Cyclin-Dependent-Kinase (CDK) 4/6 Inhibitors for the Treatment of Breast Cancer: A Review of Pre-Clinical and Clinical Data
Abstract
For millions of women, breast cancer remains a potentially life-threatening diagnosis. With advances in research, new therapies targeted to tumor biology are emerging to treat the most common form of this disease. Cyclin-dependent-kinase (CDK) 4/6 inhibitors are a new class of therapeutics that have the potential to improve the outcomes of patients with hormone receptor positive (HR+) breast cancer. Three CDK 4/6 inhibitors have been investigated for the treatment of HR+ breast cancer, including palbociclib (PD 0332991), ribociclib (LEE011), and abemaciclib (LY2835219). Palbociclib recently received accelerated FDA approval for the treatment of HR+ metastatic breast cancer in combination with letrozole, and recent data suggest improved outcomes when combined with fulvestrant. This article reviews the mechanism of action of CDK 4/6 inhibitors, pre-clinical studies examining their efficacy, ongoing clinical trials in breast cancer, and toxicity profiles.
Keywords
Breast Cancer, CDK 4/6 Inhibitors, Palbociclib, Ribociclib, Abemaciclib, Hormone Receptor Positive Breast Cancer
Introduction
Breast cancer affects millions of women annually worldwide and remains the most common cancer diagnosis in women. Although treatment options for patients with breast cancer have improved over time, metastatic breast cancer remains incurable. Hormone receptor positive (HR+) disease is the most common subtype of both early and late-stage breast cancer. With scientific advances, new targeted therapies are emerging to address specific areas of tumor biology. These targeted therapies have the potential to improve patient outcomes by delaying the onset of, or overcoming, tumor resistance.
Cyclin-dependent-kinase (CDK) 4/6 inhibitors, which affect cell cycle progression to halt tumor growth, are an exciting new direction for the treatment of HR+ breast cancer. This class of drugs is being investigated extensively in both pre-clinical and clinical studies. Palbociclib (PD0332991) recently received accelerated FDA approval for the treatment of HR+ metastatic breast cancer, in combination with letrozole, with recent data suggesting improved outcome in combination with fulvestrant. This article will discuss the mechanism of action of this class of drugs, preclinical data pertaining to their use, ongoing clinical trials with these drugs in patients with breast cancer, and their toxicity profiles.
Discussion
CDK 4/6 Inhibitors and the Cell Cycle: Mechanism of Action
The cell cycle is a process which normally results in cell division, necessary for growth and preservation of the human body. Normal cell replication progresses from the G1 (first growth period), to S (DNA replication), G2 (second growth period), and M (mitosis period). This process is regulated by a number of proteins including CDKs, a group of serine/threonine kinases, and cyclins. Within the cell cycle, CDK 4/6 forms a complex with cyclin D. This complex phosphorylates the retinoblastoma protein (Rb), which deactivates this tumor suppressor protein, resulting in gene transcription and cell cycle progression from the G1 to S period, ultimately causing cell division. Other CDKs help regulate the cell cycle including CDK 2, which forms a complex with cyclin E, also resulting in the phosphorylation and subsequent inactivation of Rb, similarly leading to cell cycle progression. Intrinsic CDK inhibitors and tumor suppressor proteins such as p16 and Rb serve as repressors of cell replication. In addition, p16, also known as Ink4a, is an inhibitor of CDK 4/6.
Cell Cycle Dysregulation in Cancer Cells
In cancer cells, the process of cell division becomes unregulated, resulting in uncontrolled growth that leads to the development of a tumor. A number of mechanisms contribute to the dysregulation of the cell cycle in malignant cells, including the amplification and hyperactivity of CDK 4/6 or their genomic instability, which may cause CDK 4/6 to become oncogenic drivers of cell replication. By usurping these mechanisms, cancer cells can continue to replicate by triggering the G1 to S phase transition. This process appears to be facilitated by a shortening of the G1 phase. In a cancer cell, CDK 4/6 antagonizes intrinsic tumor suppression mechanisms including cell senescence and apoptosis, which further augments the growth of a tumor. Cancer cells also upregulate other CDKs and cyclins and decrease suppressive mechanisms such as intrinsic CDK inhibitors and tumor suppressor proteins. The overall effect of this type of cell cycle dysregulation is malignant cell proliferation and the development of cancer.
Effect of CDK 4/6 Inhibitors on the Cell Cycle
CDKs play an important role in cell cycle dysregulation in malignant cells, resulting in uncontrolled cell growth. In breast cancer, CDK gene mutations have been identified among potentially actionable genomic alterations resulting in the development of malignancy. Initial attempts at targeting CDKs involved less potent and non-specific inhibitors, with limited clinical efficacy. Flavoperidol, an inhibitor of CDK 1, 2, 4, 6, 7, and 9, showed activity in hematologic cell lines, but was not very effective in treating chronic lymphocytic leukemia clinically. The subsequent development of potent and selective inhibitors that target cyclin D1 to block the formation of the CDK 4/6-cyclin D1 complex by inhibiting the binding site of cyclin D1 and destabilizing the complex increased interest in targeting this pathway in cancer. These novel CDK 4/6 inhibitors block the phosphorylation of Rb, resulting in cell cycle arrest. The cell cycle arrest induced by these CDK 4/6 inhibitors is hypothesized to be at least somewhat specific to cancer cells, due to the dysregulation of CDK 4/6 in malignancy.
Three CDK 4/6 inhibitors are currently being studied in clinical trials in advanced breast cancer, including palbociclib, ribociclib (LEE011), and abemaciclib (LY2835219).
CDK 4/6 Inhibitors in Pre-Clinical Models
CDK 4/6 Inhibitors as a Single Agent Treatment
Numerous pre-clinical studies have investigated the use of CDK 4/6 inhibitors. One of the first studies with a targeted CDK 4/6 inhibitor, PD 0183812, showed the agent induced G1 cell cycle arrest in cell lines expressing Rb, which correlated with the dephosphorylation of Rb, highlighting the dependence of Rb on the action of CDK 4/6 inhibitors. A CDK 4 inhibitor, Naphtho [2, 1-α] pyrrolo [3, 4-c] carbazole-5, 7 (6H, 12H)-dione (NPCD), was shown to induce cell growth arrest and apoptosis in breast cancer cells. Decreased Rb phosphorylation was observed, consistent with blockade of the cyclin D1-CDK 4 pathway.
Palbociclib has undergone extensive testing in vitro in breast cancer cell lines. Similar to the CDK 4/6 inhibitors previously described, palbociclib causes cell cycle arrest by impacting Rb protein and the cyclin D-CDK 4/6 complex. Dean and colleagues studied the effects of palbociclib on breast cancer cell lines. Cell lines with detectable Rb protein were sensitive to palbociclib, with cell cycle inhibition, whereas those cell lines deficient in Rb protein were not inhibited by palbociclib. Cell lines resistant to palbociclib also did not express cyclin D1. These findings suggest that palbociclib’s mechanism of action is dependent on the cyclin D-CDK 4/6 complex and Rb protein. Palbociclib dephosphorylates the Rb protein, which represses transcription of the E2F gene. One product of the E2F gene includes CDK 2, which promotes further cell cycle progression. Resistance to palbociclib is driven by chronic loss of Rb, which promotes the activity of CDK 2, which then drives cell cycle progression.
Ribociclib, another selective CDK 4/6 inhibitor in clinical development, was also tested in pre-clinical models. This agent has been studied in liposarcoma cell lines and primary tumor xenografts, resulting in decreased cell growth, with decreased Rb phosphorylation and decrease in CDK 4. Similar findings with ribociclib in neuroblastoma cell lines have been noted, again with a decrease in phosphorylated Rb.
P276-00, a flavones compound which selectively inhibits cyclin D1-CDK 4, has also been tested in cell lines including those of human breast and lung cancer, and once again, the downregulation of cyclin D1 and a decrease in phosphorylation of Rb were determined as the mechanism of action.
Several pre-clinical studies using CDK 4/6 inhibitors as a single agent treatment in various cell lines have demonstrated that the mechanism of action of this class of drugs is dependent on the cyclin D-CDK 4/6 complex and Rb protein, as described above. Furthermore, CDK 4 mediated phosphorylation of Smad3, a protein in the transforming growth factor β pathway, inhibits Smad3, releasing G1 arrest, leading to cell proliferation, based on in vitro findings in cyclin D over-expressing breast cancer cells. However, when these cells were treated with a CDK inhibitor, an increase in the G1 cell population was noted, suggesting that CDK 4 inhibitors may be effective in the treatment of breast cancer cell lines over-expressing cyclin D.
Palbociclib has been studied in a variety of well-characterized breast cancer cell lines. In vitro studies with palbociclib have shown a significant decrease in cell cycle progression in HR+ breast cancer cell lines, Zr-75-1, T-47D, and MCF-7, while results in HR- cell lines have been mixed with some HR- cell lines, Hs578t and MDA-MB-231, responding to palbociclib, and others, MDA-MB-468 and BT-549, displaying resistance to this agent. Finn and colleagues treated forty-seven breast cancer cell lines with palbociclib and noted the greatest growth inhibition in luminal HR+ breast cancer lines and the least inhibition in non-luminal/basal cells, with the exception of those with HER2 amplification, where palbociclib also had significant anti-tumor activity. These results suggest that CDK 4/6 inhibitors are more potent in HR+ breast cancer, with intriguing data in HER2 positive cell lines.
Estrogen causes cell cycle progression by triggering CDKs and cyclins. An in vitro study showed that MCF-7 breast cancer cells, an HR+ cell line, treated with an estrogen antagonist went into G1 arrest. However, subsequent treatment with estradiol enabled progression to the S phase, was associated with an increase in activity of CDK 4, increased cyclin D1 activity, and hyperphosphorylation of Rb, amongst other changes in CDK and cyclin activity. Dysregulation of the CDK pathway in HR+ breast cancer may explain why CDK inhibitors appear to have increased activity in HR+ cell lines.
CDK 4/6 Inhibitors in Combination with Hormone Therapy
Anti-estrogen mediated cell cycle inhibition is due in part to a decrease in cyclin D1-CDK 4/6 activity, and an increase in CDK inhibitor activity. Anti-estrogen treated MCF-7 cells showed decreased CDK 4 activity mediated by p21(WAF1/Cip1), a CDK inhibitor, leading to G1 arrest. A study of mice deficient in the gene for p18(Ink4c), which codes for a naturally present CDK 4/6 inhibitor that is repressed in luminal A type tumors, showed that these mice developed HR+ tumors. These results suggest that naturally present CDK 4/6 inhibitors suppress luminal cell tumor formation. Another study showed that cyclin E over-expressing breast cancer cells had both anti-estrogen resistance and reduced sensitivity to CDK 4 inhibition. CDK 2 inhibition of cyclin E overexpressing and tamoxifen resistant cells re-established sensitivity to both hormonal and CDK 4 inhibition therapies. Resistance to anti-estrogen therapy and CDK 4 inhibitors appears to occur through a common mechanism of cyclin E over-expression.
These data predict potential synergy between CDK inhibition and hormonal therapy, which was confirmed in cell line studies. Finn and colleagues showed that palbociclib had a synergistic effect in combination with tamoxifen in HR+ cell lines. In breast cancer cell lines with conditioned resistance to HR inhibition, palbociclib increased sensitivity to treatment with tamoxifen.
Synergistic Effects with CDK 4/6 Inhibitors and Other Targeted Therapy Drug Combinations
In addition to the interaction with anti-estrogen therapies, CDK 4/6 inhibitors appear to have synergistic relationships with other classes of drugs. In HER2+ cells, the HER2/Akt (protein kinase B) pathway is a negative regulator of the CDK inhibitor p57 (Kip2), leading to increased cell proliferation. Palbociclib combined with trastuzumab appears to have more than an additive effect on cell death in HER2+ cells.
In breast cancer xenografts, CDK 4/6 inhibitors improve the response to phosphatidylinositol 3-kinase (PI3K) inhibitors; this combination of drugs has the potential to overcome resistance in mutant xenografts with decreased PI3K sensitivity. In leukemia cells, CDK inhibitors in combination with PI3K inhibitors have been shown PD-0332991 to increase cell mitotic arrest and apoptosis.