Polo-like kinase 1
Polo-like kinases (PLK) are serine/threonine kinases with major role in cell cycle and mitosis. Since overexpression of Plk1 has been observed in different cancers, Plk1 inhibitors have emerged as targeted therapies for cancer.
The PLK kinase family is characterized depending on C- terminal polo-box domains (PBD) that facilitates the protein interaction and regulates N-terminal serine/threonine kinase domain (KD)
There has been 5 groups of PLK identified in humans. All of the family members of this kinase have a common N-terminal kinase, and one or more PBD responsible from binding to the substrate. Plk1 is the first polo-like kinase that is identified and has critical role in cell division.
Other PLK’s are identified in two different subgroups. Plk2, 3 and 5 are included in the PLK2 subgroup. Plk2 expression and activation peaks during the S phase and its inhibition results in abnormal centriole formation. However, since its function can be done by other PLK’s, it doesn’t have a critical role for the cell. Plk3 has important role in G1/S phase transition. It increases the DNA replication with inducing Cyclin E accumulation and activation of Cdc25A. Plk5 is the newest member of the group.
Plk5 expression has been shown to be associated with cell cycle arrest in G1 phase and as a result of this decreased DNA synthesis and apoptosis. Plk5 doesn’t need kinase domain for its function. Plk4 is another group of PLK having a major role in centriole duplication. Impaired Plk4 function leads to abnormalities in centriole biogenesis and aberrant cell division and mitosis.
Plk1 is the oldest and mostly studied Plk family member and its association with cancer has been well-defined.
Function of Plk1 (polo-like kinase 1)
Plk1 was discovered as a kinase with important function in mitosis and it has a critical role in cell cycle progression, centrosome maturation, kinetochore-mictorubule binding, bipolar spindle formation and cytokinesis.
Cell cycle is the event leading cell division formed by G1 (Gap 1), S (Synthesis), G2 (Gap2) and M (Mitosis) phases. These phases are tightly regulated by cyclins and cyclin dependent kinases (cdk). Plk1 expression has been found to be associated with these phases of cell cycle. Plk1 starts to accumulate in S phase and peaks at G2-M transition. It makes the plateau during mitosis and rapidly falls down after mitosis.
Plk1 interacts with various protein for its effect on cell cycle. Plk1 controls G2/M transition through its interaction with Cdk1/cyclinB complex. For the activation of Cdk1/cyclinB complex, its inhibitory phosphatases have to be removed by the Cdc25 phosphatase. Plk1 directly phosphorylates and activates Cdc25 then this activates Cdk1/cyclin B complex which facilitates transition of G2 phase to mitosis. Additionally, Plk1 phosphorylates and inhibits Wee1 which negatively controls Cdk1. So Plk1 has a critical role in G2/M phase.
Plk1 also regulates nuclear envelope breakdown (NEBD) which is an important step in transition from G2 phase to mitosis. Its regulation on NEBD is through phosphorylation of p150Glued phosphorylation which is a part of dynein/dynactin complex having an important role in prophase. Plk1 has phosphorylates CLIP-170 in cells entering the mitosis and facilitates the kinetochore- microtubule binding in prometaphase.
In this phase, Plk1 also phosphorylates Sgt1 and leads to stabilization of MIS12 in kinetochore and increases microtubule binding with NDC80-MIC12 complex formation. During anaphase, in addition to its regulation on Cdk1/cyclin B, Plk1 phosphorylates and activates the APC/C (Anaphase-promoting complex/cyclosome) complex which regulates the exit from mitosis and plays another role in the last phase of mitosis.
Plk1 function is also regulated by different mechanisms. Plk1 transcription is regulated by transcription factors like p53, E2F family and forkhead box protein M1. Activation of Plk1 is through the phosphorylation od Threonin residue (Thr210) in the loop domain by kinases like Bora/Aurora A. Additionally, various kinases/phosphatases effecting PBD domain play role in regulating Plk1 activity.
Association of Plk1 and cancer
Cell cycle abnormalities are seen commonly in cancer cells. Once the critical role of Plk1 in cell cycle was identified, its role in cancer has been investigated. Overexpression of Plk1 has been observed in different solid tumors and leukemia. Plk1 overexpression has also been to be associated with poor prognosis and survival in cancers like lung cancer and head and neck cancer.
In the cancer cells, Plk1 interacts with vital signaling pathways, transcriptional factors, and DNA damage proteins in addition to its role in cell cycle. So Plk1 was seen as an appropriate target for cancer and Plk1 inhibitors have emerged.
Inhibition of Plk1 in cancer
Downregulation of Plk1 expression with siRNA (small interfering RNA) in normal and cancer cells leads to apoptosis in cancer cells while having no effect on normal cells. This suggested that Inhibition of Plk1 could be an effective method in cancer treatment and small molecule inhibitors of Plk1 have been developed. These inhibitors target adenosine-5 triphosphate (ATP) binding sites in kinase domain and PBD.
GSK461364A is a thiophene derivative which is potent inhibitor of ATP binding site of Plk1. Since its effect on solid and liquid cancer cell lines have been shown, a phase 1 study was done. The major side effects were venous thromboembolism and myelosuppression. GSK461364A was also shown to increase radiosensitivity in glioblastoma cell lines.
GW843682 is a ATP-competitive inhibitor of Plk1. It was shown to lead to apoptosis and G2/M arrest in solid tumor cell lines.
BI2536 is another ATP-competitive inhibitor of Plk1. It also has inhibitory effect on Plk2 and Plk3. However BI2536 was also shown to have inhibitory effects on other kinases. The clinical effect of BI2536 hasn’t been proven and patients had neutropenia as major side effect in several clinical trials.
BI6727 is in the same chemical group of BI2536 and has similar mechanism of action with BI2536. However, it is more potent and the side effect profile is better. Preclinical studies have shown the effect of BI6727 in both solid and liquid tumors and has minimal effect on normal cell lines. So clinical studies have been designed and it was tolerated in different phase I and phase II studies with some antitumor activity.
ON01910 is a non-competitive ATP inhibitor with dual Plk1 and PI3 kinase inhibitor effect. The ED50 of this drug was shown below 200nM in in vitro studies for multiple different cancer cell lines. However its effect on Plk1 is not very potent and has multiple kinase targets other than PI3 kinase. Phase I studies showed that ON01910 was well in different clinical trials. However, it didn’t show significant effect in a phase III trial of myelodysplastic syndrome and a phase III trial of combination with gemcitabine for pancreatic cancer.
TKM-080301 has a different mechanism of action and is a nanoparticule compound with a siRNA targeting Plk1. Preclinical studies showed in vitro and in vivo effect and phase I studies are ongoing.
In addition to these inhibitors targeting kinase domains, PBD domain inhibitors have also been developed. PLHS-Pmab, purpurogallini and poloxin are some of these inhibitors.
Inhibition of Plk1 in anaplastic thyroid cancer
Since the Plk1 was identified as an important protein in cell cycle, its expression in various cancers has been investigated. One of the first studies done on thyroid cancers, identified association of Plk1 overexpression in papillary thyroid carcinoma. 43.7% of papillary thyroid carcinomas were found to have Plk1 overexpression. 11.3% of anaplastic carcinomas had Plk1 overexpression in this study. In another study investigating the gene expression profile of anaplastic thyroid cancer, upregulation of 54 genes involved in cell cycle and chromosome segregation was found. Plk1 overexpression was among these genes identified. Plk1 was found to be negatively regulated by p53 in anaplastic thyroid cancer. Silencing of Plk1 with RNA interference caused cell cycle arrest and cell death in anaplastic thyroid cancer cells.
Following the development of Plk1 inhibitors, some of the inhibitors were tried in anaplastic thyroid cancer in preclinical studies. BI2536 was the first drug tested in anaplastic thyroid cancer. BI2536 inhibited cell proliferation in vitro and in vivo. It also induced cell cycle arrest and mitotic cell death.
In a recent study done in our laboratory, another Plk1 inhibitor GSK461364A was tested in anaplastic thyroid cancer. It was found to have inhibitory effect in vitro and in vivo in anaplastic thyroid cancer. GSK461364A induced G2/M arrest, however Plk1 inhibition in cell lines with PI3K activation resulted in escaping growth arrest and subsequent mitotic catastrophe through mitotic slippage. As the PI3K activation is common in anaplastic thyroid cancer, there is risk of generating polyploid, genetically unstable cell populations by targeting these tumors with a Plk1 inhibitor. So combination of PI3K and Plk1 inhibitor might be effective in PI3K active tumors.