Targeting CDK4 and CDK6 in cancer

Abstract

Cyclin-dependent kinase 4 (CDK4) and CDK6 are critical mediators of cellular transition into S phase and are important for the initiation, growth and survival of many cancer types. Pharmacological inhibitors of CDK4/6 have rapidly become a new standard of care for patients with advanced hormone receptor-positive breast cancer. As expected, CDK4/6 inhibitors arrest sensitive tumour cells in the G1 phase of the cell cycle. However, the effects of CDK4/6 inhibition are far more wide-reaching. New insights into their mechanisms of action have triggered identification of new therapeutic opportunities, including the development of novel combination regimens, expanded application to a broader range of cancers and use as supportive care to ameliorate the toxic effects of other therapies. Exploring these new opportunities in the clinic is an urgent priority, which in many cases has not been adequately addressed. Here, we provide a framework for conceptualizing the activity of CDK4/6 inhibitors in cancer and explain how this framework might shape the future clinical development of these agents. We also discuss the biological underpinnings of CDK4/6 inhibitor resistance, an increasingly common challenge in clinical oncology.

Figure 1.. The CDK4 and CDK6 pathway in cancer.

a, Hypophosphorylated RB binds to and represses E2F family transcription factors (1). Negative regulation of cyclin-dependent kinase 4 (CDK4) and CDK6 (CDK4/6) activity is mediated primarily by the INK4 family of cell cycle inhibitors (INK4B (p15; encoded by cyclin-dependent kinase inhibitor 2B (CDKN2B)), INK4A (p16; encoded by CDKN2A), INK4C (p18), and INK4D (p19)), which bind to monomeric CDK4 and CDK6 to form inactive binary complexes (2). Mitogen or growth factor stimulation drives cyclin D up-regulation, leading to CDK4/6 activation (3). RB phosphorylation by cyclin D–CDK4/6 complexes promotes dissociation of RB–E2F binding (4). This in turn allows for E2F-mediated expression of genes required for cell cycle progression (5), which leads to progression through G1 phase and into S-phase. RB phosphorylation by cyclin E–CDK2 and cyclin A–CDK2 complexes (6) also promotes RB–E2F dissociation to drive progression into S phase. On the other hand, WAF1 and KIP family proteins, such as p21 (WAF1), p27 (KIP1) and p57 (KIP2), inhibit CDK2 and are important for inducing cell cycle arrest (7). Of note, p21 and p27 (p21/p27) have been shown to inhibit CDK4/6 activity in some instances and in other instances to stabilize cyclin D–CDK4/6 and thereby form an active trimeric holoenzyme. b, Major mechanisms responsible for dysregulated CDK4/6 activity in cancer include genomic alterations as well as activation of upstream signalling pathways that may up-regulate this pathway at the transcriptional, translational and post-translational levels. AR, androgen receptor; CCN, cyclin; ER, oestrogen receptor; mTORC1, mTOR complex 1.

Figure 2.. A conceptual framework to understand the effects of CDK4 and CDK6 inhibitors in cancer.

Response to cyclin-dependent kinase 4 (CDK4) and CDK6 (CDK4/6) inhibitors depends on multiple factors that affect the final outcome, including context-dependent tumour cell-intrinsic and -extrinsic features. a, RB-dependent E2F target depletion: Blockade of RB phosphorylation by cyclin D–CDK4/6 complexes leads to sustained binding to and inhibition of E2F by RB. A major consequence of E2F inactivation by RB is proliferative arrest. Moreover, E2F functions in various additional processes that are affected by CDK4/6 inhibition, including DNA damage response, chromatin remodelling, metabolism, differentiation and apoptosis. b, Senescence: CDK4/6 inhibitors may induce an RB-dependent senescent phenotype characterized by cell cycle withdrawal, chromatin remodelling, metabolic dysregulation, resistance to apoptosis and a senescence-associated secretory phenotype (SASP). Downregulation of other CDK4/6 targets, such as forkhead box protein M1 (FOXM1) and DNA methyltransferase 1 (DNMT1), is thought to enhance the senescent phenotype. In addition, p53 plays distinct and overlapping roles with RB in inducing senescence. Current evidence suggests that loss of p53 function may significantly affect senescence induced by CDK4/6 inhibitors, although additional studies have shown that CDK4/6 inactivation can still induce senescent phenotypes in the absence of wild type p53. c, Apoptosis and cytostasis: Induction of apoptosis or cytostasis appears to be cell type-dependent and may depend on differences in signalling between CDK4 and CDK6. In haematological malignancies, inhibition of CDK6–cyclin D3 complexes may lead to an RB-independent state of metabolic dysregulation that results in apoptosis. On the other hand, solid tumours such as breast cancer often depend primarily on CDK4–cyclin D1 activity for proliferation. In this case, CDK4/6 monotherapy predominantly induces RB-dependent proliferative arrest as a result of sustained E2F inhibition. d, Non-canonical RB functions: In addition to inhibiting E2F, RB has been shown to exert other functions, such as mediating chromatin remodelling and activation of other transcription factors. e, Non-RB substrates: In addition to phosphorylating and inhibiting RB, CDK4 and CDK6 phosphorylate a set of unique and overlapping targets that play important functions in numerous biological processes, including senescence, apoptosis and immunogenicity. f, CDK4/6 inhibitors have been shown to exert direct effects on multiple cell types normally present within the tumour microenvironment, including lymphocytes, fibroblasts and endothelial cells. Several studies have shown CDK4/6 inhibitors can directly and indirectly promote anti-tumour T lymphocyte effector function and inhibit immunosuppressive regulatory T (T_reg) cells. CDK4/6 inhibitors also induce senescence phenotypes in fibroblasts, which could potentially impair response to therapy by promoting tumour growth, and cell cycle arrest in endothelial cells, which has the potential to impact on clinical response. ROS, reactive oxygen species.

Figure 3.. Effect of CDK4 and CDK6 inhibition upon tumour cell-intrinsic and -extrinsic signalling pathways.

a, Inhibition of cyclin-dependent kinase 4 (CDK4) and CDK6 (CDK4/6) activity induces rewiring of multiple interconnected kinase circuits in tumour cells. First, blocking CDK4/6 inhibits RB phosphorylation (1), thereby preventing E2F-mediated cell cycle progression. However, multiple mechanisms may counteract these effects. For example, decreased phosphorylated-RB levels also lead to enhanced AKT phosphorylation by mTORC complex 2 (mTORC2) (2), which can stimulate cell survival mechanisms. Similarly, decreased CDK4/6 activity can down-regulate mTORC1 signalling via enhanced tuberous sclerosis 2 (TSC2) activity (3), which may lead to loss of negative-feedback regulation of receptor tyrosine kinase (RTK) signalling (4), resulting in up-regulated upstream PI3K–AKT pathway activity. In addition, compensatory CDK2-mediated RB phosphorylation (5) can stimulate cell cycle progression in the absence of CDK4/6 signalling. b, CDK4/6 inhibitors promote tumour cell immunogenicity via multiple mechanisms: (i) Metabolic stress and increased production of reactive oxygen species (ROS) up-regulate secretion of inflammatory chemokines CC-chemokine ligand 5 (CCL5), CXC-chemokine ligand 9 (CXCL9) and CXCL10; (ii) hypomethylation and therefore expression of endogenous retroviruses (ERVs) induces a double-stranded RNA (dsRNA) response, which leads to increased expression and secretion of type III interferon (IFN), activation of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signalling, enhanced IFN-driven gene expression, and up-regulation of major histocompatibility complex (MHC) class I expression; and (iii) chromatin remodelling facilitates IFN-mediated expression of interferon-responsive genes. 4EBP1; eukaryotic translation initiation factor 4E binding protein 1; IFNLR1, interferon λ receptor 1; IL-10RB, interleukin-10 receptor subunit β; S6K, S6 kinase; TF, transcription factor.

Figure 3.. Effect of CDK4 and CDK6 inhibition upon tumour cell-intrinsic and -extrinsic signalling pathways.

a, Inhibition of cyclin-dependent kinase 4 (CDK4) and CDK6 (CDK4/6) activity induces rewiring of multiple interconnected kinase circuits in tumour cells. First, blocking CDK4/6 inhibits RB phosphorylation (1), thereby preventing E2F-mediated cell cycle progression. However, multiple mechanisms may counteract these effects. For example, decreased phosphorylated-RB levels also lead to enhanced AKT phosphorylation by mTORC complex 2 (mTORC2) (2), which can stimulate cell survival mechanisms. Similarly, decreased CDK4/6 activity can down-regulate mTORC1 signalling via enhanced tuberous sclerosis 2 (TSC2) activity (3), which may lead to loss of negative-feedback regulation of receptor tyrosine kinase (RTK) signalling (4), resulting in up-regulated upstream PI3K–AKT pathway activity. In addition, compensatory CDK2-mediated RB phosphorylation (5) can stimulate cell cycle progression in the absence of CDK4/6 signalling. b, CDK4/6 inhibitors promote tumour cell immunogenicity via multiple mechanisms: (i) Metabolic stress and increased production of reactive oxygen species (ROS) up-regulate secretion of inflammatory chemokines CC-chemokine ligand 5 (CCL5), CXC-chemokine ligand 9 (CXCL9) and CXCL10; (ii) hypomethylation and therefore expression of endogenous retroviruses (ERVs) induces a double-stranded RNA (dsRNA) response, which leads to increased expression and secretion of type III interferon (IFN), activation of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signalling, enhanced IFN-driven gene expression, and up-regulation of major histocompatibility complex (MHC) class I expression; and (iii) chromatin remodelling facilitates IFN-mediated expression of interferon-responsive genes. 4EBP1; eukaryotic translation initiation factor 4E binding protein 1; IFNLR1, interferon λ receptor 1; IL-10RB, interleukin-10 receptor subunit β; S6K, S6 kinase; TF, transcription factor.

Figure 4.. CDK4 and CDK6 inhibitors exert differential effects in distinct immune cell populations.

a, Upon treatment with cyclin-dependent kinase (CDK4) and CDK6 (CDK4/6) inhibitors, regulatory T (T_reg) cells preferentially undergo cell cycle arrest, likely due to increased reliance on CDK4/6 signalling for cell cycle progression. b, CDK4/6 inhibition enhances CD8⁺ T-cell activation and induction of effector function via upregulation of nuclear factor of activated T-cells (NFAT) signalling. In addition, CDK4/6 inhibitors have been shown to promote memory T-cell differentiation through RB-dependent and RB-independent (increased MXD4 gene expression) mechanisms. MXD4, MAX dimerization protein 4.

Figure 5.. Novel approaches for the use of CDK4 and CDK6 inhibitors.

a, Administration of cyclin-dependent kinase 4 (CDK4) and CDK6 (CDK4/6) inhibitors after cytotoxic chemotherapy has been shown to enhance response by preventing E2F-mediated DNA damage repair after growth arrest during M-phase. Conversely, treatment with CDK4/6 inhibitors prior to chemotherapy would induce G1 cell cycle arrest, thus preventing chemotherapy-induced cytotoxicity that occurs during DNA replication or chromosome segregation. b, The CDK4/6 inhibitor trilaciclib can be used to protect hematopoietic stem and progenitor cells (HSPCs) from the cytotoxic effects of chemotherapy by inducing transient cell cycle arrest prior to treatment with chemotherapy. In this case, the target population would be patients with cancer types that are typically RB-deficient, such as small-cell-lung cancer (SCLC), so as to not interfere with cytotoxicity against tumour cells.