T-LAK cell-originated protein kinase (TOPK): an emerging target for cancer-specific therapeutics

‘Targeted’ or ‘biological’ cancer treatments rely on differential gene expression between normal tissue and cancer, and genetic changes that render tumour cells especially sensitive to the agent being applied. Problems exist with the application of many agents as a result of damage to local tissues, tumour evolution and treatment resistance, or through systemic toxicity. Hence, there is a therapeutic need to uncover specific clinical targets which enhance the efficacy of cancer treatment whilst minimising the risk to healthy tissues. T-LAK cell-originated protein kinase (TOPK) is a MAPKK-like kinase which plays a role in cell cycle regulation and mitotic progression. As a consequence, TOPK expression is minimal in differentiated cells, although its overexpression is a pathophysiological feature of many tumours. Hence, TOPK has garnered interest as a cancer-specific biomarker and biochemical target with the potential to enhance cancer therapy whilst causing minimal harm to normal tissues. Small molecule inhibitors of TOPK have produced encouraging results as a stand-alone treatment in vitro and in vivo, and are expected to advance into clinical trials in the near future. In this review, we present the current literature pertaining to TOPK as a potential clinical target and describe the progress made in uncovering its role in tumour development. Firstly, we describe the functional role of TOPK as a pro-oncogenic kinase, followed by a discussion of its potential as a target for the treatment of cancers with high-TOPK expression. Next, we provide an overview of the current preclinical progress in TOPK inhibitor discovery and development, with respect to future adaptation for clinical use.

serine/threonine kinase with sequence homology lying between the phylogenetic branches for MEK1/2 and MEK7, but with greater functional similarity to the p38activating MEKs, MKK3/6 1 . As a member of the dual specificity family of kinases, it was anticipated that TOPK would play a role in activation of JNK, ERK and Akt. Early studies suggested that TOPK facilitates transformation by upregulating and activating ERK2 through an increase in AP-1 transcriptional activity 2,3 . Indeed, a positive feedback loop between TOPK and ERK2 was found to amplify kinase signalling in HCT116 colon cancer cells. In this cell line, TOPK-mediated pathway activation was independent of B-Raf or C-Raf, leading the authors to conclude that a TOPK/ERK2 feedback loop may be contributing to transformation potential by bypassing the negative feedback loop regulating the Raf/MEK/ERK pathway 3 . This was later confirmed when TOPK was shown to activate ERK independently of MEK1/2 as part of the EGFR signalling pathway, and to contribute to MEK inhibitor resistance in MCF7 cells 4 . As a novel regulator of downstream Raf/MEK/ERK pathway activity, TOPK, therefore, provides an attractive potential target for chemotherapeutic inhibitor development.

Localisation, tissue distribution and function
TOPK expression is largely confined to tissues with high levels of proliferation. Its interest as an oncogenic target lies in the differential expression of TOPK in multiple tumour types relative to their normal tissue counterparts [5][6][7][8] . Despite this, TOPK knockout does not confer embryonic lethality in mouse models, with the only phenotype reported being an impaired inflammatory response to UV light exposure in the skin 9 . TOPK is expressed in both the nucleus and cytoplasm, however, some reports indicate that nuclear expression is exclusive to tumour cells and to those undergoing mitosis 6 , whilst others do not detect any difference in localisation 10 . TOPK mRNA is detected in several tissue types; and is most abundant in human tissues derived from testis, placenta and thymus 1,11,12 . Of these, the highest signal is measured in testicular tissue, with expression in this organ confined exclusively to spermatogenic germ cells in situ 13 . In brain tissue, TOPK-expressing cells arise from GFAP-positive neural progenitor cells in the adult sub-ependymal zone as well as in the external granule cell layer of the postnatal cerebellum 14 . Its expression is exclusive to proliferating and multipotent neurogenic precursors, and it is suppressed in mature or quiescent neurons.
TOPK expression and phosphorylation consistently increases as proliferating cells enter mitosis 13 ; and this process is regulated by Cdk1 in a Cyclin B1-dependent manner 12,15 . Although TOPK expression is closely associated with Cdk1 during the cell cycle 16 , the relationship between Cdk1 and regulation of TOPK phosphorylation is instead mediated via inactivation of protein phosphatase 1α (PP1α) through the Cyclin B1/Cdk1 complex 17 . Inactivation of PP1α causes TOPK to become phosphorylated at Thr9, promoting TOPK activation (Fig. 1). TOPK activity is primed to peak on entry into prophase, when it binds to and phosphorylates mitotic C 2 H 2 zinc finger proteins via its PDZ-binding domain 18 , and contributes to chromatin condensation by facilitating phosphorylation of histone H3 at Ser10 19 . Thus TOPK activity and expression contribute to the release of proliferating cells from midmitotic checkpoint control and maintain a timely exit from mitosis (Fig. 2).

Preclinical rationale: TOPK overexpression in cancer
High expression of TOPK has been linked to tumour aggressiveness, invasion and metastatic spread. Indeed, testing of clinical samples has revealed that a significant relationship exists between TOPK expression and poor prognosis in numerous cancers (Table 1). TOPK overexpression in cancer appears to be facilitated by suppression of microRNA-mediated regulatory control. Restoration of miR-216b-3p expression in lung cancer cell lines is sufficient to inhibit proliferation, enhance apoptotic cell death and overcome TOPK-associated downregulation of p53 and p21 20 .
Development of malignant potential is facilitated by TOPK-mediated responses to growth and migratory signalling. An increase in TOPK expression in JB6 epidermal cells accelerates cell growth and anchorage-independent colony formation in vitro, as well as promoting tumour formation in vivo 3,21 . Conversely, suppression of TOPK in cancer cell lines inhibits in vivo tumour growth 3,21,22 . In addition to its modulating effect on growth and migration, TOPK promotes epithelial-mesenchymal transition and facilitates tumour invasiveness. Anchorage-dependent growth and motility is enhanced by TOPK expression in prostate and breast cancer cell lines 5,6,10,23 , and overexpression of TOPK in metastatic prostate cancer is Activation of TOPK is initiated by Cdk1mediated dissociation of protein phosphatase 1 alpha (PP1α). TOPK activity is instrumental in the stabilisation of the Cdk1/Cyclin B complex between prophase and metaphase, which promotes spindle formation and correct chromosomal alignment. Rapid dephosphorylation of TOPK at the metaphase/anaphase border (midmitotic checkpoint) releases TOPK from Cdk1, enabling proteolytic degradation of Cyclin B by the anaphase promoting complex (APC). A drop in Cyclin B levels enables chromosomal migration to spindle poles and progression into anaphase associated with an increase in the expression of matrix metalloproteinases MMP-2 and -9 6 . These changes were attributed to nuclear localisation of β-Catenin and activation of TCF/LEF signalling at gene promotor regions. Nuclear translocation and activation of β-Catenin signalling targets MMP-7, Cyclin D1, and TCF-1 is also enhanced in hepatocellular carcinoma cell lines with increased TOPK expression, a result supported by clinical data showing correlation between high-TOPK levels and nuclear β-Catenin localisation in tissues from hepatocellular carcinoma patients 21 .
High-throughput screening has raised the possibility of increased TOPK expression in cancer being representative of stemness in tumours, however future investigations are needed to clarify the relationship between TOPK and proliferation in cancer stem cells (CSC). Nonetheless, TOPK was ranked fifth in a screen of glioblastoma and breast cancer cell lines conducted to identify potential cancer stem cell targets 24 , and was identified as a marker for proliferation potential in multipotent stromal cells derived from bone marrow 25 . Inhibition of TOPK has been effective in suppressing growth and survival in CSC subpopulations of small cell lung cancer cell lines, an outcome which is thought to be associated with suppression of FOXM1 activity-a key transcription factor regulating expression of late cell cycle control genes 26 .
TOPK expression appears to maintain haematopoietic stem cells in an undifferentiated state, as TOPK inhibition enhances production of platelets and megakaryocytes in vivo, and induces myeloid maturation in AML cell lines by downregulating STAT5 protein expression 27,28 . Hence, the interest in TOPK as a potential biomarker for patient stratification and risk assessment in cancers with high incidence of acquired treatment resistance and/or recurrence.

Therapeutic targeting of TOPK
TOPK has demonstrated potential as a therapeutic target for suppressing cancer development by overcoming treatment resistance, preventing invasion and metastatic growth, and promoting cell death signalling in oncogenic tissues. At a mechanistic level, this appears to occur through multiple interactions, and in a tumour-specific manner-predominantly due to the influence of TOPK overexpression on cancer cell physiology ( Table 2). TOPK promotes tumour dissemination by direct phosphorylation of p53-related protein kinase (PRPK) at its Ser250 residue, which in turn regulates the phosphorylation status of p53 and Akt. Through this mechanism, manipulation of TOPK expression in HCT116 colon cancer xenografts is sufficient to influence migration and metastatic growth in mouse liver tissue 29 . Upper row is stained for TOPK phosphorylated at its Thr9 residue (red). Lower row is stained for proteins bearing the phosphorylated form of the C2H2 zinc finger consensus linker sequence motif, HpTGEKP (red). Phosphorylation at this motif coordinates the dissociation of C2H2 zinc finger proteins (ZFP) from condensed chromatin, enabling mitotic progression 56 . TOPK has been identified as the primary kinase responsible for phosphorylation of HpTGEKP bearing proteins during mitosis 18 . Tubulin is counterstained green, and DNA is identified by DAPI staining (blue) TOPK T-LAK cell-originated protein kinase.
Relative expression of TOPK and its downstream target, PRPK, are also related to metastatic potential in colorectal carcinoma and squamous cell carcinoma 30,31 . Further, TOPK knockout mice are resistant to solar stimulated light-induced skin cancer 31 . For patients with acute myeloid leukaemia (AML), myeloid maturation in FTL3-ITD positive AML cells appears to be partially suppressed by a feedback loop between TOPK and the transcription factor responsible for granulocyte differentiation, CEBPA. In CD34 positive myeloblasts, inhibition of TOPK in FTL3-ITD positive AML promotes apoptotic cell death and increased myeloid differentiation 28 . TOPK activation increases during oxidative stress, and provides protection to cells in pro-inflammatory environments when overexpressed. TOPK-mediated phosphorylation of γH2AX and peroxiredoxin 1 significantly suppresses induction of apoptotic signalling cascades in melanoma cell lines, conferring resistance to arseniteinduced and UVB-induced apoptotic cell death 32,33 . Overexpression of TOPK was also found to provide protection against myocardial and neuronal ischaemiareperfusion injury by enhancing activation of the Akt signalling pathway and by upregulating expression of antioxidative proteins 34,35 . In both these investigations, inhibition of TOPK reduced cell viability and aggravated ischaemia-induced injury in vitro and in vivo.
Combination TOPK targeting in cancer therapy regimens has the potential to enhance treatment efficacy when coupled with DNA damaging agents. Suppression of TOPK activity proportionately increases the apoptotic fraction of tumour cells by upregulating pro-apoptotic p53 transcriptional targets, NOXA, BAK, FAS and Caspase 10 28 , downregulating Bcl-XL expression 36 and increasing PARP and caspase 3 cleavage via JNK and p38 signal pathway activation 22 . TOPK also confers resistance to TRAIL-induced apoptotic cell death 37 , which, along with IκBα phosphorylation and RelA nuclear translocation 38 , link TOPK activity to aberrant NF-κB signalling and cancer progression. An increase in polyploidy following doxorubicin treatment indicates that mitosis occurs aberrantly for TOPK overexpressing cells as a response to damaged DNA 39 . Indeed, TOPK knockdown or inhibition enhances radiation sensitivity in a tumour-specific manner by impeding resolution of DNA damage and increasing apoptotic cell death 40 . Taken together, increased TOPK expression in cancer cells promotes the Table 2 TOPK is a pro-oncogenic kinase with chemotherapeutic potential

Characteristic Mechanism References
Tumour dissemination PRPK phosphorylation (Ser250) by TOPK regulates p53-and Akt-mediated activation of tumour cell migration and invasion. 29 Proliferative potential, replicative immortality TOPK expression is regulated by a negative feedback loop via FLT3 expression and CEBPA phosphorylation. 28 Apoptotic resistance TOPK binds histone H2A, promoting nuclear colocalisation and phosphorylation of γH2AX. 32 TOPK and PRX1 colocalise in the nucleus. TOPK regulates PRX1 peroxidase activity by phosphorylation at Ser32. 33 Cell death signalling TOPK suppresses p53-mediated transcription of pro-apoptotic intermediates in tumour cells. 28,36 TOPK confers resistance to TRAIL-induced apoptotic cell death via NF-κB mediated transcriptional activity. 37 TOPK directly phosphorylates IκBα at Ser-32 and promotes RelA nuclear translocation. Overexpression enhances NF-κB and cIAP2 transcriptional activity. 38 Oxidative damage TOPK activation protects against cell death by enhancing the Bcl-2/Bax ratio. 34 pAkt and pTOPK colocalise in neural cells following ischaemia, increasing expression of peroxiredoxins-1 and 2, and thioredoxin-1. 35 TOPK suppresses JNK/p38 signal pathway activation during exposure to oxidising conditions. 22 DNA damage TOPK expression in cancer cell lines promotes resolution of chromosomal errors following DNA damage. 40

TOPK expression is regulated by E2F and CREB/ATF-mediated transcription. TOPK directly interacts with p53
and promotes molecular destabilisation. 39 Increased TOPK expression in cancer cells promotes the activation of multiple pathways involved in sustained growth and proliferation, evasion of cell death, invasive potential and metastatic growth. Inhibition of TOPK activity restores cellular responses to cell death signalling and replicative control mechanisms, as well as overcoming oncogenic chemoresistance by sensitising cancer cells to DNA damaging agents. Key: TOPK T-LAK cell-originated protein kinase, FLT3 fms-like tyrosine kinase 3 (CD135), CEBPA CCAAT/enhancer-binding protein alpha, NF-κB nuclear factor kappa light chain enhancer of activated B cells, TRAIL TNF-related apoptosis-inducing ligand, IκBα inhibitor of kappa B, CREB/ATF cAMP response element binding protein activating transcription factor, PRPK p53-related protein kinase, PRX1 Peroxiredoxin 1.
activation of multiple signalling pathways involved in reinforcing the hallmarks of cancer. Thus TOPK represents a master regulatory kinase whose manipulation has the potential to disrupt pro-carcinogenic processes, support the efficacy of DNA damaging agents and to overcome chemoresistance during cancer treatment.

Development of TOPK inhibitors
Three inhibitors have been developed to specifically target TOPK: HI-TOPK-032; 41 OTS514/OTS964; 27 and ADA-07 42 ( Table 3). The first of these, HI-TOPK-032, suppressed proliferation of colon cancer cells in vitro and partially suppressed colorectal and nasopharyngeal xenograft growth in vivo 22,43 . More recently, ADA-07 showed potential as a chemopreventive and therapeutic agent in the treatment of skin cancer. Inhibition of TOPK by topical application of ADA-07 suppressed development of solar ultraviolet-induced tumour development in mouse skin following exposure to solar irradiation 42 .
Two novel high potency compounds (OTS514 and its methylated derivative, OTS964) effectively inhibit lung tumour growth in murine xenografts when delivered by either oral or intravenous routes 27 . TOPK inhibition with OTS964 or OTS514 abolished ex vivo growth of patientderived ovarian cancer cells in a dose-dependent manner, and completely suppressed dissemination of ovarian malignancy in peritoneal xenografts 44 , although free OTS964 and OTS514 triggered significant weight loss and haematological toxicity during administration. Liposomal delivery was then adopted to improve tumour-specific uptake through the enhanced permeation and retention effect. This strategy prevented anaemia and leukocytopenia associated with intravenous treatment from developing, and enabled full recovery following oral delivery of the compounds. More recently, 18 F-labelled OTS964 has shown favourable pharmacokinetics and biodistribution when injected intravenously in a glioblastoma-carrying mouse model 45 . Preclinical validation of [ 18 F]FE-OTS964 is a promising first step towards the future use of TOPK inhibitors in a clinical context by enabling PET imaging of TOPK positive tumours.
Investigations into the mechanistic potential for tumour growth suppression by OTS514 in TOPK-expressing tumour cell lines have found that dose-dependent growth suppression and increased apoptotic cell death is associated with a reduction in FOXM1 activity and downregulation of TOPK and MELK expression 8,26 . These studies hint at a role for TOPK in stem cell differentiation through regulation of FOXM1-mediated transcriptional activity and MELK signalling. Indeed, whilst in vivo administration of OTS514 causes leukocytopenia, it concurrently increases the megakaryocyte subpopulation and peripheral platelets in treated mice, indicating that differentiation of haematopoietic stem cells is altered by Table 3 Characterisation of TOPK inhibitors-preclinical studies  Table 3 continued

Conclusions and future directions
Current therapeutic strategies to interfere with the proliferative potential of cancer involve either inhibition of mitogenic activating factors, or manipulation of cell cycle regulatory proteins. Of the mitogenic activators, dual specificity MEK homologues are highly prised potential targets for tackling cancers with dysregulated EGFR, Ras or Raf activity 46,47 . The small molecule inhibitors of MEK1/2 developed to date are highly potent and selective for the MEK-ERK pathway, however, systemic toxicity and acquired resistance have been limiting factors in clinical development 48 . The most promising regulatory targets are cyclin-dependent kinases (CDKs) (most recently CDK4/ 6 selective inhibitors) and checkpoint kinases (particularly WEE1, CHK1 and PLK inhibitors). However, despite demonstrating high potency in preclinical studies, early clinical trials with first and second-generation pan-CDK inhibitors have been disappointing. More highly selective CDK4/6 inhibitors have produced more encouraging outcomes, although dosing is limited by neutropoenia, and efficacy is modest when these inhibitors are used as single agents 49,50 . It appears that in order to maximise cancer treatment strategies in the future, a range of potential options will be required in the clinician's arsenal to personalise combination therapies and to combat refractory resistance. As an emerging alternative to the current cell cycle regulatory targets described above, TOPK's chemotherapeutic potential is threefold: (1) TOPK expression is enhanced in tumours arising from numerous histological subtypes and is suppressed in non-transformed cells from differentiated tissues. Dysregulation of TOPK appears to promote tumour growth and progression by enabling cancer cells to overcome cell death signalling pathways, bypass regulatory checkpoint control mechanisms and migrate beyond their point of origin. Hence, inhibition of TOPK is an attractive biochemical strategy for overcoming tumour aggression, metastatic growth and therapeutic resistance. (2) Expression in untransformed tissues is relatively low and appears to be non-essential to development or cellular function. TOPK activity rises during the progression towards mitosis and peaks in prophase, however evidence suggests that it is functionally redundant for cell division. Cells with TOPK knockdown continue to undergo growth and proliferation in vitro, and TOPK knockout mice are healthy, unlike MEK1 −/− mice, which are non-viable 51 . Consequently, TOPK inhibition is less likely to be associated with normal tissue toxicity compared to existing MEK1/2 and CDK inhibitors. (3) TOPK facilitates the fidelity and duration of mitosis in actively dividing tissues, predominantly via its influence over checkpoint control proteins. Enhanced expression of TOPK supports the demands of increased proliferative activity in carcinogenic tissue by optimising mitotic efficiency. Equally, suppression of TOPK activity weakens the regulatory efficacy of mid-mitotic signalling, with the potential to elongate the transition between prophase and anaphase and either stall, or delay, mitotic progression. Mitotic defects facilitate tumorigenesis 52 , however, complete disabling of mitotic checkpoint control has been proposed as a potential anticancer strategy [53][54][55] . Adaptation to TOPK-mediated signalling by cancer cells creates a dependence on TOPK for mitotic checkpoint control which can be exploited by chemotherapeutic inhibition in combination with DNA damaging agents. As yet, development of pharmaceutical strategies for targeting TOPK overexpression in cancer patients remain in the preclinical stage, and sensitivity and specificity for the majority of compounds developed to date is low. Of the possibilities outlined in this review, OTS514 and OTS964 are the only compounds which inhibit TOPK activity with high potency, however, these agents are particularly non-specific, with co-inhibition of other kinases demonstrated to various degrees in either case 27 . Both of these compounds have been associated with haematological toxicity in vivo, and therefore it is highly likely that their mechanism of action involves indirect targeting of multiple kinases, so their effect on tumour growth may be combinatorial. Nonetheless, novel MEK family members, such as TOPK, represent druggable targets of dysregulated signalling pathways with the potential to bypass the adverse outcomes posed by MEK1/2-targeting inhibitors. As single agents or in combination with therapeutics, selective TOPK inhibitors have the capacity to potentiate the impact of DNA damaging agents by abrogating cell cycle checkpoint control and by re-establishing proapoptotic signalling in resistant tumours.