Therapy-associated side effects in medulloblastoma have led to a concentrated search for novel therapeutic targets, particularly targets for which inhibition has radiosensitizing potential and minimal toxicities. Recent genomic studies have begun to unravel the molecular mechanisms involved in medulloblastoma, but have not yet resulted in novel therapeutic agents [2, 3, 27]. Analysis of protein kinase gene expression revealed that expression of multiple protein kinases was altered in medulloblastoma, including several components of the mitotic machinery such as aurora kinase A and PLK1 . Perturbing mitosis by disrupting the proper formation of mitotic spindles required for chromosome alignment and segregation has been shown to preferentially kill cancer cells .
It is well established that PLK1 plays an important role in cell cycle regulation by functioning in centrosome maturation, spindle formation, mitotic entry and cytokinesis. Elevated PLK1 levels have been found in many adult cancers, including breast and colorectal cancer, and in pediatric cancers, including neuroblastoma and rhabdomyosarcoma [20, 21, 29]. While PLK1 mRNA expression is upregulated in medulloblastoma, the significance of PLK1 in the pathogenesis and management of this pediatric brain tumor is not well understood.
In this study we demonstrate that PLK1 mRNA is overexpressed in two independent medulloblastoma cohorts when compared to normal cerebellum. Of note, fetal tissues expressed very high levels of PLK1 mRNA compared to adult brain tissues. This may reflect the critical role PLK1 in regulating mitosis. Indeed PLK1 is essential for progression into mitosis during embryonic development. PLK1-deficient cells displayed mitotic infidelity resulting in mitotic arrest and finally death during zebrafish embryogenesis . Furthermore, PLK1 homozygous null mice were found to be embryonic lethal and the incidence of tumors in PLK1 heterozygotes was three-fold greater than that in their wild-type counterparts, again emphasizing the importance of PLK1 in normal embryogenesis and development . Interestingly, not all tumor samples overexpressed PLK1 mRNA, further emphasizing the molecular heterogeneity of this tumor.
Decreasing the expression of PLK1 mRNA by RNAi clearly resulted in growth suppression and induction of apoptosis in medulloblastoma cells. Furthermore, we show that inhibition of PLK1 by a small molecule inhibitor, BI 2536, results in a significant reduction in the proliferation of medulloblastoma cells both in short-term and long-term assays. Importantly, IC50 values were in the low nanomolar range, which is in line with achievable therapeutic plasma concentrations demonstrated in clinical phase I/II trials of BI 2536 . Treatment with 5 nM BI 2536 in Daoy and 7.5 nM in ONS-76 medulloblatoma cells induced apoptosis, which is consistent with results found in other cancer cells .
BI 2536 (Boehringer Ingelheim, Ingelheim, Germany) is a first-in-class PLK1 inhibitor. Not only is it an ATP-competitive kinase inhibitor that inhibits the enzymatic activity of PLK1, it also shows over 1,000-fold selectivity for PLK1 against a large panel of other tyrosine and serine/threonine kinases [18, 29]. In dose-escalation Phase I trials, BI 2536 was well tolerated . Several Phase II studies are underway or have recently been completed for BI 2536 . In addition to BI 2536, there are several other inhibitors of PLK1 in development and undergoing clinical testing. These include BI 6727 (Boehringer Ingelheim, Ingelheim, Germany), GSK461364 (GlaxoSmithKline, Middlesex, UK) and HMN-214 (Nippon Shinyaku Co. Ltd, Kyoto, Japan). However, there are currently no clinical studies of PLK1 inhibitors in any pediatric cancers. Our data and those of Ackermann, et al., and Hu, et al., strongly argue for development of such studies in pediatric solid tumors [20, 21]. In particular our data show that PLK1 is a target in all subgroups of medulloblastomas making it ideal for clinical trials.
Radiation is a key component of medulloblastoma therapy. Unfortunately, it results in significant morbidity, particularly in very young patients . Thus, agents that radiosensitize medulloblastoma cells would be of great utility. Here we show that low nanomolar concentrations of BI 2536 strongly decreased the surviving fraction of tumor cells in response to radiation and increased the sensitizer enhancement ratios. These results indicate that BI 2536 can effectively enhance medulloblastoma cell radiosensitivity in vitro. These data are in accordance with previous studies showing increased radiosensitivity in malignant cells depleted of PLK1 mRNA by RNAi .
It has been hypothesized that medulloblastoma tumors contain stem cell-like tumor-initiating cells that are more resistant to therapy [33, 34]. Here we found that inhibition of PLK1 expression significantly decreased the tumor sphere size and decreased the expression of the stem cell marker SOX2. Interestingly, the decrease in stem cell markers was more pronounced in tumor spheres than in monolayer cells cultured in normal adherent conditions. Thus, there is a clear role for PLK1 in tumor-initiating cells, a finding hinted at in recent data from neuroblastoma . It will be important to elucidate in detail the specific mechanisms by which PLK1 mediates tumor-initiating cell growth.
In total, our data make a strong argument for further exploring the role of PLK1 inhibition in medulloblastoma. Regarding the PLK1 inhibitors, a second PLK1 inhibitor, BI 6727, has been used in several studies . BI 6727 is similar to BI 2536 in its in vitro activity in Daoy and ONS-76 medulloblastoma cell lines (Additional file 6: Figure S6). In in vivo studies, BI 6727 shows better toxicity and pharmacokinetic profile compared to BI 2536. The next step will be to perform carefully constructed animal studies. We plan to perform orthotopic cerebellar xenograft in vivo studies with BI 6727. We will especially take in to consideration dosing schedules and pharmacodynamic marker evaluation. These data will assist in developing future clinical trials.