Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide, and ∼85% of lung cancers are classified as nonsmall cell lung cancer (NSCLC). These malignancies can proliferate indefinitely, in part due to dysregulation of the cell cycle and the resulting abnormal cell growth. The specific activation of cyclin-dependent kinases 4 and 6 (CDK4/6) is closely linked to tumour proliferation. Approximately 80% of human tumours exhibit abnormalities in the cyclin D-CDK4/6-INK4-RB pathway. Specifically, CDK4/6 inhibitors either as monotherapy or combination therapy have been investigated in pre-clinical and clinical studies for the treatment of NSCLC, and promising results have been achieved. This review article focuses on research regarding the use of CDK4/6 inhibitors in NSCLC, including the characteristics and mechanisms of action of approved drugs and progress of pre-clinical and clinical research.

Shareable abstract

CDK4/6 inhibitors have great potential as broad-spectrum anti-tumour drugs. CDK4/6 inhibitor monotherapy or combination therapies may become a more widespread strategy for the treatment of advanced NSCLC. https://bit.ly/3SOd39V

Background

Lung cancer is a relatively common cancer and is one of the main causes of cancer deaths [1–4]. Lung cancer can be classified into two major types based on the pathological features: small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC). NSCLC in particular accounts for 80–85% of all lung cancer cases [5]. According to the World Health Organization, NSCLC has a 5-year survival rate of only 5–10% [6]. NSCLC can be further classified into three primary subtypes: adenocarcinoma (40%), squamous cell carcinoma (25–30%) and large cell carcinoma (5–10%) [7]. Most newly diagnosed patients are considered incurable because diagnosis often occurs when the cancer has reached its later stages. Although new treatments for advanced NSCLC continue to be developed, the overall benefit of NSCLC treatment remains limited. Chemotherapy has been the most commonly used anticancer treatment for decades and has been shown to be effective. However, its benefits are limited by the narrow therapeutic indices and nonselective mechanisms of action, which result in systemic toxicity. Radiotherapy is an important treatment option for NSCLC. It works by generating DNA double-strand breaks in cancer cells [8]. Despite the significant contribution of radiation to killing NSCLC cells, radiation resistance [9], metastasis and local regional disease progression persist [10]. However, recently, targeted therapies for lung cancer have yielded revolutionary breakthroughs. In particular, therapies targeting the drivers epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) have achieved positive results. Compared with chemotherapy, these therapies have significantly prolonged progression-free survival [11–13]. However, targeted therapy inevitably leads to resistance. Similarly, immunotherapies targeting programmed cell death-1 (PD-1) and programmed cell death ligand 1 (PD-L1) have made great progress, but the population that benefits from such therapies is limited. It is therefore necessary to actively search for new therapeutic targets.

Accordingly, exploring mechanisms of resistance to targeted lung cancer therapies and seeking new strategies to overcome resistance have become active areas of research. In particular, the identification of new therapeutic targets for NSCLC has attracted widespread attention. One attractive therapeutic target involves the dysregulation of the cell cycle that supports the abnormal cellular proliferation that characterises cancer [14–16]. These defects participate in malignant transformation and tumour progression, and they result in the disruption of mechanisms that controls the growth of advanced NSCLC [17–20]. In this realm, cyclin-dependent kinases 4 and 6 (CDK4/6) are key to cell cycle control. These kinases are activated in response to proliferative signalling. They induce hyperphosphorylation of the retinoblastoma (RB) tumour suppressor, which in turn activates the transcription factor 2 family of transcription factors, thus promoting the progression of the cell cycle to S phase [21–24]. As this process leads to cell growth, CDK4/6 have been found to be involved in the malignant transformation and tumour progression of lung cancer, and CDK4/6 are targets for cancer therapies [23, 25]. Notably, CDK4/6 inhibitors can not only induce cell cycle arrest, but they also increase anti-tumour immunity by stimulating the production of type III interferon, thereby enhancing the presentation of tumour antigens and inhibiting regulatory T-cells (Tregs) [26].

Pharmacological inhibition of CDK4/6 has been demonstrated to be an effective method for the treatment of breast cancer [27, 28]. The United States Food and Drug Administration (FDA) has approved several related drugs for use in combination with letrozole for the treatment of hormone receptor-positive advanced breast cancer [29–34], and the clinical potential of these inhibitors is expanding to other cancers [35, 36]; for example, inhibitors of CDK4/6 have shown significant activity against several solid tumours [37, 38]. In addition, combinations of CDK4/6 inhibitors with other targeted therapies may help overcome primary or secondary treatment resistance [23]. Though these drugs are efficacious, many patients exhibit primary resistance, meaning they gain no benefits from CDK4/6 inhibitor treatment and instead shift to chemotherapy within usually 6 months. Research is ongoing to develop better treatments for this primary resistance. Alternatively, many patients derive some benefits from the treatment initially, but eventually develop acquired resistance, which may take months or years to occur and has causes such as drug metabolism dysregulation, mutation of target proteins and changes in the tumour microenvironment. While there are no definite solutions yet, scientists are racing to find ways of dealing with the resistance, such as administering alternative drug combinations, increasing doses, or invoking immunotherapy. The FDA approved the use of sublethal CDK4/6 inhibitors, for the treatment of NSCLC. These inhibitors both induce cell cycle arrest in the G1 phase and inhibit the transcription of the gene encoding poly(ADP-ribose) polymerase 1 (PARP1). Studies have shown that CDK4/6 inhibitors sensitise cells blocked in G1 to other anticancer drugs. The effect of CDK4/6 inhibitors is not limited to the inhibition of cell growth, but treatment with these inhibitors also leads to the accumulation of DNA damage by decreasing the activity of PARP1 in oxidatively stressed cells [39].

Four selective CDK4/6 inhibitors, trilaciclib, palbociclib, ribociclib and abemaciclib, are at various stages of development in various RB tumour suppressor protein-positive tumour types, including breast cancer, melanoma, liposarcoma and NSCLC [40]. Whether used as a single agent or in combination with hormones, chemotherapy, radiation or immunotherapy, CDK4/6 inhibitors have shown significant efficacy in pre-clinical studies and clinical trials. The roles of CDK4/6 inhibitors in NSCLC in particular have also been studied on several levels, and positive results have been achieved, suggesting that this strategy may lead to new treatment methods [41, 42]. This article reviews the progress and prospects of CDK4/6 inhibitors in the treatment of NSCLC.

Mechanisms of CDK4/6 inhibitors in cancer therapyRoles of CDK4/6 in the cell cycle

The cell cycle is orchestrated by a highly regulated group of proteins. This process prevents cells from advancing inappropriately or prematurely to the next phase of the cell cycle. CDK4/6, in physical association with partner proteins cyclin D1, D2 and D3, are the key drivers of the transition from the G1 phase of the cell cycle to the S phase. Mitogenic stimuli induce the formation of complexes between cyclin D proteins and CDK4/6 [13], and this complex induces the binding of cyclin E to CDK2 (figure 1). Activated CDK2 then catalyses the hyperphosphorylation of RB, promoting its dissociation from early region 2 (E2F) and thus the activation of E2F transcription factor activity [43].

FIGURE 1

Classical model of G1–S transition regulated by cyclins and cyclin-dependent kinases (CDK). The cyclin D–CDK4/6 complex can be activated through Janus kinase–signal transducer and activator of transcription (STAT), phosphatidylinositol 3-kinase (PI3K)–protein kinase B (AKT), and Ras–Raf–extracellular signal-regulated protein kinases (ERK) signalling pathways, and is regulated by P16INK4A inhibition. Furthermore, CDK inactivation can be mediated through P21CIP1 via the P53 signalling pathway. Generally, inhibition of CDK4/6 and CDK2 leads to lower phosphorylation of retinoblastoma (RB), which in turn inhibits the expression of early region 2 (E2F) target genes. CDK2 is activated in the G1 phase with a rise in late-stage E-type cyclin levels, leading to overphosphorylation and deactivation of RB. Overphosphorylated RB is released from E2F, resulting in increased transcription of E2F target genes, which is necessary for the cell to enter the S phase. mTOR: mammalian target of rapamycin; TGF: transforming growth factor.

Importantly, the function of RB depends upon its phosphorylation state. When it is hypophosphorylated, RB prevents the advance from G1 to S by repressing the E2F family of transcription factors via interaction with their transactivation domains and recruitment of histone deacetylases [44]. Conversely, the phosphorylation of RB induces its dissociation from E2F transcription factors, liberating their transcriptional activation activity and facilitating the expression of a wide variety of genes required for DNA replication and entry into S phase [13, 23].

The activity of CDK4/6, which is key to the regulation of RB and thus E2F, is regulated by two families of endogenous inhibitory proteins, the inhibitor of CDK4 (INK4) family and the cyclin-dependent kinase inhibitor 1/kinase inhibitory protein (CIP/KIP) family [44]. Proteins of the INK4 family include p16INK4A, p15INK4B, p18INK4C and p19INK4D, and these proteins specifically bind to CDK4 and CDK6, forming binary complexes that lack kinase activity. The CIP/KIP family comprises three proteins, p27KIP1, p21CIP1 and p57KIP2. In contrast to the members of the INK4 family, the CIP/KIP proteins are able to bind to all of the CDKs involved in the cell cycle, and they have more diverse functions.

The expression of cyclin D and activity of CDK4/6 are both induced by several common oncogenic signalling pathways, such as those involving Janus kinase and signal transducers and activators of transcription [45, 46], phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) and Ras- and Raf-activated extracellular signal-regulated protein kinases (ERK) [47]. Induction by these pathways has been shown to lead to uncontrolled cell proliferation. In addition, the p53 signalling pathway also interacts with the CDK4/6–RB pathway through its regulation of the transcription of the gene encoding p21CIP1, which can inhibit the activity of the cyclin D–CDK4/6 and cyclin E–CDK2 complexes [48]. The loss of another INK4 family member, P16INK4A, has also been found to appear frequently in glioblastoma [49] (figure 1). Thus, these findings have demonstrated that CDK4/6 is a promising therapeutic target in the development of anticancer drugs.

Roles of CDK4/6 in immunity

CDK4/6 inhibitors have been found to induce anti-tumour immune responses by enhancing tumour cell antigen presentation, activating effector T-cells and inhibiting Treg cell proliferation [37, 50]. Malignant tumour cells in patients often evade immune surveillance and proliferate rapidly in the body. CDK4/6 inhibitors can increase the exposure of abnormal proteins on the cell surface, creating immune signals that can be recognised, cleared or regulated by the tumour cell cycle. This enhances the anti-tumour immune function of the body. Furthermore, CDK4/6 inhibitors can also promote the activity of negative regulator P21, inhibit the generation and proliferation of Tregs, and improve anti-tumour immune function by inhibiting DNA methyltransferase 1 of Tregs [51].

Moreover, CDK4/6 inhibition increased levels of tumour-infiltrating T-cells in vivo and was shown to augment the response to PD-1 blockade in multiple syngeneic tumour models. In agreement with these data, Schaer et al. [52] demonstrated that application of the CDK4/6 inhibitor abemaciclib combined with PD-L1 checkpoint blockade increased immune activation, as manifest in enhancing of antigen presentation on macrophages and dendritic cells, increasing of the T-cell inflamed phenotype, and a reciprocal increase in abemaciclib-dependent cell cycle gene regulation.

Other emerging data demonstrate that CDK4/6 inhibitor may promote tumour immune evasion through the induction of the expression of immunosuppressive PD-L1 on tumour cells. Zhang et al. [36] discovered that PD-L1 protein stability is regulated by the Cullin 3SPOP E3 ubiquitin ligase, which interacted with PD-L1 via SPOt-type POZ protein (SPOP). The phosphorylation of SPOP is mediated by cyclin D-CDK4, and inhibition of CDK4/6 represses SPOP phosphorylation and promotes SPOP degradation, consequently increasing the expression of PD-L1. These researchers also found in mouse tumour models that a combined treatment with a CDK4/6 inhibitor and an anti-PD-1 agent markedly promoted tumour regression and improved overall survival (figure 2). Jin et al. [53] also identify a previously uncharacterised role of CDK4/6 in promoting cancer immunity via inhibition of the transcription activity of NF-κB protein p65 and the expression of PD-L1. This effect was found to be primarily dependent on CDK4/6-mediated serine-249/threonine-252 phosphorylation of RB.

FIGURE 2

Effect of cyclin-dependent kinase (CDK)4/6 inhibition on tumour immune microenvironment. CDK4/6 inhibitors may not only have tumour-specific functions, but also affect the tumour microenvironment. In tumour cells, CDK4/6 inhibitors increase programmed cell death ligand 1 (PD-L1) expression via the E3 ligase connector protein SPOt-type POZ protein (SPOP) and enhance antigen presentation by decreasing early region 2 (E2F) target DNA methyltransferase (DNMT)1 activity. Treatment with CDK4/6 inhibitors also increases cytokine secretion in tumour and CD8+ T-cells, while inhibiting the proliferation of immunosuppressive regulatory T-cells (Tregs). ERV: endogenous retrovirus; dsRNA: double-stranded RNA; MHC: major histocompatibility complex; IFN: interferon; NFAT: nuclear factor of activated T-cells.

A combination treatment of CDK4/6 inhibitor, PD-1 monoclonal antibody and PI3K inhibitor has been shown to completely shrink the tumours of triple-negative PDX model mice and the effect can last for >1 year [54]. A phase I study using abemaciclib and pembrolizumab in hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2−) advanced breast cancer found 14.3% objective response rate with good safety after a median follow-up of 16 weeks [55]. In addition, current studies have shown that the synaptonemal complex protein (SCP)3–cyclinD1 CDK4/6 axis exists in various types of human cancers, and the SCP3–cyclinD1–CDK4/6 axis is negatively correlated with progression-free survival in patients with cervical cancer. Targeting CDK4/6 with the inhibitor palbociclib reverses multiple aggressive phenotypes in tumour cells highly immunoedited by SCP3 and leads to long-term control of the disease [56]. There are few data on the combination of CDK4/6 inhibitors with immunocheckpoint inhibitors. More large randomised clinical studies are needed to confirm the efficacy of the combination.

Roles of CDK4/6 in metabolism

Like the effects of these proteins on the cell cycle and in immunomodulation, the interface of CDK4/6 with metabolism is complex and varied. Lee et al. [57] documented that the cyclin D1–CDK4 complex controls glucose homeostasis independently of cell division. Studies have confirmed that cyclin D1–CDK4 is dysregulated in diabetic models, and that overactivation of cyclin D1–CDK4 attenuates the diabetic phenotype. These findings indicate that insulin uses components of the cell cycle mechanism in post-mitotic cells to control glucose homeostasis independently of cell division [57]. Inhibition of CDK4/6 by mechanistic target of rapamycin (mTOR) complex 1 has been shown to result in increases of both glycolytic and oxidative metabolism [58].

Pharmacological evidence also supports the role of the system in metabolism. In lung cancer patients, CDK4/6 inhibitor palbociclib was used to reduce glucose metabolism through pentose phosphate pathway by inhibiting glucose 6-phosphate dehydrogenase activity, while increasing dependence on glutamine decomposition to maintain basic mitochondrial function. These changes were also observed when treated with other CDK4/6 inhibitors, including ribociclib and abemaciclib [59]. In contrast to these findings of Franco et al. [58], Zhang et al. [60] demonstrated that oral administration of ribociclib resulted in reductions in tumour 18F-fluorodeoxyglucose uptake of ∼50% in the LP6 liposarcoma tumour xenograft model. However, the maximum standardised uptake value in tumours of vehicle-treated mice in this study was either unchanged or slightly increased, suggesting that inhibition of CDK4/6 does substantially alter tumour metabolism in the liposarcoma xenograft model.

Mechanisms of resistance to CDK4/6 inhibition

CDK4/6 inhibitors have demonstrated good efficacy in multiple clinical trials, but the problem of resistance is inevitable. Approximately 10% of patients will show primary resistance to CDK4/6 inhibitors, limiting the therapeutic effectiveness of these agents [61]. The mechanisms leading to resistance to CDK4/6 inhibition include cell cycle dysregulation and activation of carcinogenic signal transduction pathways [62].

Changes involving mediators of the cell cycle

Loss of RB is key driver of the development of resistance to CDK4/6 inhibitors [63]. The main cause of RB loss is the inactivation of the RB1 gene due to mutations [64]. The PALOMA-3 study reported that of 127 patients with advanced breast cancer who were treated with palbociclib, six cases had unique enriched RB1 alterations at the time of disease progression. The palbociclib group has unique enriched RB1 alterations in disease progression [65]. CDK6 itself has also been found to be amplified in breast cancer cells cultured in vitro, and this alteration reduces the response to CDK4/6 inhibition; conversely, in this system, knockdown of CDK6 restored drug sensitivity [66]. Loss of the tumour suppressor FAT1 is an indirect mechanism that can lead to increased CDK6 activity, and its inhibition can restore sensitivity to CDK4/6 inhibitors in the context of FAT1 inactivating mutations [67]. Because CDK6 expression is induced downstream of hippo signalling, activation of this pathway can also induce resistance to CDK4/6 inhibition [67]. The transcription factor c-Myc is another cellular component that has been found to play an important role in palbociclib sensitivity, and the c-Myc/miR-29b-3p/CDK6 has thus been identified as a key factor [68]. Another study has shown that extracellular miRNA signalling can confer CDK4/6 resistance on neighbouring cell populations [69]. Aurora kinase A (AURKA) promotes mitotic spindle assembly and regulates the G2-to-M transition in the cell cycle by phosphorylation and activation of cyclin B1–CDK1 [62, 70]. This effect means that alterations to AURKA activity can also impact sensitivity to CDK4/6 inhibition. For example, a whole-exome sequencing analysis of biopsy specimens from 59 patients treated with CDK4/6 inhibitors showed that AURKA overexpression correlated with in vitro resistance to CDK4/6 inhibition [71]. Overexpression of cyclin E1 (CCNE1), which encodes cyclin E, is another widely accepted mechanism leading to CDK4/6 inhibitor resistance [72, 73]. A recent correlation analysis of data from the PALOMA-3 study suggested that CCNE1 mRNA expression is associated with a poorer prognosis in patients treated with palbociclib [74].

Activation of oncogenic signalling pathways

Studies have shown that regulating CDK4/6 inhibitor resistance in vitro and in patient tumour specimens involves multiple carcinogenic signalling pathways [71]. For example, amplification of the gene encoding the tyrosine kinase fibroblast growth factor receptor (FGFR)1, which activates the PI3K/AKT and rat sarcoma virus (RAS)/mitogen-activated protein kinase (MEK)/ERK signalling pathways, is associated with CDK4/6 inhibitor resistance [71, 75]. In the MONALEESA-2 trial, cotreatment with the FGFR inhibitor lucitanib eliminated resistance to ribociclib [76]. In this trial, elevated FGFR1 mRNA levels were also found to be associated with significantly shorter progression-free survival (PFS) times [76]. FGFR2 has also been implicated in resistance to CDK4/6 inhibition. Baseline and end-of-treatment circulating tumour DNA sequencing in 195 patients in the PALOMA-3 randomised phase III trial revealed the presence of acquired FGFR2 mutations or amplification events at the time of disease progression in a subset of patients [65]. Other changes to oncogenic signalling have been found as well. Mutation or amplification of the proto-oncogene NRAS have been observed in cultured melanoma cells co-treated with inhibitors against MEK1 and CDK4/6 [77, 78]. Other studies have shown that amplification of the gene encoding ribosomal protein S6 kinase-1 (S6K1) occurs frequently in patients with oestrogen receptor-positive metastatic breast cancer, and the results of in vitro and in vivo studies further suggest that S6K1-induced resistance to CDK4/6 inhibitors may be mediated mainly by the c-Myc signalling cascade [79]. Deletion of the gene of phosphate and tension homology deleted on chromosome 10 (PTEN) has been found to lead to decreased sensitivity to clinically relevant doses of CDK4/6 inhibitors by initiating a signalling cascade that induces overactivation of the cyclin–CDK complex. Similarly, a variety of AKT serine/threonine-protein kinase 1 (AKT1) activation events are associated with resistance to CDK4/6 inhibition [71]. For example, increased AKT1 expression has been shown to lead to downstream activation of cyclin E1/2 and CDK2 by decreasing p27 activity [80]. Multiple activation events involving pathways associated with RAS proteins, including Kirsten rat sarcoma virus (KRAS), Harvey rat sarcoma (HRAS) and neuroblastoma rat sarcoma (NRAS), have been observed in tumour biopsy specimens from patients with CDK4/6 inhibitor-resistant cancers. RAS-activating mutations lead to activation of mitogen-activated protein kinase (MAPK) pathways, and ERK activation increased in these resistant models, suggesting that MAPK pathways play a downstream role in promoting resistance to CDK4/6 inhibition [71]. Resistance to CDK4/6 inhibition has also been shown to involve the tyrosine kinase receptor ERBB2, which has been shown to stimulate oncogenic signalling pathways in multiple models [81]. For example, enrichment of both ERBB2-activating mutations and amplification events of FGFR2 have been observed in patients with CDK4/6 inhibitor-resistant cancers, and alterations to both pathways have been shown to cause resistance to anti-oestrogen and CDK4/6 inhibitor treatments in vitro [82].

A series of phase II and III trials testing adjuvant and neoadjuvant treatments to be used with CDK4/6 inhibitors are currently underway. MONARCH2 is a phase III, randomised, double-blind, placebo-controlled study of disease progression in patients with HR+/HER2− advanced breast cancer treated with fulvestrant and prior endocrine therapy. In this trial, one group of patients was found to exhibit primary resistance to endocrine therapy [83]. There are currently three other ongoing clinical trials (clinicaltrials.gov NCT04251169, NCT03901339 and NCT04134884) evaluating the efficacy of chemotherapy following the development of CDK4/6 inhibitor resistance in patients with HR+/HER2− breast cancer. Another clinical study (clinicaltrials.gov NCT03439735) explored biomarkers that could potentially be used to predict responsiveness to endocrine therapy and to CDK4/6 inhibition in metastatic breast cancer; here, the researchers will analyse information obtained from tumour biopsies and from frequent blood sampling. The researchers hope that a deeper understanding of response to this combination and how resistance emerges will allow clinicians to better tailor treatments for various cancers.

Pre-clinical studies with CDK 4/6 inhibitors in lung cancer models

Four main CDK4/6 inhibitors are currently approved for clinical use in various parts of the world: palbociclib, trilaciclib, ribociclib and abemaciclib. Palbociclib and abemaciclib are very similar [84], but in vitro studies have demonstrated that palbociclib has similar potencies for CDK4 and CDK6, but ribociclib and abemaciclib have a preference for CDK4. All three drugs can be administered orally, and the main dose-limiting toxicity involves reactions of the digestive tract [85]. These drugs have been well studied in a series of pre-clinical and clinical studies, both singly and in combination.

CDK4/6 inhibitor monotherapyPalbociclib

Palbociclib is a small molecule inhibitor of CDK4/6, and it shows anti-tumour activity both in vivo and in vitro [86–89]. Palbociclib is a cell-permeable pyridoxine with oral bioavailability that exhibits a uniquely selective inhibition of CDK4 and CDK6 [90, 91]. Recent studies have demonstrated that palbociclib treatment leads to reprogramming of cellular metabolism. While palbociclib does not affect the glycolytic activity of lung cancer cell A549 cells, it reduces glucose metabolism through its impacts on the pentose phosphate pathway [59]. In addition, palbociclib enhances glutamine breakdown to maintain mitochondrial respiration and sensitise A549 cells to the glutaminase inhibitor CB-839 [59].

The P16 (CDKN2Aink4a) gene is an endogenous CDK4/6 inhibitor. P16 is most frequently inactivated by copy number deletion and DNA methylation in cancers. Further studies have shown that cancer cell lines with P16 methylation are more sensitive to palbociclib than those without palbociclib. Lung cancer cell lines with methylation of the P16 gene promoter are more sensitive to palbociclib than are cancer cell lines that are negative for P16 methylation; these results have also been confirmed in animal experiments [92].

RB is a key regulator of cell cycle progression and proliferation and is functionally inhibited in up to 50% of NSCLC cases. Accordingly, palbociclib has been tested for its ability to activate RB. One study used multiple isogenic NSCLC lines with active or defective RB to investigate mechanisms of cell growth inhibition and cytotoxicity of CDK4/6 inhibition in vitro and in vivo. The results showed that CDK4/6 inhibition induced pro-apoptotic transcription by inhibiting IAPsFOXM1 and Survivin, and enhanced SMAC and caspase 3 expression in a RB-dependent manner [42].

Abemaciclib

The three clinically approved CDK4/6 inhibitors, ribociclib, abemaciclib and palbociclib, have all been shown to exert cytotoxic and cytostatic effects in the G1 phase in cancer cell lines, including A549 human NSCLC cells. Among these inhibitors, abemaciclib has been demonstrated to have the strongest cytotoxic effect in NSCLC. Studies have shown that in cancer cells, abemaciclib induces a unique form of cell death that is accompanied by the swelling and dysfunction of lysosomes. This atypical cell death induced by abemaciclib is characterised by the formation of lysosome-derived cytoplasmic vacuoles [93]. In various parts of the globe, abemaciclib is undergoing clinical trials, ranging from phase 1 to phase 3, for the treatment of breast cancer and NSCLC [94].

Trilaciclib

Trilaciclib, a recently approved CDK4/6 inhibitor, was shown to reduce the incidence of chemotherapy-induced myelosuppression in adult patients with extensive-stage SCLC [95]. Trilaciclib is a small molecule, short-acting CDK4/6 inhibitor developed by G1 Therapeutics for its potential myeloprotective activity and anti-tumour efficacy, especially in combination with other chemotherapy agents. Because CDKs control cell cycle progression, treatment with trilaciclib induces transient, reversible G1 cell cycle arrest in proliferating haematopoietic stem and progenitor cells in the bone marrow, thereby protecting them from damage during chemotherapy [96]. In February 2021, trilaciclib received its first approval in the United States to decrease the incidence of chemotherapy-induced myelosuppression in adult patients when administered prior to a regimen involving platinum agents, etoposide or topotecan for extensive-stage SCLC [96].

Combination therapy with CDK4/6 inhibitors

In pre-clinical trials in lung cancer, CDK4/6 inhibitors were studied in combination with other therapies (table 1). Combinations of CDK4/6 inhibitors have been extensively studied due to the suboptimal effect of monotherapies; in-depth research on the pathogenesis of lung cancer has thus led to an increase in lung cancer treatment options (figure 3).

TABLE 1

Pre-clinical research on combination therapy of cyclin-dependent kinase (CDK)4/6 inhibitors with targeted therapies

FIGURE 3

Conceptual framework for the main combination strategies for cyclin-dependent kinase (CDK)4/6 inhibitors. a) CDK4/6 can be used in combination with chemotherapy and should be used after chemotherapy, rather than before or at the same time as chemotherapy, to avoid interfering with chemotherapy by preventing G1/S transition in advance and to prevent recovery after damage has been determined. b) CDK4/6 inhibitors can be combined with radiotherapy drugs. c) CDK4/6 inhibitors enhance the efficacy of immunotherapy by allowing the expression of immune checkpoint proteins (retinoblastoma (RB)1-dependent and -independent) and by the antiproliferative effect of immune-suppressive cells such as regulatory T-cells (Tregs) (RB1-dependent). d) In addition, CDK4/6 inhibitors can be combined with targeted agents, such as epidermal growth factor receptor (EGFR) inhibitors, MEK inhibitors, mammalian target of rapamycin (mTOR) inhibitors and phosphatidylinositol 3-kinase (PI3K)CA.

Combinations of CDK4/6 inhibitors with targeted therapiesCDK4/6 inhibitors combined with EGFR inhibitors

EGFR tyrosine kinase inhibitors (TKI) such as gefitinib are commonly targeted therapy for patients with these EGFR mutations [110, 111], and recent studies have shown that gefitinib significantly improves overall survival in patients with NSCLC [112]. However, as treatment progresses, most patients will inevitably acquire TKI resistance, which greatly limits the overall survival of patients with NSCLC [113]. Liu et al. [98] found that palbociclib potentiated gefitinib-induced growth inhibition in both EGFR-TKI-sensitive (PC-9) and EGFR-TKI-resistant (PC-9/AB2) cells by down regulating proliferation and inducing apoptosis and G0/G1 cell cycle arrest. Other studies have demonstrated that the combined use of an EGFR-TKI and palbociclib can reduce proliferation and induce apoptosis in both EGFR-TKI-sensitive and drug-resistant cell lines more potently than gefitinib alone. One study found that, as compared with lung cancer mice treated with gefitinib alone, mice treated with palbociclib and gefitinib had faster tumour regression and delayed recurrence. In this study, tumour proliferation in the experimental mice was significantly reduced by the cotreatment, apoptosis increased and angiogenesis decreased. Therefore, the combination therapy of cell cycle inhibitors and targeted drugs may prove to be effective in reversing acquired drug resistance and enhancing the effects of the targeted therapy [86].

Another study found that gefitinib combined with LY2874455 and abemaciclib showed the most potent inhibition of drug resistance in vitro and in vivo. Combined treatment with LY2874455 and abemaciclib reversed fibroblast growth factor (FGF)3/4/19/CCND1 amplification-mediated gefitinib resistance in NSCLC [98]. Conversely, Sun et al. [114] found that gefitinib could enhance the cytotoxicity of abemaciclib by inducing apoptosis and senescence in lung cancer cells.

Afatinib is approved as a second-generation TKI for the treatment of advanced/metastatic NSCLC with EGFR mutations. Despite its efficacy, acquired drug resistance limits the clinical application of afatinib. A study investigated whether the combination of palbociclib and afatinib treatment could reverse the acquired drug resistance of NSCLC cells. The results showed that this combined therapy played a synergistic effect in reducing the survival rate of cells with acquired drug resistance. In addition, the combined use of palbociclib and afatinib reduced the drug resistance that otherwise tends to lead to the recurrence of drug-resistant malignant tumours in xenograft models. Thus, the combination of palbociclib and afatinib serves as an innovative strategy to reverse acquired resistance in lung malignancies [99]. With regard to another EGFR-TKI, osimertinib, Qin et al. [100] found that CDK4 expression and RB phosphorylation were increased in osimertinib-resistant lung cancer cells. Accordingly, inhibition of CDK4/6 with palbociclib significantly enhanced the sensitivity of the resistant cells to osimertinib.

CDK4/6 inhibitors combined with MEK inhibitors

Selumetinib is an oral MEK inhibitor that is effective in inhibiting ERK phosphorylation in various types of cancer cells, including those derived from NSCLC [115]. While selumetinib has demonstrated anti-tumour activity in vitro and in several tumour xenograft models, its clinical efficacy in NSCLC as a single agent has been limited. CDK4 has a synthetic lethal interaction with the KRAS gene mutation [116]. Thus, a recent study investigated the combined effect of the MEK inhibitor selumetinib and the CDK4/6 inhibitor palbociclib in RAS-driven NSCLC [104]. The combination therapy had a synergistically enhanced growth inhibitory effect, and the number of cells arrested in the G1 phase was increased. The combination therapy of palbociclib and selumetinib has also been shown to be effective in a pre-clinical model of Ras-driven NSCLC with cyclin-dependent kinase inhibitor 2A (CDKN2A) mutations [104]. In KRAS-mutant NSCLC, inhibitory drugs that target elements of the MAPK cascade, including MEK and ERK, can act synergistically with CDK4/6 inhibitors to prevent G1 progression, thereby increasing drug dose reductions that may attenuate drug-induced therapeutic effects. While researchers recognise the potential for toxicity, the results also suggest that the judicial use of combination treatments including CDK4/6 inhibitors and inhibitors of MEK, mTOR and/or FGFR1 not only elicit longer-lasting G1 arrest, but also prevent the development of resistant variants [117].

CDK4/6 inhibitors combined with mTOR inhibitors

The serine-threonine kinase mTOR is responsible for regulating cell growth, proliferation and survival. There are two main categories of mTOR inhibitors: allosteric and ATP-competitive inhibitors [118]. Pre-clinical studies have found that mTOR pathway inhibitors produce cytotoxicity when used in combination with radiotherapy [119]. CDK4/6 inhibitors can exert inhibitory effects on a variety of lung cancer cell lines, and mTOR inhibitors can synergistically reverse resistance to CDK4/6 inhibition. Gopalan et al. [101] identified mTOR inhibitors as potent partners that synergised with palbociclib in human lung cancer cell lines with a range of palbociclib sensitivities. Western blotting analyses of these cell lines indicated that palbociclib treatment led to increased expression of cyclin D1 in some cell lines, and in these cases, the addition of everolimus decreased cyclin D1 levels. Combination treatment with a CDK4/6 inhibitor and an mTOR inhibitor enhances growth inhibition and apoptosis induction in P16-deficient NSCLC cells.

CDK4/6 inhibitors combined with inhibitors of the PI3K catalytic subunit

In lung squamous carcinoma, abnormalities in the PI3K pathway are a significant subtype, typically caused by phosphatidylinositol-4.5-bisphosphate 3-kinase catalytic subunit-α (PIK3CA) mutations, amplification and loss of PTEN. Incidence rates for these abnormalities are approximately 10–15%, 50% and 20–30%, respectively [120]. PI3K, as the upstream activation signal of CDK4/6, acts through the activation of cyclin D [121].

Dysregulation of PI3K signalling plays an important role in tumourigenesis and drug resistance of NSCLC. Combination therapy with the CDK4/6 inhibitor palbociclib and the PI3Kα inhibitor CYH33 showed synergistic activity on proliferation of CYH33-sensitive and CYH33-resistant cells, and this treatment was accompanied by enhanced G1 arrest. The results also showed that simultaneous inhibition of PI3Kα and CDK4/6 showed synergistic activity against KRAS-mutant NSCLC. These data provide a mechanistic rationale for the combined use of PI3Kα inhibitors and CDK4/6 inhibitors in KRAS-mutant NSCLC [102].

Lung squamous cell carcinoma (LUSC), the major subtype of NSCLC, is characterised by multiple genetic alterations, and alterations in components of the PI3K pathway have been identified in >50% of LUSC cases. While PI3K is therefore an attractive target, single-agent PI3K inhibitors have shown modest responses in LUSC. Therefore, novel combination therapies targeting LUSC are needed. One study found that combined inhibition of PI3K and CDK4/6 produced greater anti-tumour effects in models with the PIK3CA mutation than did monotherapy. Additionally, the combination of the two drugs achieved targeted inhibition of both PI3K and cell cycle pathways. The CDK4/6 inhibitors palbociclib or abemaciclib combined with PI3K inhibitors can significantly inhibit the growth of tumour cells more potently than can a single drug, and these treatments are typically well tolerated. The synergy of these inhibitors may be due to the activation of cyclin D through AKT and mTOR downstream of PI3K mutations, while PI3K inhibitors would then reduce cyclin D1 expression [103].

CDK4/6 inhibitors combined with other inhibitors

Polyphyllin I (PPI) is a steroid saponin extracted from the Paris polyphylla plant, with its crude grass being widely used in traditional Chinese medicine to treat various ailments, including carbuncles, snake bites, sore throats and convulsions, as well as malignancies [122]. Recent studies have shown that PPI exhibits anticancer effects, such as reducing tumour cell proliferation, inducing apoptosis and autophagy, and reversing drug resistance in cancer cells [123]. PPI was found to be an effective partner in palbociclib treatment, inhibiting NSCLC by activating the P21/CDK2/RB pathway in vitro and in vivo, and reducing NSCLC proliferation while promoting apoptosis cell apoptosis in vitro and in vivo. This effect may occur through inhibition of RB through the P21/CDK2/RB signalling pathway. Studies have also found that treatment with PPI can reverse palbociclib resistance. Thus, the combination of PPI and palbociclib has a significant synergistic anticancer ability in NSCLC [105].

Combination therapy of CDK4/6 inhibitor palbociclib with the eukaryotic initiation factor (EIF) 4A inhibitor CR-1-31-B has a synergistic effect in inhibiting the growth of cancer cells in vitro and in vivo. These findings revealed a new powerful strategy for using EIF4A inhibitors to suppress the cell cycle feedback response and thus to overcome resistance to CDK4/6 inhibition [24].

Combination treatments with CDK4/6 inhibitors with chemotherapy agents

Multiple pre-clinical studies have shown that there can be cooperation between CDK4/6 inhibitors and chemotherapy. In addition, the combination of CDK4/6 and chemotherapy is being tested in clinical trials to enhance anti-tumour efficacy and limit toxicity. The development of nonclassical effects of CDK4/6 can also provide the impetus for future research on such combined chemotherapy strategies. Emerging data suggests that different clinical strategies can be used to safely combine these drugs to improve anti-tumour efficacy and to reduce chemotherapy-induced toxicity [124].

In multiple squamous cell lung cancer models with different cancer genetic backgrounds, palbociclib in combination with taxane exhibited enhanced cytotoxicity and anti-tumour effects at clinically achievable doses. Taxane chemotherapy is the current standard treatment for these cancers, and studies have shown that pharmacological inhibition of CDK4/6 by palbociclib can improve the anti-tumour effect of treatment with taxanes [106].

Another study found that the combination of pemetrexed and ribociclib had significantly stronger anti-tumour ability than either treatment alone. In addition, it was found that ribociclib strongly enhanced the pro-apoptotic activity of pemetrexed through its effects on signalling pathways involving caspases and bcl-2. Here, ribociclib combined with pemetrexed showed a strong ability to inhibit tumour proliferation, invasion and metastasis [107]. These results indicate that CDK4/6 inhibitors can be used alone or in combination with standard cytotoxic therapies, and such combinations are currently in clinical development [125].

Combination of CDK4/6 inhibitors with radiotherapy

A number of pre-clinical studies have shown that CDK4/6 inhibitors exhibit synergistic effects with radiotherapy both in vitro and in vivo. Naz et al. [108] studied the effects of the combination of abemaciclib and ionising radiation in using methods such as delayed xenograft tumour regeneration, immunoblotting of xenograft tumour lysates and tissue section immunohistochemistry. It was found that abemaciclib and ionising radiation jointly potently inhibit the CDK4/6 pathway. It is of great significance to reduce the recurrence of tumours following treatment with ionising radiation, and the results of this study indicate that the combination therapy of abemaciclib and ionising radiation may be an effective method for the treatment of patients with early and advanced NSCLC.

Moreover, it has been reported that palbociclib may also be a new type of radiosensitiser. Fernández-Aroca et al. [126] used cell lines from colorectal cancer (HT29 and HCT116), lung cancer (A549 and H1299) and breast cancer (MCF-7) to elucidate the molecular basis of this effect. The results indicate that the presence of wild-type p53 is a strict requirement for the exertion of radiosensitisation by palbociclib, and this effect is independent of its inhibitory effect on CDK4/6. In fact, the loss of P53 blocks the radiosensitisation of palbociclib. In conclusion, palbociclib has apparently been identified as a new rad