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

Abstract

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 cancer¹ are now under way, and they may soon become an important component of combination cancer therapy.

Introduction

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.

Mechanism of Action—CDK 4/6 Pathway

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.¹ Recently developed targeted therapies take aim at cell cycle pathways, such as the CDK4/6 pathway (Figure). 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.² Approximately 15% of human breast cancers demonstrate amplification of the cyclin D1 gene, CCND1, and the majority of human breast cancers show overexpression of this protein.^3,4 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.⁵ 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.⁶

Figure 1. CDK4/6 Cell Cycle Pathway

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.^7-9 With selective CDK4/6 inhibition, normal cells can utilize other cyclin-dependent kinases, such as CDK2, to proceed with normal cell growth.¹

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.¹ Notably, some cell lines with an intact Rb protein exhibit CDK4/6 insensitivity, suggesting that other escape mechanisms exist.¹⁰Breast 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. ¹¹

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 CDK6.¹² 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.¹⁰

Their findings suggested that ER -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.¹³

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.

Clinical Trial Reports

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.^14-16 An overview of these toxicities is shown in Table 1.

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, P <.001).¹⁷ 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

CDK 4/6 Inhibitor

Palbociclib¹⁴

LEE001¹⁵

LY2835219¹⁶

Number of patients

33

132

55

Neutropenia

All grades

66%

40%

16%

Grade 3/4

24%

19%

7%

Leukopenia

All grades

88%

36%

NR

Grade 3/4

21%

12%

Anemia

All grade

67%

NR

NR

Grade 3/4

3%

Thrombocytopenia

All grades

67%

NR

NR

Grade 3/4

9%

Nausea

All grades

15-30%

35%

30%

Grade 3

3%

NR

4%

Fatigue

All grades

30-33%

27%

21%

Grade 3

3%

NR

7%

NR indicates not reported

These results were updated at the 2014 American Association for Cancer Research (AACR) meeting. The final analysis of the primary end point showed a statistically significant improvement in PFS for the palbociclib and letrozole arm (20.2 months) compared with the letrozole arm (10.2 months); (HR = 0.488; 95% CI: 0.319-0.748; P = .0004).¹⁸ Overall survival (OS) analysis favored the combination arm but did not achieve statistical significance (37.5 months vs 33.3 months, HR = 0.813; P = .2105).

Another highly selective CDK4/6 inhibitor, LEE011, developed by Novartis, showed robust antitumor activity in multiple types of cancers in preclinical studies, including breast cancer, both as single agent and in combination with other targeted therapies.^15,19,20Evidence of in vivo clinical efficacy of LEE011 was demonstrated by significant tumor growth inhibition in mice with ER-positive breast cancer treated with single agent LEE011 as well as durable tumor regression in mice treated with LEE011 in combination with hormonal therapy and PI3K inhibitors.²¹ Data from a phase 1 study was presented at the 2014 American Society of Clinical Oncology (ASCO) Annual Meeting.¹⁵ This study evaluated LEE011 in patients with advanced solid tumors and lymphomas that had an intact Rb protein. LEE011 showed preliminary clinical activity and an acceptable safety profile, with a recommended phase 2 dose of 600 mg/day on a 21/28 day schedule. Common AEs were gastrointestinal and hematologic, with 35% of patients experiencing nausea, 36% developing leukopenia, and 40% developing neutropenia (19% grade 3). Most AEs were grades 1/2 and reversible.

Lilly has also developed a potent, highly selective CDK4/6 inhibitor, LY2835219. Preclinical studies showed induction of complete cell cycle arrest and inhibition of Rb phosphorylation 24 hours after a single dose as well as a synergistic effect when combined with gemcitabine.²² A unique characteristic of LY2835219 is its ability to cross the blood-brain barrier, making it a potentially attractive treatment option for brain metastasis and primary brain tumors.²² Phase 1 data presented at the 2013 ASCO Annual Meeting demonstrated preliminary clinical activity and an acceptable safety profile of LY2835219 in patients with advanced cancer.¹⁶ Adverse events were again largely gastrointestinal and hematologic, with 52% of patients experiencing diarrhea (5% grade 3), 30% nausea (4% grade 3), 18% vomiting (2% grade 3) and 16% neutropenia (7% grade 3). At the 2014 AACR meeting, a phase 1 study evaluating single agent LY2935219 in patients with heavily pre-treated metastatic breast cancer showed significant and durable clinical activity in this group of patients with a similar side effect profile as previously observed.²³ In this study, LY2835219 was administered continuously at doses ranging from 150 to 200 mg every 12 hours.

Ongoing Clinical Trials in Breast Cancer

The PALOMA-2 trial is currently under way, comparing the combination of palbociclib and letrozole versus letrozole and placebo in postmenopausal women with ER-positive, HER-2—negative advanced breast cancer who have not received any prior systemic therapy.²⁴ PFS is the primary end point. The PALOMA-3 trial evaluates whether palbociclib in combination with fulvestrant is more effective than fulvestrant alone in women with ER-positive, HER-2—negative metastatic breast cancer whose disease has progressed after prior hormone therapy. ²⁵ PFS is the primary end point, with OS and response as secondary end points.

Palbociclib is also being studied in the adjuvant setting, in a phase 3 trial called PENELOPE-B.²⁶ This trial is designed to evaluate whether the addition of palbociclib to standard hormonal therapy provides superior invasive disease-free survival in patients who are at high risk of relapse, based on incomplete pathologic response to neoadjuvant taxane-containing chemotherapy.

A phase 3 trial, MONALEESA-2, is now under way comparing LEE011 plus letrozole versus letrozole alone among postmenopausal women with ER-positive, HER-2—negative advanced breast cancer who have received no prior therapy for advanced disease.²⁷ PFS is the primary end point while secondary end points include OS, response, clinical benefit rate, quality of life, and safety.

A phase 3 trial with LY2835219 is planned which will evaluate effectiveness with fulvestrant in the first- or second-line setting. The primary end point is PFS with OS and response rate included as secondary end points.²⁸ Current and upcoming phase 3 trials are summarized in the Table 2.

Future Directions

Other potential approaches include combining CDK4/6 inhibitors with other anti-neoplastic agents, both novel small molecules as well as more traditional cytotoxic chemotherapy. Ongoing trials are combining CDK4/6 inhibitors with mTOR inhibitors or PI3K inhibitors.^29,30There is also evidence that cell cycle modifiers could enhance the effect of cytotoxic chemotherapy and limit toxicity.³¹

Table 2. Phase 3 Trials of CDK 4/6 Inhibitors in Breast Cancer

CDK 4/6 Inhibitor

Participants

Arms

Primary Outcome Measure

Palbociclib Trial Name: PENELOPE-B

Women with ER+, HER-2 negative breast cancer with residual disease after neoadjuvant chemotherapy and surgery

Palbociclib + endocrine treatment vs placebo + endocrine treatment

Invasive disease-free survival

Palbociclib Trial Name: PALOMA-2

Postmenopausal women with ER+ HER-2 negative advanced breast cancer, no prior systemic therapy

Palbociclib + letrozole vs letrozole + placebo

Progression-free survival

Palbociclib Trial Name: PALOMA-3

Women with ER+ HER-2 negative metastatic breast cancer with progression after prior endocrine therapy

Palbociclib + fulvestrant vs fulvestrant + placebo

Progression-free survival

Palbociclib Trial Name: PEARL

Postmenopausal women with ER+ HER-2 negative metastatic breast cancer with resistance to nonsteroidal aromatase inhibitors

Palbociclib + exemestane vs chemotherapy (capecitabine)

Progression-free survival

LEE011 Trial Name: MONALEESA-2

Postmenopausal women with ER+ HER-2 negative advanced breast cancer, no prior systemic therapy

LEE011 + letrozole vs letrozole + placebo

Progression-free survival

LY2835219

Postmenopausal women with ER+ HER-2 negative advanced breast cancer

Fulvestrant + LY2835219 vs fulvestrant + placebo

Progression-free survival

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One study is evaluating the impact of adding a novel cyclin inhibitor to carboplatin and gemcitabine in metastatic triple-negative breast cancer.³² Finally, since radiation therapy requires cells to be moving through the cell cycle to exert its effect, these agents could play a role in limiting toxicity from radiation exposure in tumors that have lost an intact Rb protein by preventing noncancerous bystander cells from progressing through the cell cycle until radiation treatment is complete. The CDK4/6 inhibitor can then be stopped, allowing the cell cycle to return to normal.

As many human cancers harbor cell cycle activating mutations, it is thought that selective CDK4/6 inhibitors will have antitumor activity against a wide range of cancers with a relatively tolerable side effect profile. Trials targeting vulnerabilities in cell cycle proliferation are ongoing in a variety of malignancies including hematologic malignancies, glioblastoma multiforme, and advanced melanoma.^5,33-36

Conclusion

Inhibition of the CDK4/6 pathway has demonstrated initial promise in advanced hormone receptor—positive breast cancer. Additionally, emerging evidence suggests that novel combinations may expand the use of these agents to other breast cancer subtypes and to other roles in anti-neoplastic therapy. Notably, these small molecules appear to be relatively well tolerated. Studies looking at resistance mechanisms will be important to furthering the use of CDK4/6 inhibitors, particularly in those tumors that have an intact Rb protein but are not vulnerable to therapy. Defining gene expression patterns in tumors that confer sensitivity to these drugs will also help determine which patients will have the greatest benefit as we increasingly move toward an era of more precise, personalized medicine.

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