Polo-like kinases in AML

Abstract

New therapies targeting critical elements of the cell cycle open novel and attractive avenues for the treatment of cancer patients. At present, the number of clinical trials that are registered with the European Organization for Research and Treatment of Cancer (EORTC) and with the US National Cancer Institute, which investigate the efficacy of Polo-like kinase 1 (Plk1) inhibitors against solid tumors and different types of leukemia is growing. Plks are important regulators of mitotic progression. Plk1, the best characterized mammalian Plk, has become an attractive target for cancer drug development, because most types of cancer appear to be addicted to the non-oncogene Plk1. Here, the authors discuss the role of Plk1 and the potential tumor suppressor gene Plk2 in acute myeloid leukemia (AML).

Keywords::

• AML
• cell cycle
• polo-like kinase
• targeted therapy

Acute myeloid leukemia (AML) is a rapidly developing neoplastic disease characterized by an accumulation of immature hematopoietic cells. Enhanced self-renewal and proliferation in combination with a differentiation block are important characteristics of the disease. Intensive, cytotoxic chemotherapy and allogeneic stem cell transplantation (SCT) are the current mainstays in the treatment of AML that allow curing the disease in a sizeable proportion particularly of younger patients [1]. The vast majority of AML patients are elderly, however, and long-term outcome in these patients is dismal, as the efficacy and tolerability of intensive chemotherapy is poor and allogeneic SCT generally is not an option. In case of refractory or recurrent leukemia, results of salvage treatment are poor irrespective of age. This highlights the unmet clinical need for novel therapeutic approaches for AML that are more specific and thereby less toxic.

Recognition of the molecular heterogeneity of AML has led to the delineation of prognostically distinct AML subsets associated with therapeutically targetable genetic aberrations. Examples include the impressive responses observed with arsenic trioxide and all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL) [2] and the ability to target mutations in tyrosine kinases such as fms-like tyrosine kinase receptor-3 (FLT-3) by specific tyrosine kinase inhibitors [3]. The clinical value of these novel compounds and their optimal combination with chemotherapy remain to be determined, and there have been unexpected setbacks; for example, combined treatment with the FLT-3 inhibitor sorafenib and chemotherapy failed to improve results achieved with chemotherapy alone in patients with unselected but also FLT-3 mutated AML [4,5]. In addition, the small number of patients with leukemia of a particular, genetically defined subtype are a challenge for the clinical development of these novel treatment strategy. On the other hand, involvement of pathways such as the WNT pathway [6,7], or alterations in the epigenetic regulation of genes do not seem to be restricted to certain AML subgroups and can be ameliorated by treatments with epigenetically active compounds such as the DNA methyltransferease (DNMT) inhibitors [8]. As self-renewal and proliferation are among the hallmarks of AML development, cell cycle progression may also represent an attractive target (Figure 1).

Figure 1. Plk1 in leukemic transformation. By increasing self-renewal and proliferation leukemic transformation may increase the requirement of Plk1.

Given the proven success of antimitotic therapies that change microtubule dynamics in the clinical treatment of cancer, it is reasonable to consider non-structural components of mitosis (e.g., cell cycle kinases like cyclin-dependent kinases, Aurora kinases A/B and Plks) as potential drug targets for therapy [9]. The observation that loss-of-function alleles of the Drosophila homolog of Plk1, Polo, cause zygotic lethality indicated early on the critical role of Polo for cellular proliferation [10,11]. To date, the human serine/threonine kinase Plk1 is the most comprehensively studied member of the Plk family (Plk1 – 5) [12]. In eukaryotic cells from yeast to man, orthologs of Plk1 play essential roles during mitosis and meiosis [10,11]. In vertebrate cells, Plk1 has been implicated in multiple steps of mitotic progression including the activation of Cdk1/cyclin B1, centrosome maturation, release of cohesion from chromosomes, activation of the anaphase-promoting complex/cyclosome (APC/C), formation of bipolar spindles and the regulation of the spindle assembly checkpoint (SAC) [10,11]. Very intensive and fundamental research to understand the nature, the molecular functions and the regulations of polo-like kinases in several model systems is still continuing to this day.

Correlating with its multiple mitotic functions, Plk1 as marker of cellular proliferation is only detectable in adult tissues containing proliferating cells, for example, ovary, testis, spleen and epithelial tissues of the gastrointestinal tract [13,14]. Remarkably, the levels of Plk1 in different types of tumors correlate inversely with patient survival, providing evidence for the role of Plk1 as prognostic marker [15-19] and/or marker of metastatic disease [20,21]. Recent genome-wide RNAi screens using various normal and isogenic cancer cells confirmed that cancer cells are addicted to a high level of non-oncogenic Plk1 for their viability [22]. The recent analysis of an inducible RNAi model in transgenic mice supported the view that cancer cells and proliferating primary cells differ clearly in their dependency to Plk1 [23,24]. With the development of antisense-oligonucleotide- and RNAi-based approaches loss-of-function screens were carried out with the aim of assessing the role of Plk1 in neoplastic phenotypes [25-29]. The essential mitotic role of Plk1 in numerous types of cancer cells and its elevated expression in human cancers stimulated the validation of Plk1 as a novel drug target and the pharmaceutical development of many small molecule inhibitors [12]. Targeting of Plk1 by novel compounds was judged to be specific based on a comparison with the biologic effects observed with RNAi. Based on the analysis of cancer cells lines different small molecule inhibitors were found to recapitulate the effects of Plk1 RNAi faithfully. BI 2536 is currently one of the most intensively studied Plk1 inhibitor and the first Plk1 inhibitor that entered the clinic [30,31]. Both inhibitors, BI 2536 and BI 6727 [4,32], completed Phase I studies and demonstrated good safety margins with preliminary evidence of antineoplastic activity representing the rational for further development in Phase II studies.

Investigations on the role of Plks in hematological diseases were scarce until recently, and documented only the elevated expression of Plk1 in lymphomas. Recent data describe the overexpression of Plk1 in hematological malignancies including AML compared with normal bone marrow mononuclear cells and normal peripheral blood mononuclear cells, suggesting that Plk1 is involved in leukemogenesis and serves as a promising target for the treatment of this disease [33,34]. In contrast to normal hematopoietic stem cells, AML cell lines as well as primary human AML cells overexpress Plk1 and are sensitive to Plk1 inhibition in vitro. Remarkably, considering that the majority of the leukemic blasts are mainly blocked in G₀/G₁, the observed elevated levels of Plk1 expression in AML samples are surprising. Therefore, the role of Plk1 in proliferating and non-proliferating AML cells remains to be elucidated. Using Plk1-specific small interfering RNA (siRNA) or small molecule inhibitors like BI 2536 or GW843682X it could be demonstrated that leukemic cell lines depend on Plk1 for their proliferation and freshly isolated leukemic cells from patients including those that were refractory to conventional therapies depend on Plk1 for their clonogenic growth [33,34]. The growth inhibition of leukemic cells by Plk1 inhibition was associated with G₂/M arrest and the induction of apoptosis.

Considering the level of sequence identity of the kinase domain of Plk1 with those of Plk2, Plk3 and Plk4, it is conceivable that ATP-competitive inhibitors of Plk1 scarcely discriminate between Plk1 – 4. Still, Plk1 inhibitors that are currently in clinical trials like GSK 461364, NMS-P937, BI 2536 and BI 6727 show different selectivity profiles for the family members Plk2 and Plk3 [12]. This is an important aspect also for the treatment of AML patients, because there are some indications that Plk2 and Plk3 can act as a tumor suppressors in vivo. For example, deletions in the long arm of chromosome 5, which are frequent chromosomal abnormalities in AML and human myelodysplastic syndrome (MDS), may contribute to the pathogenesis of these diseases by the loss of one or two tumor suppressor genes. Whether this chromosomal loss includes the Plk2 gene, which maps to chromosome 5 at 5q12.1–q13.2, has not been explored yet. To date, a tumor-suppressor function of Plk2 in hematopoietic diseases has been suggested in a study of B-cell malignancies, which describes a significant downregulation of Plk2 expression in a wide range of B-cell neoplasms due to a CpG methylation-dependent transcriptional silencing of Plk2 [35]. Interestingly, the ectopic expression of Plk2 in Burkitt lymphoma cells induced apoptosis, suggesting a selective pressure to abolish the function of Plk2 in this type of cancer. The Plk2 CpG island was also found to be hypermethylated in 68.9 and 88.4% of AML and MDS cases, respectively [36].

Further support for the role of Plk2 as a tumor suppressor came from a large-scale microRNA (miRNA) expression profiling study in core binding factor AML, an AML subtype representing 10 – 20% of primary AML [37]. This study documents an overexpression of miR-126/126* that was associated with a partial demethylation of the CpG island in which miR-126/126* is located. Certainly, miR-126 has been shown to downregulate Plk2 levels and might thereby contribute to leukemogenesis. Thus, it remains to be elucidated whether ATP-competitive Plk1 inhibitors that also target Plk2 and Plk3 are optimal for cancer therapy due to their activity of inhibiting two potential tumor suppressor genes. Recent developments of small molecule inhibitors like poloxin, poloxipan, and purpurogallin. focus on Plk1 inhibition by targeting the less conserved polo-box binding domain (PBD) [38-42]. Future studies need to answer the question whether Plk1 inhibition by targeting the PBD is the better cancer strategy to hit Plk1 specifically while sparing related Plk family members.

In the light of these considerations, a novel Plk inhibitor, NMS-937, which has an improved selectivity profile for Plk1 within the Plk family and is orally available, could have better long-term safety features in regard to effects in non-proliferating cells [43]. The combination of a Plk inhibitor with cytarabine has evolved as a promising treatment strategy which prolonged survival in a xenograft model of AML [43] and has proven safe in a Phase I trial of relapsed and refractory AML [4,44].

Considering the critical role of both, Plks and Aurora kinases, in cell cycle regulation, their reported overexpression in hematologic malignancies including AML and their crosstalk, the rationale for exploring inhibitors of both of these kinase families as therapeutic strategies in AML is similar [34,45]. Several inhibitors of the Aurora kinases are in fact in early-phase clinical testing [46]. It is noteworthy that the majority of these compounds are multikinase inhibitors with activity not only against Aurora A and B but also against other kinases including ABL, FLT-3 and Janus kinase (JAK2). While these kinases play a role in hematologic malignancies, it is unclear whether the lack of selectivity typically observed with Aurora kinase inhibitors will enhance their potency or compromise their utility due to greater toxicity of multikinase inhibition. Mucositis and mucosal inflammation appear to be the major dose-limiting toxicities associated with Aurora kinase inhibitors [47]. At present, the limited data that are available from the early stage clinical trials do not permit a comparison of Plk and Aurora kinase inhibitors. Nevertheless, it is conceivable that they may have variable clinical utility in different subtypes of leukemia, depending, for example, on the expression levels of Aurora kinases and Plks or the involvement of other kinases known to be involved in disease pathogenesis, for example, in leukemias with FLT-3 mutations.

Despite the attractiveness of Plk and Aurora kinase inhibitors as therapeutic agents for often highly proliferative diseases such as AML, it is almost certain that they eventually will need to be used in combination with other agents. Clinical development strategies for combination therapy will initially be restricted to drugs that are approved for treatment of AML and offer the potential for additive or synergistic activity. Accordingly, anthracyclines such as daunorubicin and cytosine arabinoside (ara-C) are the most plausible combination partners, even though the value of combining two agents with a cell cycle directed mode of action, that is, an Aurora kinase or Plk1 inhibitor with ara-C, may appear counterintuitive. However, preliminary data from preclinical studies with NMS-P937 and a Phase I trial exploring the administration of the Plk1 inhibitor BI 6727 with low-dose ara-C are promising and so far give no indication of antagonism between these two drugs [4,43,44]. The potent myelosuppressive activity of Plk1 inhibitors makes them particularly appealing as a combination partner for induction chemotherapy, which typically is based on the combination of an anthracycline with ara-C. The tolerability of such combinations need to be determined in the setting of Phase Ib studies and will determine their feasibility during subsequent post-remission therapy.

The potential value of combining a Plk1 inhibitor with the topoisomerase inhibitor etoposide is suggested by an in vitro study demonstrating that in U937 AML cells, Plk1 participates in checkpoint recovery: inhibition of Plk1 by the GW843682X compound results in mitotic accumulation and apoptosis, and that when challenged with VP-16, inhibition of Plk1 prevented U937 cells from checkpoint exit [48]. A theoretical caveat for combining these two classes of drugs is their shared propensity for inducing clinically relevant mucositis.

The concurrent use of a Plk1 inhibitor with a hypomethylating agent such as azacytidine is intriguing as it combines a cell cycle active compound with an agent considered to have antileukemic activity at the level of the leukemic stem cell, which is typically quiescent and should thus be largely spared by drugs acting primarily at the G₂/M phase of the cell cycle. Clinical development of such a combination will need to focus particularly on the proper scheduling, as both drug classes have overlapping toxicities related to myelosuppression.

Expert opinion

In recent years, Plk1 has become one of the most attractive cancer targets in the field of antimitotics. Several Phase I/II studies using Plk1 inhibitors for monotherapy have demonstrated manageable, non-cumulative and predominately hematological side effects that reflect the inhibitor's predicted mode of action. First indications of anticancer activity are promising, but more efforts are required to improve treatment regimens, identify combinatorial agents with synergistic activity like cytarabine in AML models, and define biomarkers reflecting treatment response and patient characteristics that predict therapeutic success. Future clinical studies should include promising markers of Plk1 activity like serine 46-phosphorylation of the translational controlled tumor protein (TCTP) [49] and an enzyme-linked immunosorbent assay (ELISA) [50]. Since various lines of evidence support the correlation of p53 mutation in human cancer and cellular levels of Plk1, patient stratification will help to identify patients with high Plk1 expression, who may respond very well to therapeutical approaches aiming at Plk1 inhibition.

Declaration of interest

G Bug and OG Ottmann have each received an honorarium for participation at an advisory board meeting organized by Boehringer Ingelheim GmbH & Co. KG, Germany. No funding was received in preparation of this article. The authors have no other competing interests to declare.

Acknowledgements

T. Berg and K. Strebhardt received funding from the LOEWE-excellence cluster ‘Onkogene Signaltransduktion Frankfurt’ (OSF). Oliver G. Ottmann is an endowed Professor der German Jose Carreras Leukemnia Foundation. This work was supported by grants from the Deutsche Krebshilfe, the BANSS-Stiftung and Carls-Stiftung.

Notes