Clinical CDK4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin D-CDK4 to inhibit CDK2

Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors—palbociclib, ribociclib, or abemaciclib—immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.

Conserved Cdk inhibitors show unique structural responses to tyrosine phosphorylation

Balanced proliferation-quiescence decisions are vital during normal development and in tissue homeostasis and their dysregulation underlies tumorigenesis. Entry into proliferative cycles is driven by Cyclin/Cyclin-dependent kinases (Cdks). Conserved Cdk inhibitors (CKIs), p21Cip1/Waf1, p27Kip1 and p57Kip2, bind to Cyclin/Cdks and inhibit Cdk activity. p27 tyrosine phosphorylation, in response to mitogenic signalling, promotes activation of CyclinD/Cdk4 and CyclinA/Cdk2. Tyrosine phosphorylation is conserved in p21 and p57, although the number of sites differs. We use molecular dynamics simulations to compare the structural changes in Cyclin/Cdk/CKI trimers induced by single and multiple tyrosine phosphorylation in CKIs and their impact on CyclinD/Cdk4 and CyclinA/Cdk2 activity. Despite shared structural features, CKI binding induces distinct structural responses in Cyclin/Cdks and the predicted effects of CKI tyrosine phosphorylation on Cdk activity are not conserved across CKIs. Our analyses suggest how CKIs may have evolved to be sensitive to different inputs to give context-dependent control of Cdk activity.

Proper CycE–Cdk2 activity in endocycling tissues requires regulation of the cyclin-dependent kinase inhibitor Dacapo by dE2F1b in Drosophila

Polyploidy is an integral part of development and is associated with cellular stress, aging, and pathological conditions. The endocycle, comprised of successive rounds of G and S phases without mitosis, is widely employed to produce polyploid cells in plants and animals. In Drosophila, maintenance of the endocycle is dependent on E2F-governed oscillations of Cyclin E (CycE)–Cdk2 activity, which is known to be largely regulated at the level of transcription. In this study, we report an additional level of E2F-dependent control of CycE–Cdk2 activity during the endocycle. Genetic experiments revealed that an alternative isoform of Drosophila de2f1, dE2F1b, regulates the expression of the p27CIP/KIP-like Cdk inhibitor Dacapo (Dap). We provide evidence showing that dE2F1b-dependent Dap expression in endocycling tissues is necessary for setting proper CycE–Cdk2 activity. Furthermore, we demonstrate that dE2F1b is required for proliferating cell nuclear antigen expression that establishes a negative feedback loop in S phase. Overall, our study reveals previously unappreciated E2F-dependent regulatory networks that are critical for the periodic transition between G and S phases during the endocycle.

Altered G1 signaling order and commitment point in cells proliferating without CDK4/6 activity

Cell-cycle entry relies on an orderly progression of signaling events. To start, cells first activate the kinase cyclin D-CDK4/6, which leads to eventual inactivation of the retinoblastoma protein Rb. Hours later, cells inactivate APC/CCDH1 and cross the final commitment point. However, many cells with genetically deleted cyclin Ds, which activate and confer specificity to CDK4/6, can compensate and proliferate. Despite its importance in cancer, how this entry mechanism operates remains poorly characterized, and whether cells use this path under normal conditions remains unknown. Here, using single-cell microscopy, we demonstrate that cells with acutely inhibited CDK4/6 enter the cell cycle with a slowed and fluctuating cyclin E-CDK2 activity increase. Surprisingly, with low CDK4/6 activity, the order of APC/CCDH1 and Rb inactivation is reversed in both cell lines and wild-type mice. Finally, we show that as a consequence of this signaling inversion, Rb inactivation replaces APC/CCDH1 inactivation as the point of no return. Together, we elucidate the molecular steps that enable cell-cycle entry without CDK4/6 activity. Our findings not only have implications in cancer resistance, but also reveal temporal plasticity underlying the G1 regulatory circuit.

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

To enter the cell cycle, mammalian cells must cross a point of no return (the commitment point), after which they proceed through the cell cycle regardless of changes in external signaling. This process is tightly regulated by the cyclin-dependent kinases (CDKs) and downstream molecules such as retinoblastoma (Rb). Here we show that CDK2 activity coordinates the timing of cell-cycle commitment and DNA replication. CDK4/6 activation initiates Rb phosphorylation and E2F activity, causing a gradual increase in CDK2 activity. Once CDK2 activity reaches a threshold level, CDK2 triggers the commitment point by maintaining Rb phosphorylation and subsequently initiates DNA replication. While the timing of the commitment point is tightly coupled with DNA replication, our experiments, which acutely increased CDK2 activity, suggest that the timing of the commitment point is before DNA replication. These findings highlight how cells utilize a safety mechanism to maintain genome stability by protecting against incomplete DNA replication.

Cyclin E1 and cyclin E2 in ER+ breast cancer: prospects as biomarkers and therapeutic targets

Cyclin E1 is one the most promising biomarkers in estrogen receptor positive (ER+) breast cancer for response to the new standard of care drug class, CDK4/6 inhibitors. Because of its strong predictive value, cyclin E1 expression may be used in the future to triage patients into potential responders and non-responders. Importantly, cyclin E1 is highly related to cyclin E2, and both cyclin E1 and cyclin E2 are estrogen target genes that can facilitate anti-estrogen resistance and can be highly expressed in breast cancer. However cyclin E1 and E2 are often expressed in different subsets of patients. This raises questions about whether the expression of cyclin E1 and cyclin E2 have different biological drivers, if high expressing subsets represent different clinical subtypes, and how to effectively develop a biomarker for E-cyclin expression. Finally, several pan-CDK inhibitors that target cyclin E-CDK2 activity have reached Phase II clinical trials. In this review, we outline the data identifying that different cohorts of patients have high expression of cyclins E1 and E2 in ER+ cancer and address the implications for biomarker and therapeutic development.