Rationale For CDK4&6 Inhibition in Breast Cancer

¹Breast cancer is the fifth leading cause of cancer death in the United States, with more than 40,000 estimated deaths in 2017.²Many therapies for patients with breast cancer focus on targeting cancer cell proliferation, with a goal to divert cells to a more senescent phenotype.³Deregulation of the cyclin-dependent kinase (CDK) 4&6—retinoblastoma (Rb) axis has been demonstrated to contribute to unrestricted growth that is characteristic of many cancers, including breast cancer. “CDKs interact with cyclin D proteins to play an integral role in cell cycle progression, and represent attractive therapeutic targets in many tumors, including breast cancer,” noted Conleth G. Murphy, MD, of Bon Secours Hospital, Cork, Ireland, and Maura N. Dickler, MD, of Memorial Sloan Kettering Cancer Center, New York, in The Oncologist.⁴

Role in Oncogenesis

A hallmark of cancer cells, unrestricted growth is often associated with disordered cell cycle regulation.³The cell cycle includes steps that are highly regulated and coordinated by transient activations of cyclins and CDKs.⁵Together, these elements form bipartite complexes that phosphorylate proteins and transcription factors, which enable the cell to move through the cell cycle.^6-8CDKs are serine/threonine kinases that affect the G1-S phase transition of the cell cycle, which is mediated by an action on the Rb protein (FIGURE).^9,10

Rb and the p107 and p130 proteins have been shown to play a key role in cell cycle regulation through exerting a braking influence on cell proliferation in the early G1 phase.¹¹While hypophosphorylated, the Rb protein sequesters proliferative cellular proteins that are released via CDK4&6−cyclin D1 mediated hyperphosphorylation of Rb. These sequestered proteins include the E2F transcription factors that control gene expression that is essential for moving through the cell cycle from G1 to S phase.^12-14When Rb is hyperphosphorylated, transcription factors that initiate DNA replication are released, leading to activation of dihydrofolate reductase, histone H2A, thymidine kinase, and DNA polymerase alpha.¹⁵

The CIP/KIP and INK4 protein families mediate negative regulation of CDK4&6−cyclin D1.¹⁶The INK4 proteins inhibit D-type cyclin activity through selective interaction with CDK4 and CDK6. This inhibition mechanism involves interaction between the p16- and CDK6-charged binding domains that results in reduced kinase activity and a decreased cyclin-binding surface.^17,18Cyclin D1 is susceptible to extrinsic mitogenic stimulation, which provides a link between the external environment and cell cycle machinery.19 Because of the modulatory control over Rb and the resulting function in promoting cellular proliferation, it has been hypothesized that deregulation of cyclin D1 and CDK4&6 may promote tumorigenesis and tumor maintenance.

Many tumors have been shown to increase cyclin D−dependent activity and to avoid senescence via numerous mechanisms, including p16 inactivation, CDK4 mutation with loss of INK4 binding, CDK4 amplification, overexpression of cyclin D1, or translocation or amplification of CCND1, its encoding gene.²⁰The development and maintenance of certain breast cancers may require cyclin D1, as mice lacking cyclin D1 or CDK4 have demonstrated resistance to implanted breast tumor growth driven by human epidermal growth factor receptor 2 (HER2)/neu.^21-25

In breast cancer, multiple mechanisms are involved in frequently reported deregulation of CDK4&6−cyclin D1 activity.26,27 For example, in a cancer genome study of 482 patients with invasive breast cancer, 27.4% exhibited genetic deregulation of the cyclin D1/CDK4&6/p16INK4a pathway.²⁷Approximately half of investigated breast cancer cell lines exhibit cyclin D1 overexpression.²⁸In about 16% of breast cancers, CDK4 may be overexpressed via amplification of the CDK4 gene at 12q13-q14.29 CCND1 amplification has been reported in 38% of HER2-positive breast cancers, 29% of luminal A cancers, and in 58% of luminal B cancers.³⁰Altered function of inhibitors of CDK have also been reported in breast cancer, including p16INK4a, p21WAF1/CIP1, and p15INK4b.³¹

Murine breast cancer models have demonstrated that cyclin D1−CDK4&6 axis activation contributes to a tumorigenic phenotype and is crucial for tumorigenesis in HER2-positive breast cancer.^23,24,32-35However, viability and normal mammary development are not adversely affected when CDK2 or CDK4&6 are knocked out.

Cyclin D1 has been shown to induce chromosomal instability via transcriptional regulation of CIN-related genes.³⁶A role for cyclin D1 has also been implicated in cellular migration and invasion, DNA damage-sensing and repair, inhibition of mitochondrial metabolism, and angiogenesis enhancement.³⁷These functions are mostly independent of any CDK4&6 interactions and are not thought to be affected by CDK inhibition. Recently, however, CDK6 was reported to have a kinase-independent angiogenesis function, which suggests an alternative role for CDK6 inhibition in tumor therapy.³⁸

Estrogen receptor (ER)—positive breast cancers have been shown to be dependent on estrogen signaling for both proliferation and survival.³⁹ER inhibition has also been reported to reduce tumor cell viability and cell cycle arrest in the G1 phase.^40,41Furthermore, ER signaling upregulates cyclin D1 levels, stimulating various signaling pathways that subsequently upregulate CDK4&6 activity.^42,43Acquired resistance and lack of response to hormone-based therapies for ER-positive breast cancers may be mediated by deregulation of alternative pathways, such as HER2 and PI3K/AKT, that may induce cyclin D1−CDK4&6 signaling. Cyclin D1 has also been shown to independently activate ER, with a majority of breast cancers that overexpress cyclin D1 being ER-positive.⁴⁴

Most breast cancers maintain intact or functioning Rb, with loss of Rb expression occurring in just 20% to 35% of breast cancers.¹⁴Loss of the Rb protein is more frequently reported in triple-negative breast cancer compared with other subtypes.⁴⁵The inactivation of Rb contributes to tumor progression via proliferation control loss and conversion to a phenotype that is invasive. Generally, this is associated with poor differentiation and an increased metastatic potential. Without functional Rb, cell-to-cell adhesion is disrupted, facilitating the epithelial−mesenchymal transition, which may contribute to the increased frequency of metastases.⁴⁶In ER-positive tumors, Rb dysregulation is associated with poor prognosis, whereas it is associated with improved outcomes in ER-negative tumors.⁴⁷These reported differences in outcomes may be a result of different responses to therapy, as loss of Rb has been demonstrated to increase sensitivity to DNA-damaging agents but leads to continued proliferation with tamoxifen treatment.⁴⁸

Mechanism of Target

CDK4&6 inhibitors contribute to G1 phase cell cycle arrest by preventing the phosphorylation of Rb, which may lead to the inhibition of tumor progression in breast cancer and other cancer types. Despite oncogenic signals that are insensitive to endocrine therapy, CDK4&6 inhibitors effectively prevent cell cycle progression and mitosis.^49,50Patients who have loss of Rb expression do not benefit from CDK4&6 inhibitor treatment due to the downstream position of Rb compared with CDK4&6 in the cell cycle.¹⁴The selectivity of palbociclib (Ibrance), ribociclib (Kisqali), and abemaciclib (Verzenio)—3 FDA-approved CDK4&6 inhibitors—is thought to be related to binding to the specialized adenosine triphosphate (ATP)−binding pocket of CDK4&6 with specific residue reactions in the ATP-binding cleft.⁵¹

CDK4 and CDK6 are structurally related proteins that have biological and biochemical similarities and are considered functionally equivalent in their ability to phosphorylate Rb.^52,53CDK4 and CDK6 have overlapping functions and share nearly 75% of amino acid identity.⁵⁴CDK4&6 are expressed in most cell types and can partner with cyclin D1, D2, and D3.

Palbociclib, ribociclib, and abemaciclib bind to the ATP cleft of CDK4 and CDK6. Abemaciclib has been shown to bind more readily to the ATP cleft, forming a hydrogen bond with a conserved catalytic residue, suggesting that it binds less selectively than ribociclib and palbociclib.55 Palbociclib and ribociclib seem to have greater lipophilicity and larger side chains at the binding site compared with abemaciclib, which may reduce interactions with off-target kinase ATP-binding pockets. A study demonstrated that ribociclib exhibited significantly higher selectivity toward CDK4 and CDK6 compared with palbociclib, which interacted with more than twice as many kinases as did ribociclib.⁵⁶

Additionally, a study of palbociclib in molecularly characterized human breast cancer cell lines showed that ER-positive cell lines with luminal features were the most sensitive to palbociclib, whereas basal cell lines were the most resistant to palbociclib.⁴⁹

History of CDK Inhibition

Promising preclinical evidence demonstrating the potential therapeutic capability of targeting the cyclin D1−CDK4&6−Rb axis resulted in the development of first-generation CDK inhibitors. Flavopiridol (alvocidib), an intravenous pan-CDK inhibitor, was the first to enter clinical trials based on inhibitory activity in vitro against all human tumor cell lines contained in the tumor cell line panel from the National Cancer Institute.⁵⁷Results of clinical trials investigating flavopiridol monotherapy have been mixed.^58-60Increased antitumor efficacy was reported for the combination of flavopiridol and chemotherapy agents that promote S-phase accumulation.⁶¹In a phase I trial of patients with breast cancer and other solid tumors, encouraging results were reported following weekly administration of sequential docetaxel (Taxotere) and flavopiridol.⁶²Complex pharmacokinetics and dose-limiting toxicities have complicated the use of flavopiridol, however. Distal cellular effects associated with flavopiridol treatment include transcriptional suppression, apoptosis, autophagy, and endoplasmic reticulum stress, which have been associated with high rates of dose-limiting toxicities, including hyperglycemia, neutropenia, and cardiac and pulmonary dysfunction.^63-66Based on the low CDK specificity and the narrow therapeutic window, the development of flavopiridol was discontinued.⁵¹Seliciclib (roscovitine) as monotherapy demonstrated minimal disease stabilization in phase I studies of patients with solid tumors.⁶⁷Seliciclib inhibits CDK4&6 poorly, but also inhibits CDK1, CDK2, CDK7, and CDK9.⁶⁸

Second- and third-generation CDK inhibitors have been developed, including compounds with specificity for CDK4 and CDK6. Second-generation inhibitors started to show preferential inhibition of specific CDK subtypes, with initial efforts primarily focused on inhibition of CDK2 due to crystallographic structure availability.⁶⁹Later, highly selective inhibitors, including palbociclib, ribociclib, and abemaciclib, were evaluated through chemical screening and optimization by adding pyrido[2,3-d] pyrimidin-7-one compounds with a side chain of 2-amino pyridine at the C2 position.⁷⁰The major CDK inhibitors (palbociclib, ribociclib, and abemaciclib) currently are orally administered and have limited suppression of other CDK activities. These inhibitors have structures analogous to those of flavopiridol, but have different chemical functions.⁷¹

A Promising Approach in Breast Cancer

Cyclin D1 overexpression has been reported in the majority of breast cancers.^28,72-74Mice lacking cyclin D1 have been shown to be resistant to mammary carcinomas triggered by the oncogene ErbB-2. The crucial role for cyclin D1 in breast cancer formation is the activation of CDK4.⁷⁵The continued presence of CDK4-associated kinase activity has also been shown to be required for the maintenance of breast tumorigenesis. A study investigating knock-in mice expressing kinase-deficient cyclin D1−CDK4 complexes demonstrated that these mice develop normally; however, these mice demonstrated resistance to mammary carcinomas triggered by ErbB-2.22 “These results point to a differential requirement for cyclin D1−CDK4 kinase activity in mammary neoplasia versus in development,” noted a team led by Qunyan Yu, MD, of Dana-Farber Cancer Institute, Boston, Massachusetts, in Cancer Cell.⁷⁵“These findings suggest that the pharmacological inhibition of CDK4 kinase might preferentially target breast cancer cells. Our findings will hopefully stimulate therapeutic strategies aiming at blocking CDK4 kinase activity in patients.”

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