1 Introduction

Aberrant cell proliferation is a notable cancer hallmark which is characterized by dysregulated mitotic cell cycle progression [1]. The canonical mitotic cell cycle comprises four highly conserved phases: the mitosis (M), DNA synthesis (S), first gap (G1) and second gap (G2) phases. A variety of cyclins and cyclin-dependent kinases (CDKs), such as CDK4, CDK6 and CDK2, regulate the cell division cycle progression in the G1 and G2 phases, determining whether entering the S phase (G1-restriction point) and performing mitosis [2, 3].

CDK4/6 forms a complex with of Cyclin D family members (D1, D2 and D3). Multiple transcriptional regulatory pathways for Cyclin D are closely linked to kinases activity, which is crucial for cell cycle [4]. Specifically, the Cyclin D1–CDK4/6 complex phosphorylates retinoblastoma (Rb) and releases the transcription factor E2F, stimulating related genes transcription needed for cell cycle and for DNA replication [5]. Dysfunction in the Cyclin D1–CDK4/6–Rb axis is constantly observed in various cancers [6,7,8]. Surprisingly, researchers found that besides regulating the cell cycle and impeding tumor growth, CDK4/6 inhibitors (CDK4/6i) also augment immunogenicity of tumor cells, enhance the antigen-presenting cells (APCs) function, re-accommodate immune effector cells and immune-suppressing cells, and equilibrate senescence-associated secretory phenotype (SASP) and cytokine secretion, thus promote the aging of tumor cells and alter the tumor immune microenvironment (TIME), suggesting that antitumor immune response is vital to enhancing CDK4/6-targeted therapy efficacy.

Agents used for immune checkpoint blockade (ICB), particularly the programmed cell death protein-1 (PD-1) and programmed death - ligand1 (PD-L1) monoclonal antibodies, are approved for cancer therapy with sustainable therapeutic benefits [9]. ICB aims to eliminate tumor cells by restoring recognition and killing function of regular T cell, thus avoiding the occurrence of immune escape [10, 11]. Numerous studies have reported that CDK4/6-targeted therapy combined with ICB has an immunological effect that occurs via PD-L1 expression regulation or the elimination of innate and acquired resistance [12].

Thus, inhibition of CDK4/6 may prevent immune evasion through multiple pathways and promote broad-spectrum antitumor immunotherapy. In this review, we briefly summary the role of aberrant CDK4/6 and related signaling during tumor progression, focusing on the critical immunomodulatory roles of targeting CDK4/6 in anticancer therapy. In addition, the potential synergistic mechanism of CDK4/6i and ICB is summarized to help clinicians become more confident in their application of CDK4/6i in antitumor immunotherapy.

2 Targeting CDK4/6 in antitumor therapy

Cyclin D1 transcription is intricately related to multiple signaling pathways, and CDK4/6 serves as a sentinel that integrates diverse signaling cascades in the initiation and progression of the cell cycle [13, 14]. In noncancer cells, extracellular signals can stimulate or inhibit the complex activity of Cyclin D–CDK4/6. However, the aberrant complex activation is independent of mitotic stimulation in cancer cells, leading to uncontrolled proliferation [5].

CDK4/6i suppress the Rb phosphorylation, leading to cell cycle arrest [15]. Moreover, as reported by early studies, CDK4/6 coexists with classical tumor signaling pathways such as the Wnt, Myc and p53 pathways [16,17,18]. Ma et al. mentioned that high expression of Myc degrades Rb phosphorylation to improve CDK4/6i therapeutic efficacy [19, 20]. Zhu and his colleagues found that in PARP inhibitors-resistant triple-negative breast cancer (TNBC) cell lines, activated Wnt signaling pathway and overexpressed hyperphosphorylation of β-catenin at the Ser675 site could be strongly inhibited by combining olaparib with palbociclib [21]. Huang et al. confirmed that CDK4/6i induced DNA damage, leading to failure of cells to complete DNA replication in a p53-dependent manner [22]. Researchers further found that prolonged stimulation creates replication pressure in a panel of p53-null tumor cells, contributing to the process of cycle extension instead of complete G1 arrest [23].

Additionally, the function of the CDK4-cyclin D complex is modulated partly via the RTK signaling axis [24, 25]. For instance, the PI3K–AKT–mTOR and RAS–MEK–ERK pathways were found to be excessively activated in palbociclib-resistant BC cells [26, 27]. Kohlmeyer et al. furtherly mentioned that dual inhibition of CDK4/6-MEK emerged novel antitumor activity in Malignant peripheral nerve sheath tumors (MPNST) by inducing plasma cell-associated immune response [28]. In patient-derived organoid and xenograft ovarian cancer (OC) models, CDK4/6i combined with specific PI3K inhibitors (BYL3) and mTOR inhibitors (everolimus) showed effective synergy [29, 30], thereby restoring sensitivity to single-agent exposure, inducing stronger senescence and apoptosis and more effectively inhibiting tumor proliferation [31].

It is therefore more theoretically sound to combine CDK4/6i with other small-molecule inhibitors when treating cancers (Fig. 1). The functions of current CDK4/6i are summarized in detail (Table 1).

Fig. 1
figure 1

Mechanisms of targeting CDK4/6 combination therapy. The combination of existing CDK4/6 inhibitors with other targeted therapies related pathway such as the PI3K-AKT-mTOR pathway, RAS-RAF-MEK-ERK pathway, Wnt-β-catenin pathway and JAK-STAT pathway, as well as the function of CDK4/6 in cells

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Table 1 Classification and summary of CDK4/6 inhibitors
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3 Immunomodulatory function of targeting CDK4/6 in cancer

The TIME is a complex biosystem, comprises mainly tumor cells, surrounding immune and inflammatory cells, tumor-associated fibroblasts, and various cytokines and chemokines [32, 33]. Many clinical studies have confirmed that inhibition of CDK4/6 has an immunomodulatory effect via its prevention of tumor immune evasion and regulation of the TIME, affecting the combined efficiency. Here, we discuss mainly the different immune response processes induced by targeting CDK4/6 therapy in the TIME.

3.1 Effects on the process of antigen presentation

In addition to inducing cell cycle arrest, CDK4/6i also enhance T cell-mediated antigen presentation by modulating major histocompatibility complex (MHC) I and II expression, APCs function, and costimulatory molecules secretion, thereby inhibiting immune evasion [34].

3.1.1 MHC-I/II

On the tumor cell surface, the lack of MHC expression and molecular presentation function are the most critical factors that promote tumor cell immune evasion [35, 36]. MHC class I and II present polypeptides respectively recognized by CD8+ T-cells and CD4+ T-cells, binding with T cell receptors and activating killing functions [37, 38]. Multiple relative and absolute quantitative immunopeptidomics analyses also revealed alterations in the MHC-I repertoire caused by inhibition of CDK4/6 [39]. This finding has been confirmed in several tumor cell lines, such as BC, melanoma, primary effusion lymphoma (PEL) and Epstein-Barr virus-positive Burkitt lymphoma (BL). Abemaciclib, palbociclib, and ribociclib can upregulate MHC-I/II expression on the surface, making tumor cells easier to identify by an immunological surveillance system [40, 41].

Moreover, Lu and colleagues proved that Rb acts in rescuing interferon (IFN)-γ-induced MHC-II gene transcription in BC cells [42]. Zhu et al. furtherly demonstrated that in mouse embryonic fibroblasts (MEFs), the induction of IFN-γ at the MHC-II Aβ locus is dependent on Rb, inducing antigen presentation to CD4+ T-cells, while other genes, such as Aα and Eβ, are independent of Rb [43, 44]. Liu and colleagues studied the mechanisms underlying palbociclib-induced immune responses in OC. Palbociclib caused an increase in antigen presentation-related gene expression levels, which was partially reversed by small interfering STING RNA [45, 46], and decreased the level of endogenous DNA damage [47]. Thus, palbociclib induces an immune response partially dependent on STING signaling.

3.1.2 APCs and costimulatory molecules

APCs including dendritic cells (DCs) and B cells, macrophages, among others, are carriers that coactivate T-cell recognition ability due to costimulatory molecules [48]. Charles et al. detected positive T-cell responses by ELISpot after T-cell stimulation with peptide-pulsed APCs, founded CDK4/6i-induced human leukocyte antigen (HLA) ligands and maturation of monocytic DCs had a higher capacities for T-cell recognition [49, 50]. Adoptively transferring differentiated DCs in vitro achieved restoration of the DCs compartment in mice treated with CDK4/6i and ICB therapy, resulting in potent tumor inhibition [51]. The study also revealed an inclination towards an increased level of colony-stimulating factor 2 (CSF2) during abemaciclib treatment, which is crucial in promoting M1 polarization and MHC-II expression, as well as the mature of DCs [52].

Consistently, Wu and colleagues further reported that incubation of T cells with CDK4/6i-treated PEL and BL cell lines significantly upregulated the surface costimulatory molecules ICAM-1 and B7-2 (CD86, ligand of cytotoxic T lymphocyte-associated antigen-4, CTLA-4), thus enhancing T-cell activation and recognition [41]. Meanwhile, in melanoma cells, CDK4/6i alone or together with MEK inhibitors increased the T-cell costimulatory molecules CD103 and CD137 expression [53]. Furthermore, a comprehensive understanding from two independent phase I clinical trials (NCT02778685, NCI02648477) analyzed subjects’ peripheral blood mononuclear cells by high-parameter flow cytometry to assess baseline immune subset composition and longitudinal changes after therapy and found that palbociclib-treated patients had increased levels of CD83 and human leukocyte antigen-DR (HLA-DR), indicating enhanced antitumor maturity and cDC antigen-presenting capacity [54] (Fig. 2).

Fig. 2
figure 2

Functions of targeting CDK4/6 in antigen presentation. Inhibition of CDK4/6 up-regulates cell-surface MHC moleculesby stimulating IFN-γ secretion or activating the ubiquitination pathway and the STING pathway, as well as facilitates the antigen presentation to CD8 + T cells and CD4 + T cells under the action of various stimulating factors

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Therefore, CDK4/6i increase the immunogenicity of tumor cells surface and improve the presentation function of APCs by simulating costimulatory molecules, providing new ideas for activating CDK4/6i in immunotherapy.

3.2 Function and distribution of immune cells

In addition to promoting antigen presentation in T-cells, inhibition of CDK4/6 can indirectly or directly affect immune cells and promote corresponding immune responses. TCGA analysis indicated a negative relevance between immune cell abundance and CDK4/6 expression in cytotoxic cells, Th1 cells, CD8+ T cells, NK cells, and CD56 bright cells [45]. Here, we discuss mainly the immune cell modulatory mechanisms of targeting CDK4/6 therapy from immune effector and suppressing cells.

3.2.1 Immune effector cells

Several studies have shown that CDK4/6i induce CD8+ T cell, CD4+ T cell, and B-cell activation and recruitment in TIME [55, 56]. A clinical retrospective study (NCT02499770) showed that brief exposure to trilaciclib during chemotherapy not only preserves and increases peripheral blood lymphocyte counts but also significantly predicts prognosis and survival by increasing CD8+ T cells and CD4+ T cells in SCLC patients [57, 58]. Chimeric Antigen Receptor T-Cell Therapy (CAR T-cell therapy) plays a novel adoptive antitumor immunomodulatory role by arming T cells or increasing the immunogenicity of tumor cells. Thus CDK4/6i could be a promising adjuvant for CAR T-cell therapy because of its immunomodulatory effects.

Deng and colleagues isolated CD4+CD25− T cells from tumor-bearing and naïve mice under CD3 and CD28 stimulation and assessed IL-2 production from T-cells to further explore effector T-cell regulatory mechanisms. Finally, inhibition of CDK6 results in high activation of NFAT4 signaling in relation to IL-2 secretion and upregulation of activation markers of T-cells, such as 4-1BB (Tnfrsf9), Icosl, GITR (Tnfrsf18), CD40, and CD86 [59]. Specifically, NFAT4 is needed to activate T-cells and transcriptionally regulating IL-2, while CDK6 can phosphorylate and inactivate NFAT4 [60].

In addition, authors also observed an increased persistence and memory effect in parallel with CD8+ tumor-infiltrating lymphocyte (TIL) enrichment, which occurs regardless of whether APCs activate T-cells [56]. Higher expression of TCF1, CD127, and CD147, as memory markers, and CD8+ TILs was the most effective effector cell [61, 62]. Veiga-Fernandes and colleagues further demonstrated the mechanism of memory cell formation. The G0/G1 status of memory T-cells is different from that of naïve cells, as it is characterized by lower p27Kip1 expression and higher CDK6 kinase activity [63]. Heckler et al. also considered that it not only relies on arrest of cell cycle but is also related to MXD4-mediated Myc inhibition after CDK4/6i treatment [64]. Notably, although CDK4/6i can promote effector T-cell infiltration and activation, they suppress cell proliferation, indicating that these targeted agents may have complementary effects when used with antitumor immunotherapy.

3.2.2 Immune-suppressing cells

Immune suppressing cells consist of regulatory T-cell (Treg), M2 macrophages, regulatory B cell (Breg), among others. A series of studies demonstrated that treating metastatic BC patients with CDK4/6i led to a global reduction in Tregs (CD4+CD25+FOXP+), polymorphonuclear bone and monocyte marrow-derived suppressor cells (CD66b+HLA−DR−), thereby attenuating the immunosuppressive TIME [65]. Additionally, after 12 weeks of treatment with ribociclib (NCT03096847), immune-related signature gene expression analysis and deep T-cell receptor profiling showed that Helios+ Tregs declined more significantly than did Helios− Tregs [66]. This demonstrated that an activated phenotype of Tregs was more easily induced by CDK 4/6 inhibitors, which more effectively inhibit the proliferation of autoreactive splenocytes.

To explore how CDK4/6 is involved in the downregulation of Tregs, Xu and colleagues reported that when mice were treated IL-33, CDK4 and Cyclin D1 expression significantly increased, and hepatic Tregs proliferated, indicating that IL-33-induced Treg cell proliferation was caused by overexpression of CDK4 and Cyclin D1 [67]. Additionally, according to the TCGA data, CCND1 is also linked to an increased ratio of immune suppressor cells. CCND1-amplified melanoma murine models were also found to have notably reduced expression of CD8, Gzm, B2M, and Tap1, which resulted in decreases in the number of plasma cell, CD8+ T cell, T follicular helper cell and an increase in Treg [68].

Overall, after targeted CDK4/6 therapy, the proportion of Treg/CD8+ T cells significantly declined, indicating a shift toward antitumor immunity in the immune balance. The short-term exposure to CDK4/6i facilitated long-term effect of endogenous antitumor T-cell immunity, thereby augmenting the persistence and therapeutic efficacy of CAR T-cell therapy.

3.3 Modulation of cytokines in the TIME

Cytokines are key elements that coordinate TME and control tumor-immune cell interactions. A series of studies demonstrated that CDK4/6i can modulate the TIME by influencing cytokine expression and secretion, thereby augmenting the responsiveness of antitumor immunotherapy [69]. For instance, in OC patient peripheral blood treated with abemaciclib, immune effector cytokines, including IL9, IL3, CXCL16, CRG-2, TNFSF8, and CXCL13, etc. were up-regulated, while three immune suppressive factors (TCK-1, MMP3, SDF1-α) were downregulated [55, 70]. (Fig. 3).

Fig. 3
figure 3

The role of CDK4/6 inhibitors in remodeling TIME. Targeting CDK4/6 promotes immune effector cells through the Mxd4-Myc pathway, stimulates IL-2 secretion by NFAT4, and inhibits immune suppressive cells through the Rb-dependent pathway and the IL-33-MyD88 axis, promoting the secretion of some stimulating factors or inhibiting some factors release to remodel the TIME

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3.3.1 Aging-related secretory phenotypes

A typical biological endpoint of CDK4/6 inhibition therapy is induced aging. Senescent cells undergo stable cell cycle arrest and fail to proliferate but can initiate complex proinflammatory responses and the enhanced expression of several secretory cytokines, known as SASP [71]. In A673, SK-ES-1, SK-N-MC, and Ewen's sarcoma (ES) cell lines, abemaciclib can upregulate the expression of CCL2/MCP-1 and IL8, which are biomarkers of therapy-induced senescence (TIS) [72], and stimulate the interferon pathway to promote the inflammatory immune response and remodel the inflammatory microenvironment [73, 74]. Interestingly, Lee et al. found that CDK4/6i consistently induce more SASP secretion and maintain higher expression than DNA-damaging agents-induced cells [75]. Anna and colleagues also showed that in melanoma PDX models, inhibition of CDK4/6 confers CCL5 secretion in an NF-κB-dependent manner and facilitates the recruitment of tumor-infiltrating leukocytes (TILs), finally leading to senescence [71, 76].

3.3.2 Chemokines

Cytokines and their receptors have been extensively studied as poteintial anti-tumor therapeutic strategies. CDK4/6 inhibition therapy can enhance the activity and chemotaxis of immune effector cells by inducing chemokine secretion. In an experiment by Uzhachenko et al., CDK4/6i were beneficial to T cell therapies like adoptive T cell transfers and T cell activating antibodies by activating chemokines CCL5, CXCL9, and CXCL10, thus recruiting activated CD8+ T-cells in BC mouse models [77]. According to Peuker et al., ribociclib downregulates immunosuppressive chemokines (CCL2, CCL7, CCL22) and then drives immunosuppressive cell chemotaxis and differentiation, including Tregs, MDSCs, and macrophages [66, 78]. Moreover, secretion of these immunosuppressive chemokines also indicates a poor prognosis in BC patients [79].

Taken together, the TIME is induced to a favorable immune-prohibitory tumor microenvironment with a more immune initiation state and improved patient survival. CDK4/6i may be a reliable clinical tool either as adjuvants to reinforce antitumor function or as immunomodulatory agents such as targeting cytokine and its receptor therapy.

4 CDK4/6 inhibition augments ICB sensitivity

Currently, anti-PD-1/PD-L1 monoclonal antibody (mAb) has made an achievement worthy of celebration in tumor immunotherapy. Despite the improvements in progression-free survival, the response efficacy in some patients remains unsatisfactory [80, 81]. Emerging evidence suggests that CDK4/6i could augment anti-PD-L1 agent sensitivity and overcome either innate or acquired resistance to ICB therapy. Combining CDK4/6i with ICB has shown decent therapeutic effects in existing clinical trials, which is summarized as follows (Table 2).

Table 2 Clinical trials of combination CDK4/6 inhibitors with ICB
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4.1 Synergistic effect of CDK4/6 inhibitors and ICB

In combination with anti-PD-1 therapy, abemaciclib has synergistic effects according to meaningful factor analysis and survival analysis [55, 82]. Abemaciclib monotherapy in a CT26 BALB/c mouse model has demonstrated the capacity to impede tumor growth by augmenting the inflammatory signature of T-cells within the TIME. Combined with anti-PD-L1 mAb, it can generate complete regression of tumors, generate immune memory, and enhance cell cycle control [74]. Subsequently, Jang et al. also claimed that combination therapy results in a more pronounced tumor inhibition and superior survival time compared to anti-PD-1 mAb monotherapy [83].

A series of studies related to the regulatory mechanism noted that the CyclinD1-CDK4/6-Rb axis participates in the direct or indirect regulation of PD-L1 expression and thus has an auxiliary effect during immunotherapy. Jin et al. knocked down Rb with two independent shRNAs in PC-3 cells and found that phosphorylated Rb can directly bind to NF-κB and its promoter and then promote PD-L1 transcription [80]. Moreover, in the prostate cancer cell line LNCaP, NF-κB and Rb phospho-mimetic peptides activated the expression of the inflammatory factor TNF-α, thereby indirectly upregulating PD-L1 mRNA levels [84, 85]. A combination treatment regimen of avelumab, palbociclib, and cetuximab is well tolerated in patients with R/M HNSCC in a phase I study and warrants further study [90]. All this evidence suggests that combining CDK4/6i and ICB may confer a greater benefit than either agent alone.

4.2 CDK4/6 inhibitors eliminate both innate and acquired resistance to ICB

4.2.1 Innate resistance

Regulator of chromosome condensation 1 (RCC1) has been implicated by Zeng et al. in the mechanism of inherent ICB resistance in lung adenocarcinoma. In their in vitro trials, knocking down RCC1 hindered the proliferation of H1299 and A549 cells and downregulated CDK4 and p-Rb protein expression levels, thus upregulating PD-L1 expression and reducing resistance to ICB therapy [86]. However, whether using CDK4/6i in PD-L1-resistant tumors with high RCC expression can block RCC's regulation of PD-L1, resulting in excellent combination efficacy, remains to be investigated. RNA-Seq analyses have also suggested that CDK4 abnormalities may also contribute to aberrant PD-L1 expression, as evidenced by PD-L1-positive rates for CDK4-gain patients being lower than those for CDK4-normal patients. The authors further explored AMC-3 (CDK4 gain) and SK-MEL-5 (CCND1 gain) melanoma cell lines and found that treatment with palbociclib increased PD-L1 protein levels [87].

4.2.2 Acquired resistance

Wang et al. reported that in NSCLC, acquired drug resistance to anti-PD-1 mAb may be attributed to the acquisition of a homozygous deletion of B2-microglobulin (B2M), an essential protein for MHC-I antigen presentation, resulting in loss of surface MHC-I expression [88, 89]. Ribas et al. also demonstrated that this kind of resistance is due to the lack of memory T-cells and re-exhausted Tex cells [90, 91]. However, tumors with CCND1 amplification exhibited either a reduction in B2M expression or a remarkable increase in quiescent CD4+ memory T cell [68]. Therefore, inactivating Cyclin D–CDK4/6 complex may weaken or abrogate acquired drug resistance when combined with ICB, which needs further experiments to verify.

In summary, CDK4/6i have synergistical effects with ICB that occur through multiple pathways, including the promotion of immune regulation via CDK4/6 and the reduction or elimination of resistance to ICB, indicating excellent research value and providing good combination therapy prospects (Fig. 4).

Fig. 4
figure 4

CDK4/6 inhibitors augment the sensitivity of ICB. Targeting CDK4/6 abrogates the resistance of anti-PD-L1 mAb by down-regulating PD-L1 expression through phosphorylating Rb and regulating B2M expression. It also enhances the sensitivity by down-regulating PD-L1 related to the NF-κB pathway and phosphorylating SPOP. And facilitating the occurrence of DDR, which mTOR regulates

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5 Conclusion and perspectives

Although CDK4/6i promote the immune response, it remains to be further elucidated. Notably, apoptosis of tumor cells is a critical factor contributing to immune evasion. Rb has at least 16 CDK phosphorylation sites, the effects on apoptosis vary when stimulated or phosphorylated at different locations, and this may be associated with the Fas/FasL system activated by TNF. Hence, further investigation is warranted to determine whether CDK4/6i has disparate effects on Rb phosphorylation sites that impact tumor cell apoptosis and prevent immune evasion.

CDK4/6i are majorly accredited in breast cancer, the clinical feasibility in other cancer types need to be further tested. The intricate pathway crosstalk related research provide theoretical basis for CDK4/6i combination therapy. Particularly in solid malignant tumors, preoperative concurrent chemoradiotherapy and immunotherapy are chosen by more patients. CDK4/6i as radio-sensitizer has been well demonstrated in ER+ BC, HNSCC (NCT02499120), and Lung squamous cell carcinoma (LSCC) [92, 93]. Trilaciclib (G1T28) is a short-acting therapeutic agent designed specifically to protect hematopoietic stem cells from DNA-damaging chemotherapeutic agents that has been widely used in chemotherapy (NCT02499770, NCT02978716) [94, 95]. Moreover, G1T28 showed good pharmacological effect in healthy volunteers and could temporarily inhibit the proliferation of bone marrow hematopoietic stem cells and progenitor cells (HSPC) [96]. Examining drug effect and general toxicity profiles will be a critical first step. The results of these trials are eagerly awaited.

Our review demonstrates that CDK4/6i have wider applications in immunotherapy beyond conventional antitumor treatments, providing a rationale and novel therapeutic strategy for the implementation of target–immune combinations. Despite many clinical trials supporting the feasibility of target–immune combination therapy, there are still complications. In both early and advanced HR+ BC, the combination therapy resulted in a treatment-related adverse events (AEs) of hepatitis and pneumonitis, which likely caused by proinflammatory cytokine production and suppression of Treg proliferation [97]. Surprisingly, the application of CDK4/6i before surgery to create a reservoir of memory CD8+ T cells using ICB may subsequently alleviate the synergistic toxicity derived from simultaneous application. It is expected as adjuvant immunotherapy to reduce local tumor progression efficaciously, improve patient survival and prognosis and has maximal efficacy in the neoadjuvant therapy of a variety of potentially operable tumors. Thus, further investigation is warranted to optimize the therapeutic schedule of CDK4/6-based combination immunotherapy while minimizing potential complications.