Neelima Vidula, MD

San Francisco, Division of Hematology-Oncology,

Therapies targeted to tumor biology hold the promise of improved outcomes for patients with breast cancer. Cyclin-dependent kinase (CDK) 4/6 inhibitors are being investigated for the treatment of this disease. In preclinical studies, CDK inhibitors have been found to be effective at inducing cell cycle arrest in breast cancer cells, and to have a synergistic effect when combined with antiestrogen therapy. Results from early clinical studies testing CDK 4/6 inhibitors in combination with hormone therapy or as single agents in patients with advanced-stage breast cancer have yielded promising results, and late-stage trials are ongoing. This article discusses both the preclinical and clinical data for CDK 4/6 inhibitors in breast cancer, including palbociclib (PD 0332991), LEE011, and abemaciclib (LY2835219).

Breast cancer is the most common cancer diagnosis in women, with hormone receptorpositive (HR+) disease being the most predominant subtype in both the early and late stages. While treatment options for patients with breast cancer have improved, metastatic breast cancer remains incurable. Therapies targeted to specific aspects of tumor biology offer the promise of improved efficacy, particularly in combination with standard treatment. Combinations driven by preclinical data may help to achieve the goal of circumventing or delaying the development of resistance.

Targeting cyclin-dependent kinases (CDKs) has been the focus of considerable basic science and clinical research. CDKs are a class of serine/ threonine kinases that help govern the cell cycle,1 a process that results in cell division.

In a normal cell, replication occurs via progression through 4 different phases: G1 (growth period); S (DNA replication period); G2 (growth period); and M (mitosis or cell division period).

CDKs, in conjunction with cyclins, another group of proteins, are involved in the regulation of this process.

CDK 4/6 generates a complex with cyclin D, which in turn allows for the phosphorylation and inactivation of the retinoblastoma protein (Rb), a tumor suppressor protein, enabling gene transcription. Ultimately, this results in the progression of a cell from the G1 to the S period of the cell cycle, and eventually leads to cell division.

Another tumor suppressor protein, p16, inhibits this process.

The cell cycle is also regulated by other CDKs, including CDK 2, which forms a complex with cyclin E that phosphorylates and inactivates Rb, causing cell cycle progression from the G1 to the S period.

CDK 2 also helps regulate the G2 period of the cell cycle. These normal cell regulatory checkpoints are often deranged in cancer cells, allowing for uncontrolled growth. Cancer cells may have upregulated CDKs and cyclins, or downregulated Rb, p16, or intrinsic CDK inhibitors.

Ultimately, these aberrations in a cancer cell result in excessive cell proliferation, leading to tumor growth and resistance.

Preclinical Studies on CDK 4/6 Inhibitors in Breast Cancer

The CDK 4/6 inhibitors are a novel class of therapeutics that target the CDK 4/6 pathway. While early- generation CDK inhibitors were nonspecific to several CDKs and therefore had limited clinical efficacy,

a new class of oral CDK 4/6 inhibitors that specifically target CDK 4/6 and block the phosphorylation of Rb holds greater promise as a therapeutic option.

are CDK 4/6 inhibitors that are being investigated in clinical trials. In this article, we review the preclinical and clinical studies of CDK 4/6 inhibitors in breast cancer, as well as directions for future development.

CDK 4/6 inhibitors have been investigated extensively in preclinical models. As early as 2001, Fry et al tested the CDK inhibitor PD 0183812 in cell lines, noting G1 arrest in lines expressing Rb protein, correlating with a decrease in Rb phosphorylation.

A recent study tested Naphtho [2,1-&alpha;]pyrrolo[3, 4-c] carbazole-5,7(6H, 12H)-dione (NPCD), a novel CDK 4 inhibitor, in breast cancer cell lines and reported cell growth arrest and apoptosis, which persisted for several days after treatment discontinuation.

Decreased phosphorylation of Rb was observed, mediated by the cyclin D1-CDK 4 complex.

Dean et al tested palbociclib in vitro and reported a significant decrease in cell cycle progression specifically in estrogen receptor (ER)+ breast cancer cell lines Zr-75-1, T-47D, and MCF-7 without induction of cell death.

Some ER cell lines, including Hs578t and MDA-MB-231, were also susceptible to the effects of palbociclib, although other ER&ndash; cell lines, MDA-MB-468 and BT-549, were resistant to this agent.

In addition, palbociclib had no impact on cell lines deficient in the Rb protein, including those that were ER, whereas Rb protein was detectable in cell lines inhibited by palbociclib, highlighting the importance of Rb protein on the cell cycle&rsquo;s effect of CDK 4/6 inhibition.

Additionally, a lack of cyclin D1 was seen in cell lines nonresponsive to palbociclib. Based on these findings, it is reasonable to conclude that palbociclib depends on the cyclin D-CDK 4/6 complex and Rb protein to effect a response in cell cycle inhibition. Furthermore, the authors showed that palbociclib causes dephosphorylation of Rb protein, which then represses E2F gene transcription, whose products include CDK 2. This then modulates subsequent cell cycle progression.

Resistance to palbociclib may develop through chronic loss of Rb, driving the activity of CDK 2, which promotes cell cycle progression.

In breast cancer, the antiproliferative effect of CDK 4/6 inhibitors is the most promising in ER+ and human epidermal growth factor receptor 2 positive (HER2+) cell lines.

Finn et al tested palbociclib in 47 breast cancer cell lines and found the greatest growth inhibition in luminal ER+ breast cancer cells and the least inhibition in nonluminal cells.8 A synergistic effect in combination with tamoxifen was noted in ER+ cells, as well as in conjunction with trastuzumab in HER2+ cells.8 Another study noted that in mice deficient in the p18(Ink4c) gene, coding for a natural CDK 4/6 inhibitor that is often repressed in luminal A type tumors, may spontaneously develop ER+ tumors, suggesting that p18(Ink4c) may suppress tumor formation in luminal cells.

There are also data suggesting that antiestrogenmediated cell cycle inhibition may be due in part to a decrease in cyclin D1-CDK 4/6 activity and to an increase in CDK inhibitor activity.

CDK 4/6 inhibitors have been found to be effective at inducing cell cycle arrest in preclinical studies of breast cancer cells, and may have a synergistic effect with antiestrogen therapy

CDK 4/6 inhibitors, including palbociclib (PD 0332991), LEE011, and abemaciclib (LY2835219), are being evaluated in clinical trials with promising outcomes

The most common toxicity from CDK 4/6 inhibition is self-limited and generally uncomplicated neutropenia. Gastrointestinal toxicity has also been observed, as well as QTc prolongation. Diarrhea is more common with abemaciclib, with relatively less myelosuppression; this drug is also the only agent given continuously, compared with the intermittent dosing schedules employed for palbociclib and LEE011

While CDK 4/6 inhibitors appear to have a synergistic activity with antiestrogens, there are limited preclinical data on the combination of this class of drugs with conventional chemotherapy. One study performed by Roberts et al evaluated palbociclib and carboplatin in mice, including those with breast cancer.

The combination was associated with less antitumor activity in Rb-competent mice, and no effect in Rb-deficient mice. However, the combination of palbociclib and carboplatin was associated with

an increase in all lineage blood counts. Based on these data, the authors concluded that CDK 4/6 inhibitors should not be used in conjunction with carboplatin in tumors that are dependent on CDK 4/6, but they hypothesized that there could be a possible role for the combination in CDK 4/6 independent tumors to minimize chemotherapy-induced hematologic toxicity.

Clinical Studies on CDK 4/6 Inhibitors in Breast Cancer

In summary, the preclinical studies on CDK 4/6 inhibitors in breast cancer cell lines suggest that these agents are effective in inducing cell growth arrest and are dependent on Rb. Suppression of tumor cell growth appears to be specific for ER+ and HER2+ cell types, and these studies support a possible synergistic effect of CDK 4/6 inhibition with antiestrogen therapy.

Of the CDK 4/6 inhibitors currently being investigated in clinical trials, palbociclib is the most studied to date. Palbociclib is a potent oral agent that can be administered at doses of 200 mg daily for 14 days of a 21-day cycle, or 125 mg daily for 21 days of a 28- day cycle.

It is a very selective inhibitor of CDK 4 with a half maximal inhibitory concentration (IC50) of 0.011 &mu;mol/L, and CDK 6 with an IC50 of 0.016 &mu;mol/L.

In a phase I dose escalation study, Flaherty et al treated 41 patients with Rb-positive advanced cancers with once-daily palbociclib (75 mg, 125 mg, and 150 mg) for 21 out of 28 days in repeated cycles.

The maximum tolerated dose was 125 mg once daily, with neutropenia being the dose-limiting toxicity. Twenty-seven percent of patients had stable disease after a minimum of 4 cycles.

A phase I study established the safety of palbociclib administered to 33 patients with Rb-positive advanced tumors or refractory non-Hodgkin&rsquo;s lymphoma. 20 In this study, patients were treated with 200 mg of palbociclib orally once daily for 2 weeks out of every 3-week cycle. The major toxicity experienced by patients was myelosuppression without infection, and 9/31 patients had stable disease with treatment.

A phase II trial investigated single-agent palbociclib 125 mg by mouth once daily using the same schedule in patients with advanced breast cancer positive for Rb protein expression.

completed treatment. Four out of 18 (23%) patients with HR+/HER2 disease had a partial response or stable disease for greater than 6 months. High-grade toxicities noted were primarily myelosuppressive in nature, requiring dose reduction in 46% of patients and treatment interruption in 25% of patients. Based on these findings, it appears that single-agent palbociclib has evidence of clinical efficacy in heavily pretreated, Rb+ and HR+/HER2&ndash; advanced breast cancer, and is relatively well tolerated.

Palbociclib is currently being studied in combination with paclitaxel in a phase I study of patients with Rb+ metastatic breast cancer.

Fifteen patients have been treated thus far, with 11 patients having a partial remission or stable disease. The combination appears to be well tolerated, although 10 patients experienced grade 3 or 4 uncomplicated neutropenia, requiring either a dose reduction or interruption of palbociclib dosed on a 5-day schedule. A phase I dose-expansion study is now ongoing to determine the safety of a 3-day schedule of palbociclib.

To evaluate the potential synergistic effect of palbociclib in combination with antiestrogen therapy found in preclinical studies, the phase II PALOMA-1 trial randomized patients with untreated ER+/ HER2 advanced breast cancer to receive the nonsteroidal aromatase inhibitor letrozole with or without palbociclib.

Letrozole was given continuously and palbociclib was given at 125 mg a day for 3 out of every 4 weeks. The trial employed a 2-part design, enrolling 66 patients with ER+/HER2 disease in the first part. The second part, which enrolled 99 patients, also required amplification of cyclin D1 and/or loss of p16 in archived tumor, consistent with amplification of the pathway inhibited by this agent. The primary endpoint was progression-free survival (PFS); the trial was powered to detect a 50% improvement in median PFS from 9 to 13.5 months with 80% power and a 1-sided alpha of 10%.

a significant improvement was seen in PFS, but the exploratory biomarkers for cyclin D1 gains or p16 loss did not add to ER as a predictive factor for response. Hence, subsequent analyses combined the 2 parts of the trial. At the second interim analysis,25 there were 61 PFS events with continued improvement in the palbociclib arm (HR = 0.37;

<.001). The final analysis included 100 PFS events and was reported at the American Association for Cancer Research (AACR) annual meeting in March 2014.

Outcome was significantly improved in patients receiving palbociclib in addition to letrozole, with PFS 10.2 months for letrozole alone, increasing to 20.2 months in patients receiving palbociclib (HR 0.49;

= .0004). Objective response and clinical benefit were also improved, although complete responses were rare.

Overall survival was similar between the 2 arms and was not statistically different, at 37.5 and 33.3 months for letrozole and palbociclib compared with letrozole alone, with a hazard ratio of 0.81. Toxicity was similar to that seen in the phase I and II trials of single-agent palbociclib, with a 54% rate of grade 3/4 neutropenia without an increase in febrile neutropenia; other toxicities were primarily grade 1. Dose interruptions due to adverse events occurred in 33% of patients; 45% had cycle delays and 40% had dose reductions. Despite this, only 13% discontinued treatment due to adverse events.

Based on these compelling findings, the PALOMA- 2 phase III trial was initiated with a similar design as PALOMA-1, randomizing postmenopausal women with treatment-na&iuml;ve, metastatic ER+/ HER2 breast cancer to palbociclib/letrozole versus placebo/letrozole using a 3:1 randomization.

This trial completed accrual in May of 2014 after randomizing over 650 patients; first results are expected in 2015.

PALOMA-3 is a second phase III study that is randomizing patients with HR+/HER2 metastatic breast cancer that has progressed on hormonal therapy to the combination of palbociclib and fulvestrant, an estrogen receptor antagonist, versus fulvestrant and placebo in a 2:1 ratio.

The results of both PALOMA-1 and PALOMA-2 will be used for palbocilib registration, which has been given breakthrough drug status by the US Food and Drug Administration (FDA). The FDA has allowed submission of the phase II data, so it is possible that palbociclib could be approved as early as the first part of 2015, pending the results from PALOMA-2.

Several other trials are evaluating palbociclib in a variety of settings. PEARL is studying the combination of palbociclib and exemestane versus capecitabine in ER+ advanced disease that did not respond to prior aromatase inhibitor therapy.

In early-stage disease, a phase II neoadjuvant study is treating patients with stage 2 or 3 ER+/HER2 breast cancer with palbociclib in combination with anastrozole.

The international phase III PENELOPE-B trial will randomize patients with high-risk HR+ disease and residual cancer following neoadjuvant chemotherapy to hormone therapy in combination with palbociclib or placebo.

In the adjuvant setting, a phase II feasibility study will strive to determine the safety of administering palbociclib in combination with letrozole, anastrozole, or exemestane for 2 years to postmenopausal patients with HR+ stage 2/3 invasive breast cancer.

This study&rsquo;s primary endpoint is to determine the treatment discontinuation rate at 2 years, with a planned cutoff rate for feasibility of less than 33.3%. Investigators plan to enroll 120 patients to determine the primary endpoint with 92% power, and a one-sided alpha of 0.025. Treatment related adverse events and medication compliance will be examined as secondary endpoints.

Based on the currently available data, it is likely that palbociclib may be combined with antiestrogen therapy in the near future to treat patients with metastatic breast cancer with an augmented response compared with hormonal therapy alone.

LEE011 is an orally administered small-molecule inhibitor of CDK 4/6 at the nanomolar level.

In a phase I trial, 70 patients with advanced solid tumors or lymphomas were treated with LEE011 at doses ranging from 70 to 1200 mg daily for 21 days in a 28-day cycle.

The drug was found to be active at 600 mg or above, with a maximum tolerated dose of 900 mg daily for 21 of 28 days. Ten out of 70 patients had stable disease on treatment, and one patient with ER+ breast cancer had a partial response. Toxicity included myelosuppression, nausea, diarrhea, and QTc prolongation without arrhythmias.

Based on these encouraging data, a phase III trial is under way with a similar design to PALOMA 2. In MONALEESA-2, 500 patients with previously untreated advanced HR+/HER2 breast cancer will be randomized to letrozole 2.5 mg daily with either LEE011 600 mg daily for 3 out of every 4 weeks or placebo.

In early-stage disease, MONALEESA-1 is a phase II trial randomizing patients with HR+/HER2 grade 2 or 3 invasive breast cancer to treatment with LEE011 and letrozole or letrozole alone prior to surgery.

The primary outcome measure for this study is the cell cycle response rate, defined as the fraction of patients at the time of surgery who have a natural logarithm of Ki-67 (proliferative index on tumor tissue) percentage of baseline that is <1. The secondary endpoint is to determine the safety of this combination.

Table 1. Ongoing Phase II and III Clinical Trials With CDK 4/6 Inhibitors for Patients With Breast Cancer

Multiple other combination studies are being evaluated (see Table 1) and are reviewed in this article. Based on preclinical evidence of dependency of CDK inhibition on the phosphatidylinositol-3 kinase (PI3K) pathway, and synergy between CDK and PI3K inhibitors,

a phase Ib/II trial of patients with ER+/ HER2 metastatic breast cancer or locally advanced breast cancer is currently ongoing, in which patients are randomized to LEE011 and letrozole, BYL719 (a PI3K alpha inhibitor) and letrozole, or the triple combination of LEE011, BYL719, and letrozole.

LEE011 is being combined with BYL719 in one arm of this study, as a synergistic effect with these agents has been observed in preclinical studies. Once the dose escalation for these arms is determined in the phase Ib portion of the study, 300 patients will be randomized in a 1:1:1 ratio to the arms above in a phase II component. Thus far, 6 patients have received LEE011 600 mg in combination with letrozole, and only one instance of neutropenia was noted.

The safety and efficacy of the combination therapy has not yet been reported, but preliminary data is expected by late 2014 or early 2015.

Additional studies capitalizing on this potential synergy are under way. One phase Ib/II trial is studying the combination of LEE011 with everolimus and exemestane for postmenopausal patients with ER+/ HER2 metastatic breast cancer.

In the phase Ib component, patients will either receive escalating doses of LEE011 with everolimus and exemestane or a fixed dose of LEE011 and exemestane to determine the phase II dose of LEE011. In the phase II component, they will then be randomized to either the triple combination of LEE011, everolimus, exemestane versus LEE011 and exemestane, or everolimus and exemestane, to compare relative safety and efficacy. Preliminary results from 5 patients treated with the combination of LEE011 200 mg daily for 3 weeks on and 1 week off, everolimus 2.5 mg daily, and exemestane showed that the combination was well tolerated, with mild to moderate hematologic toxicity.

LEE011 is also being studied in combination with fulvestrant and BYL719 (PI3K-alpha inhibitor) or BKM120 (PI3K pan-class I-inhibitor) in a phase Ib/ II trial. Once the safety of the combinations are established in the phase Ib component, patients with ER+/HER2 metastatic or locally advanced breast cancer will be randomized to LEE011 and fulvestrant alone or in combination with BKM120 or BYL719.

Abemaciclib is another CDK 4/6 inhibitor with activity in ER+ breast cancer, with a differential toxicity profile as well as a different administration schedule. Abemaciclib is given continuously without a break, unlike the other CDK inhibitors, and has less hematologic and more gastrointestinal toxicity. A phase I trial investigating abemaciclib in various solid tumors was recently presented,12 with a focus on patients with metastatic breast cancer. As an expansion cohort, 47 patients with metastatic breast cancer who had failed several prior therapies were treated with abemaciclib 150 to 200 mg twice daily in a 28-day cycle.

In the entire population, 9/47 patients had a partial response and 14/47 patients had stable disease, for a total of 48.9% of patients with a clinical benefit. In the subset of patients with HR+ disease, 9/36 patients had a partial response, and 13/36 patients with HR+ cancer had stable disease, for a total of 61.1% of patients with clinical benefit. The doselimited toxicity for abemaciclib is unique in that it is associated with diarrhea, managed by dose interruption, dose reduction, and prophylaxis with antidiarrheals. Additional reported toxicities included nausea, fatigue, vomiting, and neutropenia.

Neutropenia was still the most common grade 3 toxicity.

The combination of abemaciclib and fulvestrant was then studied in patients with HR+ metastatic breast cancer as an expansion of the aforementioned phase I trial.

Eighteen patients were treated with intramuscular fulvestrant 500 mg once monthly and abemaciclib 200 mg twice daily in a 28-day cycle. Thirteen patients were noted to have controlled disease, for a clinical benefit ratio of 72.2%.

These findings are compelling and warrant further investigation to determine whether or not abemaciclib may improve outcomes in patients with HR+ metastatic breast cancer, both as a single agent and in combination with fulvestrant. The most common all-grade toxicity experienced by patients treated with the combination of abemaciclib and fulvestrant was diarrhea, followed by fatigue, nausea, neutropenia, vomiting, anorexia, abdominal pain and dehydration,

and the most common grade 3 toxicity was neutropenia.

Additional phase I trials with abemaciclib in patients with advanced cancer are ongoing.

One study is evaluating the combination of abemaciclib and clarithromycin, to determine how the pharmacokinetics of abemaciclib are affected by clarithromycin, a potent cytochrome P450 3A enzyme inhibitor.

A phase I trial with abemaciclib in conjunction with standard hormonal therapy is also ongoing.

A phase II trial to study abemaciclib in patients with HR+/HER2 breast cancer refractory to prior treatment has also been approved, although it is not yet active.

A phase III trial with abemaciclib in combination with fulvestrant in patients with HR+/HER2 advanced breast cancer will start in mid 2015.

The toxicity profile of CDK 4/6 inhibitors is variable depending on the agent studied, with neutropenia being common among all drugs in this class.

Grade 3 neutropenia has been noted in up to 50% of patients but is generally uncomplicated, without subsequent febrile neutropenia. In addition, dose delays and reductions result in prompt recovery. Interestingly, anemia and thrombocytopenia are less common. This effect is thought to be due to growth phase arrest of bone marrow cells. As with many agents used to treat advanced disease, fatigue has been reported.

Additional reported toxicities include primarily grade 1 and 2 nausea, vomiting and diarrhea, and, with LEE011, uncomplicated QTc prolongation.

For abemaciclib, the most commonly reported all-grade toxicity is diarrhea, although the most common grade 3 toxicity is still neutropenia. These side effects are less severe than those commonly seen with chemotherapy, but are important complications requiring careful monitoring for patients who are undergoing treatment with this class of drugs. The potential for QTc prolongation exists with agents of this class, and baseline monitoring has been incorporated into all clinical trials to identify those patients at potential risk. In addition, many commonly used agents have the potential to further increase the QTc interval, so careful control of concomitant drugs and drug monitoring is critical.

In summary, there are promising data on the potential efficacy of CDK 4/6 inhibitors in treating HR+ advanced breast cancer. These oral agents offer the hope of improved response and response duration to hormone therapy, with modest toxicity that appears to be easily controlled with minimal to modest intervention. Further clinical research is needed to validate the findings of phase I and phase II trials, and several phase III trials are under way with results expected in 2015. Table 1 summarizes current phase II and III clinical trials with CDK 4/6 inhibitors in breast cancer. If the data seen in phase II trials hold up in the phase III trials, CDK 4/6 inhibitors will clearly be incorporated into the treatment algorithms for breast cancer, possibly as early as 2015. Preclinical data suggesting synergy between CDK 4/6 inhibition, inhibitors of the PI3K pathway, and hormone therapy are intriguing and are currently being explored in clinical trials. Studies have already been started to evaluate the impact of these agents in early-stage, high-risk disease.

Zardavas D, Baselga J, Piccart M. Emerging targeted agents in metastatic breast cancer.

Dickson MA. Molecular pathways: CDK4 inhibitors for cancer therapy.

Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York, NY: Garland Science, 2002.

Fry DW, Harvey PJ, Keller PR, et al. Specific inhibition of cyclindependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts.

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Therapies targeted to tumor biology hold the promise of improved outcomes for patients with breast cancer.

Neelima Vidula, MD | Authors

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Regaining Control of the Cell Cycle With CDK 4/6 Inhibitors: A Promising Targeted Therapy for Breast Cancer