Historical Background: Cyclin-Dependent Kinase 4 (CDK4) in Cell Cycle Control and Regulation
The mammalian cell cycle is typically divided into four phases, G1, S, G2, and M, and within the last two decades, there have been a series of discoveries that have provided us with a better understanding of the control mechanisms that regulate cell cycle progression. It is apparent that the order and timing of the cell cycle is critical for accurate transmission of genetic information, and consequently, a number of biochemical pathways have evolved to ensure that initiation of a particular cell cycle event is dependent on the accurate completion of another. These biochemical pathways have been termed “checkpoints.”
Mitogenic growth factors bind to their cognate receptors and initiate a cascade of events that culminate in the expression and assembly of different kinase holoenzymes composed of a regulatory subunit, called a cyclin, and a catalytic subunit, termed a cyclin-dependent kinase (CDK) (reviewed in Morgan 1997). Cyclin-dependent kinases (CDKs) are serine/threonine kinases that are inactive when underphosphorylated and monomeric (reviewed in Morgan 1997). The primary mechanism of CDK activation is its association with a regulatory cyclin partner. Complete activation of most CDKs also requires phosphorylation of a conserved threonine (Thr) residue located in the T-loop, by CAK1/CDK7, a cyclin-dependent kinase that has been shown to phosphorylate the catalytic subunit of various CDKs at the equivalent residue Thr161 of CDC2. In CDK4, this phosphorylation occurs at Thr172 (Kato et al. 1994).
One of the major breakthroughs in our understanding of cell cycle regulation was the discovery of the cdc2+ and cdc28 genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Both proteins code for related CDKs, and their activities are required during the G1/S and G2/M transitions. While a single Cdk triggers the major transitions of the yeast cell division cycle, mammalian cells encode multiple CDC2-related genes. In mammalian cells, CDK4/6 associate with D-type cyclins and mediate progression through the G1 phase when the cell prepares to initiate DNA synthesis. Activation of CDK4/6/cyclin D complexes contributes to hyperphosphroylation of the retinoblastoma (RB) protein and its related proteins, p107 and p130. The hypophosphorylated form of pRB binds to and sequesters several cellular proteins, and its phosphorylation results in the release of these protein factors. One key binding partner is the transcription factor E2F-1, which appears to positively activate the transcription of genes whose products are required for S-phase progression. E2F1 and other members of the E2F family are known to bind to pRB and heterodimerize with DP-1 and -2, an interaction that is required for their DNA-binding capacity (reviewed in Morgan 1997; Harbour and Dean 2000). Once the cell has made the G1/S transition, CYCLIN E/CDK2 phosphorylates the remaining residues on the RB family proteins that are critical for E2F activation. The activation of E2F-mediated transcription allows the cell to transit into S phase and initiate DNA replication, which is controlled, in part, through CYCLIN A/CDK2. Cyclin A/Cdk2 ultimately forces the cell through the G2 phase prior to the assembly of the CYCLIN B/CDK1 and the initiation of mitosis (reviewed in Harbour and Dean 2000).
Regulation of CDK4 Activity
A key response to growth factors in many cell types is the activation of CDK4 (or CDK6) by members of the CYCLIN D family (D1, D2, and D3). Although D-type cyclins are absent in quiescent cells, they are important integrators of mitogenic signaling. A fully active CDK/CYCLIN complex can be turned off by at least two different mechanisms. Regulatory kinases can phosphorylate the CDK subunit at inhibitory sites near the N-terminus, or, CYCLIN/CDK complexes can be negatively controlled in a tissue-restricted manner by two families of cyclin kinase inhibitors (CKIs), the INK4 and CIP/KIP families of proteins (reviewed in Gil and Peters 2006; Blain 2008; Denicourt and Dowdy 2004).
The CIP/KIP family (p21CIP1, p27KIP1, p57KIP2) of proteins binds and inactivates CDK2/CYCLIN E, CDK2/CYCLIN A, and CDK1/CYCLIN B complexes. Structure/function analysis of the p21 and p27 proteins show that their N-termini contain two key domains, one that is required for cyclin binding and another that is required for association with the CDK subunit. A majority of p27KIP1 in proliferating cells is thought to be associated with CYCLIN D-CDK4 complexes that possess kinase activity, suggesting that this interaction does not result in an inhibition of CDK4 (reviewed in Blain 2008; Denicourt and Dowdy 2004). In this case, p27 appears to stabilize CYCLIN D-CDK4 complexes, as increased expression of p27 has been shown to result in increased CDK4 kinase activity. Hence, CYCLIN D-CDK4/6 complexes exhibit a noncatalytic function, whereby their association with p21 and p27 in the G1 phase sequesters these CKIs and prevents their binding to CYCLIN E/CDK2 to allow progression through G1. Both p21 and p27 have also been shown to inhibit the CYCLIN D/CDK4/6 complex under certain growth conditions. p27 levels increase dramatically in response to certain antiproliferative signals and under these conditions, cyclin D-CDK4 complexes are inactive (reviewed in Blain 2008; Denicourt and Dowdy 2004).
One of the most studied G1 CYCLIN/CDK substrates is RB, which is phosphorylated in a cell cycle–dependent manner. RB is hypophosphorylated in quiescent cells and becomes phosphorylated on Ser780 and Ser795 by CDK4/CDK6 during mid to late G1. The hypophosphorylated form of pRB associates with several cellular proteins, and its phosphorylation results in the disassociation of RB from its binding partners (Endicott 1999; Kitagawa et al. 1996; Connell-Crowley et al. 1997; Grafstrom et al. 1999). One such protein is the transcription factor E2F-1, which activates the transcription of genes whose products are required for S-phase progression. Most of the E2F-responsive genes identified so far are required for G1 transition to the S phase of the cell cycle, being transcriptionally activated in a period of G1 that coincides with passage through the restriction point. The two other RB-related genes that encode pocket proteins with similar biochemical activity, p107 and p130, are also substrates of CYCLIN/CDK complexes. In addition to these proteins, other CDK4 substrates include (but are not limited to) SMAD3, CDT1 MARCKS, FOXM1, and PRMT5. Several of these proteins have been shown to serve as substrates for other CDKs as well. Interestingly, CDK4 does not phosphorylate p27 or histone H1, a canonical CDK substrate, and when compared to other CDKs, the number of bona fide CDK4 substrates is relatively small (reviewed in Baker and Reddy 2012). Crystal structures of CDK4/CYCLIN D complexes suggest that the active conformation of CDK4 is highly dependent on binding to both substrate and cyclin (Takaki et al. 2009).
Role of CDK4 in Mammalian Development and Cancer
CDK4 Knockout Mice
Mice that are nullizygous for the Cdk4 allele exhibit a diabetic-like phenotype, with a 90% reduction in glucose levels, polyuria, polydipsia, and dramatic reductions in the size and number of pancreatic ß-islet cells (Rane et al. 1999). Both male and female mice are infertile, with males exhibiting testicular atrophy due to meiotic abnormalities. While females ovulate normally, embryos fail to undergo implantation (reviewed in Baker and Reddy 2012; Rane et al. 1999). Females also display pituitary hypoplasia that is characterized by a reduction in the number of prolactin-producing lacotrophic cells. In addition to these overt phenotypes, Cdk4 null-mutant mice are also prone to neurological defects such as impaired locomotion, staggering, and hyperactivity; have abnormalities in thymocyte maturation and allergen response; and exhibit impaired adipocyte differentiation and function (reviewed in Baker and Reddy 2012).
While Cdk4 null-mutant mice underscore a role for the gene in normal cell development, this animal model has also shed light on the role that this kinase plays in the genesis and progression of cancer, particularly that of the mammary gland. As discussed above, a key response to growth factors in many cell types is the activation of CDK4 or CDK6 by D-type CYCLINS. Approximately 50% of human mammary carcinomas express abnormally high levels of CYCLIN D1 that are maintained throughout subsequent stages of breast cancer progression from in situ carcinoma to invasive carcinomas (Buckley et al. 1993). CDK4 is also amplified or overexpressed in a variety of tumor types, including sarcomas, gliomas, lymphomas, and those of the breast (reviewed in Malumbres and Barbacid 2009).
The absence of CDK4 expression in the mammary gland results in defective mammary gland development, with the mammary glands of virgin female CDK4-null mice displaying defects in ductal outgrowth, a reduction in the number of mammary ducts, and a complete absence of alveoli. Expression of the MMTV-driven Neu or mutant Ras oncogenes in wild-type animals results in the appearance of infiltrating hyperphastic and dysplastic nodules in the mammary gland that is considerably reduced in the absence of CDK4 expression. Cdk4-null females also fail to show any of the proliferative disturbances that are otherwise normally observed as a result of Neu or Ras expression. As a result, the onset and incidence of mammary carcinoma in MMTV-Neu-Cdk4−/− and MMTV-Ras-Cdk4−/− mice are delayed and substantially reduced, respectively (reviewed in Baker and Reddy 2012).
Although Cdk4-null-mutant mice highlight the importance of the CDK4/CYCLIN D1 complex in breast tumors and provide evidence to suggest that small molecule inhibitors of CDK4 kinase activity could be effective in the treatment of human disease, the importance of mutations in the CDK4 locus in human cancer was first underscored by discoveries which showed that germline mutations in this gene which abolish the ability of the encoded protein to bind to p16INK4A resulted in a predisposition of individuals to the development of melanoma (Wolfel et al. 1995; Zuo et al. 1996). The CDK4-Arg24Cys (R24C) mutation was also detected in sporadic melanomas, suggesting that a CDK4 gene containing this mutation could act as a dominant oncogene that is resistant to inhibition by p16INK4A.
CDK4R24C Knockin Mice
CDK4R24C mice develop a variety of spontaneous primary and metastatic tumors (reviewed in Baker and Reddy 2012; Rane et al. 1999); however, the major pathological abnormality observed in these animals is the onset of pancreatic islet cell hyperplasia during the first three months of life. CDK4R24C mice are also susceptible to an increase in the development of pituitary tumors arising either in the pars intermedia or the pars distalis with characteristic angiomatous areas or dilated “blood-filled lakes” of various sizes. Skin tumors, including melanomas and papillomas, also develop with a very short latency period in CDK4R24C heterozygous and homozygous mice exposed to carcinogens or UV radiation. In addition to pancreatic, thyroid, and skin tumors, CDK4R24C female mice develop severe mammary duct dilation and a high incidence of mammary tumors, which are often extremely aggressive with large tumor burden (reviewed in Baker and Reddy 2012).
Interestingly, coexpression of CDK4R24C and vHa-ras in mammary epithelial cells delays the onset of tumorigenesis due to the activation of senescence pathways and the subsequent induction of apoptotic and DNA damage pathways. Loss of CDK4 expression also results in the senescence of preneoplastic cells in the lung and blocks the development of lung tumors in mouse models. Given that oncogenic mutations can result in a tumor cell’s dependence on CDK4, and that CDK4 regulates tumor cell stemness, these studies suggest that the development of CDK4-specific inhibitors may be beneficial in the treatment of cancer types that rely predominantly on CDK4 expression Baker and Reddy 2012).
Role of CDK4 in Human Cancer
It is now believed that a vast number of human tumors exhibit deregulation of the CDK4/6-CYCLIN D-INK4-RB pathway by multiple mechanisms (reviewed in Malumbres and Barbacid 2009). For example, CDK4/6 is hyperactivated in a number of human cancers as a result of overexpression of positive regulators such as CYCLIN D, inactivation of INK4 and CIP/KIP inhibitors, or deletion and/or epigenetic alterations of substrates such as RB (reviewed in Malumbres and Barbacid 2009). Hyperactive CDK4 has been reported in epithelial malignancies in the endocrine tissues and mucosa, while CDK6 activation was reported in certain mesenchymal tumors such as sarcomas and leukemias (reviewed in Malumbres and Barbacid 2009). Mutations and chromosomal translocations in the CDK4 and CDK6 loci have also been described. One of the best examples is the CDK4R24C mutation that results in insensitivity to INK4 family inhibitors and was first described in patients with familial melanoma (Wolfel et al. 1995; Zuo et al. 1996). Finally, CDK4/6 amplification or overexpression has also been observed in a wide spectrum of tumors, including gliomas, sarcomas, lymphomas, melanomas, carcinomas of breast, squamous cell carcinomas, and leukemias (reviewed in Malumbres and Barbacid 2009).
Targeting CDK4 for Cancer Therapy
Early evidence that CYCLIN D and CDK4 and CDK6 activities are upregulated in certain tumor cell types led to concerted efforts to develop small molecule inhibitors for these kinases. The first generation of CDK inhibitors, e.g., flavopiridol and roscovitine, were potent CDK4 inhibitors but were nonselective, inhibited multiple kinases, and caused severe toxic side effects when these molecules entered clinical trials (reviewed in VanArsdale et al. 2015).
In an attempt to overcome the toxicity profile of pan-CDK inhibitors, several groups undertook the initial development of next generation CDK inhibitors that are specific for individual CDKs. Some of these compounds exhibited a high degree of selectivity toward CDK4/6 by targeting the ATP binding site of CDK4/6-CYCLIN D complexes. Of these, one CDK4/6 selective compound palbociclib (PD-0332991, Ibrance®) (Fig. 1), a pyrido[2,3-d]pyrimidine derivative, is exquisitely specific for CDK4 and CDK6, inhibiting these two kinases with IC50 values of 0.011 and 0.015 μmol/, respectively (Fry et al. 2004; Toogood et al. 2005). In its early stages of preclinical development, the efficacy of this compound was extensively studied in tissue culture model systems as well as in mouse xenograft models of colorectal cancer, mantle cell lymphoma (MCL), and disseminated myeloma, where it showed excellent efficacy (Fry et al. 2004; Toogood et al. 2005). Palbociclib causes G1 arrest in cultured tumor cell lines and inhibits tumor growth in xenograft models of RB-positive human tumor cell lines derived from multiple tumor types (reviewed in Baker and Reddy 2012). Therapeutic doses of palbociclib resulted in a marked reduction of both phosphorylated RB and the proliferative marker Ki-67 in the tumor tissue and the downregulation of E2F-target genes. Based on these very promising results, this compound entered Phase I clinical trials in 2004, with early results indicating tolerable side effects (reviewed in Baker and Reddy 2012). Unfortunately, the efficacy profile of this compound as a single agent was somewhat disappointing, resulting in disease stabilization rather than regression. However, results from Phase II and III trials testing palbociclib in combination therapy were far more encouraging. One Phase III trial, PALOMA-3, was a randomized, double-blind two-arm study that examined the efficacy of palbociclib in combination with fulvestrant in patients with ER+HER2− breast cancer who had failed to respond or relapsed during previous treatment with endocrine therapy (reviewed in VanArsdale et al. 2015). The design and success of this trial, which ultimately resulted in the approval of palbociclib by the US Food and Drug Administration (FDA) in 2015, was based on the results of solid preclinical basic science, namely experiments evaluating the effects of CDK4/6 inhibition in combination with estrogen receptor (ER) antagonists in ER+ breast tumor models. These cell lines and tumors, which express functional RB, responded remarkably well to dual inhibition when compared to use of either inhibitor as a single agent (reviewed in VanArsdale et al. 2015) as evidenced by loss of pRB phosphorylation, a reliable marker of response to CDK4 inhibition both in vitro and in the clinic. Today, palbociclib is widely used in the treatment of advanced hormone receptor-positive (HR+) metastatic breast cancer in combination with both letrozole and fulvestrant (reviewed in VanArsdale et al. 2015). Owing to the success of palbociclib, two additional CDK4 inhibitors with low nanomolar IC50 values against CDK4/6, ribocilib (LEE011), and abemaciclib (LY2835219) (Fig. 1) are currently being evaluated in preclinical studies of HR+ breast cancers with encouraging results (reviewed in VanArsdale et al. 2015).
CDK4/6 is a key mediator of cell cycle progression through the G1 phase, the time when a cell prepares to initiate DNA synthesis. The cell’s reliance on this protein as well as its CYCLIN D binding partner and downstream target RB for proliferation underscores why the CDK4/CYCLIN D/RB signaling module is often deregulated in transformed cells. Palbociclib’s success in the treatment of HR+ breast cancer has paved the way for ongoing clinical studies evaluating the utility of CDK4 inhibitors combination with those of other signaling pathways (such as but not limited to BRAF, PI3K, and MEK) in other tumor types that exhibit reliance on CYCLIN D1/CDK4/RB (reviewed in VanArsdale et al. 2015). The success of these trials should provide an answer as to whether selective inhibitors of CDK4/6 can provide therapeutic benefit in a broader array of cancers.
This work was supported by grants from the NIH (P01CA-130821), (NIH-RO1-CA-158209) to EPR.
Conflict of Interest Statement
The authors declare no conflicts of interest with respect to the authorship and publication of this article.