The concept of targeting mitotic cell division to halt the progression of rapidly dividing cancer cells has long been a staple of oncology therapy, yet chemotherapy agents that are the prime examples of this approach are nonselective in their action and can kill normal and malignant cells alike.
The concept of targeting mitotic cell division to halt the progression of rapidly dividing cancer cells has long been a staple of oncology therapy, yet chemotherapy agents that are the prime examples of this approach are nonselective in their action and can kill normal and malignant cells alike. Now, novel targeted approaches focusing on the inhibition of mitotic kinases are showing significant promise.
Among them are agents targeting polo-like kinase 1 (Plk1), which plays an important role throughout mitosis. Despite some clinical disappointment, a greater understanding of the intricate molecular mechanisms underlying the progression of the cell cycle and the respective roles of the PLK kinase family has offered renewed hope.
The lead agent in this category is volasertib, which has achieved breakthrough therapy and orphan drug designations from the FDA, and expectations are high for late-stage clinical testing that may result in the first approved agent in this class.
One of the hallmarks of cancer is unchecked cell division and, as such, targeting mitosis in these rapidly dividing cells has become a validated therapeutic approach. In recent years, researchers have identified various “checkpoints” that ensure cell division occurs at the appropriate time—and that may become dysregulated in human cancers.
Many of these checkpoints are controlled by protein kinases, among them the PLKs, a family of serine/threonine kinases. There are five members of the PLK family: Plks 1 through 5. Although they have different functions and tissue locations, most share a similar structure. At one end of the protein is the serine/threonine kinase domain (except for Plk5 which seems to lack a functional kinase domain) that is responsible for catalytic activity, while at the other end is a polo-box domain (PBD). The PBD is a regulatory domain that controls kinase activity; it prevents the kinase domain from binding to its target proteins until it receives the appropriate activation signal.
Plk1 is the most well characterized member of the family. The Plk1 protein is only expressed in dividing cells and its levels fluctuate during the course of the cell cycle. Its activity peaks during mitosis (M) phase, where it is involved in almost every step of mitotic cell division via the phosphorylation of a wide variety of downstream targets, including cyclin B1 and CDC25, which trigger entry into mitosis. Plk1 also regulates other mitotic events such as spindle formation and chromosome segregation.
Polo-like kinase 1 (Plk1) is activated throughout the process of mitotic regulation and cell division, with its peak expression during the G2/M phase of the cycle, as illustrated above.
G1 indicates growth 1 phase of the cell cycle; G2, growth 2; M, mitosis.
Donaldson MM, et al. The mitotic roles of polo-like kinase. J Cell Sci. 2001;114(13):2357-2358. jcs.biologists.org. Adapted with permission.
Plk1 and other mitotic kinases act as cell cycle checkpoints, so that entry into mitosis and subsequent cell division and proliferation is prevented in the absence of appropriate signals. High levels of Plk1 essentially permit the cancer cell to pass through these checkpoints unimpeded, contributing to the hallmark unchecked cell division and proliferation observed in tumors. Although evidence that Plk1 as an oncogene is lacking, it is frequently overexpressed in many different cancer types, including non—small cell lung cancer (NSCLC), melanoma, colorectal carcinoma, and prostate cancer, and high levels of Plk1 expression often correlate with poor prognosis.
The cellular roles of the other PLKs are less clear. However, several studies have suggested that Plk2 and Plk3 may in fact have opposing functions to the proliferative role of Plk1 and act as tumor suppressors or at the very least have functions that are unrelated to cancer cell proliferation. The PLK2 gene has been shown to be frequently methylated and silenced in B cell malignancies, while reduced expression of Plk3 proteins has been observed in several cancer types.
Since Plk1 is highly expressed in cancer cells, but not in nondividing healthy cells, it represents an attractive means to target mitosis in a more specific manner than chemotherapeutics. There has been significant research and development in this area and a number of Plk1-targeting agents have been investigated.
A common method of inhibiting protein kinases is to develop compounds that target the adenosine triphosphate (ATP) binding site. As the name implies, these ATP-competitive inhibitors compete with ATP for binding to the kinase and thus prevent its activation. The first Plk1 inhibitors were designed, as such, to be ATP-competitive inhibitors.
Among the first generation of Plk1 inhibitors was Boehringer Ingelheim’s BI2536, a dihydropteridinone class of compound. Phase II trials were completed in a variety of different cancer types, but development was subsequently halted upon discovery of a second member of this class, volasertib (BI6727), which displayed an improved pharmacokinetic profile and increased efficacy and safety.
Volasertib is the most advanced Plk1 inhibitor in clinical development. It potently inhibits Plk1 with an IC50 of 0.87 nM. This agent has advanced through to phase III where it is currently being evaluated in patients with acute myeloid leukemia (AML).
In phase II trial results recently reported in Blood, Döhner et al found that a combination of volasertib and low-dose cytarabine (LDAC) demonstrated an objective response rate of 31% (13 of 42 patients) compared with just 13.3% (6 of 45 patients) in the LDAC monotherapy arm; a response was defined as complete remission or complete remission with incomplete blood count recovery.
Median event-free survival, measured from the date of randomization to the date of progression, relapse or death, was 5.6 months for the combination versus 2.3 months with monotherapy while relapse-free survival was 18.5 months versus 10 months, respectively. These responses resulted in a median overall survival for the volasertib arm of 8.0 months compared with 5.2 months for LDAC alone (HR = 0.63; 95% CI, 0.40-1.00; P = .047), As a result of these promising results, the phase III POLO-AML-2 trial was initiated, evaluating this combination in AML patients aged 65 years and over who are frequently ineligible for the standard of care treatment option for AML, intensive remission induction therapy. AML is an aggressive form of cancer with among the lowest survival rates for all leukemias and this age group in particular represents a significant unmet therapeutic need. As a reflection of this, the FDA recently awarded breakthrough therapy and orphan drug designations to volasertib to further support its development for patients with AML.
A second ATP-competitive PLK inhibitor has reached late-stage clinical testing. Rigosertib is a dual targeting inhibitor of both Plk1 and phosphatidylinositol-3- kinase (PI3K), an important signaling molecule in many cellular proliferation pathways. Many researchers in the field class rigosertib as a PI3K inhibitor and do not consider it a “real” Plk1 inhibitor, although the National Cancer Institute maintains the Plk1 definition.
Nonetheless, this agent is the subject of several phase III trials. Rigosertib is the lead compound of Onconova Therapeutics, and the company is pursuing its development in a variety of cancers despite several recent phase III setbacks.
In December 2013, the company halted the ONTRAC study of intravenous rigosertib plus gemcitabine in frontline metastatic pancreatic cancer after a planned interim analysis showed the experimental combination was unlikely to demonstrate a statistically significant improvement in overall survival compared with gemcitabine alone.
The relationship of the tumor suppressor protein p53 to Plk1 activity has drawn considerable research interest.
Louwen F, Yuan J. Battle of the eternal rivals: restoring functional p53 and inhibiting Polo-like kinase 1 as cancer therapy. Oncotarget. 2013;4(7):958-971. http://goo.gl/6eBRs9. Reprinted with permission.
The ONTIME trial also failed to meet its primary endpoint in patients with high-risk myelodysplastic syndromes (MDSs) who had progressed on, failed, or relapsed after prior therapy with hypomethylating agents (HMAs).
However, a subsequent subset analysis including only patients who were nonresponders to prior HMA treatment (184 of 299 enrolled patients) demonstrated a statistically significant improvement in median overall survival, suggesting potential activity in these patients. As a result, Onconova said it has met with the FDA to design an approval-track trial for this subset of patients. Another phase III trial in MDS, which frequently develops into AML, is ongoing, as are several phase I and II trials in a variety of different cancer types.
Several other ATP-competitive Plk1 inhibitors have been developed. Nerviano Medical Science reports that it is continuing development of NMS-1286937, although the only clinical trial registered on Clinical- Trials.gov has been completed and results have not yet been disclosed. Two other pharmaceutical companies conducted studies of other agents, but are apparently no longer actively pursuing these compounds following completion of phase I trials.
The drawback to using ATP-competitive inhibitors is that they can lack specificity. Although the Plk1 inhibitors discussed above most potently inhibit Plk1, they also have inhibitory activities on other members of the PLK family. This could pose a significant problem, given that Plk2 and Plk3 are believed to have opposing functions to Plk1. At the very least this may be limiting the efficacy of Plk1 inhibitors.
As a result, researchers have also evaluated other means of targeting the Plk1 protein. The PBD is unique to PLKs and, since it also regulates PLK activity by preventing substrate binding to the kinase domain, it represents an attractive drug target to improve the specificity of Plk1 inhibition.
The phytochemical compound thymoquinone inhibits Plk1 through the PBD. Since thymoquinone binds a range of other kinases, a synthetic derivative that more specifically inhibited Plk1 called poloxin was generated. However, this compound was significantly less active than thymoquinone and, although it has proved extremely useful for studying the biological functions of Plk1, it has not progressed into clinical testing. Agents targeting both the PBD and other non-ATP competitive methods of targeting Plk1 continue to be pursued.
A particularly interesting aspect of Plk1 biology that has been uncovered in recent years is its relationship to a number of important oncogenes. Numerous studies have now demonstrated an interaction between Plk1 and the tumor suppressor protein p53, often dubbed the “guardian of the genome” for its vital role in maintaining genome stability. It is believed that the two proteins negatively regulate each other; for example, Plk1 phosphorylates p53 and down-regulates its activity, while PLK1 expression is in turn down-regulated by p53-driven transcriptional repression.
There has, therefore, been a great deal of interest in determining whether the status of p53 in a tumor would impact the efficacy of Plk1-targeted agents. The TP53 gene is among the most frequently altered in cancer, mutated in more than half of all cancers, making this an important question to answer. Research has shown that TP53-mutant cancer cells are more sensitive to Plk1 inhibitors than those with wild-type TP53.
A relationship between Plk1 and the RAS pathway has also been uncovered. Members of the RAS protein family form central regulatory nodes in a variety of cell signaling pathways and are highly mutated in a number of different cancers. Researchers have sought for more than a quarter of a century to develop small molecule inhibitors of the RAS family and have come up empty.
More recently, the focus has turned to using RNA interference screens to identify proteins that are synthetically lethal with RAS mutations, wherein the combination of the two mutations drives cell death. RAS-mutant cells were shown to be highly sensitive to the additional loss of genes with mitotic functions; in particular these cells were highly sensitized to Plk1 inhibition.
These and other findings, including the potential for synthetic lethality with the tumor suppressor protein phosphatase and tensin homolog (PTEN) and the results of a recent preclinical study reported at the 2014 American Society for Clinical Oncology meeting, which suggested that Plk1 may play a role in antiestrogen resistance in breast cancer, demonstrate that Plk1 still holds significant future clinical potential in the treatment of a wide range of particularly challenging forms of cancer.
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Onconova announces results from phase 3 ONTIME trial of rigosertib in higher risk myelodysplastic syndromes (MDS) [news release]. Newtown, PA: Onconova Therapeutics, Inc; February 19, 2014. http:// investor.onconova.com/releases.cfm. Accessed July 12, 2014.
Volasertib, Boehringer Ingelheim’s investigational oncology compound, is granted orphan drug designation for acute myeloid leukemia in the U.S. and EU [news release]. Ridgefield, CT: Boehringer Ingelheim Pharmaceuticals, Inc; April 17, 2014. http://goo.gl/6hbepD. Accessed September 3, 2014.
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