	Cyclin-dependent kinase (CDK) 4/6 inhibitors are novel agents that have shown promising results in the treatment of breast cancer. CDK4 and CDK6 are proteins that are part of a cell cycle regulatory pathway that also includes p16, cyclin D, and the retinoblastoma (Rb) protein. CDK4/6 inhibitors bind to CDK4 and CDK6, preventing phosphorylation of the Rb protein. This halts cell cycle progression and induces G1 cell cycle arrest. Malignant cells frequently acquire mutations in the CDK4/6 pathway, either activating mutations of CDK4/6 or mutations in CDK4/6 regulatory mechanisms, thereby conferring a growth advantage. By inhibiting CDK4/6, tumor cells are unable to exploit this pathway for cell proliferation.

	In vitro studies show robust activity of CDK4/6 inhibitors in multiple types of cancer, including breast cancer. Estrogen receptor (ER)-positive luminal breast cancer cell lines were shown to be particularly sensitive to CDK4/6 inhibition. Early clinical studies have demonstrated improvement in progression-free survival with use of these agents in patients with ER-positive breast cancer. With the success of CDK4/6 inhibitors in early clinical trials, several phase 3 trials evaluating these agents in breast cancer

 are now under way, and they may soon become an important component of combination cancer therapy.

One of the most promising novel therapies for breast cancer involves interrupting cell cycle progression through cyclin-dependent kinase (CDK) inhibition. Several agents acting on this pathway are in early- and late-phase trials. This review describes the biologic pathways involved in this therapy, reviews the status of current agents and trials, and looks at future directions for this class of drugs.

Normal cell cycle progression is tightly regulated by several mechanisms that control cell growth and division. Alterations in these mechanisms contribute to malignant transformation of cells and uncontrolled cell proliferation. Cyclin-dependent kinases play a key role in the progression of the cell cycle from the first growth phase (G1) through the DNA synthesis (S) phase, the second growth phase (G2), and ultimately, mitosis, or (M) phase.

 Recently developed targeted therapies take aim at cell cycle pathways, such as the CDK4/6 pathway (

). Proteins in this cell proliferative pathway include p16, an endogenous suppressor of CDK4/6, cyclin D1, the regulatory subunit of CDK4/6, and retinoblastoma (Rb) protein, a tumor suppressor.

Cyclin D1 was the first described G1 phase cyclin.

Approximately 15% of human breast cancers demonstrate amplification of the cyclin D1 gene, 

, and the majority of human breast cancers show overexpression of this protein.

Cyclin D1 is a key component of cell cycle progression and interacts closely with the Rb protein. In the hypo-phosphorylated state, Rb protein acts as a tumor suppressor and contributes to cell cycle regulation at the G1 to S checkpoint by suppressing gene transcription that is required for entry into the S phase. The cell cycle is then arrested in G1. In response to mitogenic signals, CDK4 and CDK6 form a complex with their regulatory subunit, cyclin D1, which phosphorylates the Rb protein, reducing its ability to suppress gene transcription. Controlled phosphorylation and deactivation of the Rb protein by the CDK4/6 complex is essential to progression of the normal cell cycle.

 In malignant cells, unrestricted CDK4/6 pathway activity can result from alterations in the expression of cyclin-dependent kinases and their regulatory mechanisms. This unhindered cell cycle stimulation yields a growth advantage and uncontrolled cell proliferation.

Recognition of CDK4/6 pathway proteins as novel targets in anti-cancer therapy led to the development of pan-CDK inhibitors which non-selectively inhibit the cell cycle and affect normal cells, leading to high rates of adverse events (AEs) and excessive toxicity.

 With selective CDK4/6 inhibition, normal cells can utilize other cyclin-dependent kinases, such as CDK2, to proceed with normal cell growth.

	Role of Cyclin-Dependent Kinases in Breast Cancer

Several selective CDK4/6 inhibitors are currently in development, and these agents appear to be better tolerated. By binding to CDK4 and CDK6, these inhibitors prevent phosphorylation of the Rb protein, resulting in G1 cell cycle arrest. This mechanism of action requires that the malignant cell have a normally functioning Rb protein. If there is loss of the intact Rb protein, then the G1 to S checkpoint becomes unrestricted, making the proliferation of malignant cells CDK4/6-independent, and thus resistant to treatment with CDK4/6 inhibitors.

 Notably, some cell lines with an intact Rb protein exhibit CDK4/6 insensitivity, suggesting that other escape mechanisms exist.

Breast cancer cells, like other malignant cells, develop genetic alterations that result in loss of normal mechanisms that control growth. Results from the Cancer Genome Atlas show that aberrations in the cell cycle pathway are attractive targets for a large portion of mammary carcinomas, with cell cycle alterations particularly enriched in the luminal breast cancer subtypes. 

Hormone-sensitive tumors have the additional feature that their cell proliferation is regulated in part by endocrine hormones acting on their receptors. Cyclin D1 is a target gene of the estrogen bound receptor and is essential to estrogen-induced cell proliferation partly through its interaction with CDK4 and CDK6.

 In 2009, a study published by Finn and colleagues evaluated whether a selective CDK4/6 inhibitor developed by Pfizer, then called PD0332991 (now palbociclib), had in vitro activity against breast cancer and also identified genes that predicted PD0332991 responsiveness.

 -positive luminal breast cancers, including those that were also human epidermal growth factor receptor 2 (HER-2) positive, were the most sensitive to inhibition by PD0332991. When PD0332991 was combined with tamoxifen, or trastuzumab for HER-2 positive tumors, a synergistic effect was noted. Elevated expression of cyclin D1 and Rb protein and reduced p16 expression were found to be associated with sensitivity to PD0332991. There was also evidence that PD0332991 may be able to overcome acquired resistance to tamoxifen. Earlier studies have shown that cyclin D1 can activate the ER in the absence of estrogen, suggesting a possible mechanism of how CDK4/6 inhibition may overcome hormone therapy resistance.

Notably, Finn and colleagues found 3 cell lines with detectable Rb protein that were resistant to PD0332991 inhibition, raising the possibility of another mechanism of Rb phosphorylation or of CDK4/6 mutations that resist the binding of an inhibitor.

With promising preclinical data, clinical trials began evaluating CDK4/6 inhibitors. Single-agent phase 1 studies showed class activity with frequent, manageable hematologic AEs as well as common, generally low-grade systemic side effects.

 An overview of these toxicities is shown in 

Subsequent trials have looked largely at combination therapy. The PALOMA-1 study was a phase 1/2 trial designed to assess the combination of palbociclib and the aromatase inhibitor letrozole versus letrozole alone in postmenopausal women with ER-positive, HER-2—negative, locally advanced or newly diagnosed metastatic breast cancer. The phase 1 portion determined the recommended combination dosage to be letrozole 2.5 mg continuous daily plus palbociclib 125 mg daily for 3 weeks with 1 week off. The phase 2 portion was a 2-part study. Part 1 enrolled postmenopausal women with ER-positive, HER-2 negative advanced breast cancer while part 2 included the same criteria while also including an exploratory component, requiring that tumors demonstrate cyclin D1 amplification and/or loss of p16 by FISH. In total, 66 patients were randomized to part 1 and 99 patients to part 2. Interim analysis reported at the San Antonio Breast Cancer Symposium in 2012 revealed that the primary end point, median progression-free survival (PFS), was 26.1 months with letrozole plus palbociclib compared with 7.5 months for letrozole alone (hazard ratio [HR] = 0.37; 95% CI, 0.21-0.63,

 The combination resulted in an objective response rate of 34% compared with 26% for letrozole alone. The clinical benefit rate, defined as complete response, partial response, and stable disease at 24 weeks or more, was 70% for the combination versus 44% for single agent letrozole. Most AEs were grade 1 or 2. The most common grade 3/4 AEs were hematologic, with 51 of 83 patients on the combination arm developing grade 3/4 neutropenia and 14 of 83 developing grade 3/4 leukopenia. No febrile neutropenia was reported, and growth factor support was not utilized. Nausea, fatigue, arthralgia, and hot flushes were also more prevalent in the combination group, but were largely low grade.

		Table 1. Common Adverse Events Reported in Single-Agent Phase 1 Trials

Articles

Cyclin-dependent kinase (CDK) 4/6 inhibitors are novel agents that have shown promising results in the treatment of breast cancer. 

Alexandra Thomas, MD

Articles

Targeting Cell Cycle Progression: CDK4/6 Inhibition in Breast Cancer