 cells were plated in a 10 cm dish (353003, Falcon) with 10 ml 174 media (plating density = 4.4 x 10 3 per cm 2 ). For the THP1 suspension line, cells were pelleted 175 by centrifugation at 300 g for 5 minutes, resuspended in growth media and counted, before 176 1 x 10 6 cells were seeded in 10 ml media in a 10 cm dish. Cells were then incubated for 6 or 177 12 hours (see figure legends) before drug was added.    As the second cycle after release is less likely to be influenced by the physiological 282 challenges of induction synchrony, it is often desirable to monitor this second cycle, rather 283 than the first cycle after release [1]. We therefore extended our assessments to monitor

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Recent studies suggest that adaptation is unlikely to occur after the second cycle after 515 release [59]. The approach is also not effective in many lines when the level of control 516 exerted by Cdk4/6 vs Cdk2 is tipped more heavily in favour of Cdk2 control [10,41,47- and Cdk4-CyclinD away from the Hsp90 chaperone system to reduce the number of molecules that can form an active complex with p27 [41,53,70]. The lower affinity of Cdk6 531 for the Hsp90 chaperone complex enables it to more readily assemble into active trimers 532 that have no affinity for the inhibitors than Cdk4 does. This has prompted the suggestion 533 that the predominance of Cdk6-Cyclin D-p27 trimers depletes p27 from the pools of Cdk2 to 534 elevate Cdk2 activities and confer palbociclib resistance in cancers in which Cdk6 expression 535 is elevated [53,71,72]. Thus, cell lines that rely upon Cdk6 rather than Cdk4 to drive passage 536 through the gateway into the cycle will have reduced sensitivity to Cdk4/6 inhibitors. In such 537 lines, a Cdk6 specific PROTAC may reduce reliance upon Cdk6 to impose sensitivity to Cdk4/6 538 inhibition and so support synchronisation with the inhibitors [73]. Finally, because many cell 539 lines will bypass the requirement for Cdk4/6 by exploiting Cdk2 to drive cells into cycle, 540 partial inhibition of Cdk2 in these lines [10,[74][75][76] should sensitize cells to Cdk4 inhibition.

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One challenge with this approach is the accompanying risk that strong Cdk2 inhibition will 542 impact not only upon DNA replication in the preceding cycle, but upon the regulation of 543 mitotic commitment as Cdk2-Cyclin A has been tied directly to regulation of Wee1 and the 544 G2/M transition [10,[77][78][79].

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Given the variety of means by which resistance to Cdk4/6 inhibitors arises, unless the 547 genomics indicate a clear resistance to Cdk4/6 inhibition, such as loss of Rb, it is perhaps 548 easiest to empirically test whether a line will be amenable to Cdk4/6i induction synchrony.

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One of two simple assessments will identify a line as being Cdk4/6i induction synchrony 550 compliant. The arrest/release into nocodazole that we show in Figures 1 and 4 will indicate 551 competence of most lines to synchronise by Cdk4/6i induction synchrony: a line will 552 synchronise if a population accumulates 2N DNA content one doubling time after inhibitor 553 addition, before switching to 4N DNA content a further doubling time after release into 554 nocodazole. However, this assay relies upon the strength of the spindle assembly 555 checkpoint (SAC) [58], that can be so weak in some lines that it fails to impose a long cell cycle arrest with 4N DNA content [80]. The second approach is independent of SAC integrity.

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Cells are released from palbociclib into 1μM EdU before entrance into the next cycle is 558 blocked by re-addition of palbociclib 12 hours after release. If EdU is incorporated into most 559 genomes throughout a population, that has arrested cell cycle progression with a 2N DNA 560 content in response to the second dose of palbociclib, then the line will be competent for