CDK4 and CDK6 kinases: from basic science to cancer therapy

Abstract

Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and their activating partners, D-type cyclins, link the extracellular environment with the core cell-cycle machinery. Constitutive activation of cyclin D-CDK4/6 represents the driving force of tumorigenesis in several cancer types. Small-molecule inhibitors of CDK4/6 have been used with great success in the treatment of hormone receptor-positive breast cancers, and are in clinical trials for many other tumor types. Unexpectedly, recent work indicates that inhibition of CDK4/6 affects a wide range of cellular functions, such as tumor cell metabolism or anti-tumor immunity. In this Review, we discuss how recent advances in understanding CDK4/6 biology open new avenues for future use of cyclin D-CDK4/6 inhibitors in cancer treatment.

Summary sentence:

In this Review, Fassl et al. discuss basic biology of CDK4 and CDK6 kinases, studies that led to identification of CDK4/6 as excellent anti-cancer targets, the growing application of CDK4/6 inhibitors in cancer treatment and the promise these compounds hold for future therapies.

PRINT SUMMARY

BACKGROUND

Cyclins and cyclin-dependent kinases (CDKs) drive cell division. Of particular importance to the cancer field are D-cyclins, which bind and activate CDK4 and CDK6. In normal cells, the activity of cyclin D-CDK4/6 is controlled by the extracellular pro-proliferative or inhibitory signals. In contrast, in many cancers, cyclin D-CDK4/6 kinases are hyperactivated and become independent of the mitogenic stimulation, thereby driving uncontrolled tumor cell proliferation. Mouse genetic experiments established that cyclin D-CDK4/6 kinases are essential for growth of many tumor types, and they represent potential therapeutic targets. Genetic and cell culture studies documented the dependence of breast cancer cells on CDK4/6. Chemical CDK4/6-inhibitors were synthesized and tested in pre-clinical studies. Introduction of these compounds to the clinics represented a breakthrough in breast cancer treatment, and will likely have a major impact on the treatment of many other tumor types.

ADVANCES

Small-molecule CDK4/6-inhibitors (palbociclib, ribociclib and abemaciclib) have shown impressive results in clinical trials for patients with hormone receptor-positive breast cancers. Addition of CDK4/6-inhibitors to standard endocrine therapy substantially extended the median progression-free survival, and prolonged median overall survival. Consequently, all three CDK4/6-inhibitors received ‘Breakthrough Therapy’ designation status from U.S. Food and Drug Administration, and have been approved for the treatment of women with advanced/metastatic hormone receptor-positive breast cancers. In the last few years, the renewed interest in basic CDK4/6 biology has yielded several surprising discoveries. The emerging concept is that CDK4/6 kinases regulate a much wider set of cellular functions than previously anticipated. Consequently, CDK4/6-inhibitors, beyond inhibiting tumor cell proliferation, affect tumor cells and the tumor environment through mechanisms that are only beginning to be elucidated. For example, inhibition of CDK4/6 affects anti-tumor immunity acting both on tumor cells as well as on the host immune system. Importantly, CDK4/6-inhibitors were shown to greatly enhance the efficacy of immune checkpoint blockade in pre-clinical mouse cancer models. These new concepts are now being tested in clinical trials.

OUTLOOK

Palbociclib, ribociclib and abemaciclib are now being tested in over 300 active or recruiting clinical trials for over 50 tumor types, such as other sub-types of breast cancers (HER2-positive, triple-negative), as well as colon, lung, liver, uterine and ovarian cancers, glioblastoma and various lymphoid malignancies. These trials test CDK4/6-inhibitors in combination with a wide range of therapeutic compounds that target other cancer-relevant pathways. Several other combination treatments were shown to be efficacious in pre-clinical studies and will enter clinical trials in the near future. Another CDK4/6-inhibitor, trilaciclib, is being tested for its ability to shield normal, non-transformed cells of the host from cytotoxic effects of chemotherapy. New CDK4/6-inhibitors have been developed and are being tested in pre-clinical and clinical trials. The major impediment in the therapeutic use of CDK4/6-inhibitors is that patients who initially respond to treatment often develop resistance and eventually succumb to the disease. Moreover, a substantial fraction of tumors shows pre-existing, intrinsic resistance to CDK4/6-inhibitors. One of the main challenges will be to elucidate the full-range of resistance molecular mechanisms. Even with the current, limited knowledge, one can envisage the principles of new, improved approaches which would overcome known resistance mechanisms. Another largely unexplored area is the possible involvement of CDK4/6 in other pathologic states, beyond cancer. This area will be the subject of intense studies and it may extend the utility of CDK4/6-inhibitors to the treatment of other diseases.

Cyclin D1, the activator of CDK4 and CDK6, was discovered in the early 1990s (1, 2). The role of cyclin D1 in oncogensis was evident already at the time of its cloning, as it was also identified as the PRAD1 oncogene which is rearranged and overexpressed in parathyroid adenomas (3), and the BCL1 oncogene that is rearranged in B-lymphocytic malignancies (4). Subsequently, the remaining two D-type cyclins, D2 and D3, were identified based on their homology to cyclin D1 (1).

Cyclins serve as regulatory subunits of CDKs (5). Shortly after the discovery of D-cyclins, CDK4 and CDK6 were identified as their kinase partners (6). Mouse gene knockout studies revealed that CDK4 and CDK6 play redundant roles in development, and combined ablation of CDK4 and CDK6 resulted in embryonic lethality (7). Essentially, the identical phenotype was seen in cyclin D-knockout mice, thereby confirming the role of D-cyclins as CDK4/6 activators in vivo (8). Surprisingly, these analyses revealed that many normal non-transformed mammalian cell types can proliferate without any cyclin D-CDK4/6 activity (7, 8).

CDK4 and CDK6 are expressed at constant levels throughout the cell-cycle. In contrast, D-cyclins represent labile proteins that are transcriptionally induced upon stimulation of cells with growth factors. For this reason, D-cyclins are regarded as links between the cellular environment and the cell-cycle machinery (6).

An important role in regulating the activity of cyclin D-CDK4/6 is played by cell-cycle inhibitors (Fig. 1). The INK inhibitors (p16^INK4A, p15^INK4B, p18^INK4C, p19^INK4D) bind to CDK4 or CDK6 and prevent their interaction with D-type cyclins, thereby inhibiting cyclin D-CDK4/6 kinase activity. In contrast, KIP/CIP inhibitors (p27^KIP1, p57^KIP2, p21^CIP1), which inhibit the activity of CDK2-containing complexes, serve as assembly factors for cyclin D-CDK4/6 (6, 9). This was demonstrated by the observation that mouse fibroblasts devoid of p27^KIP1 and p21^CIP1 fail to assemble cyclin D-CDK4/6 complexes (10).

Fig. 1. Molecular events governing progression through the G1 phase of the cell-cycle.

Mammalian-cell cycle can be divided into G1, S (DNA-synthesis), G2 and M (mitosis) phases. During G1 phase, cyclin D (CycD)-CDK4/6 kinases together with cyclin E (CycE)-CDK2 phosphorylate the retinoblastoma protein, RB1. This activates the E2F transcriptional program, and allows entry of cells into S-phase. INK family of inhibitors (p16^INK4A, p15^INK4B, p18^INK4C and p19^INK4D) inhibit cyclin D-CDK4/6; KIP/CIP proteins (p21^CIP1, p27^KIP1 and p57^KIP2) inhibit cyclin E-CDK2. Cyclin D-CDK4/6 complexes use p27^KIP1 and p21^CIP1 as ‘assembly factors’ and sequester them away from cyclin E-CDK2, thereby activating CDK2. Proteins that are frequently lost or downregulated in cancers are marked with green arrows, overexpressed proteins with red arrows.

p27^KIP1 can bind cyclin D-CDK4/6 in an inhibitory or non-inhibitory mode, depending on p27^KIP1 phosphorylation status. Cyclin D-p27^KIP1-CDK4/6 complexes are catalytically inactive, unless p27^KIP1 is phosphorylated on tyrosines 88 and 89 (11). Two molecular mechanisms may explain this switch. First, Y88/89 phosphorylation may dislodge the helix of p27^KIP1 from the CDK active site and allow ATP-binding (12). Second, the presence of tyrosine-unphosphorylated p27^KIP1 within the cyclin D-CDK4 complex prevents the activating phosphorylation of CDK4’s T-loop by the CDK-activating kinase (CAK) (12). Brk has been identified as a physiological kinase of p27^KIP1 (13); Abl and Lyn can phosphorylate p27^KIP1 in vitro, but the in vivo significance remains unclear (11, 14).

The activity of cyclin-CDK4/6 is also regulated by proteolysis. Cyclin D1 is an unstable protein with half-life of less than 30 minutes. At the end of G1 phase, cyclin D1 is phosphorylated at threonine-286 by GSK3β (15). This facilitates association of cyclin D1 with nuclear exportin CRM1, and promotes export of cyclin D1 from the nucleus to cytoplasm (16). Subsequently, phosphorylated cyclin D1 becomes polyubiquitinated by E3 ubiquitin-ligases, thereby targeting it for proteasomal degradation. Several substrate-receptors of E3 ubiquitin-ligases have been implicated in recognizing phosphorylated cyclin D1, including F-box proteins FBXO4 (along with αB crystallin), FBXO31, FBXW8, SKP2, β-TrCP1/2, and SKP2 (17). The anaphase promoting complex/cyclosome (APC/C) was also proposed to target cyclin D1, while F-box proteins FBXL2 and FBXL8 target cyclins D2 and D3 (17, 18). Surprisingly, the level and stability of cyclin D1 was unaffected by depletion of several of these proteins, indicating that some other E3 plays a rate-limiting role in cyclin D1 degradation (19). Indeed, recent studies reported that D-cyclins are ubiquitylated and targeted for proteasomal degradation by the E3 ubiquitin-ligase termed CRL4, which uses AMBRA1 protein as its substrate-receptor (20–22).

Cyclin D-CDK4/6 in cancer

Genomic aberrations of the cyclin D1 gene (CCND1) represent frequent events in different tumor types. The t(11;14)(q13;q32) translocation juxtaposing CCND1 with the immunoglobulin heavy-chain (IGH) locus represents the characteristic feature of mantle-cell lymphoma, and is frequently observed in multiple-myeloma or plasma cell leukemia (23, 24). Amplification of CCND1 is seen in many other malignancies, for example in 13–20% of breast cancers (23, 24), over 40% of head and neck squamous cell carcinomas, and over 30% of esophageal squamous cell carcinomas (23). A higher proportion of cancers, e.g., up to 50% of mammary carcinomas, overexpresses cyclin D1 protein (24). Also cyclins D2, D3, CDK4 and CDK6 are overexpressed in various tumor types (5, 9). The cyclin D-CDK4/6-kinase can also be hyperactivated through other mechanisms, for example deletion or inactivation of INK-inhibitors, most frequently p16^INK4A (5, 9, 23). Altogether, a very large number of human tumors contain lesions that hyperactivate cyclin D-CDK4/6 kinase (5).

The oncogenic role for cyclin D-CDK4/6 has been supported by mouse cancer models. For example, targeted overexpression of cyclin D1 in mammary glands of transgenic mice led to development of mammary carcinomas (25). Also overexpression of cyclin D2, D3, or CDK4, or loss of p16^INK4a resulted in tumor formation (9).

Conversely, genetic ablation of D-cyclins, CDK4 or CDK6 decreased tumor-sensitivity (9). For instance, Ccnd1-, or Cdk4-null mice, or knock-in mice expressing kinase-inactive cyclin D1-CDK4/6, are resistant to develop HER2-driven mammary carcinomas (26–29). An acute, global shutdown of cyclin D1 in mice bearing HER2-driven tumors arrested tumor growth and triggered tumor-specific senescence, while having no obvious impact on normal tissues (30). Likewise, an acute ablation of CDK4 arrested tumor cell proliferation and triggered tumor cell senescence in KRAS-driven non-small cell lung cancer (NSCLC) mouse model (31). These observations indicated that CDK4 and CDK6 might represent excellent therapeutic targets in cancer treatment.

CDK4/6 functions in cell proliferation and oncogenesis

The best-documented function of cyclin D-CDK4/6 in driving cell proliferation is phosphorylation of the retinoblastoma protein, RB1, and RB-like proteins, RBL1 and RBL2 (5, 6) (Fig. 1). Unphosphorylated RB1 binds and inactivates or represses E2F transcription factors. According to the prevailing model, phosphorylation of RB1 by cyclin D-CDK4/6 partially inactivates RB1, leading to release of E2Fs and upregulation of E2F-transcriptional targets, including cyclin E. Cyclin E forms a complex with its kinase partner, CDK2 and completes full RB1-phosphorylation, leading to activation of the E2F transcription program, and facilitating S-phase entry (5, 6). In normal, non-transformed cells, the activity of cyclin D-CDK4/6 is tightly regulated by the extracellular mitogenic milieu. This links inactivation of RB1 with the mitogenic signals. In cancer cells carrying activating lesions in cyclin D-CDK4/6, the kinase is constitutively active, thereby de-coupling cell division from proliferative and inhibitory signals (5).

This model has been questioned by the demonstration that throughout G1-phase RB1 exists in a mono-phosphorylated state, and becomes inactivated in late G1-phase by cyclin E-CDK2, which ‘hyperphosphorylates’ RB1 on multiple residues (32). Still, recent single-cell analyses revealed that cyclin D-CDK4/6 activity is required to hyperphosphorylate RB1 throughout G1-phase, while cyclin E/A-CDK maintains RB1-hyperphosphorylation in S-phase (33). Moreover, phosphorylation of RB1 by cyclin D-CDK4/6 was shown to be required for normal cell-cycle progression (34).

In addition to this kinase-dependent mechanism, upregulation of D-cyclins and formation of cyclin D-CDK4/6 complexes causes re-distribution of KIP/CIP inhibitors from cyclin E-CDK2 complexes (which are inhibited by these proteins) to cyclin D-CDK4/6 (which use them as assembly factors), thereby activating the kinase activity of cyclin E-CDK2 (6). Cyclin E-CDK2, in turn, phosphorylates RB1 and other cellular proteins and promotes cell-cycle progression.

Cyclin D1-CDK4/6 directly phosphorylates and stabilizes and activates transcription factor, FOXM1. This promotes cell-cycle progression and protects cancer cells from entering senescence (35). Cyclin D-CDK4 also phosphorylates and inactivates SMAD3, which mediates TGF-β anti-proliferative response. CDK4/6-dependent phosphorylation of SMAD3 inhibits its transcriptional activity, and disables the ability of TGF-β to induce cell-cycle arrest (36). FZR1/CDH1, an adaptor protein of the APC complex, is another phosphorylation substrate of CDK4. Depletion of CDH1 in human cancer cells partially rescued the proliferative block upon CDK4/6-inhibition, and it cooperated with RB1-depletion in restoring full proliferation (37).

Cyclin D-CDK4/6 also phosphorylates and inactivates TSC2, a negative regulator of mTORC1, thereby resulting in mTORC1-activation. Conversely, inhibition of CDK4/6 led to decreased mTORC1 activity and reduced protein synthesis in cells representing different human tumor types. It was proposed that through TSC2-phosphorylation, activation of cyclin D-CDK4/6 couples cell-growth with cell-division (38). Consistent with this, the anti-proliferative effect of CDK4/6-inhibition was reduced in cells lacking TSC2 (38).

MEP50, a co-regulatory factor of protein arginine-methyltransferase 5 (PRMT5) is phosphorylated by cyclin D1-CDK4. Through this mechanism, cyclin D1-CDK4/6 increases the catalytic activity of PRMT5/MEP50 (39). It was proposed that deregulated cyclin D1-CDK4 kinase in tumor cells, by increasing PRMT5/MEP50 activity, reduces expression of a component of the E3 ubiquitin-ligase complex, CUL4, and stabilizes CUL4 targets, such as CDT1 (39). In addition, by stimulating PRMT5/MEP50-dependent arginine-methylation of p53, cyclin D-CDK4/6 suppresses the expression of key anti-proliferative and pro-apoptotic p53-target genes (40). Another study proposed that PRMT5 regulates splicing of the transcript encoding MDM4, a negative regulator of p53. CDK4/6-inhibition reduces PRMT5 activity and alters the pre-mRNA splicing of MDM4, leading to decreased levels of MDM4 protein, and resulting in p53 activation. This, in turn, upregulates expression of a p53 target p21^CIP1, which blocks cell-cycle progression (41).

During oncogenic transformation of hematopoietic cells, chromatin-bound CDK6 phosphorylates NFY and SP1 transcription-factors and induces expression of p53-antagonists such as PRMT5, PPM1D and MDM4 (42). Also, in acute myeloid leukemia cells expressing constitutively activated FLT3, CDK6 binds the promoter region of the FLT3 gene as well as the promoter of PIM1 pro-oncogenic kinase and stimulates their expression. Treatment of FLT3-mutant leukemic cells with a CDK4/6-inhibitor decreased FLT3 and PIM1-expression, and triggered cell-cycle arrest and apoptosis (43).

It should be noted that the relevance of these various mechanisms in the context of human tumors is unclear and requires further studies.

Mechanism of action of CDK4/6-inhibitors

Three small-molecule CDK4/6-inhibitors have been extensively characterized in preclinical studies: palbociclib and ribociclib, which are highly-specific CDK4/6-inhibitors, and abemaciclib, which inhibits CDK4/6 and other kinases (Table 1). It has been assumed that these compounds act in vivo by directly inhibiting cyclin D-CDK4/6 (9). This simple model has been recently questioned by observations that palbociclib inhibits only cyclin D-CDK4/6 dimers, but not trimeric cyclin D-CDK4/6-p27^KIP1 (44). However, it is unlikely that significant amounts of cyclin D-CDK4 dimers ever exist in cells, because nearly all cyclin D-CDK4 in vivo is thought to be complexed with KIP/CIP-proteins (11, 14, 44). The authors also showed that palbociclib binds monomeric CDK4 (44). Surprisingly, treatment of cancer cells with palbociclib for 48 hours failed to inhibit CDK4-kinase, despite cell cycle arrest, but it inhibited CDK2 (44). Hence, palbociclib might prevent formation of active CDK4-containing complexes (through binding to CDK4) and indirectly inhibit CDK2 by liberating KIP/CIP-inhibitors. This model needs to be reconciled with several observations. First, treatment of cells with CDK4/6-inhibitors results in a rapid decrease of RB1 phosphorylation on cyclin D-CDK4/6-dependent sites, indicating an acute inhibition of CDK4/6 (45–47). Moreover, CDK4/6 immunoprecipitated from cells can be inhibited by palbociclib (48) and p21^CIP-associated cyclin CDK4/6-kinase is also inhibited by treatment of cells with palbociclib (49). Lastly, CDK2 is dispensable for proliferation of several cancer cell lines (50, 51), hence the indirect inhibition of CDK2 alone is unlikely to be responsible for cell-cycle arrest.

Table 1. Currently available CDK4/6-inhibitors.

This table lists major inhibitors of CDK4 and CDK6, half maximal inhibitory concentration (IC₅₀) for different cyclin D-CDK4/6 complexes (if known), other known targets and the stage of clinical development.

Name of compound	IC50	Other known targets	Stage in clinical development
Palbociclib (PD-0332991)	D1-CDK4 – 11 nM; D2-CDK6 – 15 nM; D3-CDK4 – 9 nM		FDA-approved for HR-positive/HER2-negative advanced breast cancer in combination with endocrine therapy; Phase 2/3 trials for several other tumor types
Ribociclib (LEE011)	D1-CDK4 – 10 nM; D3-CDK6 – 39 nM		FDA-approved for HR-positive/HER2-negative advanced breast cancer in combination with endocrine therapy; Phase 2/3 trials for several other tumor types
Abemaciclib (LY2835219)	D1-CDK4 – 0.6–2 nM; D3-CDK6 – 8 nM	cyclin T1-CDK9, PIM1, HIPK2, CDKL5, CAMK2A, CAMK2D, CAMK2G, GSK3α/β, and - at higher doses - cyclin E/A-CDK2 and B-CDK1	FDA-approved for early (adjuvant) and advanced HR-positive/HER2-negative breast cancer in combination with endocrine therapy; FDA-approved as monotherapy in advanced HR-positive/HER2-negative breast cancer; Phase 2/3 trials for several other tumor types
Trilaciclib (G1T28)	D1-CDK4 – 1 nM; D3-CDK6 – 4 nM		FDA-approved for small cell lung cancer to reduce chemotherapy-induced bone marrow suppression; Phase 2/3 trials for other solid tumors
Lerociclib (G1T38)	D1-CDK4 – 1 nM; D3-CDK6 – 2 nM		Phase 1/2 trials for HR-positive/HER2-negative advanced breast cancer and EGFR-mutant non-small cell lung cancer
SHR6390	CDK4 – 12 nM; CDK6 – 10 nM		Phase 1/2/3 trials for HR-positive/HER2-negative advanced breast cancer and other solid tumors
PF-06873600	CDK4 – 0.13 nM (Ki), CDK6 – 0.16 nM (Ki)	CDK2 – 0.09 nM (Ki)	Phase 2 trials for HR-positive/HER2-negative advanced breast cancer and other solid tumors
FCN-437	D1-CDK4 – 3.3 nM; D3-CDK6 – 13.7 nM		Phase 1/2 trials for HR-positive/HER2-negative advanced breast cancer and other solid tumors
Birociclib (XZP-3287)	Not reported		Phase 1/2 trials for HR-positive/HER2-negative advanced breast cancer and other solid tumors
HS-10342	Not reported		Phase 1/2 trials for HR-positive/HER2-negative advanced breast cancer and other solid tumor
CS3002	Not reported		Phase 1 trial for solid tumors

Palbociclib, ribociclib and abemaciclib were shown to block binding of CDK4 and CDK6 to CDC37, the kinase-targeting subunit of HSP90, thereby preventing access of CDK4/6 to the HSP90-chaperone system (52). Since the HSP90-CDC37 complex stabilizes several kinases (53), these observations suggest that CDK4/6-inhibitors, by disrupting the interaction between CDC37 and CDK4 or CDK6, might promote degradation of these CDKs. However, depletion of CDK4/6 is typically not observed upon treatment with CDK4/6-inhibitors (54). More studies are needed to resolve these conflicting reports and to establish how CDK4/6-inhibitors affect the cell-cycle machinery in cancer cells.

Validation of CDK4/6-inhibitors as anti-cancer agents

Consistent with the notion that RB1 represents the major rate-limiting substrate of cyclin D-CDK4/6 in cell-cycle progression (55–57), palbociclib, ribociclib, and abemaciclib were shown to block proliferation of several RB1-positive cancer cell lines, but not cell lines that have lost RB1-expression (46, 58, 59). Breast cancer cell lines representing the luminal, estrogen receptor-positive (ER+) subtype were shown to be most susceptible to arrest cell proliferation upon palbociclib-treatment (45). Palbociclib, ribociclib, abemaciclib and another CDK4/6-inhibitor, lerociclib were demonstrated to display potent antitumor activity in xenografts of several tumor types, including breast cancers (46, 60–62). Palbociclib and abemaciclib cross the blood-brain barrier and inhibit growth of intracranial glioblastoma (GBM) xenografts, with abemaciclib being more efficient in reaching the brain (63, 64). Recently, additional CDK4/6-inhibitors were shown to exert therapeutic effects in mouse xenograft models of various cancer types, including SHR6390 (65), FCN-437 (66), and compound 11 (67); the latter two reported to cross the blood-brain barrier. While in most in vivo studies the therapeutic effect was dependent on expression of intact RB1 protein in tumor cells (46, 63), anti-tumor effects of palbociclib were also reported on bladder cancer xenografts independently of RB1-status and attributed to decreased phosphorylation of FOXM1 (68).

Tumor cell senescence upon CDK4/6-inhibition

In addition to blocking cell proliferation, inhibition of CDK4/6 can also trigger tumor cell senescence (63), which depends on RB1 and FOXM1 (35, 54). The role of RB1 in enforcing cellular senescence is well-established (69). In addition, cyclin D-CDK4/6 phosphorylates and activates FOXM1, which has anti-senescence activity (35, 70).

It is not clear what determines the extent of senescence upon treatment of cancer cells with CDK4/6-inhibitors. A recent study showed that inhibition of CDK4/6 leads to RB1-dependent increase in reactive oxygen species (ROS) levels, resulting in activation of autophagy, which mitigates the senescence of breast cancer cells. (71). Co-treatment with palbociclib plus autophagy-inhibitors strongly augmented the ability of CDK4/6-inhibitors to induce tumor cell senescence and led to sustained inhibition of cancer cell proliferation in vitro and xenograft growth in vivo (71). Decreased mTOR signaling following long-term CDK4/6-inhibition was shown to be essential for the induction of senescence in melanoma cells, and activation of mTORC1 overrode palbociclib-induced senescence (72). Others postulated that expression of the chromatin-remodelling enzyme ATRX, and degradation of MDM2 determines the choice between quiescence and senescence upon CDK4/6-inhibition (73). Inhibition of CDK4 causes dissociation of de-ubiquitinase HAUSP/USP7 from MDM2, thereby driving autoubiquitination and proteolytic degradation of MDM2, which, in turn, promotes senescence. Importantly, this mechanism requires ATRX, suggesting that expression of ATRX can be used to predict the senescence response (73). Two additional proteins that play a role in this process are PDLIM7 and type II cadherin CDH18. Importantly, expression of CDH18 correlated with a sustained response to palbociclib in a phase 2 trial for patients with liposarcoma (74). Indeed, senescence represents a preferred therapeutic outcome to cell cycle arrest, as it may lead to a durable inhibition of tumor growth.

Markers predicting response to CDK4/6-inhibition

Only tumors with intact RB1 respond to CDK4/6-inhibitor treatment by undergoing cell-cycle arrest or senescence (9, 58). In addition, ‘D-cyclin-activating features’ (CCND1 translocation, CCND2 or CCND3 amplification, CCND1–3 3’UTR loss, and deletion of FBXO31 encoding an F-box protein implicated in cyclin D1-degradation) were shown to confer a strong response to abemaciclib in cancer cell lines (58). Moreover, co-deletion of CDKN2A and CDKN2C (encoding p16^INK4A/p19^ARF and p18^INK4C, respectively) confer palbociclib-sensitivity in glioblastoma (75). T172-phosphorylation of CDK4 and Y88-phosphorylation of p27^KIP1 (both associated with active cyclin D-CDK4) correlate with sensitivity of breast cancer cell lines or tumor-explants to palbociclib (76, 77). Surprisingly, in PALOMA-1, PALOMA-2 and PALOMA-3 trials (78–80), and in another independent large-scale study (81), CCND1 gene amplification or the elevated levels of cyclin D1 mRNA or protein were not predictive of palbociclib-efficacy. On the other hand, overexpression of CDK4, CDK6 or cyclin E1 are associated with resistance of tumors to CDK4/6-inhibitors (discussed further below).

Synergy of CDK4/6-inhibitors with other compounds

Several pre-clinical studies documented additive or synergistic effects of combining CDK4/6-inhibitors with inhibitors of receptor tyrosine-kinases, PI3K, RAF or MEK (Table 2). This synergism might be explained because these pathways impinge on the cell-cycle machinery through cyclin D-CDK4/6 (82–86). In some cases, the effect was seen in the presence of specific genetic lesions, such as EGFR, BRAF^V600E, KRAS and PIK3CA mutations (59, 87–89) (Table 2). When comparing different dosing regimens, continuous treatment with a MEK-inhibitor with intermittent palbociclib resulted in more complete tumor responses than other combination schedules (90). Treatment with CDK4/6-inhibitors sensitized cancer cells to ionizing radiation (63) or cisplatin (68). The synergism with platinum-based chemotherapy was attributed to the observation that upon treatment, CDK6 phosphorylates and stabilizes the FOXO3 transcription factor, thereby promoting tumor cell survival. Consequently, inhibition of CDK6 increases platinum-sensitivity by enhancing tumor cell death (91).

Table 2. Combination treatments that demonstrated synergy with CDK4/6-inhibitors in pre-clinical studies.

TNBC, triple-negative breast cancer; ER+, estrogen receptor-positive; T-ALL, T-cell acute lymphoblastic leukemia; HER2+, human epidermal growth factor receptor 2-positive; PI3K, phosphoinositide 3-kinase; EGFR, epidermal growth factor receptor.

CDK4/6 inhibitor	Synergistic Target	Inhibitor	Disease
Palbociclib	PI3K	taselisib, pictilisib	PIK3CA mutant TNBC
	AR	enzalutamide	androgen receptor-positive TNBC
	EGFR	erlotinib	TNBC, esophageal squamous cell carcinoma
	RAF	PLX4720	BRAF-V600E mutant melanoma
	MEK	trametinib	KRAS mutant colorectal cancer
	MEK	PD0325901 (mirdametinib)	KRAS or BRAFV600E mutant colorectal cancer
	MEK	MEK162 (binimetinib)	KRAS mutant colorectal cancer
	MEK	AZD6244 (selumetinib)	pancreatic ductal adenocarcinoma
	PI3K/mTOR	BEZ235 (dactolisib), AZD0855, GDC0980 (apitolisib)	pancreatic ductal adenocarcinoma
	IGF1R/InsR	BMS-754807	pancreatic ductal adenocarcinoma
	mTOR	temsirolimus	pancreatic ductal adenocarcinoma
	mTOR	AZD2014 (vistusertib)	ER+ breast cancer
	mTOR	MLN0128 (sapanisertib)	intrahepatic cholangiocarcinoma
	mTOR	everolimus	melanoma, glioblastoma
Ribociclib	PI3K	GDC-0941 (pictilisib), BYL719 (alpelisib)	PIK3CA mutant breast cancer
	PDK1	GSK2334470	ER+ breast cancer
	EGFR	nazartinib	EGFR-mutant lung cancer
	RAF	encorafenib	BRAF-V600E mutant melanoma
	mTOR	everolimus	T-ALL
	inflammation	glucocorticoid dexamethasone	T-ALL
	gamma secretase	Compound E	T-ALL
Abemaciclib	HER2	trastuzumab	HER2+ breast cancer
	EGFR and HER2	lapatinib	HER2+ breast cancer
	RAF	LY3009120, vemurafenib	KRAS mutant lung or colorectal cancer, NRAS or BRAF-V600E mutant melanoma
		alkylating agent temozolomide	glioblastoma

In several instances, co-treatment with CDK4/6-inhibitors prevented development of resistance to other compounds, or inhibited proliferation of resistant tumor cells. Co-treatment of melanoma patient-derived xenografts (PDX) with ribociclib plus the RAF-inhibitor encorafenib delayed or prevented development of encorafenib-resistance (92). PDX that acquired encorafenib-resistance remained sensitive to combination of encorafenib plus ribociclib (59). Treatment of BRAF^V600E-mutant melanoma xenografts with palbociclib plus BRAF^V600E-inhibitor PLX4720 prevented development of resistance (89). BRAF^V600E-mutant cell lines which acquired resistance to BRAF^V600E-inhibitor vemurafenib remained sensitive to palbociclib or abemaciclib, and xenografts underwent senescence and tumor regression upon CDK4/6-inhibition (72, 93). Treatment of ALK-mutant, ALK kinase inhibitor-resistant neuroblastoma xenografts with palbociclib restored the sensitivity to these compounds (94). Combination of PI3K- and CDK4/6-inhibitors overcame the intrinsic and acquired resistance of breast cancers to PI3K-inhibitors, and resulted in regression of PIK3CA-mutant xenografts (88).

Upregulation of cyclin D1 was shown to mediate acquired resistance of HER2+ tumors to anti-HER2 therapies in a mouse breast cancer model (95). Treatment of mice bearing trastuzumab-resistant tumors or PDX of resistant HER2+ mammary carcinomas with abemaciclib restored the sensitivity of tumors to HER2-inhibitors, and inhibited tumor cell proliferation. Moreover, in case of treatment-naïve tumors, co-administration of abemaciclib significantly delayed the development of resistance to anti-HER2 therapies (95).

Several anti-cancer treatments, such as chemotherapy, target dividing cells. Since CDK4/6-inhibitors block tumor cell proliferation, they might impede the effects of chemotherapy. Indeed, several reports documented that co-administration of CDK4/6-inhibitors antagonized anti-tumor effects of compounds which act during S-phase (doxorubicin, gemcitabine, methotrexate, mercaptopurine) or mitosis (taxanes) (96, 97). However, some authors reported synergistic effects (98, 99), but the molecular underpinnings are unclear.

A recent report documented that administration of CDK4/6-inhibitors prior to taxanes inhibited tumor cell proliferation, and impeded the effect of taxanes (100). By contrast, administration of taxanes first (or other chemotherapeutic compounds that act on mitotic cells or cells undergoing DNA-synthesis), followed by CDK4/6-inhibitors had a strong synergistic effect. The authors showed that by repressing the E2F-dependent transcriptional program, CDK4/6-inhibitors impaired expression of genes required for DNA-damage repair via homologous-recombination. Since treatment of cancer cells with chemotherapy triggers DNA-damage, the impairment of DNA-damage repair induced cytotoxicity, thereby explaining the synergistic effect (100).

Cells with impaired homologous-recombination rely on poly-(ADP-ribose)-polymerase (PARP) for double-stranded DNA-damage repair, which renders them sensitive to PARP-inhibition. Indeed, a strong synergistic effect has been demonstrated in PDX-derived cell lines between of CDK4/6- and PARP-inhibitors (100). Such synergy was also reported for ovarian cancer cells (101). Another study found that inhibition of CDK4/6 results in downregulation of PARP-levels (102).

Protection against chemotherapy-induced toxicity

Administration of palbociclib to mice induced reversible quiescence of hematopoietic stem/progenitor cells (HSPC). This effect protected mice from myelosuppression following total-body irradiation. Moreover, treatment of tumor-bearing mice with CDK4/6-inhibitors together with irradiation mitigated radiation-induced toxicity without compromising the therapeutic effect (103). Co-administration of a CDK4/6-inhibitor trilaciclib with cytotoxic chemotherapy (5-FU, etoposide) protected animals from chemotherapy-induced exhaustion of HSPC, myelosuppression and apoptosis of bone marrow (60, 104). These observations led to phase 2 clinical trial, which evaluated the effects of trilaciclib administered prior to etoposide and carboplatin for treatment of small cell lung cancer. Trilaciclib improved myelopreservation while having no adverse effect on anti-tumor efficacy (105). A similar phase 2 clinical trial investigating trilaciclib in combination with gemcitabine and carboplatin chemotherapy in patients with metastatic triple-negative breast cancer (TNBC) did not observe a significant difference in myelosuppression. However, this study demonstrated an overall survival benefit of the combination therapy (106, 107).

Metabolic function of CDK4/6 in cancer cells

The role of CDK4/6 in tumor metabolism is only starting to be appreciated (Fig. 2A). Treatment of pancreatic cancer cells with CDK4/6-inhibitors was shown to induce tumor cell metabolic reprogramming (108). CDK4/6-inhibition increased the numbers of mitochondria and lysosomes, activated mTOR and increased the rate of oxidative phosphorylation, likely through an RB1-dependent mechanism (108). Importantly, combined inhibition of CDK4/6 and mTOR strongly suppressed tumor cell proliferation (108). Moreover, CDK4/6 can phosphorylate and inactivate TFEB, the master-regulator of lysosomogenesis, and through this mechanism reduce lysosomal numbers. CDK4/6-inhibition activates TFEB and increases the number of lysosomes (109). Another mechanism linking CDK4/6 and lysosomes was provided by the observation that treatment of TNBC cells with CDK4/6-inhibitors decreased mTORC1 activity and impaired recruitment of mTORC1 to lysosomes (110). Consistent with the idea that mTORC1 inhibits lysosomal biogenesis, CDK4/6-inhibition increased the number of lysosomes in tumor cells. Since an increased lysosomal biomass underlies some cases of CDK4/6-inhibitor resistance (see below) (111), stimulation of lysosomogenesis by CDK4/6-inhibitors might limit their clinical efficacy by inducing resistance.

Fig. 2. CDK4 and CDK6 – more than cell cycle kinases.

While the role of CDK4 and CDK6 in cell-cycle progression has been well-documented, both kinases regulate several other functions that are only now starting to be unraveled. (A) Inhibition of CDK4/6 (CDK4/6i) affects lysosome and mitochondrial numbers, as well as oxidative phosphorylation. Cyclin D3-CDK6 phosphorylates glycolytic enzymes 6-phosphofruktokinase (PFKP) and pyruvate kinase M2 (PKM2), thereby controlling ROS levels via the pentose phosphate (PPP) and serine pathways. (B) Inhibition of CDK4/6 affects the anti-tumor immunity, acting both within cancer cells as well as on the immune system of the host. In tumor cells, inhibition of CDK4/6 impedes expression of an E2F-target, DNA methyltransferase (DNMT). DNMT-inhibition reduces methylation of endogenous retroviral genes (ERV), and increases intracellular levels of double-stranded (ds) RNA (114). In effector T-cells, inhibition of CDK4/6 stimulates NFAT transcriptional activity and enhances secretion of IFNγ and interleukin 2 (IL2) (115).

Lastly, CDK4/6-inhibition impaired lysosomal function and the autophagic flux in cancer cells. It was argued that this lysosomal dysfunction was responsible for the senescent phenotype in CDK4/6-inhibitor-treated cells (110). Since lysosomes are essential for autophagy, the authors co-treated TNBC xenografts with abemaciclib plus an AMPK-activator A769662 (which induces autophagy), and found that this led to cancer cell death and subsequent regression of tumors (110).

Cyclin D3-CDK6 phosphorylates and inhibits two rate-limiting glycolytic enzymes, 6-phosphofruktokinase and pyruvate kinase M2. This re-directs glycolytic intermediates into the pentose phosphate- (PPP) and serine-pathways. Through this mechanism, cyclin D3-CDK6 promotes production of NADPH and GSH and helps to neutralize ROS (112). Treatment of tumors expressing high levels of cyclin D3-CDK6 (such as leukemias) with CDK4/6-inhibitors reduces the PPP- and serine-pathway flow, thereby depleting antioxidants NADPH and glutathione. This increases ROS levels and triggers tumor cell apoptosis (112).

Another link between cyclin D-CDK4/6 in metabolism and cancer was provided by the observation that livers of obese/diabetic mice upregulate cyclin D1 (113). Treatment of these mice with anti-diabetic compound metformin reduced liver cyclin D1 levels and largely protected mice against development of hepatocellular carcinoma. Also genetic ablation of cyclin D1 protected obese/diabetic mice from liver cancer, while administration of palbociclib inhibited liver cancer progression. Importantly, these treatments had no effect on tumors in non-obese animals (113). These observations raise the possibility of using anti-diabetic compounds with CDK4/6-inhibitors for treatment of liver cancers in obese patients.

CDK4/6-inhibitors and anti-tumor immune responses

Several recent reports have started to unravel how inhibition of CDK4/6 influences anti-tumor immune responses, acting both on tumor cells as well as on the tumor immune-environment (Fig. 2B). Treatment of breast cancer-bearing mice or breast cancer cells with abemaciclib activates expression of endogenous-retroviral elements in tumor cells, thereby increasing the levels of double-stranded RNA. This, in turn, stimulates production of type-III interferons and increases presentation of tumor antigens. Hence, CDK4/6-inhibitors, by inducing viral genes, trigger the anti-viral immune response which helps to eliminate the tumor by the immune system (114).

Inhibition of CDK4/6 also affects the immune system by impeding proliferation of CD4⁺FOXP3⁺ regulatory T-cells (Treg), which normally inhibit the anti-tumor response. Because cytotoxic CD8⁺ T-cells are less affected by CDK4/6-inhibition, abemaciclib-treatment decreases the Treg/CD8⁺ ratio of intratumoral T-cells and facilitates tumor cell-killing by cytotoxic CD8⁺ T-cells (114).

Inhibition of CDK4/6 also results in activation of T-cells, due to de-repression of NFAT-signaling. NFAT4 (and possibly other NFATs) are phosphorylated by cyclin D3-CDK6. (115). Inhibition of CDK4/6 decreases phosphorylation of NFATs, resulting in their nuclear translocation and enhanced transcriptional activity. This causes upregulation of NFAT-targets, resulting in T-cell activation, which enhances the anti-tumor immune response. In addition, CDK4/6-inhibitors increase infiltration of effector T-cells into tumors, likely because of elevated levels of chemokines CXCL9 and CXCL10 following CDK4/6-inhibitor treatment (115). Abemaciclib-treatment also induced T-cell inflammatory and activated phenotype in tumors and upregulated expression of immune-checkpoint proteins CD137, PD-L1 and TIM-3 on CD4⁺ and CD8⁺ cells (116).

CDK4/6-inhibition also causes upregulation of PD-L1 protein in tumor cells (117, 118). This effect was shown to be independent of RB1 status in the tumor. Mechanistically, CDK4/6 phosphorylates and stabilizes SPOP, which promotes PD-L1-polyubiquitination and degradation (118). Cyclin D-CDK4 also represses expression of PD-L1 through RB1. Specifically, cyclin D-CDK4/6-mediated phosphorylation of RB1 on S249/T252 promotes binding of RB1 to NF-κB protein p65, and this represses expression of a subset NF-κB-regulated genes, including PD-L1 (119).

These observations prompted testing of the efficacy of combining CDK4/6-inhibitors with antibodies that elicit immune-checkpoint blockade. Indeed, treatment of mice bearing autochthonous breast cancers, or cancer allografts, with CDK4/6-inhibitors together with anti-PD-1/PD-L1-antibodies enhanced the efficacy of immune-checkpoint blockade and led to complete tumor regression in high proportion of animals (114, 115, 118). Conversely, activation of cyclin D-CDK4-pathway by genomic lesions in human melanomas correlated with the resistance to anti-PD-1 therapy (117).

Some authors did not observe synergy when abemaciclib was administered concurrently with immune checkpoint-inhibitors in allograft tumor models (116, 120). However, a strong synergistic anti-tumor effect was detected when abemaciclib was administered first (and continued), while anti-PD-L1 antibody was administered later. The combined treatment induced immunological memory, as mice that underwent tumor-regression were resistant to re-challenge with the same tumor (116). Abemaciclib plus anti-PD-L1-treatment increased infiltration of CD4⁺ and CD8⁺ T-cells into tumors, and increased expression of MHC-I and MHC-II on tumor cells and on macrophages and MHC-I on dendritic cells (116). In the case of anti-CTLA-4 plus anti-PD-1 treatment in melanoma allograft model, the synergistic effect was observed when immune checkpoint-inhibitor treatment was started first, followed by abemaciclib (120).

The synergistic anti-tumor effect of PI3K- and CDK4/6-inhibitors in TNBC is mediated, in part, by enhancement of tumor-immunogenicity (121). Combined treatment of TNBC cells with ribociclib plus the PI3K-inhibitor apelisib synergistically upregulated expression of immune-related pathways in tumor cells, including proteins involved in antigen presentation. Co-treatment of tumor-bearing mice also decreased proliferation of CD4⁺FOXP3⁺ Treg cells, increased activation of intratumoral CD4⁺ and CD8⁺ T-cells, increased the frequency of tumor-infiltrating NKT-cells, and decreased the numbers of intratumoral immunosuppressive myeloid-derived suppressor cells. Moreover, combined treatment strongly augmented response to immune-checkpoint therapy with PD-1 and CTLA-4 antibodies (121).

Single-cell RNA-sequencing of human melanomas identified an immune resistance program expressed by tumor cells that correlates with T-cell exclusion from the tumor mass and immune evasion by tumor cells. The program can predict the response of tumors to immune checkpoint inhibitors. Importantly, treatment of human melanoma cells with abemaciclib represses this program in an RB1-dependent fashion (120).

Together, these findings indicate that CDK4/6-inhibitors may convert immunologically “cold” tumors into “hot” ones. The most pressing issue is to validate these findings in a clinical setting. The utility of combining CDK4/6-inhibitors with PD-1 or PD-L1 antibodies is currently being evaluated in several clinical trials. It should be noted that the effects of CDK4/6-inhibition on the immune system of the host are independent of tumor cell RB1 status, raising the possibility of utilizing CDK4/6-inhibitors to boost the immune response also against RB1-negative tumors.

CDK4/6-inhibitors in clinical trials

Table 3 summarizes major clinical trials with CDK4/6-inhibitors. Given early pre-clinical data indicating that breast cancers, in particular the hormone receptor-positive ones, are very sensitive to CDK4/6 inhibition (as discussed above), many clinical trials focused on this cancer type. Most studies evaluated CDK4/6-inhibitors administered together with anti-estrogens (aromatase-inhibitors letrozole or anastrozole, or estrogen receptor-antagonist fulvestrant) for treatment of advanced/metastatic HR+/HER2− breast cancers in postmenopausal women. Addition of CDK4/6-inhibitors significantly extended median progression-free survival (78, 122–130) and prolonged median overall survival (131–134). Moreover, abemaciclib has shown clinical activity when administered as a single agent (135). Consequently, palbociclib, ribociclib and abemaciclib have been approved by the US Food and Drug Administration (FDA) for treatment of patients with advanced/metastatic HR+/HER2− breast cancer (Box 1). A recent phase 3 clinical trial MonarchE evaluated abemaciclib plus standard endocrine therapy in treatment of patients with early-stage, high-risk lymph node-positive, HR+/HER2− breast cancer. Addition of abemaciclib reduced the risk of breast cancer recurrence (136). This is in contrast to the similar PALLAS study reported this year, which found no benefit of adding palbociclib to the endocrine therapy for women with early-stage breast cancer (137). Analysis of patient populations in these two trials may help to explain the different outcome. It is also possible that the favorable outcome of the MonarchE study reflects a broader spectrum of kinases inhibited by abemaciclib. The utility of CDK4/6-inhibitors in early-stage breast cancer remains unclear, and is being addressed in ongoing clinical trials (PALLAS, PENELOPE-B, EarLEE-1, MonarchE) (138).

Table 3. Major past clinical trials with CDK4/6-inhibitors.

ER+, estrogen receptor-positive; HER2−, human epidermal growth factor receptor 2-negative; HR+, hormone receptor-positive. PFS, progression-free survival.

Inhibitor	Trial name	Trial details	T reatment	Patients	Outcome	Reference	Other outcomes
Palbociclib	PALOMA-1	randomized phase 2	aromatase inhibitor letrozole alone (standard of care) versus letrozole plus palbociclib	postmenopausal women with advanced ER+/HER2-breast cancer who had not received any systemic treatment for their advanced disease.	Addition of palbociclib dramatically increased median progression free survival (PFS) from 10.2 months in the letrozole group to 20.2 months in the palbociclib plus letrozole group.	ref. 78	Based on this result, palbociclib received a ‘Breakthrough Therapy’ designation status from the FDA and was granted accelerated approval, in combination with letrozole, for the treatment of ER+/HER2-metastatic breast cancer.
Palbociclib	PALOMA-2	double-blind phase 3	palbociclib plus letrozole as first-line therapy	postmenopausal women with ER+/HER2-breast cancer	Addition of palbociclib strongly increased the median PFS: 14.5 months in the placebo-letrozole group versus 24.8 month for the palbociclib-letrozole group.	ref. 123	Palbociclib was equally efficacious in patients with luminal A and B breast cancers, and there was no single biomarker associated with the lack of clinical benefit, except for RB1 loss. CDK4 amplification was associated with endocrine resistance, but this was mitigated by addition of palbociclib. Tumors with high levels of FGFR2 and ERBB3 mRNA displayed greater PFS gain after addition of palbociclib (79).
Palbociclib	PALOMA-3	randomized phase 3	estrogen receptor antagonist fulvestrant plus placebo versus fulvestrant plus palbociclib	women with HR+/HER2-metastatic breast cancer that had progressed on previous endocrine therapy	The study demonstrated a substantial prolongation of median PFS in the palbociclib-treated group: 4.6 months in the placebo plus fulvestrant group versus 9.5 months in the palbociclib plus fulvestrant group. Addition of palbociclib also extended median overall survival from 28.0 months (placebo-fulvestrant) to 34.9 months (palbociclib-fulvestrant). The estimated rate of survival at 3 years was 41% vs. 50%, respectively.	ref. 124, 125, 135
Palbociclib	NeoPalAna	palbociclib in an neoadjuvant setting (i.e., prior to surgery)	compared the effects of an aromatase inhibitor anastrozole versus palbociclib plus anastrozole on tumor cell proliferation	women with newly diagnosed clinical stage II/III ER+/HER2-breast cancer	Addition of palbociclib enhanced the anti-proliferative effect of anastrozole.	ref. 161
Palbociclib	PALLAS	randomized phase 3	palbociclib plus standard endocrine therapy versus endocrine therapy alone	patients with early (stage 2 or 3), HR+/HER2-breast cancer	Preliminary results indicate that the trial is unlikely to show a statistically significant improvement of invasive disease-free survival.	ref. 138
Palbociclib	PENELOPE-B		palbociclib in patients with early breast cancer at high risk of recurrence		ongoing
Ribociclib	MONALEESA-2	randomized phase 3	ribociclib plus letrozole versus placebo plus letrozole	fist-line treatment for postmenopausal women with HR+/HER2-recurrent or metastatic breast cancer who had not received previous systemic therapy for advanced disease	At 18 months, the progression free-survival rate was 42.2% in the placebo-letrozole group and 63.0% in the ribociclib-letrozole group.	ref. 126
Ribociclib	MONALEESA-3	phase 3	ribociclib plus fulvestrant	patients with advanced (metastatic or recurrent) HR+, HER2-breast cancer who have either received no treatment for the advanced disease, or previously received a single line of endocrine therapy for the advanced disease	Addition of ribociclib significantly extended the median PFS, from 12.8 months (placebo-fulvestrant) to 20.5 months (ribociclib-fulvestrant). The overall survival at 42 months was also extended from 45.9% (placebo-fulvestrant) to 57.8% (ribociclib-fulvestrant).	ref. 127, 133
Ribociclib	MONALEESA-7	phase 3 randomized, double-blind	ribociclib versus placebo together with an antiestrogen tamoxifen or an aromatase inhibitor (letrozole or anastrozole)	premenopausal and perimenopausal women with HR+/HER2-advanced breast cancer who had not received previous treatment with CDK4/6 inhibitors	Ribociclib significantly increased median PFS from 13.0 months in the placebo-endocrine therapy group to 23.8 months in the ribociclib-endocrine therapy group. The overall survival was also strongly prolonged in the ribociclib group (the estimated overall survival at 42 months was 46.0% for the placebo group and 70.2% in the ribociclib group).	ref. 128, 132
Ribociclib	EarLEE-1	phase 3 trial	ribociclib in the treatment of early-stage, high risk HR+/HER2-breast cancers		ongoing
Abemaciclib	MONARCH 1	phase 2 trial	abemaciclib as a single agent	women with HR+/HER2-metastatic breast cancer who had progressed on or after prior endocrine therapy and had 1 or 2 chemotherapy regimens in the metastatic setting	Abemaciclib exhibited promising activity in these heavily pretreated patients with poor prognosis; median PFS was 6.0 months and overall survival 17.7 months.	ref. 136	The most common adverse events were diarrhea, fatigue and nausea (136).
Abemaciclib	MONARCH 2	double-blind phase 3	abemaciclib in combination with fulvestrant	women with HR+/HER2-breast cancer who had progressed while receiving endocrine therapy, or while receiving first line endocrine therapy for metastatic disease	Addition of abemaciclib significantly increased PFS from 9.3 months in the placebo-fulvestrant to 16.4 in the abemaciclib-fulvestrant group. The median overall survival was also extended from 37.3 months to 46.7 months.	ref. 129, 134
Abemaciclib	MONARCH 3	randomized, phase 3 double-blind	abemaciclib plus an aromatase inhibitor (anastrozole or letrozole)	postmenopausal women with advanced HR+/HER2-breast cancer who had no prior systemic therapy in the advanced setting	Addition of abemaciclib prolonged the PFS from 14.8 months (in the placeboaromatase inhibitor group) to 28.2 months (abemaciclib-aromatase inhibitor group).	ref. 130, 131
Abemaciclib	MonrarchE	phase 3 study	endocrine with or without abemaciclib	patients with HR+/HER2-lymph node-positive, high-risk early breast cancer	Preliminary analysis indicates that addition of abemaciclib resulted in a significant improvement of invasive disease-free survival and of distant relapse-free survival.	ref. 137
Trilaciclib		randomized phase 2 study	chemotherapy alone (gemcitabine and carboplatin), versus concurrent administration of trilaciclib plus chemotherapy, versus administration of trilaciclib prior to chemotherapy (to mitigate the cytotoxic effect of chemotherapy on bone marrow)	patients with recurrent or metastatic triple-negative breast cancer who had no more than two previous lines of chemotherapy	Addition of trilaciclib did not offer detectable myeloprotection, however it resulted in increased overall survival from 12.8 months in the chemotherapy only group to 20.1 months in concurrent trilaciclib and chemotherapy group and 17.8 month in trilaciclib before chemotherapy group.	ref. 162	The most common adverse events were neutropenia, thrombocytopenia and anemia (162).

Box 1. Clinical use of CDK4/6 inhibitors.

Palbociclib

Approved by the FDA in 2016, in combination with fulvestrant for the treatment of hormone receptor-positive (HR+), HER2-negative (HER2−) advanced or metastatic breast cancer in women with disease progression following endocrine therapy. Approved in 2017 for the treatment of HR+, HER2− advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine-based therapy in postmenopausal women.

Palbociclib dose is 125 mg (given orally) daily for 3 weeks followed by 1 week off, or 200 mg daily for 2 weeks followed by 1 week off. The rate-limiting toxicities are neutropenia, thrombocytopenia and anemia.

Ribociclib

Approved by FDA in 2017, in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with HR+, HER2− advanced or metastatic breast cancer. In 2018, the FDA expanded the indication for ribociclib in combination with an aromatase inhibitor for pre/perimenopausal women with HR+, HER2− advanced or metastatic breast cancer, as initial endocrine-based therapy. FDA also approved ribociclib in combination with fulvestrant for postmenopausal women with HR+, HER2− advanced or metastatic breast cancer, as initial endocrine-based therapy or following disease progression on endocrine therapy.

Ribociclib is administered at a dose of 600 mg (given orally) daily for 3 weeks followed by 1 week off. The main toxicities are neutropenia and thrombocytopenia.

Abemaciclib

Approved by FDA in 2017 in combination with fulvestrant for women with HR+, HER2− advanced or metastatic breast cancer with disease progression following endocrine therapy. In addition, abemaciclib was approved as monotherapy for women and men with HR+, HER2− advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting. Approved by FDA in 2018 in combination with an aromatase inhibitor as initial endocrine-based therapy for postmenopausal women with HR+, HER2− advanced or metastatic breast cancer. Approved by FDA in 2021 for adjuvant treatment of early-stage HR+, HER2− breast cancer in combination with endocrine therapy.

Abemaciclib is administered at a dose of 200 mg (given orally) every 12 hours. The dose-limiting toxicity is fatigue. Neutropenia is observed as well, but is not rate-limiting. Other severe side effects include diarrhea and nausea.

Currently, palbociclib is being used in 164 active or recruiting clinical trials, ribociclib in 69 trials and abemaciclib in 98 trials for over 50 tumor types (139). These trials evaluate combinations of CDK4/6-inhibitors with a wide range of compounds (Table 4). Trials with trilaciclib test the benefit of this compound in preserving bone marrow and the immune system.

Table 4. Ongoing clinical trials testing new combinations with CDK4/6-inhibitors.

HR+, hormone receptor-positive; LHRH, luteinizing hormone-releasing hormone; ER+, estrogen receptor-positive; PD-1, programmed cell death protein 1; PD-L1, programmed cell death 1 ligand 1; AR+, androgen receptor-positive; TNBC, triple-negative breast cancer; EGFR, epidermal growth factor receptor; HER2+, human epidermal growth factor receptor 2-positive; FGFR, fibroblast growth factor receptor; IGFR, insulin-like growth factor receptor; VEGF, vascular endothelial growth factor receptor; PI3K, phosphoinositide 3-kinase; NSCLC, non-small cell lung cancer; ALL, acute lymphoblastic leukemia; SCLC, small cell lung cancer.

CDK4/6 inhibitor	Additional target	Inhibitor	Immune checkpoint inhibitor	Tumor type	Trial identifier
Palbociclib	aromatase	letrozole, anastrozole, exemestane		HR+ breast cancer, HR+ ovarian cancer, metastatic breast cancer, metastatic endometrial cancer	NCT04130152; NCT03054363; NCT03936270; NCT04047758; NCT02692755; NCT02806050; NCT03870919; NCT02040857; NCT04176354; NCT02028507; NCT03220178; NCT02592083; NCT02603679; NCT04256941; NCT03425838; NCT02894398; NCT02297438; NCT02730429; NCT02142868; NCT02942355
	LHRH	LHRH agonists: goserelin, leuprolide		HR+ breast cancer	NCT03969121; NCT03423199; NCT01723774; NCT02917005; NCT02592746; NCT03628066
	ER	ER-antagonists: fulvestrant, tamoxifen		HR+ breast cancer, metastatic breast cancer	NCT02668666; NCT02738866; NCT03184090; NCT04526028; NCT02513394; NCT03560856; NCT02760030; NCT03079011; NCT03227328; NCT03809988; NCT02764541; NCT03007979; NCT03633331
	ER	selective estrogen receptor degraders (SERD): G1T48, ZN-c5, SAR439859, AZD9833, GDC-9545		HR+ breast cancer	NCT03455270; NCT04546009; NCT04436744; NCT04478266; NCT03560531; NCT03616587; NCT03284957; NCT03332797
	ER	selective estrogen receptor modulator (SERM): bazedoxifene		HR+ breast cancer	NCT03820830; NCT02448771
	aromatase + PD-1	letrozole, anastrozole	pembrolizumab, nivolumab	stage IV ER+ breast cancer	NCT02778685; NCT04075604
	PD-1		nivolumab, pembrolizumab, MGA012	liposarcoma	NCT04438824
	PD-L1		Avelumab	AR+ breast cancer, TNBC, ER+/HER2-metastatic breast cancer	NCT04360941; NCT03147287
	EGFR + PD-L1	cetuximab	Avelumab	squamous cell carcinoma of the head and neck	NCT03498378
	HER2	tucatinib, trastuzumab, pertuzumab, T-DM1, ZW25		HER2+ breast cancer	NCT03530696; NCT03054363; NCT02448420; NCT03709082; NCT03304080; NCT02947685
	EGFR/HER2	neratinib		advanced solid tumors with EGFR mutation/amplification, HER2 mutation/amplification, HER3/4 mutation, or KRAS mutation	NCT03065387
	EGFR	cetuximab		metastatic colorectal cancer, squamous cell carcinoma of the head and neck	NCT03446157; NCT02499120
	FGFR	erdafitinib		ER+/HER2-/FGFR-amplified metastatic breast cancer	NCT03238196
	FGFR1–3	rogaratinib		FGFR1–3+/HR+ breast cancer	NCT04483505
	IGF-1R	ganitumab		Ewing sarcoma	NCT04129151
	VEGF1–3 receptors + PD-L1	axitinib	Avelumab	NSCLC	NCT03386929
	RAF	sorafenib		leukemia	NCT03132454
	MEK	PD-0325901, binimetinib		KRAS mutant NSCLC, TNB, KRAS and NRAS mutant metastatic or unresectable colorectal cancer	NCT02022982; NCT03170206; NCT04494958; NCT03981614
	ERK	ulixertinib		advanced pancreatic and other solid tumors	NCT03454035
	PI3K	copanlisib		HR+ breast cancer	NCT03128619
	PI3K	taselisib, pictilisib, GDC-0077		PIK3CA mutant advanced solid tumors, PIK3CA mutant and HR+ breast cancer	NCT02389842; NCT04191499; NCT03006172
	PI3K/mTOR	gedatolisib		metastatic breast cancer, advanced squamous cell lung, pancreatic, head & neck cancer and other solid tumors	NCT02684032; NCT03065062; NCT02626507
	mTOR	everolimus, vistusertib		HR+ breast cancer	NCT02871791
	AKT	ipatasertib		HR+ breast cancer, metastatic breast cancer, metastatic gastro-intestinal tumors, NSCLC	NCT03959891; NCT04060862; NCT04591431
	BTK	ibrutinib		mantle cell lymphoma	NCT03478514
	BCL-2	venetoclax		ER+/BCL-2+ advanced or metastatic breast cancer	NCT03900884
	AR	AR-antagonists: bicalutamide		AR+ metastatic breast cancer	NCT02605486
	lysosome + aromatase	hydroxychloroqu ine + letrozole		ER+ breast cancer	NCT03774472
	proliferating cells	standard chemotherapy		stage IV ER+ breast cancer	NCT03355157
	proliferating cells	radiation		stage IV ER+ breast cancer	NCT03870919; NCT03691493; NCT04605562
	BCR-ABL	bosutinib		HR+ breast cancer	NCT03854903
Ribociclib	aromatase	letrozole, anastrozole, exemestane		HR+ breast cancer, metastatic breast cancer, ovarian cancer	NCT04256941; NCT03425838; NCT03822468; NCT02712723; NCT03673124; NCT02941926; NCT03248427; NCT03671330; NCT02333370; NCT01958021; NCT03425838;
	LHRH	LHRH agonists: goserelin, leuprolide		HR+ breast cancer	NCT03944434
	ER	ER-antagonists: fulvestrant		HR+ breast cancer, advanced breast cancer	NCT03227328; NCT02632045; NCT02632045; NCT03555877
	PD-1		spartalizumab	breast cancer and ovarian cancer, recurrent and/or metastatic head and neck squamous cell carcinoma, melanoma	NCT03294694; NCT04213404; NCT03484923
	HER2	trastuzumab, pertuzumab, T-DM1		HER2+ breast cancer	NCT03913234; NCT02657343
	EGFR	nazartinib (EGF816)		EGFR mutant NSCLC	NCT03333343
	RAF	encorafenib, LXH254		NSCLC, BRAF mutant melanoma	NCT02974725; NCT03333343; NCT04417621; NCT02159066
	MEK	binimetinib		BRAF V600-dependent advanced solid tumors, melanoma	NCT01543698; NCT02159066
	PI3K	alpelisib		breast cancer with PIK3CA mutation	NCT03439046
	mTOR	everolimus		advanced dedifferentiated liposarcoma, leiomyosarcoma, glioma, astrocytoma, glioblastoma, endometrial carcinoma, pancreatic cancer, neuroendocrine tumors	NCT03114527; NCT03355794; NCT03834740; NCT03008408; NCT02985125; NCT03070301
	mTOR + inflammation	everolimus + dexamethasone		ALL	NCT03740334
	SHP2	TNO155		advanced solid tumors	NCT04000529
	AR	AR-antagonists: bicalutamide, enzalutamide		TNBC, metastatic prostate carcinoma	NCT03090165; NCT02555189
	HDAC	belinostat		TNBC, ovarian cancer	NCT04315233
	proliferating cells	standard chemotherapy		ovarian cancer, metastatic solid tumors, soft tissue sarcoma, hepatocellular carcinoma	NCT03056833; NCT03237390; NCT03009201; NCT02524119
Abemaciclib	aromatase	letrozole, anastrozole, exemestane		HR+ breast cancer, metastatic breast cancer, endometrial cancer	NCT04256941; NCT03425838; NCT04227327; NCT04393285; NCT04305236; NCT03643510; NCT03675893; NCT04352777; NCT04293393; NCT02057133
	ER	ER-antagonists: fulvestrant		advanced breast cancer, low-grade serous ovarian cancer	NCT03227328; NCT03531645; NCT04158362; NCT01394016
	PD-1		nivolumab, pembrolizumab	head and neck cancer, gastroesophageal cancer, nScLC, HR+ breast cancer	NCT04169074; NCT03655444; NCT03997448; NCT02779751
	ER + PD-L1	ER-antagonists: fulvestrant	atezolizumab	HR+ breast cancer, metastatic breast cancer	NCT03280563
	AKT + ER + PD-L1	ipatasertib + ER-antagonists fulvestrant	atezolizumab	HR+ breast cancer	NCT03280563
	PD-L1		LY3300054	advanced solid tumors	NCT02791334
	HER2	trastuzumab		HER2+ metastatic breast cancer	NCT04351230
	receptor tyrosine kinases	sunitinib		metastatic renal cell carcinoma	NCT03905889
	IGF-1/IGF-2	xentuzumab		HR+ breast cancer	NCT03099174
	VEGF-A	bevacizumab		Glioblastoma	NCT04074785
	PI3K	copanlisib		HR+ breast cancer, metastatic breast cancer	NCT03939897
	PI3K/mTOR	LY3023414		metastatic cancer	NCT01655225
	ERK1/2	LY3214996		tumors with ERK1/2 mutations, glioblastoma, metastatic cancer	NCT04534283; NCT04391595; NCT02857270
Trilaciclib	proliferating cells	chemotherapy		SCLC. This trial evaluates the potential clinical benefit of trilaciclib in preventing chemotherapy-induced myelosuppression in patients receiving chemotherapy.	NCT04504513
	proliferating cells + PD-L1	carboplatin + etoposite	Atezolizumab	SCLC. This trial investigates the potential clinical benefit of trilaciclib in preserving the bone marrow and the immune system, and enhancing antitumor efficacy when administered with chemotherapy.	NCT03041311
	proliferating cells	topotecan		SCLC. This trial investigates the potential clinical benefit of trilaciclib in preserving the bone marrow and the immune system, and enhancing chemotherapy antitumor efficacy when administered prior to chemotherapy.	NCT02514447
	proliferating cells	carboplatin + gemcitabine		metastatic TNBC. This study investigates the potential clinical benefit of trilaciclib in preserving the bone marrow and the immune system, and enhancing chemotherapy antitumor efficacy when administered prior to chemotherapy.	NCT02978716
Lerociclib	ER	ER antagonist: fulvestrant		HR+/HER2-metastatic breast cancer	NCT02983071
	EGFR	osimertinib		EGFR mutant NSCLC	NCT03455829
SHR6390	ER	ER antagonist: fulvestrant		HR+/HER2-recurrent/metastatic breast cancer	NCT03481998
	aromatase	letrozole, anastrozole		HR+/HER2-recurrent/metastatic breast cancer	NCT03966898; NCT03772353
	EGFR/HER2	pyrotinib		HER2+ gastric cancer, HER2+ metastatic breast cancer	NCT04095390; NCT03993964
	AR	AR-antagonists: SHR3680		metastatic TNBC	NCT03805399
PF-06873600	endocrine therapy	single agent and then in combination with endocrine therapy		HR+/HER2-metastatic breast cancer, ovarian and fallopian tube cancer, TNBC and other tumors	NCT03519178
FCN-473c	aromatase	Letrozole		ER+/HER2-advanced breast cancer	NCT04488107

Resistance to CDK4/6-inhibitors

Although CDK4/6-inhibitors represent very effective agents in cancer treatment, nearly all patients eventually develop resistance and succumb to the disease. Moreover, a substantial fraction of tumors shows an intrinsic resistance to treatment with CDK4/6-inhibitors (Fig. 3).

Fig. 3. Mechanisms of cancer cell resistance to CDK4/6-inhibition.

Known mechanisms include loss of RB1, activation of pathways impinging on cycD-CDK4/6, amplification of the CDK4/6 genes and overexpression of CDK6 protein, activation of CycE-CDK2, lysosomal sequestration of CDK4/6-inhibitors. Blank pieces of the puzzle denote additional mechanisms that remain to be discovered.

The best-documented mechanisms of pre-existing and acquired resistance is the loss of RB1 (71, 81, 140). Acquired RB1-loss has been detected in PDX (141), circulating tumor DNA (ctDNA) (142, 143) and in tumors from patients treated with CDK4/6-inhibitors (144, 145). However, RB1-mutations are likely subclonal and seen in only 5–10% of patients (143, 145).

Increased expression of CDK6 was shown to underlie acquired resistance to CDK4/6-inhibitors. Amplification of the CDK6 gene and the resulting overexpression of CDK6 protein was found in abemaciclib-resistant ER+ breast cancer cells (146), and in ctDNA of patients with ER+ breast cancers that progressed during treatment with palbociclib plus endocrine therapy (147). Also CDK4 gene amplification confers insensitivity to CDK4/6-inhibition in GBM and sarcomas (148–150), while overexpression of CDK4 protein was associated with resistance to endocrine therapy in HR+ breast cancers (79). Resistant breast cancer cells can also upregulate CDK6 through suppression of the TGF-β/SMAD4-pathway by miR-432–5p. In this mechanism, exosomal expression of miR-432–5p mediates the transfer of the resistance phenotype between neighboring cell populations (151). Another mechanism of CDK6-upregulation in ER+ breast cancers is the loss of FAT1, which represses CDK6 expression via the Hippo-pathway. Loss of FAT1 triggers upregulation of CDK6 expression by Hippo-pathway effectors TAZ and YAP. Moreover, genomic alterations in other components of the Hippo-pathway, although rare, are also associated with reduced sensitivity to CDK4/6-inhibitors (81).

Genetic lesions that activate pathways converging on D-type cyclins can cause resistance to CDK4/6-inhibitors. These include: (a) FGFR1/2 gene amplification or mutational activation, detected in ctDNA from patients with ER+ breast cancers which progressed on treatment with palbociclib plus endocrine therapy (147); (b) Hyperactivation of the MAPK-pathway in resistant prostate adenocarcinoma cells, possibly due to increased production of EGF by cancer cells (152); (c) Increased secretion of FGF in palbociclib-resistant KRAS-mutant NSCLC cells, which stimulates FGFR1-signaling in an autocrine or paracrine fashion, resulting in activation of ERK1/2 and mTOR, and upregulation of D-cyclins, CDK6 and cyclin E (153). Analyses of longitudinal tumor biopsies from a melanoma patient revealed an activating mutation in the PIK3CA gene that conferred resistance to ribociclib plus MEK-inhibitor treatment (154). It is possible that these lesions elevate the cellular levels of active cyclin D-CDK4/6 complexes, thereby increasing the threshold for CDK4/6-inhibition.

Formation of a non-canonical cyclin D1-CDK2 complex was shown to represent another mechanism of acquired CDK4/6-inhibitor resistance. Such a complex was observed in palbociclib-treated ER+ breast cancer cells and was implicated in overcoming palbociclib-induced cell-cycle arrest (141). Also depletion of AMBRA1 promotes interaction of D-cyclins with CDK2, resulting in resistance to CDK4/6-inhibitors (20, 22); it remains to be seen whether this represents an intrinsic or acquired resistance mechanism in human tumors.

Genetic analyses revealed that activation of cyclin E can by-pass the requirement for cyclin D-CDK4/6 in development and tumorigenesis (155, 156). Hence, it comes as no surprise that increased activity of cyclin E-CDK2 is responsible for a large proportion of intrinsic and acquired resistance to CDK4/6-inhibitors. Several different mechanisms can activate cyclin E-CDK2-kinase in resistant tumor cells, including: (a) Downregulation of KIP/CIP-inhibitors (54, 157); (b) Loss of PTEN expression, which activates AKT-signaling, leading to nuclear exclusion of p27^KIP1. This, in turn, prevents the access of p27^KIP1 to CDK2, resulting in increased CDK2-kinase activity (144); (c) Activation of PI3K/AKT-pathway causing decreased levels of p21^CIP1. Importantly, co-treatment of melanoma PDX with MDM2-inhibitors (which upregulate p21^CIP1 via p53) sensitized intrinsically resistant tumor cells to CDK4/6-inhibitors (158). (d) Upregulation of cyclin D1 levels triggers sequestration of KIP/CIP-inhibitors from cyclin E-CDK2 to cyclin D-CDK4/6, thereby activating the former (158). (e) Amplification of the CCNE1 gene and increased levels of cyclin E1 protein (141); (f) mTOR-signaling was shown to upregulate cyclin E1 (and D1) in KRAS-mutated pancreatic cancer cells; CDK2-activity was essential for CDK4/6-inhibitor resistance (159); (g) Upregulation of PDK1 and the resulting activation of the AKT-pathway, which increases the expression of cyclins E and A and activates CDK2 (160); (h) In CDK4/6-inhibitor resistant melanoma cells, high levels of RNA-binding protein FXR1 increase translation of amino-acid transporter SLC36A1. Upregulation of SLC36A1 expression activates mTORC1 which, in turn, increases CDK2 expression (161). All these lesions are expected to allow cell proliferation despite CDK4/6-inhibition, due to activation of the downstream cell-cycle kinase, CDK2.

The role for cyclin E-CDK2 in CDK4/6-inhibitor resistance has been confirmed in clinical trials. In patients with advanced ER+ breast cancer treated with palbociclib and letrozole or fulvestrant, the presence of proteolytically-cleaved cytoplasmic cyclin E in tumor tissue conferred strongly shortened progression-free survival (71). Moreover, analyses of PALOMA-3 trial for patients with ER+ breast cancers revealed lower efficacy of palbociclib plus fulvestrant in patients with tumors displaying high cyclin E mRNA levels in metastatic biopsies (80). Amplification of the CCNE1 gene was detected in ctDNA of patients with ER+ breast cancers that progressed on palbociclib plus endocrine therapy (147). Also amplification of the CCNE2 gene (encoding cyclin E2) was seen in a fraction of CDK4/6-inhibitor-resistant resistant HR+ mammary carcinomas (145, 162).

Collectively, these analyses indicate that resistant cells may become dependent on CDK2 for cell-cycle progression. Indeed, depletion of CDK2 or inhibition of CDK2-kinase activity in combination with CDK4/6-inhibitors blocked proliferation of CDK4/6-inhibitor resistant cancer cells (111, 141, 158–161). Recently, two CDK2-specific inhibitors PF-07104091 (163) and BLU0298 (164) have been reported. The former compound is now being tested in a phase 2 clinical trial in combination with palbociclib plus antiestrogens. Another recent study identified a novel compound, PF-3600, that inhibits CDK4/6 and CDK2 (165). Importantly, PF3600 had potent anti-tumor effects against xenograft models of intrinsic and acquired resistance to CDK4/6 inhibition (165). A clinical phase 2 trial is currently evaluating this compound as a single agent and in combination with endocrine therapy in patients with HR+, HER2− breast cancer and other cancer types.

Whole-exome sequencing of 59 HR+, HER2− metastatic breast tumors from patients treated with CDK4/6-inhibitors and anti-estrogens revealed eight alterations that likely conferred resistance: RB1-loss, amplification of CCNE2 or AURKA, activating mutations or amplification of AKT1, FGFR2, or ERBB2, activating mutations in RAS genes, loss of ER-expression. The frequent activation of AURKA (in 27% of resistant tumors) raises a possibility of combining CDK4/6-inhibitors with inhibitors of Aurora A kinase to overcome resistance (145).

In contrast to ER+ mammary carcinomas, TNBC are overall resistant to CDK4/6-inhibition (45). A subset of TNBCs display high numbers of lysosomes, which causes sequestration of CDK4/6-inhibitors into the expanded lysosomal compartment, thereby preventing their action on nuclear CDK4/6. Importantly, pre-clinical studies revealed that lysosomotropic agents that reverse the lysosomal sequestration (such as chloroquine, azithromycin, or siramesine) render TNBC cells fully sensitive to CDK4/6-inhibition (71, 111). These observations need now to be tested in clinical trials for TNBC patients.

Outlook

Although D-cyclins and CDK4/6 were discovered almost 30 years ago, several aspects of cyclin D-CDK4/6 biology, such as their role in anti-tumor immunity, are only now starting to be appreciated. The full-range of cyclin D-CDK4/6 functions in tumor cells remains unknown. It is likely that these kinases play a much broader role in cancer cells than currently appreciated. Hence, the impact of CDK4/6-inhibition on various aspects of tumorigenesis requires further studies. Also, treatment of patients with CDK4/6-inhibitors likely affects several aspects of host physiology, which may be relevant to cancer progression.

In the next years, we will undoubtedly witness development and testing of new CDK4/6-inhibitors. Since activation of CDK2 represents a frequent CDK4/6-inhibitor resistance mechanism, compounds that inhibit CDK4/6 and CDK2 may prevent or delay the development of resistance. Conversely, selective compounds which inhibit CDK4, but not CDK6, may allow more aggressive dosing, as they are expected not to cause bone marrow toxicity caused by CDK6-inhibiton. New, less basic CDK4/6-inhibitor compounds (111), may escape lysosomal sequestration, and may be efficacious against resistant cancer types, such as TNBC. Degrader compounds, which induce proteolysis of cyclin D, rather than inhibit cyclin D-CDK4/6-kinase may have superior properties, as they would extinguish both CDK4/6-dependent and -independent functions of D-cyclins in tumorigenesis. Moreover, dissolution of cyclin D-CDK4/6 complexes is expected to liberate KIP/CIP-inhibitors, which would then inhibit CDK2. D-cyclins likely play CDK-independent functions in tumorigenesis, for example by regulating gene expression (166). However, their role in tumor biology and the utility of targeting these functions for cancer treatment remain largely unexplored.

An important challenge will be to test and identify combinatorial treatments involving CDK4/6-inhibitors for the treatment of different tumor-types. CDK4/6-inhibitors trigger cell cycle arrest of tumor cells and, in some cases, senescence. It will be essential to identify combination treatments which would convert CDK4/6-inhibitors from the cytostatic compounds to cytotoxic ones, and would unleash the killing of tumor cells. Genome-wide high-throughput screens along with analyses of mouse cancer models and PDX will help to address this point. Another largely unexplored area of cyclin D-CDK4/6 biology is the possible involvement of these proteins in other pathologies, such as metabolic disorders. Research in this area may extend the use of CDK4/6-inhibitors to treatment of other diseases. All these unresolved questions assure that CDK4/6 biology will remain an active area of basic, translational and clinical research for several years to come.

Print Figure. Targeting cyclin D-CDK4/6 for cancer treatment.

D-cyclins (CycD) activate CDK4 and CDK6 in G1 phase of the cell cycle. CycD-CDK4/6 contribute to cell-cycle progression by phosphorylating the retinoblastoma protein, RB1. RB1 inhibits E2F transcription-factors; phosphorylation of RB1 activates E2F-driven transcription. In many cancers CycD-CDK4/6 is constitutively activated and drives uncontrolled cell proliferation. The development of small-molecule CDK4/6-inhibitors provided a therapeutic tool to repress constitutive CycD-CDK4/6 activity and inhibit cancer cell proliferation. As with several targeted therapies, many tumors eventually develop resistance and resume cell proliferation despite CDK4/6-inhibition. New combination treatments, involving CDK4/6-inhibitors plus inhibition of other pathways, are being tested in the clinics to delay or overcome the resistance.