Targeting Echinococcus multilocularis Stem Cells by Inhibition of the Polo-Like Kinase EmPlk1

Abstract

BackgroundAlveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation.Methodology/Principal findingsWe herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation.Conclusions/SignificanceWe conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy.

Recent advances in Echinococcus genomics and stem cell research

“…Promising drug targets do not have to be specifically expressed in germinative cells but they should 'also' be expressed in these cells. If promising targets are exclusively expressed in germinative cells, such as the Polo-like kinase EmPlk1 (Schubert et al, 2014), respective drug treatment can be combined with benzimidazole therapy in order to affect a broad range of Echinococcus cells. As an aid for targeted drug design, we are currently carrying out, in cooperation with the Wellcome Trust Sanger Institute, extensive transcription profiling of all E. multilocularis genes in various life cycle stages such as primary cells, metacestode vesicles with and without brood capsules, and protoscoleces before and after activation with low pH/pepsin (all preparations in triplicate).…”

“…In a different study with a more direct connection to possible clinical applications, metacestodes and primary cells of E. multilocularis where treated in vitro for weeks with low concentrations (5-100 nM) of BI 2536, a highly specific inhibitor for polo-like kinase 1 (PLK-1) (Schubert et al, 2014). PLK-1 is an important regulator of mitosis, and as such, was validated as an anti-cancer drug target (Archambault and Glover, 2009).…”

“…PLK-1 is an important regulator of mitosis, and as such, was validated as an anti-cancer drug target (Archambault and Glover, 2009). Furthermore, the E. multilocularis ortholog emplk-1 is specifically expressed in germinative cells, and upregulated in primary cell cultures (Schubert et al, 2014). Treatment with BI 2536 at concentrations as low as 25 nM resulted in an almost complete elimination of proliferating cells in metacestode vesicles and drastically blocked the regeneration of new metacestode vesicles from primary cells.…”

“…Furthermore, due to their importance in malignant transformation, the biochemistry of kinases is exceptionally well studied and research has already brought up a plethora of inhibitory compounds, many of which are currently in use to treat various forms of cancer (Brehm, 2014). Several prominent kinase inhibitors such as Imatinib, directed against Abl-kinases, BI 2536 (Polo-like kinase) or pyridinylimidazoles (p38 MAPK) have already been shown to exert anti-Echinococcus activities in vitro by inhibition of the orthologous parasite factors (Gelmedin et al, 2008;Hemer et al, 2014;Schubert et al, 2014) and are thus promising lead compounds for the design of related small molecule compounds that show a high specificity for parasite kinases (Brehm, 2014). Interestingly, genomic and transcriptomic analyses also revealed why some drugs are not effective against larval cestodes.…”

The role of fibroblast growth factor signalling in Echinococcus multilocularis development and host-parasite interaction

“…Since primary cell preparations are characterized by a much higher content of germinative (stem) cells than metacestode vesicles [6], these expression patterns could indicate that emfr3 is stem cell specifically expressed. We therefore carried out semi-quantitative RT-PCR experiments on cDNA preparations from in vitro -cultivated metacestode vesicles (MV) versus metacestode vesicles after treatment with hudroxyurea (MV-HU) or the Polo-like kinase inhibitor BI 2536 (MV-BI), in which the stem cell population had been selectively depleted [6,42]. While emfr1 was well expressed in MV as well as MV-HU and MV-BI, both emfr2 and emfr3 transcripts were barely detectable in MV-HU and MV-BI (data not shown).…”