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Introduction

Cyclin-dependent kinase (CDK)4/6 plays a key role in cell cycle regulation, where aberrant activation of the cyclin D-CDK4/6-retinoblastoma (Rb) gene axis often leads to unrestrained cell proliferation, one of the central mechanisms of tumorigenesis (1). Notably, CDK4/6 inhibitors, such as palbociclib, ribociclib and abemaciclib, have shown marked efficacy in the treatment of patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast cancer (HR+/HER2− MBC) (2). Traditionally, CDK4/6 inhibitors inhibit cell proliferation by inhibiting Rb phosphorylation and inducing pre-DNA synthesis (G1) cell cycle arrest in tumor cells (3). However, it has also been shown that CDK4/6 inhibitors are essential in modulating tumor immunity. For example, CDK4/6 inhibitors can promote tumor cytotoxic T-cell-mediated clearance by affecting tumor cells and can reduce the activity of E2F-targeted DNA methyltransferase 1 (DNMT1) in regulatory T cells (Tregs). In addition, CDK4/6 inhibitors can enhance the therapeutic effects of immune checkpoint inhibitors (ICIs) by upregulating antigen presentation mechanisms and programmed death-ligand 1 (PD-L1) expression, thus exhibiting good synergistic potential and providing new perspectives for tumor immunotherapy (4).

Despite their potential, CDK4/6 inhibitors still face challenges in the clinic. Clinical and preclinical studies have shown that ~20% of patients with breast cancer (BC) have innate resistance to CDK4/6 inhibitors (5), and >30% of patients gradually develop acquired resistance during ongoing treatment (6), severely limiting the durability of their efficacy and widespread use. The mechanisms of resistance, whether innate or acquired, involve both compensatory activation of cell cycle-related pathways and multiple non-cell cycle escape strategies (7). In addition, the hematological toxicity of CDK4/6 inhibitors should not be ignored. In the PALOMA-3 study, 55.3% of patients developed grade 3 neutropenia and 9.7% developed grade 4 neutropenia, suggesting the importance of regular patient monitoring and dose adjustment to ensure safety (8). Therefore, future studies should focus on overcoming resistance to CDK4/6 inhibitors, searching for predictive biomarkers of therapeutic sensitivity, optimizing dosing regimens, and improving patient monitoring and dose adjustment to improve therapeutic efficacy and safety.

Based on the aforementioned information, the present study aims to systematically review the mechanism of CDK4/6 inhibitors in cell cycle regulation and tumor immunomodulation, and to thoroughly elaborate their drug resistance mechanisms, including the compensatory activation of cell cycle-related pathways and multiple non-cell cycle escape strategies. The review also discusses the recent progress and future directions in overcoming drug resistance and optimizing clinical application, with the aim of providing a theoretical basis and practical guidance for the future use of precision therapy with CDK4/6 inhibitors.

Fundamental mechanisms of action and cell cycle regulation of CDK4/6 inhibitors

The CDK4/6-Rb-E2F pathway

CDK4/6 are key regulators of the cell cycle via the CDK4/6-Rb-E2F pathway, with CDK4/6 assuming a pivotal role in the G1-S checkpoint of the cell cycle, which oversees genome replication (9). The Rb suppressor protein represents a principal cell cycle target of CDK4/6. In normal cells, the phosphorylated state of Rb is typically the consequence of a sequential interaction between CDK4/6 and the D-type cyclin subunit, inhibiting Rb activity (10). The D-type cyclin subunit is generated by stimulating activation signals, including growth factors and cell adhesion molecules. The phosphorylation of Rb results in the deregulation of E2F inhibition, thereby enabling its activation of downstream targets. This, in turn, facilitates the recruitment of transcriptional activators and the irreversible entry of quiescent cells into the DNA-synthesis (S) phase of the cell cycle (1,11). CDK4/6 inhibitors induce complete Rb dephosphorylation, resulting in the sequestration of the transcription factor E2F and subsequent inhibition of cell cycle progression, thereby causing cell cycle arrest in G1 (12). Common CDK4/6 inhibitors include palbociclib, ribociclib and abemaciclib. The fundamental mechanism of CDK4/6 inhibitors and their role in regulating the cell cycle are elucidated in the present review.

CDK4/6 inhibitors prevent the formation of an active CDK4/6 complex by binding to CDK4/6. The most significant function of cyclin D-CDK4/6 in promoting cellular proliferation is the phosphorylation of Rb, which results in a rapid reduction in the phosphorylation of Rb1 at cyclin D-CDK4/6-dependent sites. This indicates that CDK4/6 is subject to acute inhibition (13). Palbociclib, ribociclib and abemaciclib have been demonstrated to impede the binding of CDK4 and CDK6 to cell division cycle 37, the kinase-targeting subunit of heat shock protein 90 (HSP90). This prevents CDK4/6 from entering the HSP90-chaperone system (14). CDK4/6 inhibitors have demonstrated considerable efficacy in treating HR+/HER2− MBC by binding to CDK4/6 (15). In addition to this mechanism, the upregulation of cyclin D expression and formation of the cyclin D-CDK4/6 complex results in the redistribution of kinase inhibitor protein/CDK inhibitor protein inhibitors from the cyclin E-CDK2 complex to cyclin D-CDK4/6, which activates the kinase activity of cyclin E-CDK2 and facilitates cell-cycle progression. This pathway can also be blocked by CDK4/6 inhibitors (13).

The cell cycle is divided into interphase and mitosis (M). Interphase encompasses G1, S and pre-division (G2) phases. The cell cycle regulates intricate, orchestrated interactions between cyclin-regulating proteins and cell CDKs (16). CDK4/6 are two major kinases promoting the G1-S phase transition by phosphorylating and inactivating Rb, facilitating S-phase entry and DNA replication. While the level of CDK remains constant throughout the cell cycle, the expression of cyclin D is subject to dynamic regulation at multiple levels (17). Targeting CDK4 and CDK6 with CDK4/6 inhibitors blocks the binding of cyclin D to CDKs, thereby halting the cell cycle at the G1 phase and preventing cell proliferation (18). The uncontrolled proliferation of cells, driven by aberrant cell cycle progression, represents a pivotal characteristic of cancerous growth. Consequently, the pursuit of pharmacological agents that impede the cell cycle represents a rational strategy for the treatment of cancer (19). The cell cycle-blocking effect of CDK4/6 inhibitors has markedly benefited the utilization of these pharmaceutical agents in treating cancer (Fig. 1).

Figure 1 The CDK4/6-Rb-E2F pathway blocks the cell cycle in G1 phase. Created with MedPeer (medpeer.cn). This diagram illustrates the mechanisms of action of key molecular pathways and related inhibitors in cell cycle regulation. The left side of the diagram illustrates the mechanisms of action of inhibitors, such as palbociclib, ribociclib and abemaciclib, which act on the CDK4/6-cyclin D complex. The right side of the diagram is further refined to present the inhibition of Rb1 phosphorylation by the CDK4/6-cyclin D complex and the interaction of other related proteins, such as CDC37, HSP90, p21, p27, p57 and CDK2, in cell cycle progression. As illustrated in the lower left of the image, E2F is a transcription factor that, under normal conditions, is inactive due to its binding to the Rb1 protein. Upon receiving a growth signal, the CDK2-cyclin E complex is activated, resulting in the activation of CDK2, a cell cycle protein-dependent kinase, through its binding to cyclin E. The activated CDK2-cyclin E complex can phosphorylate the Rb1 protein, causing the dissociation of the phosphorylated Rb1 from E2F. The released E2F is then activated, which in turn initiates the transcription of a series of genes related to growth and survival. These genes drive the cell into specific stages of the cell cycle and promote cell proliferation. The lower arrows signify the mechanism through which CDK4/6 inhibitors impede the progression of cells from the G1 phase to the S phase, a process that is intricately linked to cell proliferation and survival. CDC37, cell division cycle 37; CDK, cyclin-dependent kinase; HSP90, heat shock protein 90; Rb1, retinoblastoma 1.

The role of estrogen and estrogen receptors (ERs) in regulating the expression of cyclin D

The hormones estrogen and progesterone have been shown to trigger the process of breast morphogenesis through a mechanism of action on a specific subset of mammary epithelial cells that express their cognate receptors, known as ERα and progesterone receptor (PR). Cyclin D1, a critical oncogene in BC, is frequently amplified in ER- and PR-positive BC, which is associated with a poor patient prognosis. Cyclin D1 is specifically amplified in ERα+ BC and is a progesterone target gene (20); thus, the process of progesterone-induced cell proliferation may manifest through the expression of cyclin D1. The binding between PR and progesterone leads to cyclin D1 expression, which is connected to intracellular signaling and activation of the mitotic cell cycle (21). Research findings have demonstrated that in animals injected with ERα− cells, a substantial decrease in tumor volume is observed following treatment with adiponectin; conversely, in animals injected with ERα+ cells, tumor growth is increased. In the absence of ERα, the process of auto linker-mediated downregulation of cyclin D1 has been shown to involve the recruitment of co-stressors; by contrast, in the presence of ERα, the expression of cyclin D1 was revealed to be induced by the activation complex (22). Cyclin D1 has been demonstrated to regulate PR expression, thereby enhancing responses to estrogen and progesterone. Meanwhile, progesterone can also regulate cyclin D1 through genetic effects or positive feedback loops. It has been demonstrated that estrogen treatment of cyclin D1-transgenic mice can lead to an increase in PR expression and the induction of mammary hyperplasia. This process is stimulated by progesterone and blocked by progesterone antagonists. Conversely, this previous study observed a decrease in PR levels in cyclin D1-knockout mice. The mechanism by which cyclin D1 regulates PR expression involves a novel estrogen- and cyclin D1-response enhancer located in the DNA-coding portion of the 3′untranslated region of the PR gene. The expression of PR has been shown to be reduced in human BC cells by small inhibitory RNA which targets at cyclin D1, which also diminishes ER binding to the 3′-enhancing region. Given the established role of estrogen and progesterone in regulating cyclin D1, the involvement of cyclin D1 in the feed-forward loop may potentially increase the risk of BC associated with the combination of estrogen and progesterone (23). Furthermore, the ability of cyclin D1 to regulate PRs suggests a novel function for cyclin D1 in ovarian hormonal control of breast development and breast carcinogenesis (23).

The PI3K/AKT/mTOR pathway of HR+/HER2− MBC

In addition to CDK4/6-Rb1 signaling, the PI3K/AKT/mTOR signaling pathway is a vital growth stimulation pathway in HR+/HER2− MBC (24). The PI3K family of proteins is comprised of three classes of lipid kinases that catalyze the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate. The PI3K class 1 family, a heterodimer with a regulatory and catalytic subunit, is the most commonly involved with human cancer. Somatic mutations in genes encoding components of the PI3K pathway occur in >70% of cases of BC; these include mutations or amplifications of subunit p110α (protein encoded by PIK3CA), subunit p110β and subunit p85α, which are the catalytic subunits of PI3K, or mutations in PI3K modulators, such as phosphatase and tensin homolog (PTEN), AKT and mTOR (25). The most common mechanism underlying PI3K pathway activation is via PIK3CA mutations or amplifications, in which p110α (PIK3CA) serves a vital role in BC tumorigenesis through extra-nuclear ER signaling and is often responsible for endocrine therapy resistance (26). mTOR is a serine/threonine protein kinase and a downstream effector of AKT, which consists of two functionally distinct complexes: mTOR complex (mTORC)1 and mTORC2. mTORC1 belongs to a network of regulatory feedback loops; once activated, it limits the transmission of upstream effectors, such as platelet-derived growth factor receptors α and β, to transmit proliferative signals. This, in turn, results in the attenuation of PI3K/AKT activity. Conversely, mTORC2 is implicated in the regulation of AKT phosphorylation at S473 and the organization of the cellular actin cytoskeleton. Furthermore, mTORC1 activation results in a direct decrease in mTORC2 activity. The mTOR downstream substrate S6 kinase can phosphorylate and activate functional structural domains of ERs, consequently resulting in ligand-independent receptor activation (27). Molecularly, estrogenic activity has been shown to induce insulin-like growth factor, and activate the PI3K/AKT and MAPK pathways, which have been observed to downregulate ERs and PR gene expression at the cell surface. The PI3K/AKT/mTOR pathway is the most frequently altered in MBC, and the upregulation of this pathway has been demonstrated to promote ER-dependent and -independent transcriptional activity, which contributes to anti-estrogen resistance, leading to tumor cell proliferation, survival, motility and metabolism. Drugs targeting this pathway have demonstrated encouraging results in HR+/HER2− MBC when combined with anti-estrogens (28). In addition, it has been shown that PI3K and mTOR inhibition can enhance sensitivity to endocrine therapies in vivo, providing a compelling rationale for the combination of these two therapeutic modalities (29). Similarly, mTORC1/2 inhibitors have demonstrated efficacy in enhancing the sensitivity of drug-resistant HR+ BC cell lines to CDK4/6 inhibitors (30).

The mechanisms facilitate the understanding of the use of CDK4/6 inhibitors in clinically relevant tumors and in the treatment of HR+/HER2− MBC, as mentioned in the present review.

Impact of CDK4/6 inhibitors on tumor immunity

CDK4/6 inhibitors affect tumor immunity by altering the immune microenvironment and modulating immune checkpoints (Fig. 2).

Figure 2 Impact of CDK4/6 inhibitors on the tumor microenvironment. Created with MedPeer (medpeer.cn). This image illustrates the mechanism of action of CDK4/6 inhibitors in the tumor microenvironment. CDK4/6 inhibitors have the capacity to affect a wide range of immune cells and tumor cells. They have been observed to act on CD8+ T cells, prompting them to secrete IFN-γ and IL-2, thereby enhancing immune responses. Additionally, they have been shown to have an inhibitory effect on Tregs, thus regulating immune homeostasis. Regarding tumor cells, the expression of MHC1, PD-L1, ROS and type III IFN is enhanced. Furthermore, they have been observed to enhance the expression of macrophages, DCs and NK cells, among others, thereby remodeling the tumor microenvironment and enhancing the immune surveillance and tumor cell-killing ability. DC, dendritic cell; IFN, interferon; MHC, major histocompatibility complex; NK, natural killer; PD-L1, programmed death-ligand 1; ROS, reactive oxygen species; SASP, senescence-associated secretory phenotype; Tregs, regulatory T cells.

Changes in the immune microenvironment

CDK4/6 inhibitors exert a direct effect on tumors by influencing the cell cycle, in addition to affecting cells within the tumor immune microenvironment. CDK4/6 inhibitors enhance cytokine secretion in both tumor cells and cytotoxic T lymphocytes (CD8+ T cells), while the proliferation of immunosuppressive Tregs is inhibited (31). In addition, CDK4/6 inhibitors have been demonstrated to influence the functionality of immune cells, including natural killer (NK) cells and macrophages, which can consequently impact the tumor microenvironment.

T cells

Although CDK4/6 inhibitors have been demonstrated to inhibit T-cell proliferation, they have also been shown to enhance tumor infiltration and effector T-cell activation. Additionally, tumor antigen-experienced T cells demonstrate a heightened sensitivity to CDK4/6 inhibition relative to naive T cells (32). Nuclear factor of activated T cells 4 (NFAT4) is a well-documented regulator of IL-2 secretion, controlled by CDK6, and IL-2 is a well-established marker of cellular activation. It has been demonstrated that brief exposure to CDK4/6 inhibitors impedes CDK6-induced phosphorylation of NFAT4, facilitates its nuclear translocation and augments its transcriptional activity. This ultimately promotes IL-2 synthesis, T-cell activation and cytokine production while reinforcing defense against tumors (33). In a mouse lung cancer model, CDK4/6 inhibitors have been observed to increase the percentage of effector cells within the tumor microenvironment, thereby enhancing the functionality of effector T cells. The capacity of CDK4/6 inhibitors to induce long-term immune T-cell memory in both mouse models and humans provides a robust foundation for treating tumor recurrence (34).

Secondly, it has been observed that CDK4/6 inhibitors exert a considerable inhibitory effect on the proliferation of Tregs. Tregs can impede the proliferation of effector T cells and release cytokines, thereby facilitating tumor cell immune evasion. It has been demonstrated that abemaciclib enhances downstream p21 production via the Rb-E2F axis. p21 can then compete with cyclin D for binding to CDK2, thereby regulating the G1-S transition of the cell cycle and mediating cell cycle arrest in Tregs. CDK4/6 inhibitors reduce immunosuppressive Treg populations by inhibiting their proliferation. By inhibiting the Rb-E2F axis and thereby reducing recombinant DNMT1 expression, CDK4/6 inhibitors induce hypomethylation of genes that regulate immune function. Selective inhibition of Treg proliferation may be associated with the observation that Treg cells express higher levels of Rb1 (4,34).

Administration of CDK4/6 inhibitors has been observed to reduce the number of circulating Tregs, the proportion of Tregs in tumors and their cellular activity. Notably, Tregs have been observed to exhibit elevated CDK6 expression relative to other T-cell subtypes (35). This observation suggests that heightened levels of CDK6 and potential increased reliance on CDK6 in Tregs could contribute to their heightened sensitivity to CDK4/6 inhibitors. Furthermore, CDK2 has been demonstrated to function as a negative regulator that CDK4/6 can modulate. CDK4/6 inhibitors influence cells by regulating CDK2 levels in FOXP3+ Tregs, thereby affecting Treg formation (31). In addition, it has been demonstrated that p21 can competitively bind to CDK2, thereby regulating the G1-S transition of the cell cycle and mediating the cycle block of Tregs. CDK4/6 inhibitors affect Treg cycle block via the Rb-E2F-DNMT1 pathway, inhibiting Treg proliferation, reducing cytotoxic T-lymphocyte depletion and enhancing antitumor immunity (34).

Consequently, CDK4/6 inhibitors can modify the tumor immune microenvironment, namely by enhancing the activation of effector T cells, the capacity to generate immune T-cell memory over time and the inhibition of Tregs proliferation.

NK cells, macrophages and other immune cells

CDK4/6 inhibitors have been demonstrated to enhance antigen presentation. The genes encoding major histocompatibility complex (MHC) class I molecules in cancer cells are upregulated by CDK4/6 inhibitors, thereby strengthening the ability of immune cells to process and present antigens. Inhibition of the CDK-Rb-E2F axis by CDK4/6 inhibitors has been shown to attenuate methylation of endogenous retroviral genes, increase levels of double-stranded RNA and enhance type III interferon (IFN) synthesis. Notably, elevated levels of type III IFN stimulate the expression of IFN-sensitive genes and promote the transcription of downstream genes encoding MHC class I molecules, thereby enhancing the antitumor capacity of immune cells (36). It has been postulated that CDK4/6 inhibitors may exert direct immunomodulatory effects on tumor cells. The number of MHC class I proteins on the surface of tumor cells is increased by genes involved in peptide cleavage and transport. This also enhances the activation of antigen-presenting cells (APCs) and strengthens their ability to deliver antigens (34). Drug-resistant tumors adopt an immunosuppressive microenvironment characterized by elevated MHC II-low macrophages and increased PD-L1 expression on tumor cells. This phenomenon can be partially attributed to low MHC-II levels in macrophages, which result in decreased antigen presentation and T-cell activation (37). CDK4/6 inhibitors have the potential to impede immune evasion by enhancing antigen presentation to resistant tumors, thereby increasing MHC-II levels in macrophages. Concurrently, CDK4/6 inhibitors elevate PD-L1 levels, which counteract the antitumor impact. Consequently, the requirement for concomitant administration of ICIs arises.

The recruitment of NK cells in a senescence-dependent manner has been demonstrated to mediate tumor regression. Cellular senescence is a form of stable cell cycle arrest, and a marked proportion of cancer cells retain the ability to undergo this process. Consequently, the activation of senescence represents a promising new approach to cancer intervention. Palbociclib induces cell cycle arrest and leads to cellular senescence (38). Following dilution or washout from the extracellular medium, palbociclib is released from intracellular acidic vesicles; this reversible drug storage in acidic vesicles is often called lysosomal trapping. It has been demonstrated that brief exposure of cells to palbociclib is sufficient to produce stable cell cycle arrest and long-term senescence (39). NK cells can recognize and eliminate senescent cells that express activating receptors, such as NK cell group 2D and DNAM-1 ligand, by releasing granules containing perforin and granzyme. Combining CDK4/6 inhibitors with a targeted drug (trametinib) and hormone (hepcidin) after endogenous tumor formation has been shown to result in a notable reduction in Rb phosphorylation and tumor cell proliferation, accumulation of SA-β-gal+ senescent tumor cells, induction of senescence-associated secretory phenotype factors (including TNF-α and intercellular cell adhesion molecule-1) and subsequent NK cell recruitment in a KP GEMM mouse model of lung adenocarcinoma (38).

Serum amyloid A1 (SAA1) serves a notable role in inflating tumor cell senescence induced by palbociclib (4). This acute-phase protein is a serum factor that exerts differential effects on the neutrophil phenotype, enhancing the inflammatory response when neutrophils arrive at the site of injury and promoting resolution during the resolution phase of inflammation (40). Senescent cells coordinate inflammatory processes and their resolution, partly by regulating SAA1, through a coordinated program involving the transition of neutrophils from an 'N1' to an 'N2' phenotype (41). Following tissue injury, induced senescence prevents the propagation of damage by preventing the proliferation of damaged cells (42). Furthermore, it causes tissue remodeling by providing large amounts of bioactive factors in the microenvironment. This perspective elucidates the capacity of senescent cells to engage the immune system to facilitate their clearance while promoting the stem cell signature of surrounding cells to enhance tissue repair (43). Palbociclib confers reversible phenotypic senescence on tumor cells, and it induces a senescent phenotype with an inflammatory secretosome that recruits and activates neutrophils by releasing different inflammatory factors, such as IL-8 and SAA1. Meanwhile, activated neutrophils can cause phagocytosis of senescent tumor cells (44).

Among the various immunotherapeutic strategies, macrophage-based therapies have garnered marked interest in tumor immunotherapy, given that macrophages represent a prominent component of tumor-infiltrating immune cells. CDK4/6 inhibitors can induce differentiation, maturation and antitumor activation of macrophages, which can differentiate into the M1 type (antitumor type). This subsequently opens up immune-suppressive signaling pathways, as evidenced by an increase in the expression of PD-L1 and colony-stimulating factor 1 receptor, and a dependence on lipid metabolism (45). Macrophages are also implicated in the clearance of senescent cells. The repolarization of macrophage subtypes from M2 to M1 demonstrates a capacity to reverse tumor immunosuppression and promote antitumor immunity within the tumor microenvironment (46). Additionally, macrophages have a prolonged half-life in the circulation and can migrate specifically to tumors. Consequently, macrophages can be utilized as drug carriers for tumor therapy. It has been demonstrated that the selective loading of the photosensitizer black phosphorus quantum dots and abemaciclib into artificially assembled macrophages can confer immunomodulatory and tumor-killing effects within the microenvironment of malignant peripheral nerve sheath tumors (47).

CDK4/6 inhibitors can enhance the antitumor effect of DCs. However the loss of dendritic cells (DCs) within the tumor microenvironment due to the damage by the tumor represents a notable impediment to antitumor immunity following the administration of CDK4/6 inhibitors and immune checkpoint blockade (ICB). Consequently, restoring the DC compartment through the adoptive transfer of ex vivo-differentiated DCs has been shown to result in potent tumor suppression in CDK4/6 inhibitors- and ICB-treated patients of BC. The addition of DCs can facilitate tumor localization and induction of systemic CD4 T-cell responses in patients treated with the CDK4/6 inhibitors-ICB-DC combination, which is characterized by enrichment of programmed death-1 (PD-1), and activating phenotype-1-negative T helper (Th)1 and Th2 cells. Depletion of CD4 T cells can abrogate the antitumor benefit of the CDK4/6 inhibitors-ICB-DC combination, and growing tumors exhibit an increased proportion of terminally depleted CD8 T cells. Furthermore, the restoration of DC function through the adoptive transfer of exogenous mature bone marrow-derived DCs has been reported to result in an enhanced tumor response to CDK4/6 inhibitors and ICB therapy. These findings provide substantial evidence that the limited efficacy of CDK4/6 inhibitors and ICB combination therapy observed in clinical trials may be attributable to the inhibitory effect of CDK4/6 inhibitors on DCs. Furthermore, DC therapy may be viable for augmenting CDK4/6 inhibitors and immunotherapy responses in clinical settings (48).

Taken together, CDK4/6 inhibitors can modify the tumor immune microenvironment by enhancing the capacity of immune cells, including NK cells and macrophages, to present antigens, induce tumor cell senescence and recruit neutrophils for tumor destruction. Additionally, they can repolarize macrophage subtypes from M2 to M1, thereby augmenting antitumor activity. Additionally, DC therapy has been demonstrated to enhance the efficacy of CDK4/6 inhibitors.

Immune checkpoint modulation

The combination of CDK4/6 inhibitors with ICIs can enhance antitumor immune responses

The administration of a single CDK4/6 inhibitor has been demonstrated to elevate PD-L1 levels, thereby impeding the response of the immune system to tumor cells. The molecular mechanisms underlying this process have been the subject of investigation. It has been reported that the abundance of the PD-L1 protein is regulated by the cyclin D-CDK4 and cullin 3 speckle-type POZ protein (SPOP) E3 ubiquitin ligase via proteasome-mediated degradation (49). In vivo, the inhibition of CDK4/6 has been observed to increase PD-L1 protein levels, mainly by inhibiting cyclin D-CDK4-mediated phosphorylation of SPOP, which in turn promotes SPOP degradation by anaphase-promoting complex/cyclosome bound to the regulatory subunit Cdh1 (49). It has been established that PD-1 is a pivotal immune checkpoint that modulates the threshold of T- and B-cell response to antigens. As a crucial regulatory checkpoint for T cells, PD-1 exerts a central role in governing their cellular function. The interaction between PD-L1 and PD-1 induces T-cell depletion, thereby promoting immune evasion (50). The immunoreceptor tyrosine-based switch motif (ITSM) is an essential site of PD-1 biological function, and PD-1 is phosphorylated by binding to PD-L1 and further induces immunosuppression by activating a series of intracellular pathways. The specific mechanisms by which PD-1 exerts its immunosuppressive effects differ between T and B lymphocytes (51). Two signaling pathways have been identified as being involved in the immune response induced by T cells after pathogen invasion. The first pathway is the binding of MHCs on the surface of APCs to the T-cell receptor (TCR) (52). The second pathway is the binding of immunostimulatory ligands expressed by APCs to the TCR (52). Consequently, this results in the transduction of activating or inhibitory signals to T cells, modulating immune responses such as T-cell activation and exhaustion. The engagement of PD-L1 and PD-1 in T cells leads to the recruitment of Src homology 2-containing tyrosine phosphatase 1/2 (SHP-1/2) to the C-terminus of ITSM. Subsequently, SHP-2 functions by dephosphorylating TCR-associated CD-3ζ and ZAP70, inhibiting downstream signaling. Specifically, the PI3K pathway has been shown to be inhibited, and the expression of the cell survival gene BCL-XL is reduced.

PD-1 has also been shown to impede TCR-induced PI3K/AKT pathway activation by stimulating PTEN. Moreover, PD-1 has been shown to restrict the proliferation of T cells by hindering the activation of the RAS/MEK/ERK pathway. Concurrently, PD-1 suppresses the activation of PKCδ, thereby diminishing the secretion of cytokines, such as IFN-γ and IL-2, by T cells (53). Furthermore, PD-1 signaling orchestrates T-cell metabolism by impeding glycolysis while promoting lipolysis and fatty acid oxidation (54).

The PD-1/PD-L1 interaction has also been demonstrated to inhibit B-cell activation. The binding of PD-L1 to PD-1 results in the phosphorylation of two tyrosines located within the ITSM of PD-L1 by the B-cell receptor. This, in turn, leads to the recruitment of SHP-2 to the C-terminus of PD-1. Subsequently, SHP-2 undergoes phosphorylation. Consequently, the dephosphorylation of SHP-2 by the C-terminus of PD-1 leads to acute Ca2+ disruption and long-term growth arrest. Consequently, the PD-1-mediated suppression of the immune response of B cells to antigens is impaired (55).

Notably, combining CDK4/6 inhibitors with PD-1/PD-L1 inhibitors has been demonstrated to augment the antitumor immune response. The antitumor response has been reported to be further enhanced when anti-PD-1 and anti-CTLA-4 are used in combination with CDK4/6 and PI3K inhibitors, resulting in a more robust antitumor effect in humans. This combination therapy can result in enhanced T-cell activation and a reduction in the number of Tregs, thereby enhancing cancer cell death (4). Schaer et al (56) demonstrated that the combination of abemaciclib and anti-PD-L1 treatment results in the increased expression of genes involved in antigen presentation and the innate immune response, which in turn leads to an increase in the expression of MHC class I and class II molecules in tumor cells. In a BC mouse model, Goel et al (4) demonstrated that the combination of CDK4/6 inhibitors and PD-1/PD-L1 inhibitors directly activates T cells and modulates innate immunity by enhancing antigen presentation. The combination therapy has been demonstrated to present superior efficacy in inhibiting tumor growth compared with abemaciclib monotherapy. Furthermore, combining CDK4/6 inhibitors with anti-PD-1 antibodies can enhance tumor control in most mouse models (34). Preclinical in vivo BC studies have indicated that CDK4/6 inhibitors may enhance tumor sensitivity to PD-1/PD-L1 blockers. Notably, Schaer et al observed that treatment with a CDK4/6 inhibitor followed by the staged use of anti-PD-L1 agents results in more complete remission (31). In conclusion, further investigation into the potential of combining CDK4/6 inhibitors with ICIs to enhance antitumor immune responses is warranted (Fig. 3).

Figure 3 Combining CDK4/6 inhibitors with immune checkpoint (PD-1/PD-L1) inhibitors has been demonstrated to enhance antitumor immune responses. Created with MedPeer (medpeer.cn). As illustrated on the left side of the image, the TCR on the surface of CD8+ T cells recognizes MHC on the tumor cells. Concurrently, PD-1 binds to PD-L1 on the tumor cells, thereby inhibiting T-cell activity. This inhibitory signaling can be blocked with anti-PD-1 drugs. The upper right part of the diagram illustrates the signaling pathways in T cells, including the RAS/MEK/ERK and PI3K/AKT pathways, as well as inhibitory signaling through SHP1 and SHP2 after PD-1 activation. The lower right part of the diagram illustrates the signaling pathways within tumor cells. Notably, PD-L1-related signals have the capacity to influence various critical processes within tumor cells, such as the proliferation of tumor cells and the regulation of other processes. This influence can be mitigated by the intervention of related inhibitors within the signaling pathways. MHC, major histocompatibility complex; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; PTEN, phosphatase and tensin homolog; SHP, Src homology 2-containing tyrosine phosphatase; TCR, T-cell receptor.

The impact of CDK4/6 inhibitors on immune evasion within the tumor microenvironment

PD-1 on immune cells and PD-L1 on tumor cells can facilitate immune escape. The expression of PD-L1 on tumor cells can be promoted by CDK4/6 inhibitors, which may encourage immune evasion in a variety of tumor models (34). CDK4/6 inhibitors have been demonstrated to elevate PD-L1 levels, thereby enhancing the interaction of PD-L1 with the PD-1 receptor on the surface of T cells and inhibiting T-cell activity (57); this allows tumor cells to evade the immune system. Concurrently, CDK4/6 inhibitors also elevate the number of negative immunodetection sites, PD-1 and CTLA-4, on T lymphocytes, thereby exerting an adverse immunomodulatory influence. However, CDK4/6 inhibitors can also induce cell cycle blockade, trigger T cells, enhance antigen presentation, stimulate the immune system, inhibit the growth of Tregs and reduce the secretion of inhibitory cytokines. This results in a decrease in the inhibitory effect on immune cells and an increase in the immune response against tumor cells. Consequently, the impact of CDK4/6 inhibitors on immune evasion within the tumor microenvironment is multifaceted. It is of the utmost importance to exercise caution when developing antitumor drugs that utilize CDK4/6 inhibitors to prevent immune evasion by tumor cells.

Current research progress on CDK4/6 inhibitors

Approved CDK4/6 inhibitors

Given the prevalence of cell cycle gene abnormalities in solid tumors, developing and approving CDK4/6 inhibitors for treating several solid tumors in recent years has been a significant advancement in oncological therapy. This section lists the most used CDK4/6 inhibitors approved for clinical use.

Palbociclib (Ibrance®; Pfizer) was first synthesized in 2001 and was the original CDK4/6-specific inhibitor. In February 2015, the U.S. Food and Drug Administration (FDA) granted accelerated approval of palbociclib for postmenopausal women with advanced or metastatic HR+/HER− MBC as a first-line therapy (58). Palbociclib was approved for use with aromatase inhibitors (AIs) based on PALOMA-1 and PALOMA-2 trial findings (6,59). Palbociclib was also approved in combination with fulvestrant based on the results of the PALOMA-3 trial (60). In 2019, the FDA expanded the use of palbociclib in combination with an AIs or fulvestrant to include men (61). The agent in question has been demonstrated to reduce the proliferation of ER-positive BC cell lines in vitro by blocking Rb phosphorylation, leading to G1 phase arrest (62).

Ribociclib (Kisqali®; Novartis), another oral CDK4/6 small-molecule inhibitor, was approved by the US FDA in March 2017 in combination with AIs for first-line endocrine therapy in postmenopausal patients with HR+/HER− MBC. This is based on the progression-free survival (PFS) and overall survival (OS) benefits demonstrated with ribociclib in the MONALEESA program (63-65). On July 18, 2018, the FDA expanded its indications and approved ribociclib combined with fulvestrant for treating postmenopausal patients with HR+/HER− MBC. On the same day, the FDA also approved ribociclib in combination with AIs as initial endocrine therapy for premenopausal, perimenopausal and postmenopausal patients with HR+/HER2− MBC (66). On September 17, 2024, based on the results of the NATALEE study (67), the FDA approved ribociclib in combination with AIs as adjuvant therapy for HR+/HER2− stage II and III early BC in adults with a high risk of recurrence.

Abemaciclib (Verzenios®; Eli Lilly and Company) was approved by the FDA in September 2017 in combination with fulvestrant as initial endocrine therapy based on the results of the MONARCH-2 trial (68); it is suitable for postmenopausal patients with HR+/HER2− MBC. In February 2018, based on the results of the MONARCH-3 study, the FDA approved abemaciclib for combination therapy with AIs (69). In addition, the MONARCH-1 study showed the benefit of abemaciclib as monotherapy in severely pretreated patients (70). Abemaciclib is the first CDK4/6 inhibitor approved for HR+/HER2− early-stage BC with a high recurrence risk (71). The FDA approved abemaciclib in combination with endocrine therapy (tamoxifen or AIs) in October 2021 as adjuvant therapy for adult patients with HR+/HER2−, node-positive, early-stage BC with a high risk for recurrence and a Ki-67 score ≥20% (72). In March 2023, the FDA expanded the indications for abemaciclib, eliminating the requirement for a Ki-67 score (73).

In three large randomized phase III trials, the CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib were investigated when each was combined with AIs in the first-line treatment of postmenopausal patients with HR+/HER2− MBC (59,65,69). The three CDK4/6 inhibitors resulted in marked increased in PFS compared with AI treatment alone, exhibiting nearly identical hazard ratios for PFS and potentially analogous efficacy. In another three other large randomized phase III trials, PALOMA-3 (palbociclib), MONALEESA-3 (ribociclib) and MONARCH-2 (abemaciclib), the combinations of the three CDK4/6 inhibitors with fulvestrant were investigated in patients with HR+/HER2− MBC as the second line of therapy (63,74,75). In the CDK4/6 inhibitor group, all three drugs showed prolonged PFS and OS benefits compared with fulvestrant alone. The results from pertinent clinical studies are provided in Table SI.

Palbociclib, ribociclib and abemaciclib have been widely used in clinical practice, mostly in combination with endocrine therapy for HR+/HER2− MBC. In addition, extensive comparative studies on the efficacy and safety of these three drugs have been conducted. Desnoyers et al (76) performed a meta-analysis, which showed that the OS and PFS of the three drugs in the treatment of patients with HR+/HER2− MBC are similar, and supplemented the relative safety and tolerability of the three drugs, which may help guide clinicians in the decision-making of drug selection. The results of this previous study showed that ribociclib and abemaciclib are associated with significantly lower rates of grade 3-4 neutropenia, but significantly higher gastrointestinal toxicity when compared with palbociclib. In addition, abemaciclib is less well tolerated, and patients treated with this drug were reported to have a significantly higher incidence of treatment interruptions due to adverse events (AEs) compared with the other two drugs. In 2024, a meta-analysis by Kappel et al (77) also showed similar results. Some additional findings from this study were that palbociclib was shown to be associated with a significantly higher incidence of neutropenia, but a substantially lower risk of grade 3-4 infections than ribociclib and abemaciclib. Cases of grade 3-4 transaminitis and grade 3-4 neutropenia were also reported to be significantly lower with abemaciclib than with ribociclib.

CDK4/6 inhibitors in clinical trials

The preliminary preclinical data indicate that BC, particularly the HR+ form, is highly susceptible to CDK4/6 inhibitors. Consequently, numerous clinical trials have concentrated on this specific cancer type (6,78,79). A substantial body of clinical evidence supports the use of CDK4/6 inhibitors in the treatment of postmenopausal women with advanced or HR+/HER2− MBC, demonstrating a marked prolongation of patient survival (74,78,79). Consequently, the FDA has approved several CDK4/6 inhibitors in clinical settings, including palbociclib, ribociclib and abemaciclib. In recent years, a number of novel CDK4/6 inhibitors have commenced clinical investigation. Table SII provides an overview of the drugs subjected to clinical studies (80-84).

G1T38 is a novel CDK4/6 inhibitor developed by G1 Therapeutics, Inc. G1T38 has been demonstrated to block cells in the G1 phase by reducing Rb phosphorylation and inhibiting cell proliferation in CDK4/6-dependent oncogenic cell lines, including those derived from BC, melanoma and leukemia. Furthermore, G1T38 has been shown to possess selective and oral bioavailability characteristics, with good selectivity. It is currently undergoing phase II clinical trials and may become an effective CDK4/6 inhibitor in the clinic in the future (81).

Narazaciclib (ON123300) is a CDK4/6 inhibitor developed by Onconova Therapeutics, Inc. and is undergoing phase I/II clinical studies. A previous study has shown that it exhibits relatively high antitumor activity in mantle cell lymphoma cell lines (82).

PF-06873600, a CDK2/4/6 inhibitor developed by Pfizer, has been identified as a potential therapeutic agent for cancer treatment and has been subjected to phase I clinical trials for the treatment of patients with HR+/HER2− BC, metastatic triple-negative BC (TNBC) or advanced cisplatin-resistant epithelial ovarian/tubal carcinoma (83).

ETH-155008, developed by Shengke Pharmaceutical (Jiangsu) Ltd., represents a novel dual inhibitor that may enable effective acute myeloid leukemia (AML) therapy through dual targeting of FLT3 and CDK4/6. By further blocking tumor cells at the G1 stage, ETH-155008, through a synergistic effect, to some extent avoids the development of CDK4/6 inhibitor resistance. Notably, it shows a favorable safety and efficacy profile in phase I clinical studies (84).

AMG925 (FLX925), developed by Amgen, has been shown to act on FLT3 and CDK4/6 selectively. It is currently in phase I clinical trials, primarily for the treatment of AML (80).

In addition to new CDK4/6 inhibitors entering clinical studies, various drugs approved for clinical use, including palbociclib, ribociclib and abemaciclib, are also under investigation. In addition to HR+ BC, the clinical studies of these drugs have primarily focused on other solid tumors, including non-small cell lung cancer and prostate cancer. In addition, further in-depth research may be conducted on the therapeutic effect, safety, pharmacokinetics, dosage regimen and other pertinent factors of these drugs. Table SIII provides a comprehensive listing of clinical trials related to CDK4/6 inhibitors, as listed on ClinicalTrials.gov (85-92).

Mechanism of CDK4/6 inhibitors resistance

Resistance to CDK4/6 inhibitors is an almost inevitable biological event. From a therapeutic point of view, it has become urgent to find biomarkers of sensitivity and resistance, to understand the mechanisms of resistance to CDK4/6 inhibitors, and to identify strategies to overcome them according to the target. A trial of patients with HR+/HER2+ MBC treated with a combination of CDK4/6 inhibitors and endocrine therapy, and analyzed by biopsy whole exome sequencing, identified eight different mechanisms of CDK4/6 inhibitor resistance (93): i) Rb1 deletion, ii) AKT1 mutation/amplification, iii) RAS activation, iv) fibroblast growth factor receptor (FGFR) activation, v) HER2 activation, vi) cell cycle protein-E activation, vii) Aurora kinase A (AURKA) amplification and viii) estrogen signaling deletion. These diverse mechanisms (including alterations in cell cycle-specific or non-specific mechanisms) have been observed in two-thirds of resistant tumors examined. These mechanisms can be further subdivided into the following categories: Rb function deficiency, abnormalities in cell cycle regulatory factors, activation of bypass signaling pathways, and dysfunction of other transcription factors and hormones. The identification of these mechanisms has the potential to yield novel therapeutic interventions for patients with these tumors.

Alterations in specific mechanisms of the cell cycle

Rb deletion and E2F amplification

CDK4/6 inhibitors inhibit cell proliferation through a mechanism of action dependent on Rb. Specifically, CDK4/6 inhibitors block CDK4/6 activity and prevent phosphorylation of the Rb protein, which in turn maintains the inhibition of Rb on the E2F transcription factor and prevents the cell cycle from moving from the G1 phase into the S phase (94,95). However, it has been reported that a variety of BC cell lines resistant to palbociclib possess reduced levels of Rb expression compared with cells that had not received the drug (96). This implies that CDK4/6 inhibitors treatment may reduce Rb protein levels while inhibiting proliferation. This phenomenon has been further validated in clinical practice, where up to 10% of patients with disease progression treated with CDK4/6 inhibitors have been shown to possess alterations or deletions in both copies of the Rb1 gene, a feature not seen in patients treated with endocrine therapy only (97). Rb1 loss is a proven resistance mechanism in vitro and in patient-derived tumor xenograft models (24). The level of Rb1 protein decline is further exacerbated by multiple molecular causes (such as mutation, silencing or hyperphosphorylation) (98). Ultimately, the constitutive progression of the cell cycle continues through the activation of other cell cycle mechanisms (such as E2F amplification) and is not driven in a manner that relies on CDK4/6 (99). However, a simple decline in Rb levels is not sufficient to activate E2F transcriptional activity necessary for cell cycle progression, nor is it alone adequate to directly drive cell cycle deregulation (100). In the process of developing CDK4/6 inhibitor resistance, upregulation of mitogenic or hormonal signaling, or activating mutations are also required to stabilize c-Myc levels, which mediate the amplification of E2F transcriptional activity, bypassing the inhibitory effects of CDK4/6 inhibitors (96). Therefore, for this group of patients, restoring their Rb levels or inhibiting c-Myc to reduce E2F activity may be two critical methods to overcome drug resistance.

Abnormalities in cell cycle regulators

Amplification of CDK4/6

Amplification of CDK4 or CDK6 is considered one of the essential mechanisms of CDK4/6 inhibitor resistance, as this amplification can drive cell cycle progression through activation of the cyclin D-CDK4/6-Rb pathway, thereby impairing the effect of CDK4/6 inhibitors on cell cycle arrest (101). CDK4 is widely expressed in various cancer types and CDK4-upregulated cells have reduced sensitivity to CDK4/6 inhibitors. In glioma cells, CDK4 overexpression can even lead to complete resistance to CDK4/6 inhibitors (102). In addition, CDK4 overexpression has been identified as a potential biomarker for predicting resistance to conventional chemotherapy in patients with osteosarcoma (103). Moreover, the overexpression of CDK4 may be attributed to the enhancement of CDK4 activation by the cell-cycle regulator CDK7 through T-loop phosphorylation (104). Coombes et al (105) reported that the CDK7 inhibitor samuraciclib exerts some clinical activity with an objective remission rate of 36% (9/25 patients with HR+/HER2− BC). In a population of patients previously treated with a CDK4/6 inhibitor in combination with fluvastatin, samuraciclib was associated with a median PFS time of 3.7 months (105). These data suggest that samuraciclib may have potential clinical benefit in managing disease progression associated with CDK4/6 inhibitor resistance (105).

It has also been reported that the CDK6 gene locus is de novo amplified in a cellular model of resistance to palbociclib. By contrast, knockdown of CDK6 can restore sensitivity to CDK4/6 inhibitors, suggesting that the level of CDK6 activity also serves a critical role in CDK4/6 inhibitor resistance (106,107). Multiple mechanisms may lead to elevated CDK6 levels. On one hand, mutations in the upstream gene FAT1, and accumulation of YAP and TAZ transcription factors at the CDK6 promoter, lead to Hippo pathway-mediated CDK6 upregulation, which promotes resistance to CDK4/6 inhibition (108). On the other hand, Cornell et al (109) reported that, in BC, increased expression of the microRNA (miR)-432-5p exosome may downregulate SMAD4 to weaken the TGF-β signaling pathway, thereby upregulating the expression level of CDK6. During this process, the development of resistance to CDK4/6 inhibitors is due to the highly expressed CDK6 enhancing the activity of the cyclin D-CDK6 complex, phosphorylating Rb protein, driving the cell cycle process and bypassing the blocking effect of CDK4/6 inhibitors. In addition, miR-432-5p may be delivered by exosomes and can thus diffuse the phenotype of CDK6 upregulation in surrounding cancer cells, which further enhances the drug resistance of the population.

Hyperactivation of the cyclin E-CDK2 complex

The cyclin E-CDK2 complex is another key regulator of the G1/S phase transition and is capable of phosphorylating the Rb protein independently (110). Notably, amplification of cyclin E1 and cyclin E2 leads to increased CDK2 activity, which in turn promotes the degradation of the CDK inhibitor p27Kip1, thereby facilitating cell cycle progression (111). Under treatment with CDK4/6 inhibitors, aberrant activation of cyclin E and of the CDK2 complex downstream of CDK4/6 can bypass the inhibitory effects of CDK4/6, resulting in complete phosphorylation of Rb and the release of E2F, which triggers the subsequent cell entry into S phase (112,113). Herrera-Abreu et al (24) reported that amplification or overexpression of the cyclin E1 gene is significantly associated with palbociclib resistance and that knockdown of cyclin E1 restores cellular sensitivity to this inhibitor. In another correlative analysis of the PALOMA-3 trial (114), 302 patients underwent tumor tissue analysis. The results showed lower efficacy of palbociclib in patients with high vs. low CNE1 mRNA expression (median PFS: palbociclib group, 7.6 vs. 14.1 months; placebo group, 4.0 vs. 4.8 months). In addition, the preoperative palbociclib randomized clinical trial independently validated the role of high cyclin E1 mRNA as a palbociclib resistance marker in regulating Rb phosphorylation and antiproliferative response to palbociclib. Therefore, overexpression of cyclin E is considered one of the potential mechanisms of CDK4/6 inhibitor resistance. Notably, the overexpression of cyclin E was shown to disappear when CDK2 is inhibited, a finding that suggests that the combined CDK2/4/6 inhibition strategy may inhibit cell proliferation through a synergistic effect, thus providing a potential therapeutic tool for the prevention of resistance to CDK4/6 inhibitors (115).

In addition to the cyclin E complex, Cai et al (19) showed that the long noncoding RNA (lncRNA) EILA, which interacts with cyclin E1, is upregulated in CDK4/6 inhibitor-resistant BC cells and promotes CDK4/6 inhibitor resistance by stabilizing cyclin E1. By contrast, silencing EILA reduces cyclin E1 protein and restores CDK4/6 inhibitor sensitivity in vitro and in vivo (19). lncRNAs may thus serve as another potential therapeutic target for overcoming drug resistance.

AURKA amplification

AURKA is a kinase that serves a vital role in cell division, and has been associated with intrinsic and acquired resistance to CDK4/6 inhibitors and endocrine therapies (93). AURKA amplification promotes cell cycle progression directly or indirectly. For example, AURKA can phosphorylate Rb proteins, weakening their inhibitory effect on E2F transcription factors, thereby restoring E2F transcriptional activity and driving the cell cycle from G1 to S phase (116). In addition, AURKA is a key regulatory component of the p53 pathway, particularly in the checkpoint-response pathway necessary for oncogenic transformation through phosphorylation and stabilization of p53, which confers tumor cell resistance to CDK4/6 inhibitors (117). Together, these mechanisms diminish the cell cycle-blocking effects of CDK4/6 inhibitors. AURKA amplification is also highly associated with CDK4/6 inhibitor treatment failure in clinical samples. Wander et al (93) demonstrated that the proportion of AURKA overexpression tends to be higher in patients with disease progression after treatment with CDK4/6 inhibitors than in patients with effective treatment. This finding suggests that AURKA amplification may serve as a potential biomarker of CDK4/6 inhibitor resistance to inform patient stratification and treatment decisions.

Alterations in nonspecific mechanisms of the cell cycle Activation of bypass signaling pathways

Activation of FGFR1

The FGFR family consists of highly conserved transmembrane receptors (FGFR1-4) involved in a number of key mechanisms that lead to cancer, such as proliferation, differentiation and cell survival (118). Post-progression analysis of circulating tumor DNA from 34 patients treated with palbociclib in combination with letrozole has been reported to show alterations in FGFR, including FGFR1 amplification, FGFR2 amplification, and activating FGFR1 and FGFR2 mutations (119). Amplification of FGFR1 is the most common genomic alteration in BC, occurring in ~10% of BC cases (120), and FGFR1 has been identified as a potential mechanism of acquired resistance to endocrine therapy in combination with CDK4/6 inhibitors (121,122). The development of this resistance mechanism may be mediated by FGFR1 amplification, which activates the PI3K/AKT/mTOR and RAS/MEK/ERK signaling pathways (123). Genetic analysis of the MONALEESA-2 study showed that patients with amplification of the FGFR1/ZNF703 fusion gene had a PFS of 10.61 months, compared with 24.84 months for patients with wild-type FGFR1/ZNF703. In addition, patients with high FGFR1 mRNA expression (50% of the median mRNA expression) had a median PFS time of 22.21 months, whereas patients with low FGFR1 mRNA expression did not reach a median PFS after 32 months of follow-up (65). These results indicate that FGFR1 amplification and high expression may be predictors for a poor prognosis in patients, and CDK4/6 inhibitors combined with blocking the FGFR pathway may be a potential strategy used to overcome drug resistance and improve the prognosis of patients.

Aberrant PI3K/AKT/mTOR signaling

PI3K/AKT/mTOR is hyperactivated or altered in numerous types of cancer and regulates a wide range of cellular processes, including proliferation, growth, metabolism, angiogenesis and metastasis (124). In BC, inhibition of CDK4/6 promotes the adaptive rewiring of the PI3K/AKT/mTOR signaling pathway in BC cells, which enhances their dependence on this axis, promoting phosphorylation and activity of the PI3K/AKT/mTOR pathway (101). Notably, overactivation of the PI3K/AKT/mTOR pathway induces cyclin D1 and CDK4 protein upregulation, whereas direct inhibition of the signaling pathway silences cyclin D1 and CDK4, and leads to cell cycle arrest. These findings confirm that activation of the PI3K/AKT/mTOR pathway and resistance to CDK4/6 inhibitors may be interrelated (125). On the other hand, elevated levels of phosphorylated AKT have been associated with prolonged expression of the cyclin E2-CDK2 complex of cell cycle proteins, which suggests that tumor cells can bypass the cyclin D-CDK4/6-dependent mechanism of cell cycle regulation via an alternative pathway to maintain cell cycle progression (24).

Abnormal MAPK signaling pathway

The MAPK signaling pathway, or RAS/MEK/ERK pathway, is a signaling pathway downstream of FGFR1. Activation of the RAS family of oncogenes (KRAS, HRAS, NRAS) has been observed in tumor biopsies collected from patients with HR+ BC resistant to CDK4/6 inhibitors (93). Most patients with KRAS-mutant malignancies exhibit elevated levels of expression of cyclin D1 (126). Patients with high levels of cyclin D1 protein expression tend to have a worse prognosis than those with low expression levels (median OS 41.7 vs. 3.5 months) (127). This may be explained by the fact that KRAS produces aberrant growth signals in the presence of CDK4/6 inhibitors, leading to cellular resistance. In addition, KRAS and RAF kinases regulate ERK1/2 via MEK. ERK1/2 is also highly represented in the oncogenic profile of patient RNAseq data related to the occurrence and development of cancer (128). Based on this, there have been several clinically tested pharmacological agents that target the MAPK pathway. For example, the FDA has approved MEK inhibitors for certain types of cancer (such as melanoma) (128). In BC, the MEK1/2 inhibitor selumetinib, in combination with fulvestrant and palbociclib, can effectively inhibit the proliferation of BC cells resistant to CDK4/6 inhibitors (119). These trials have shown an association between acquired resistance to CDK4/6 inhibitors and induced MAPK pathway activity, indicating that selecting the correct target for targeted therapy is an effective measure to address resistance.

Abnormal expression of other cytokines and hormones

In preclinical models, acquired CDK6 amplification not only confers abemaciclib resistance by bypassing the activation of alternative cell cycle checkpoints, but also leads to a notable reduction in the mRNA and protein levels of ER and PR (106,111). Furthermore, it can suppress the expression of ER-regulated genes, such as XBP1 and MYC (106). Reduced ER/PR levels impair estrogen-dependent induction of cyclinD-CDK4/6 complex activity (129). Notably, the absence of ER/PR expression is closely associated with acquired insensitivity to hormonal agents, such as the ER antagonists fulvestrant and tamoxifen (130), suggesting that the dual mechanisms of drug resistance involve cell cycle dysregulation and endocrine therapy escape.

The AP-1 family is a key transcription factor for cyclin D1, and includes Jun, Fos, and their related subfamilies of activating transcription factors (ATF2, LRF1/ATF3 and B-ATF) (131). In 20-40% of human patients with BC, c-Jun exhibits high levels of activation. It has been reported that c-Jun activation inhibits ER activity and upregulates cyclin D1 transcription, thereby inducing resistance to CDK4/6 inhibitors (132). To the best of our knowledge, the efficacy of AP-1 inhibitors in patients with CDK4/6 inhibitor-resistant BC has not yet been thoroughly investigated. However, the results of this exploration are of great clinical value.

MYC is a widespread oncogenic transcription factor that promotes cancer cell progression (133). MYC-acquired mutations have been identified in 8.8 and 20.1% of patients with BC in phase III MONARCH-3 (NCT02246621) and phase II Next MONARCH-1 clinical trials (NCT02747004) respectively (134,135). The Next MONARCH-1 clinical trial has shown that acquired MYC genomic aberrations were enhanced after abemaciclib monotherapy or combination therapy with a nonsteroidal AI in patients with HR+ BC (136). In addition, Pandey et al (137) has validated the upregulation of MYC in palbociclib-resistant cells by reverse transcription-quantitative PCR, suggesting that the MYC upregulation may be another marker of acquired resistance to CDK4/6 inhibitors.

Despite the diversity of drug resistance mechanisms, there are some commonalities and cross-regulatory relationships among different drug resistance mechanisms. The commonality of such a variety of drug resistance mechanisms lies in the imbalance of the cell cycle regulation system. Whether through loss of Rb function, amplification of CDK4/6 or activation of the cyclin E-CDK2 complex, this ultimately leads to the bypass of the G1-phase blocking signal. This co-imbalance makes the cells no longer dependent on the CDK4/6-driven pathway, thus reducing the effectiveness of CDK4/6 inhibitors (138). In addition, FGFR1 activation and the abnormal transduction of upstream growth factor signaling pathways (such as PI3K/AKT/mTOR and RAS/MAPK) affected by FGFR1 can not only directly compensate for the loss of cell cycle regulation, but also often interact with the regulation of intracellular cyclin expression. This means that even when CDK4/6 is inhibited, these alternative pathways can activate downstream cell proliferation mechanisms (139). Part of the resistance mechanism, while largely attributable to genetic mutations (such as loss of Rb), is often also accompanied by extensive epigenetic regulatory changes, such as the regulation of AP-1 and MYC transcription factor activity. This interaction at the genetic and epigenetic levels further underlies the commonality between drug resistance mechanisms (140). These commonalities also suggest that drug resistance can be predicted in a timely manner by monitoring Rb status, cyclin E-CDK2 activity and related signaling pathway changes in the early stage, so as to provide personalized treatment. Combined treatment of CDK4/6 inhibitors with endocrine therapy, chemotherapy and targeted therapy can simultaneously target multiple pathways and may effectively overcome the problem of drug resistance.

Combination therapy with CDK4/6 inhibitors

Targeting CDK4/6 is under active research in cancer therapy as a promising cancer treatment. The present study previously reviewed the resistance to CDK4/6 inhibitors in cancer therapy, and addressing the resistance to CDK4/6 inhibitors has profound implications for realizing their potential in cancer therapy. Effective combination therapies involving CDK4/6 inhibitors have been shown to delay resistance while reducing the side effects of other treatments, leading to favorable antitumor effects (138). Despite the current lack of effective remedies for low Rb1 expression, which is the most fundamental cause of resistance, several drug combination strategies have shown the potential to improve the drug sensitivity of CDK4/6 inhibitors (141). Drug combination strategies involving CDK4/6 inhibitors may include endocrine therapy, chemotherapy, targeted therapy and immunotherapy.

CDK4/6 inhibitors in combination with endocrine therapy

Endocrine therapy is one of the main treatments for HR+ BC, and developing novel endocrine therapies in combination with CDK4/6 inhibitors offers new hope for patients with BC and other types of cancer (142). As aforementioned, palbociclib, ribociclib and abemaciclib were approved by the FDA for HR+/HER− MBC based on multiple clinical studies combined with AIs or fulvestrant. In 2017, the PALOMA-2 clinical trial (NCT01740427) by Finn et al (143) showed that the combination of letrozole and a CDK4/6 inhibitor (PFS, 27.6 months) prolonged PFS by ~13.1 months compared with letrozole alone (PFS, 14.5 months). For selective ER degraders (SERDs), a clinical trial in advanced BC (NCT01942135) showed that the combination of fulvestrant and a CDK4/6 inhibitor (PFS, 34.9 months) prolonged PFS by 5.2 months compared with fulvestrant alone (PFS, 29.7 months) (75). Another phase II clinical study, MAINTAIN (NCT02632045), had similar results (144). The phase III EMERALD trial (NCT03778931) by Bidard et al (145) and the phase II SERENA-2 trial by (NCT04214288) Oliveira et al (146) showed that SERD management has a PFS benefit. On January 27, 2023, the next-generation SERD elacestrant was approved by the FDA for patients with metastatic HR+ BC and ESR1 mutations (147). Notably, two ongoing clinical trials are testing the selective androgen receptor modulator enobosarm alone (NCT04869943) and in combination with abemaciclib (NCT05065411) in patients with ER+/HER2− MBC (138).

Currently, most international guidelines recommend first-line CDK4/6 inhibitors for patients with newly diagnosed HR+/HER2− MBC (148). However, the optimal timing of CDK4/6 inhibitors has not been fully elucidated. The randomized phase III SONIA trial (NCT03425838), conducted in the Netherlands, randomized 1,050 patients who had not previously received treatment for advanced BC to receive a CDK4/6 inhibitor in either the first-line or second-line setting. This study aimed to address whether delaying CDK4/6 inhibitors to second-line therapy after initial endocrine therapy monotherapy affects efficacy, and also assessed safety, quality of life and cost-effectiveness (149). The study aimed to assess whether all patients should start treatment with CDK4/6 inhibitors. The findings indicated that more research is required to identify patients who truly benefit from first-line CDK4/6 inhibitor therapy and those who do better when deferred to second-line treatment. More recently, Sonke et al (150) found no statistically significant benefit of using CDK4/6 inhibitors as first-line therapy compared with second-line therapy (median 31.0 and 26.8 months, respectively; hazard ratio, 0.87; 95% confidence interval, 0.74-1.03; P=0.10). Health-related quality of life was also similar in the two groups. In addition, first-line CDK4/6 inhibitor use was associated with a longer duration of CDK4/6 inhibitor treatment and more grade ≥3 AEs than second-line use. This study challenges past perceptions of the need for first-line use of CDK4/6 inhibitors and points to the need for robust sequencing trial data to determine the best use of available therapies.

Combination chemotherapy

For patients with HR+/HER2− BC resistant to endocrine therapy in combination with CDK4/6 inhibitors, the application of combination chemotherapy with CDK4/6 inhibitors may be considered (151). Recently, in a phase III TROPiCS-02 trial (NCT03901339) conducted by Rugo et al (152), patients with ER+/HER2− BC were evaluated for the efficacy of chemotherapy after CDK4/6 inhibitor resistance. Chemotherapy was indicated to be a viable treatment option after these patients demonstrated resistance to CDK4/6 inhibitors combined with endocrine therapy.

The commonly used chemotherapeutic agent paclitaxel regulates microtubule proteins during cell cycle division, while CDK4/6 inhibitors prevent phosphorylation of Rb1 during the G0 phase of the cell cycle (153). The combination of paclitaxel and CDK4/6 inhibitors can co-regulate the cell cycle and has been shown to reduce the side effects of paclitaxel treatment, such as hematopoietic stem cell death, and to improve its impact on TNBC cells (154). Another study has shown that CDK4/6 inhibitors and paclitaxel synergistically work together by inducing apoptosis in KRAS-mutant lung adenocarcinoma cells (155).

Platinum compounds, another widely used chemotherapeutic agent for cancer treatment, often have side effects such as acute kidney injury (AKI) when applied alone. Kim et al (156) demonstrated that CDK4/6 inhibitors can alleviate platinum-associated AKI in an Rb1-dependent manner, but the exact mechanism is unclear. Liu et al (157) reported that CDK4/6 inhibitors can inhibit cell proliferation and induce apoptosis to reverse the acquired resistance to platinum compounds in lung cancer cells. The combination of the two in therapy provides a new therapeutic strategy for patients with platinum compound-resistant lung cancer.

Five studies have reported the efficacy of CDK4/6 inhibitors in neoadjuvant therapy, NeoPalAna (158), New MONARCH (74), N007 (159), CORALLEEN (160) and NeoPAL (161). Studies such as NeoPalAna, New MONARCH and N007 mainly focused on the evaluation of the biological effects of CDK4/6 inhibitors combined with endocrine therapy on tumors, such as the reduction of Ki-67. These three studies indicated that CDK4/6 inhibitors such as palbociclib and abemaciclib combined with endocrine therapy can significantly reduce Ki-67 levels and inhibit tumor proliferation, showing a good biological response, and are potentially effective options for neoadjuvant treatment of HR+/HER2− BC. Although the treatments evaluated in these studies (74,158,159) were not directly compared with chemotherapy, they laid the groundwork for subsequent comparative trials, such as CORALLEEN (160) and NeoPAL (161), which directly assessed CDK4/6 inhibitors plus endocrine therapy vs. standard chemotherapy. The latter two trials randomly assigned patients to receive neoadjuvant endocrine therapy plus a CDK4/6 inhibitor or neoadjuvant chemotherapy to investigate whether the addition of a CDK4/6 inhibitor to letrozole would provide at least as much clinical benefit as standard chemotherapy. Overall, these two studies showed that in selected patients with HR+/HER2− BC, neoadjuvant therapy with a CDK4/6 inhibitor plus letrozole may achieve some equivalent efficacy to conventional chemotherapy, but gaps remain in endpoints such as the rate of pathological complete response (160,161).

Combination ICIs

Increased PD-L1 expression in patients treated with CDK4/6 inhibitors is a clinical problem currently being encountered. It may be one of the potential mechanisms leading to resistance to CDK4/6 inhibitors through evasion of immune-surveillance checkpoints (162).

CDK4/6 inhibitors can be used in combination with ICIs to transform immunologically 'cold' tumors into 'hot' tumors. 'Hot' tumors are more susceptible to immune cell infiltration and are more easy to treat (163). Results from preclinical studies have suggested that ICIs can be a therapeutic strategy to overcome CDK4/6 resistance in BC (164,165). Meanwhile, ICI resistance is associated with cyclin D1 amplification and CDKN2A deletion, which can also be overcome by CDK4/6 inhibition (166-168). Schaer et al (56) and Jerby-Arnon et al (169) observed synergistic effects exhibited by sequential administration of CDK4/6 inhibitors and ICIs in allogeneic tumor models. Schaer et al (56) showed that continuous administration of abemaciclib followed by ICIs, divided into 28 days of treatment, resulted in improved antitumor activity than 14-day treatment or monotherapy. Jerby-Arnon et al (169) reported that, as compared with other treatment approaches (vehicle, monotherapy, ICI + abemaciclib Q3D, abemaciclib QD followed by ICI), staged combination therapy (ICIs followed by ICIs plus abemaciclib) resulted in higher rates of tumor suppression and survival in melanoma models.

Consistent with most preclinical data, combination therapy with CDK4/6 inhibitors and ICIs have shown potential in patients with tumors. Rugo et al (170), in a phase Ib study of pembrolizumab combined with abemaciclib, found that this combination showed good potential in preliminary clinical efficacy and safety, although further validation is still needed. In an early clinical study involving patients with metastatic disease, Yuan et al (171) revealed that palbociclib in combination with pembrolizumab showed a complete remission rate of up to 31% in patients, with median PFS and OS durations of 25.2 and 36.9 months, respectively. Additionally, a previous case report demonstrated that a patient with refractory SMARCA4-deficient small cell carcinoma of the ovary hypercalcemic type experienced disease progression despite multiple treatment regimens, including immunotherapy. Still, the combination of a CDK4/6 inhibitor and ICIs demonstrated efficacy (172). The combination of a CDK4/6 inhibitor and ICIs has also been reported to be effective in treating SMARCA4-deficient cancers and has demonstrated potential (173). Bose et al (174) similarly reported a case in which a patient who resisted an ICI alone developed a profound and long-lasting response to combination therapy with a CDK4/6 inhibitor and an ICI.

Because combination therapies with CDK4/6 inhibitors and ICIs have been validated in preclinical studies in overcoming drug resistance and have shown potential in cancer treatment, a number of clinical trials evaluating the safety and efficacy of CDK4/6 inhibitors in combination with ICIs for the treatment of cancer are ongoing (Table SIV) (170,171,175-180). The PACE trial, a multicenter, randomized, open-label phase II trial prospectively evaluated the efficacy of palbociclib in combination with fluvastatin and nivolumab in patients with ER+/HER2− MBC after progression following treatment with CDK4/6 inhibitor and AI combination therapy. The results demonstrated that combination therapy with the PD-L1 antibody avelumab and CDK4/6 inhibitors significantly improved PFS (176).

However, combination therapy with CDK4/6 inhibitors and ICIs still has some limitations. Noticeable toxic reactions, especially hematological toxicity such as neutropenia, have become a hindrance limiting the potential of this therapy (171,181,182). Rugo et al (170) investigated the safety and efficacy of abemaciclib plus pembrolizumab with/without anastrozole in patients with HR+/HER− MBC and reported a spectrum of toxicities with the combination therapy, including an increased incidence and severity of hematological toxicities (such as neutropenia) and immune-related AEs (such as hepatotoxicity and pneumonia). Preliminary reports have indicated that the toxicity-related symptom burden, including but not limited to diarrhea and fatigue, results in a reduction of physical activity scores in the short term. However, these symptoms can be managed effectively, thereby restoring quality of life to a certain extent. Another phase II clinical study reported that the combination of nivolumab and abemaciclib, which has demonstrated greater clinical activity compared with other CDK4/6 inhibitors, resulted in severe and prolonged immune-associated AEs (183). A phase I clinical study by Patnaik et al (180) combined LY3300054, a PD-L1 inhibitor, with multiple targeted agents, including abemaciclib. The study reported the impact of hematological toxicity on the long-term prognosis of patients, with some patients discontinuing treatment as a result. Combination therapies with CDK4/6 inhibitors and ICIs currently present both risks and opportunities. If toxicity can be effectively managed, combination therapies have the potential to improve outcomes. However, toxicity management strategies for this combination therapy remain understudied and require further clinical investigation.

It is also worth noting that a previous study (180) revealed that LY3300054 was well tolerated by patients when taken concomitantly with abemaciclib. However, the introduction of abemaciclib prior to combining LY3300054 resulted in hepatotoxicity. These findings suggest that the optimal timing of the combination of CDK4/6 inhibitors and immunotherapy should be explored to maximize efficacy and reduce toxicity. However, to the best of our knowledge, there are currently no studies suggesting the optimal timing of drugs in this combination therapy, and further future clinical trials evaluating this combination therapy are needed to determine the optimal timing and dosage of administration.

Combination targeted therapy

Targeted therapies target specific cancer sites (such as genes or protein molecules in tumor cells), thereby interfering with tumor metastasis, inhibiting tumor cell proliferation and differentiation, and inducing apoptosis of tumor cells (184).

CDK4/6 inhibitors and drugs targeting the PI3K/AKT/mTOR pathway

Extensive preclinical studies have demonstrated that cyclin D1 acts as a common node to achieve crosstalk between CDK4/6 and the PI3K/AKT/mTOR pathway (182). The binding of cyclin D1 to CDK4/6 induces phosphorylation of Rb and its subsequent uncoupling from E2F, which contributes to the progression of the cell cycle in the G1-S phase (181). Aberrant PI3K/AKT/mTOR pathway activation contributes to CDK4/6 inhibitor resistance (141). Clinical trials have examined the effects of combining PI3K inhibitors and CDK4/6 inhibitors in cancer therapy (Table SV) (185-187). Notably, one trial showed that the triple combination of the PIK3CA-isoform-specific inhibitor alpelisib or the pan-PI3K inhibitor buparlisib with the CDK4/6 inhibitor ribociclib and fulvestrant had high toxicity (182). Another phase Ib trial tested a triple combination therapy consisting of the CDK4/6 inhibitor palbociclib, the PIK3CA-isoform-specific inhibitor taselisib, and fulvestrant, which showed promising efficacy in ER+/HER2− advanced BC with PIK3CA mutations and demonstrated tolerability at pharmacologically active doses (188).

AKT acts as a bridge between the PI3K and mTOR signaling pathways. The TAKTIC study showed that AKT inhibitor therapy in combination with CDK4/6 inhibitors and endocrine therapy was efficacious in some patients and showed good tolerability (189). Table SV includes information three ongoing clinical trials on AKT inhibitors (NCT04862663, NCT04920708 and NCT03959891). Whether AKT inhibitors can be one of the therapeutic options after CDK4/6 inhibitor resistance still awaits validation in clinical trials.

The mTOR pathway is an important signaling pathway downstream of PI3K, and thus, mTOR inhibitors have received as much attention as PI3K inhibitors (190). Everolimus is a typical mTOR inhibitor that has shown promising therapeutic efficacy in the phase III, double-masked, randomized international BOLERO-2 trial (median PFS extension of 4.6 months; P<0.0001) (191). For patients resistant to CDK4/6 inhibitors, mTOR inhibitors may provide satisfactory results.

CDK4/6 inhibitors and poly ADP-ribose polymerase (PARP) inhibitors

PARP is a key enzyme involved in the repair of single-stranded DNA gaps. Preclinical studies have shown that the combination of PARP inhibitors, such as olaparib, and CDK4/6 inhibitors can synergistically inhibit the Rb1-E2F1 signaling pathway at both the transcriptional and post-translational levels, leading to cell cycle arrest and downregulation of the E2F1 gene target, ultimately achieving inhibition of tumor cell proliferation, induction of apoptosis and blocking of neuroendocrine differentiation (192-194). Zhu et al (195) demonstrated that overexpression of β-catenin, especially hyperphosphorylation of its Ser675 site, activates the Wnt signaling pathway. This process mediates olaparib resistance, which palbociclib significantly attenuates. Currently, several PARP inhibitors, such as olaparib, have been approved by the FDA for cancer treatment, and the potential of combination therapies of PARP inhibitors and CDK4/6 inhibitors in treating cancers such as TNBC has been reported (196).

CDK4/6 inhibitors and other targeted agents

Histone deacetylase (HDAC) inhibitors are also a targeted agent that has received attention. A phase III clinical trial (ACE; NCT02482753) evaluated the efficacy and safety of chidamide combined with exemestane in the treatment of postmenopausal patients with HR+ advanced BC. The results showed that the modified PFS time was longer with chidamide combined with exemestane (7.4 months) than with placebo combined with exemestane (3.8 months) (197). The San Antonio Breast Cancer Symposium meeting in 2021 reported that entinostat, another HDAC inhibitor, was able to improve the PFS time in patients with HR+/HER2− BC (198). HDAC inhibitors may therefore offer new options for combination therapy with CDK4/6 inhibitors and targeted agents.

BCL2 inhibitors are another option for combination therapy with CDK4/6 inhibitors. An early phase I clinical trial demonstrated that venetoclax, a BCL2 inhibitor, was highly tolerated in combination with tamoxifen, and the high level of BCL2 expression in ~70% of patients with ER+ tumors led to encouraging activity in both ER+ and BCL2+ metastatic BC (199). However, a phase II clinical trial (Veronica; NCT03584009) showed that venetoclax did not significantly improve PFS in patients who progressed after treatment with CDK4/6 inhibitors (200). There is also an ongoing clinical trial (NCT03900884) designed to evaluate the efficacy, safety and tolerability of a BCL2 inhibitor (venetoclax) in combination with fulvestrant in patients with ER+/HER2− MBC. More clinical studies are still needed regarding the therapeutic efficacy of BCL2 inhibitors in combination with endocrine therapy and CDK4/6 inhibitors.

Table SV provides information on combination therapy with CDK4/6 inhibitors and targeted agents. However, combining CDK4/6 inhibitors and targeted agents still has some limitations (201). The optimal timing of including these agents in the therapeutic continuum remains unclear (for example, as part of first-line therapy or at disease progression). As with immunotherapeutic combination therapies, there are still no studies that propose the optimal practical application of drugs in such combination therapies. Further clinical trials evaluating such combination therapies are needed to determine the timing and dosage of administration of the specific therapy in question.

Combination radiation therapy (RT)

RT is one of the most widely used local therapeutic options, with palliative RT and ablation commonly used for advanced BC (202). Numerous preclinical studies have shown that CDK4/6 inhibitors can enhance the radiosensitivity of human cancer cell lines by inhibiting the repair mechanism of double DNA scaffold breaks, enhancing apoptosis and blocking cell cycle progression (203-207). This implies that the combination of CDK4/6 inhibitors with RT may increase the antitumor effect of RT but provides a theoretical basis for clinical application.

The NATALEE study (NCT03701334) was a phase III trial that demonstrated the benefit of this combination in patients with stage II and stage III HR+/HER2− early BC at risk of recurrence, including patients without lymph node involvement (208). Currently published data on the feasibility of concomitant RT and CDK4/6 inhibitors are based on small-scale retrospective series with low evidence and a lack of prospective clinical data (209). There are currently several ongoing clinical trials evaluating the efficacy of combination therapy with CDK4/6 inhibitors and RT in BC (NCT03691493, NCT03870919, NCT03750396, NCT04563507, NCT05664893 and NCT04923542), almost all of which are in phase I or II. Of these, the prospective phase II ASPIRE trial (NCT03691493) is evaluating RT in combination with palbociclib and hormonal therapy in patients with BC who have developed bone metastases. The PALATINE prospective trial (NCT03870919) will evaluate localized breast RT in combination with CDK4/6 inhibitors in advanced BC.

There is a need to characterize the safety issues of combining CDK4/6 inhibitors and RT, and to help guide therapeutic decisions. A systematic evaluation and meta-analysis has previously been conducted, encompassing 15 studies with a total of 1,133 patients diagnosed with HR+/HER2− BC, 617 of whom received CDK4/6 inhibitors in conjunction with RT. The results indicated a 29.4% incidence of severe hematological toxicity, a 2.8% incidence of non-hematological toxicity, a 24.0% CDK4/6 inhibitor dose reduction rate and a 2.3% discontinuation rate. These findings are analogous to the toxicity rates observed when CDK4/6 inhibitors are used as monotherapy, suggesting that combination therapy does not appear to markedly augment toxicity. However, it is important to note that the majority of the data were derived from retrospective studies, which are subject to certain limitations (202). Another study analyzed 373 patients with MBC who received CDK4/6 inhibitors and RT, and found similar results: The overall toxicity of this combination therapy was shown to be limited, and it may be safe and feasible (210). An observational study revealed that the combination therapy led to an elevated occurrence of grade ≥3 AEs up to 6 weeks following RT. However, it exhibited a higher rate of local control, indicating that the combination therapy achieves a balance between efficacy and toxicity (211). Conversely, a multicenter cohort study observed frequent moderate toxicity reactions to RT with CDK4/6 inhibitors, thereby underscoring the necessity for caution regarding this combination therapy (212).

However, the preclinical rationale suggesting that combined RT and CDK4/6 inhibitors may be unsafe is derived from the cytostatic and immunomodulatory effects reported in studies testing combination therapy in in vitro and in vivo models (209). Selective inhibition of CDK4/6 may exert its effect on the cell cycle by interfering with the transition from G1 to S phase, decreasing the level of phosphorylation of Rb proteins, and inducing G1-phase cell cycle arrest. Irradiated normal cells may exhibit delayed progression through the G1, S and G2 phases. Cells demonstrate robust radiation tolerance in the G0, early G1 and late S phases of the cell cycle, while exhibiting heightened sensitivity in the late G1, G2 and M phases. The combination of CDK4/6 inhibitors and RT has been shown to induce a higher proportion of cells to remain in the G2/M phase of the cell cycle (1,32,210,213,214).

Preclinical rationale and clinical data on the potential synergistic toxicity of RT and CDK4/6 inhibitor are limited and conflicting, and data on the feasibility are based on low-level evidence from small retrospective series and are somewhat heterogeneous; therefore, caution is recommended in the application of this combination therapy (215).

Potential of combined chimeric antigen receptor (CAR)-T cell therapy

CAR-T cell therapy is a novel and personalized immunotherapeutic approach that has shown promising results in treating hematological malignancies (216). However, the application of this approach in solid tumors has been limited by a variety of factors that may be related to antigenic escape and heterogeneity, limited CAR-T cell transport and infiltration, T-cell depletion and lack of persistence, associated with the immunosuppressive microenvironment in solid tumors (217). Combination therapies may be able to address these limitations. A preclinical study by Lelliot et al (218) has shown that inhibition of CDK4/6 activity improves the self-renewal and viability of CD4+ and CD8+ CAR-T memory stem cells, which can enhance the durability and therapeutic efficacy of CAR-T cell therapies. This provides novel insight into the field of CDK4/6 inhibitor combination therapies. This idea is still in preclinical development, and clinical trials combining CDK4/6 inhibitors and CAR-T cell therapy for cancer treatment are lacking.

Safety and tolerability of CDK4/6 inhibitors in combination therapy

With the increasing use of CDK4/6 inhibitors in combination with endocrine therapy, immunotherapy and targeted therapies, clinicians must be aware of associated AEs and their management. Drug-induced AEs often lead to dose reduction and discontinuation, which may affect their efficacy. The safety profile of some of the FDA-approved CDK4/6 inhibitors has been described in the present review.

Nausea, vomiting, fatigue, and cytopenia are common AEs of all CDK4/6 inhibitors (73,79,219,220). Neutropenia has been reported to be a particularly significant AE in the cases of palbociclib and ribociclib. In the PALOMA-2 trial, neutropenia was identified as the most prevalent grade 3/4 AE associated with palbociclib in combination with letrozole for the treatment of advanced BC (59). The MONALEESA-2 study revealed a substantial increase in neutropenia in the treatment of HR+/HER− advanced BC with ribociclib in combination with letrozole, with grade 3/4 AEs most frequently characterized by neutropenia (65). The most prevalent AE associated with the administration of abemaciclib was diarrhea, as documented in the MONARCH 2 trial. In this trial, abemaciclib was administered in combination with fulvestrant to patients diagnosed with HR+/HER− MBC (68). Additionally, QTc prolongation and hepatobiliary toxicity were identified as significant AEs associated with ribociclib, as reported in the MONALEESA-2 trial (221) of ribociclib in combination with letrozole for the treatment of patients with HR+/HER2− MBC, and in the MONALEESA-7 trial (219), respectively. Ribociclib in combination with endocrine therapy has been evaluated in premenopausal women with HR+ advanced BC. Although the rate of diarrhea was reported to be high among patients receiving abemaciclib, it was usually managed with antidiarrheal medications or dose reductions without discontinuation (222). Abemaciclib has also been reported to be associated with a higher incidence of venous thromboembolism events and a reversible increase in serum creatinine levels (68,71). Hematological AEs are common with CDK4/6 inhibitors, especially in response to combination therapy. CDK6 is particularly important in promoting the proliferation of hematologic precursors, and its inhibition results in decreased production of hematopoietic cells (59,223). Unlike chemotherapy-induced neutropenia, hematological AEs, such as neutropenia, caused by CDK4/6 inhibitors can be rapidly reversed, reflecting cytostatic effects on bone marrow neutrophil precursors (224). Most hematological abnormalities caused by CDK4/6 inhibitors can be adequately managed with standard supportive care and dose modifications (68). Currently, clinical management strategies for the treatment of potential toxicities associated with CDK4/6 inhibitors have been suggested. However, more research is needed to better understand the toxicity profile, and to develop management strategies to minimize drug interruptions and thus optimize the highest treatment effect for patients with BC (223).

Discussion

CDK4/6 inhibitors are cell cycle-targeting drugs designed to prevent tumor cell proliferation while restoring normal cell cycle regulation (225). Guidelines have recommended several CDK4/6 inhibitors as first- or second-line drugs for various tumors. By exploring the clinical application of the aforementioned CDK4/6 inhibitors, it may be concluded that CDK4/6 inhibitors have notable potential in tumor immunotherapy. This is particularly evident when combined with endocrine therapy, chemotherapy, ICIs, targeted therapy, RT and CAR T-cell therapy, as well as other drug combinations, where the combination of related treatments can notably enhance therapeutic efficacy.

However, CDK4/6 inhibitor resistance remains a marked challenge in clinical practice, with several potential resistance mechanisms (138). To address this critical challenge, future research should assess the full scope of the resistance mechanisms mediated by CDK4/6 inhibitors and the potential mechanisms of their synergistic effects with various other therapies (such as endocrine therapy). In addition, treatment options should be sought based on current advances and the resistance mechanisms of CDK4/6 inhibitors in combination with endocrine therapy should be determined through appropriate clinical validation.

Protein degraders can increase selectivity, improve efficacy and overcome drug resistance; they may also provide a solution to the problem of CDK4/6 inhibitor resistance, which limits the use of these inhibitors (226). The degradation products of CDK can induce the ubiquitination and proteasomal degradation of CDK, thereby reducing the expression levels and stability of CDKs and achieving the purpose of CDK inhibition. Currently, the results of preclinical studies have shown that in ER+/HER2− BC models, compared with clinically approved CDK4/6 inhibitors, CDK4/6 degraders exhibit excellent single-agent antitumor activity both in vitro and in vivo (227). However, clinical trials using CDK4/6 degraders to overcome CDK4/6 inhibitor resistance are at an early stage. ARV-471 is a selective, orally available proteolysis-targeting chimera protein degrader targeting wild-type and mutant ER. In the phase I/II VERITAC trial (NCT04072952), ARV-471 demonstrated clinical activity in patients with advanced HR+/HER2− BC who had received prior treatment with a CDK4/6 inhibitor (228). In addition, the phase III VERITAC-2 trial (NCT05654623) is ongoing to compare the efficacy of ARV-471 with fulvestrant in patients who have failed treatment with a CDK4/6 inhibitor (229). CDK4/6 degraders show potential clinical value in overcoming drug resistance, but their clinical application still requires more research and trials to verify their safety and efficacy.

The development and testing of novel CDK4/6 inhibitors are also key research directions in the future. Given that the activation of CDK2 represents a common mechanism of resistance to CDK4/6 inhibitors, the development of compounds that inhibit CDK4/6 and CDK2 may prevent or delay the occurrence of resistance (13). Furthermore, a novel CDK4/6 inhibitor compound with lower alkalinity may evade lysosome isolation and be effective against drug-resistant cancer types, such as TNBC (230). Novel CDK inhibitor compounds from different synthetic frameworks are currently under investigation, examples of such derivatives include amidoyl, pyrazole, quinazoline and pyrimidine derivatives (231). In the forthcoming years, we may observe the advent of novel CDK4/6 inhibitors that have exhibited substantial promise in preclinical studies and clinical trials.

In addition, CDK4/6 inhibitors possess inherent limitations in clinical practice. The most notable challenge confronting these novel targeted therapies is the absence of predictive molecular biomarkers. The clinical efficacy of CDK4/6 inhibitors in treatment is contingent upon developing predictive biomarkers and biologically reasonable combination therapies (232). In actual clinical settings, the AEs caused by CDK4/6 inhibitors are also worth consideration. Although there are currently clinical management recommendations regarding the potential toxicity of CDK4/6 inhibitors, further research is still needed to understand their toxicological characteristics and to continuously optimize management strategies comprehensively. This will contribute to reducing the occurrence of treatment interruptions and improving the treatment efficacy and compliance of patients with BC (223).

Conclusion

The present study reviewed the regulatory role of CDK4/6 inhibitors in tumor immunity and the potential value of tumor immunotherapy. CDK4/6 inhibitors affect tumors by regulating the cell cycle, changing the immune microenvironment and regulating immune checkpoints, and they have garnered attention as clinical drugs for the treatment of various types of cancer. Du et al (233) thoroughly explored their clinical application in malignant solid tumors, alongside strategies for combining these inhibitors with endocrine or targeted therapies. Sun et al (234) delved into the immunomodulatory effects of targeting CDK4/6 in cancer, whereas Glaviano et al (138) offered an in-depth analysis of resistance mechanisms associated with CDK4/6 inhibitors. By contrast, the present review provides a more systematic account of recent advances in CDK4/6 inhibitors in tumor immunity and resistance mechanisms, with a comprehensive review of recent advances in clinical trials. This integrated perspective may reflect current research dynamics more comprehensively than the existing literature, providing researchers with a unified frame of reference.

In response to the problem of patient resistance to CDK4/6 inhibitors, the current review further proposed a variety of combination treatment strategies to enhance drug sensitivity and improve efficacy. This direction has been less explored in the existing literature and clinical trials. Although CDK4/6 inhibitors show promising applications, the review concludes with an objective analysis of current challenges and a clear direction for future development, such as identifying predictive molecular biomarkers and addressing safety issues, which need to be validated in more clinical trials.

In summary, the present study systematically reviewed the latest progress of CDK4/6 inhibitors in the field of tumor immunity, proposed an innovative combination therapy strategy and provided a clear direction for future research, showing its unique contribution. These innovations not only provide novel ideas for overcoming drug resistance and improving the effects of tumor immunotherapy, but also provide suggestions for future follow-up research and verification.

Supplementary Data

Availability of data and materials

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Authors' contributions

JX and YW drafted and revised the manuscript. FH, QZ, YC, SG, YX and RS collected the relevant papers and helped to revise the manuscript. FH, QZ and YC designed the tables and charts. JX and YW reviewed the article. Data authentication is not applicable. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no known competing interests.

Acknowledgments

Not applicable.

Funding

This research was supported by the National Natural Science Foundation of China (grant no. 82103328), the Scientific Research Project of Jiangsu Maternal and Child Health Association (grant no. FYX202348 and the Jiangsu Graduate Research Innovation Program (grant no. KYCX18_1453).

References