The mammalian cell cycle is a tightly regulated, ordered process that goes through four distinct phases termed G1, S, G2 and M (Figure 1). Between each phase is a controlled checkpoint that is regulated by a variety of cyclins and cyclin-dependent kinases (CDKs).
Cyclin-dependent kinases (CDKs): Family of serine/threonine protein kinases that interact with specific cyclins to promote cell cycle progression (as well as other functions)
Cyclins: Diverse family of proteins divided into four classes (A, B, D and E) with multiple members within each class (e.g. cyclin D1, D2 and D3)
During the G1 phase of the cell cycle, cells must overcome the restriction point, a checkpoint that once passed, commits a cell to the S phase and another cycle of cell division. Progression through G1 and the restriction point requires synthesis of cyclin D and the formation of a cyclin D:CDK4/6 complex.
A key function of the cyclin D:CDK4/6 complex is to overcome the inhibitory function of the retinoblastoma (Rb) protein that binds and sequesters the transcription factor E2F. Cyclin D:CDK4/6 complexes phosphorylate Rb reducing its ability to bind E2F and enabling E2F to drive the transcription of genes required for the S phase of the cell cycle (Figure 2). These include cyclin E that binds CDK2 and promotes progress through the S phase and genes involved in DNA replication. Interestingly, cyclinD:CDK4 has been shown to directly promote other pathways involved in cell proliferation, migration and the DNA damage response while CDK6 may play a kinase-independent role in angiogenesis (Murphy & Dickler, 2015; Finn et al., 2016).
Escape of senescence and progression through the cell cycle is essential for cancer development and progression. A number of strategies are utilised by breast cancer cells to achieve this, many of which resulting in an increase in cyclin D-dependent activity. Amplification of CDK4 and cyclin D1 have been observed in between 15–25% of breast cancers while overexpression of cyclin D1 has been reported in over half of all breast cancers (Finn et al., 2016). Interestingly, amplification of cyclin D1 and CDK4 genes differs between breast cancer subtypes. Cyclin D1 amplification was seen in 29%, 58% and 38% of luminal A, luminal B and HER2 subtypes, respectively. Meanwhile, CDK4 amplification was seen in 14%, 25% and 24% of luminal A, luminal B and HER2 subtypes, respectively. However, reduced expression/mutation/loss of Rb protein is more common in the basal subtype where changes in cyclin D and CDK4 were less common (Finn et al., 2016).
Upstream signalling is also capable of driving increased cyclin D:CDK4/6 activity. Oestrogen activation of ER upregulates cyclin D1 levels culminating in increased activation of CDK4/6 and cell proliferation. Mitogenic signalling pathways can also promote cyclin D1:CDK4/6 signalling. Interestingly, cyclin D1 can independently activate ER suggesting a possible mechanism for ER+ cells becoming resistant to hormone therapy and the oestrogen-independence of some ER+ cancers (Finn et al., 2016).
For many years, it was believed that the cyclins and CDKs would be essential to cellular proliferation and normal embryonic development. However, this changed with the discovery that knockout mice lacking individual cyclins or CDKs were viable and double knockouts of CDK4/6 and triple knockouts of cyclin D1, D2 and D3 develop normally until mid/late gestation. Furthermore, breast cancer cells that become oestrogen-independent and resistant to hormone therapy are still reliant upon cyclin D:CDK4/6 signalling to drive proliferation (Finn et al., 2016; Iwata, 2017). With these insights and the likelihood that CDK4/6 may be dispensable for some normal cell function, in addition existing knowledge on inhibiting kinase activity, CDK4/6 became increasingly interesting targets for drug discovery (Finn et al., 2016).
However, many of the initial attempts at CDK inhibition resulted in poor activity and an undesirable adverse event profile. These first generation of CDK inhibitors were essentially pan-CDK inhibitors (Murphy & Dickler, 2015; Finn et al., 2016).
The most studied of the first-generation of CDK inhibitors is flavopiridol. Intravenously administered, it targets CDKs 1, 2, 4/6 and 7 and induces G0 and G1 arrest (Murphy & Dickler, 2015). In phase I and II studies, monotherapy showed limited efficacy but several side effects including infusion site reactions, gastrointestinal toxicity and severe neutropenia. Unacceptably high rates of neutropenia were a particular issue when administered to patients with mBC.
An analogue of staurosporine, this kinase inhibitor was shown to have broad activity against CDKs as well as against Akt, Chk1 and protein kinase C. While pre-clinical studies identified promising efficacy, Phase I trials identified several dose-limiting toxicities while the results in a Phase II trial in metastatic triple negative breast cancer were unimpressive (Ma et al., 2013; Finn et al., 2016).
Following the poor outcomes associated with pan-CDK inhibition, CDK-specific inhibitors were developed. While CDK2 was an initial target for many groups looking to develop inhibitors, the focus has shifted to inhibition of CDK4/6. In recent years, the first CDK inhibitors targeting CDK4/6 have reached the market (Figure 3) (Murphy & Dickler, 2015).
All current treatments are CDK4/6 inhibitors, although ribociclib and abemaciclib show stronger activity towards CDK4 than CDK6 in in vitro studies whereas palbociclib has similar activity against both kinases. Furthermore, abemaciclib appears to show modest activity against a range of other CDKs (Table 1), although it appears unlikely that this has a significant in vivo effect given the similar specificity towards Rb-positive cells versus Rb-negative cells seen with all the CDK4/6 inhibitors (Klein et al., 2018).
Table 1: IC50 of CDK4/6 inhibitors in Cell-Free Assays (Adapted from Fry et al., 2004; Gelbert et al., 2014; Tripathy et al., 2017).
The role of CDK4/6 in cell proliferation has been extensively studied and it was assumed that inhibition of CDK4/6 would simply prevent cell cycle progression. However, the mechanism of action of CDK4/6 inhibitors may be more complicated than this and involve additional non-cell cycle related effects (Klein et al., 2018).