MELK Research Articles

Maternal Embryonic Leucine Zipper Kinase (MELK) Reduces Replication Stress in Glioblastoma Cells

Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21(WAF1/CIP1), an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies.

Structural basis for the regulation of maternal embryonic leucine zipper kinase.

MELK (maternal embryonic leucine zipper kinase), which is a member of the AMPK (AMP-activated protein kinase)-related kinase family, plays important roles in diverse cellular processes and has become a promising drug target for certain cancers. However, the regulatory mechanism of MELK remains elusive. Here, we report the crystal structure of a fragment of human MELK that contains the kinase domain and ubiquitin-associated (UBA) domain. The UBA domain tightly binds to the back of the kinase domain, which may contribute to the proper conformation and activity of the kinase domain. Interestingly, the activation segment in the kinase domain displays a unique conformation that contains an intramolecular disulfide bond. The structural and biochemical analyses unravel the molecular mechanisms for the autophosphorylation/activation of MELK and the dependence of its catalytic activity on reducing agents. Thus, our results may provide the basis for designing specific MELK inhibitors for cancer treatment.

Orally administrative melk (maternal embryonic leucine zipper kinase)-targeting small molecule inhibitor suppresses the growth of various types of human cancer.

e13569 Background: We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also highly upregulated in multiple types of cancer including prostate, pancreas and lung cancers, and plays indispensable roles in cancer cell survival, particularly in the maintenance of tumor-initiating cells. In this study, we attempted to identify novel substrates of MELK and develop the small-molecule MELK inhibitor. Methods: To elucidate the MELK signaling pathway in breast cancer cells, we screened MELK substrates by 2D-PAGE and mass spectrometric analysis, and characterized two of them for their roles in mammary carcinogenesis. Furthermore, we conducted a high-throughput screening of a compound library followed by structure-activity relationship studies. We investigated the growth suppressive effect of a MELK inhibitor OTSSP167 using xenograft models in mice and a mammosphere-formation assay. Results: We identified two novel MELK substrates, DBNL and PSMA1, which plays critical roles in invasiveness of cancer cells and maintenance of mammary tumor-initiating cells. We successfully obtained a highly potent MELK inhibitor OTSSP167 with IC50 of 0.41 nM. OTSSP167 inhibited the phosphorylation of these two substrates by MELK as well as mammosphere formation of breast cancer cells, and exhibited strong tumor-growth suppressive effects on xenografts of human breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. Conclusions: Orally administrative MELK inhibitor OTSSP167 is a promising compound to treat various types of human cancer. This compound can inhibit the phosphorylation of MELK substrates and also suppresses the formation of cancer stem cells in breast cancer cells, providing a novel strategy to cure cancer.

A phase I/II study of multiple peptides cocktail vaccine for advanced/recurrent ovarian cancer.

TPS3116 Background: Despite improvement in chemotherapy including the molecular targeting agents, advanced or recurrent ovarian cancer (A/ROC) are still incurable. Some tumor associated proteins, hypoxia-inducible protein 2 (HIG2), and tumor related vascular endothelial growth factor receptor 1,2 (VEGFR1,VEGFR2) were found to be candidates as new targets, and their epitope peptides have been shown to have the ability to induce specific cytotoxic T lymphocyte ( CTL) responses. We conducted a phase I/II study of these peptides cocktail vaccine (PCV) in patients with A/ROC in order to evaluate toxicity, immunological response, and tumor response. Methods: 23 patients positive for human leukocyte antigen (HLA)-A2402 (A24) and 15 patients with HLA-A0201(A02) were enrolled in this study. Enrollment is still on going up to 30 evaluable patients in each group. PCV for A24 comprises FOXM1(Forkhead Box M1), MELK(maternal embryonic leucine zipper kinase), HJURP(Holliday junction recognition protein), VEGFR1 and VEGFR2. As for A02- PCV comprises HIG2, VEGFR1and VEGFR2. Cocktails were made at a dose of 1mg of each peptide with GMP grade-adjuvant, MONTANIDE ISA51. Vaccination schedule included weekly subcutaneous administration for first 12 weeks, then bi-weekly administration for next 16 weeks, then further vaccination was done monthly for 8 times and 3-6 months as a maintenance step according to patient’s need. Pre- and post-vaccination blood samples were obtained from the patients for toxicity assessment and immunological evaluation. Clinical responses were evaluated every three months by RECIST v 1.1. Results: PCV were generally well tolerated with no major adverse events, and most of the patients developed specific CTL responses. One patient showed complete response, two showed partial response and 10 showed stable disease in 22 evaluable patients. At the time of the analysis, median overall survival was 5 months (from 30 to 623 days) and 9 months(from 54 to 921 days),in A24 and A02, respectively. 11 patients remained alive with median follow up of 9 months. Conclusions: These findings suggest these peptides cocktail vaccines are safe and applicable for advanced/recurrent ovarian cancer. Clinical trial information: UMIN000003862, UMIN000003860.

Tumor-Specific Activation of the C-JUN/MELK Pathway Regulates Glioma Stem Cell Growth in a p53-Dependent Manner

Accumulated evidence suggests that glioma stem cells (GSCs) may contribute to therapy resistance in high-grade glioma (HGG). Although recent studies have shown that the serine/threonine kinase maternal embryonic leucine-zipper kinase (MELK) is abundantly expressed in various cancers, the function and mechanism of MELK remain elusive. Here, we demonstrate that MELK depletion by shRNA diminishes the growth of GSC-derived mouse intracranial tumors in vivo, induces glial fibrillary acidic protein (+) glial differentiation of GSCs leading to decreased malignancy of the resulting tumors, and prolongs survival periods of tumor-bearing mice. Tissue microarray analysis with 91 HGG tumors demonstrates that the proportion of MELK (+) cells is a statistically significant indicator of postsurgical survival periods. Mechanistically, MELK is regulated by the c-Jun NH(2)-terminal kinase (JNK) signaling and forms a complex with the oncoprotein c-JUN in GSCs but not in normal progenitors. MELK silencing induces p53 expression, whereas p53 inhibition induces MELK expression, indicating that MELK and p53 expression are mutually exclusive. Additionally, MELK silencing-mediated GSC apoptosis is partially rescued by both pharmacological p53 inhibition and p53 gene silencing, indicating that MELK action in GSCs is p53 dependent. Furthermore, irradiation of GSCs markedly elevates MELK mRNA and protein expression both in vitro and in vivo. Clinically, recurrent HGG tumors following the failure of radiation and chemotherapy exhibit a statistically significant elevation of MELK protein compared with untreated newly diagnosed HGG tumors. Together, our data indicate that GSCs, but not normal cells, depend on JNK-driven MELK/c-JUN signaling to regulate their survival, maintain GSCs in an immature state, and facilitate tumor radioresistance in a p53-dependent manner.

Abstract LB-300: Development of an orally-administrative MELK (maternal embryonic leucine zipper kinase)-targeting small molecule inhibitor that suppresses the growth of various types of human cancer.

Abstract Breast cancer is the most common malignancy among women worldwide. Treatment for breast cancer acting on molecular targets in critical signaling pathways in cancer cells have successfully reduced the motility rate in a subset of breast cancer patients, but a significant portion of patients expect to have little benefit from these treatments. Therefore, development of new therapeutics for those patients is urgently required. We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was highly upregulated in multiple types of human cancer including breast, prostate and lung cancers, and plays indispensable roles in cancer cell survival, particularly in the maintenance of tumor-initiating cells. Although several substrates of MELK were reported, we still do not understood fully how MELK contributes to breast cancer development and/or progression. To elucidate the MELK signaling pathway in breast cancer cells, we screened and identified two novel MELK substrates, DBNL and PSMA1, which were also found to play critical roles in human carcinogenesis and mammary tumor-initiating cells. Furthermore, we conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC50 of 0.41 nM. OTSSP167 inhibited the phosphorylation of these two substrates by MELK as well as mammosphere formation of breast cancer cells, and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. Here, we report the biological function of novel substrates of MELK and the first MELK inhibitor OTSSP167 that is a promising compound to treat various types of human cancer. Citation Format: Suyoun Chung, Hanae Suzuki, Takashi Miyamoto, Naofumi Takamatsu, Ayako Tatsuguchi, Koji Ueda, Kyoko Kijima, Yo Matsuo, Yusuke Nakamura. Development of an orally-administrative MELK (maternal embryonic leucine zipper kinase)-targeting small molecule inhibitor that suppresses the growth of various types of human cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-300. doi:10.1158/1538-7445.AM2013-LB-300

Abstract 2157: Targeting the Maternal Embryonic Leucin zipper Kinase (MELK) in gastric cancers.

Abstract Gastric cancer (GC) is the fourth most common cancer, and the second leading cause of cancer-related death in the world. The high mortality rate of gastric cancer is due to late-stage diagnosis and unpredictable outcome with empiric treatments. There are no tools to stratify therapies for ERBB2-negative GC (90% of patients). Even in ERBB2-positive patients, much better therapies are desirable. To identify novel GC drivers and therapeutic targets, we conducted a whole-transcriptome RNA-seq analysis in Caucasian cancers and paired normal gastric tissues. We identified 879 differentially expressed genes with FDR<0.01 & fold change (FC) >2. We next compared our results with those of Cui J. et al. (Nucl. Acids Res. 2011) which identified differential expressions in 80 tumor-normal pairs of Asian GC. With an FDR <0.01 & FC>2, we found 308 genes overlapping, including 30 kinases. Among the kinases, we found consistent high expression of the Maternal Embryonic Leucine zipper Kinase (MELK), the only member of the member of the Snf1/AMPK serine/threonine kinase family not regulated by LKB1 kinase, but rather through autophosphorylation. MELK is putatively involved in mitotic progression, inhibition of mRNA splicing, cytokinesis, and apoptosis. MELK overexpression has been found in other cancers, including human glioblastoma and breast cancers where it promotes tumorigenesis and portends to worse outcome. The precise in vivo regulation, function and crystal/NMR structures of MELK are still unknown; however the evidence that three MELK substrates are critical regulators of the mitotic entry, underscores the hypothesis that MELK inhibition may offer important novel therapeutic strategy. We investigated MELK protein expression in a panel of 30 gastric cell lines, and found high levels of MELK protein in 14/30. Similar results were observed in a panel of 22 colon carcinoma lines. MELK blockade using siRNA affected cytokinesis with the formation of polynucleated cells and aberrant mitotic spindle. Ongoing in vitro and in vivo investigations include cells with MELK knockdown by shRNA as well as MELK -/-, MELK +/+ cells, and MELK point mutation cells generated by Zinc Finger Nuclease Technology, and cells carrying MELK kinase dead; and identification of novel small compounds inhibiting MELK activity. Because of the increased expression in a broad spectrum of cancer sites, and the putative role in orchestrating mitotic entry, we believe that MELK offers important targeted therapeutic windows: 1) it will enhance the efficacy of antineoplastic drugs targeting mitosis; 2) by hindering the ability of tumor initiating cells to proliferate, it will impact the repopulation phase following chemo-radiation therapy, and therefore could be incorporated into molecular targeted strategies circumventing resistance to conventional therapy in MELK positive cancers. Citation Format: Julie G. Izzo, Kevin N. Dalby, Han Liang, Hui Yao, Brian P. James, Saira Ahmed, Xiuping Liu, Chang-Gong Liu, Yon Hui Kim, Garth Powis. Targeting the Maternal Embryonic Leucin zipper Kinase (MELK) in gastric cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2157. doi:10.1158/1538-7445.AM2013-2157 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 4369: Compound 1, a quinoxaline-based small molecule, regulates the cancer cell proliferantion by the down-regulation of maternal embryonic leucine zipper kinase gene expression in brain tumor cells.

Abstract Maternal embryonic leucine zipper kinase (MELK), a member of the snf1/AMPK family of serine-threonine kinase, was indicated in stem cell renewal, cell cycle progression. Recently, several groups reported that MELK expression level is highly correlated with malignancy of a variety of tumors and its expression in the cancer cells is a negative factor of radiotherapy in cancer patients. Therefore, MELK can be a good target for cancer drug development. Previously, we tested the neuronal cell differentiation by the compound-1, a quinoxaline-based small molecule, with rat mesenchymal stem cells, and we found that the gene expression level of MELK is dramatically decreased in the differentiated cells in comparison with the parental stem cells. Based on this phenomenon, we checked the MELK gene expression in the several central nerve system cancer cell lines such as U87MG, XL498 and SNB19, and examined the change of the gene expression level by treatment of compound 1. All the cancer cells tested in this study expressed the MELK gene and its expression level is significantly decreased by the treatment of 20 uM compound 1. To confirm the relationship between the MELK gene expression and cell proliferation in brain tumor cells, we transfected the MELK gene to the U87MG cells and tested the cell growth rate in the presence or absence of compound 1 in comparison with parental cells. The cell growth rate of MELK gene-transfected cancer cells increased in comparison with the non-transfected control cells, and its overexpression can recover the down-regulation of the cell proliferation by treatment of compound 1. Meanwhile, the treatment of MELK gene siRNA decreased the cell proliferation rate of the U87MG cells in time dependent manner. In the cell cycle analysis, compound 1 induced both the increase of apoptotic cell rate from less than 5% to 22% and the decrease of G0/G1 phase U87MG cells in comparison with control. Meanwhile, the S and G2/M phase is not changed by the treatment of compound 1 in the U87MG cells. These results support MELK has an effect on regulation of cancer cell proliferation, and it may be a good target for cancer drug development. Citation Format: Sang-Un Choi, Chi Hoon Park, Mansoor M. Ahmed, Stephen Yoo, Shin-Young Park, Kwangrok Kim. Compound 1, a quinoxaline-based small molecule, regulates the cancer cell proliferantion by the down-regulation of maternal embryonic leucine zipper kinase gene expression in brain tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2013-4369

Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer

We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC₅₀ of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer.

The maternal embryonic leucine zipper kinase (MELK) is upregulated in high-grade prostate cancer

Loss of cell cycle control is a prerequisite for cancer onset and progression. In prostate cancer, increased activity of cell cycle genes has been associated with prognostic parameters such as biochemical relapse and survival. The identification of novel oncogenic and druggable targets in patient subgroups with poor prognosis may help to develop targeted therapy approaches. We analyzed prostate cancer and corresponding benign tissues (n = 98) using microarrays. The comparison of high- and low-grade tumors (Gleason score ≥ 4 + 3 vs. ≤ 3 + 4) revealed 144 differentially expressed genes (p < 0.05). Out of these, 15 genes were involved in the cell cycle process. The gene maternal embryonic leucine zipper kinase (MELK) was identified to be highly correlated with cell cycle genes like UBE2C, TOP2A, CCNB2, and AURKB. Increased MELK gene expression in high-risk prostate cancer was validated by qPCR in an independent patient cohort (p < 0.005, n = 79). Immunohistochemistry analysis using a tissue microarray (n = 94) revealed increased MELK protein expression in prostate cancer tissues of high Gleason scores. RNAi-based inhibition of MELK in PC3 and LNCaP cells suggested putative function in chromatin modification, embryonic development and cell migration. The concerted inhibition of MELK and other cell cycle targets by the antibiotic siomycin A strongly impaired cell viability of prostate cancer cells, and may point to a novel therapy approach for a subset of high-risk prostate cancer patients.

Novel Inhibitor Discovery through Virtual Screening against Multiple Protein Conformations Generated via Ligand-Directed Modeling: A Maternal Embryonic Leucine Zipper Kinase Example

Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand binding. Such protein conformational plasticity remains a significant challenge in structure-based virtual screening methodology and may be approximated by screening against an ensemble of diverse protein conformations. Maternal embryonic leucine zipper kinase (MELK), a member of serine-threonine kinase family, has been recently found to be involved in the tumerogenic state of glioblastoma, breast, ovarian, and colon cancers. We therefore modeled several conformers of MELK utilizing the available chemogenomic and crystallographic data of homologous kinases. We carried out docking pose prediction and virtual screening enrichment studies with these conformers. The performances of the ensembles were evaluated by their ability to reproduce known inhibitor bioactive conformations and to efficiently recover known active compounds early in the virtual screen when seeded with decoy sets. A few of the individual MELK conformers performed satisfactorily in reproducing the native protein-ligand pharmacophoric interactions up to 50% of the cases. By selecting an ensemble of a few representative conformational states, most of the known inhibitor binding poses could be rationalized. For example, a four conformer ensemble is able to recover 95% of the studied actives, especially with imperfect scoring function(s). The virtual screening enrichment varied considerably among different MELK conformers. Enrichment appears to improve by selection of a proper protein conformation. For example, several holo and unliganded active conformations are better to accommodate diverse chemotypes than ATP-bound conformer. These results prove that using an ensemble of diverse conformations could give a better performance. Applying this approach, we were able to screen a commercially available library of half a million compounds against three conformers to discover three novel inhibitors of MELK, one from each template. Among the three compounds validated via experimental enzyme inhibition assays, one is relatively potent (15; K(d) = 0.37 μM), one moderately active (12; K(d) = 3.2 μM), and one weak but very selective (9; K(d) = 18 μM). These novel hits may be utilized to assist in the development of small molecule therapeutic agents useful in diseases caused by deregulated MELK, and perhaps more importantly, the approach demonstrates the advantages of choosing an appropriate ensemble of a few conformers in pursuing compound potency, selectivity, and novel chemotypes over using single target conformation for structure-based drug design in general.

MELK (maternal embryonic leucine zipper kinase)

Review on MELK (maternal embryonic leucine zipper kinase), with data on DNA, on the protein encoded, and where the gene is implicated.

Abstract 75: Identification and characterization of novel MELK (maternal embryonic leucine zipper kinase) substrates in breast cancer cells

Abstract Breast cancer is the most common malignancy among women worldwide. Treatment for breast cancer acting on molecular targets in critical signaling pathways in cancer cells have successfully reduced the motility rate in a subset of breast cancer patients, but a significant portion of patients cannot expect to have benefits from these treatments. Therefore, development of new therapeutics for those patients urgently required. Toward the goal of cancer-specific drug development, we previously identified MELK (maternal embryonic leucine zipper kinase) as a target for development of novel breast cancer therapy through genome-wide expression profile analysis. MELK was over-expressed in the great majority of breast cancer cells, particularly in TN breast cancer cells, and knockdown of MELK significantly suppressed the growth of breast cancer cells. Concordantly, the cells, which were transiently induced MELK, promoted its proliferation. Furthermore, cells in which over-expression of MELK were likely to increase the proportion of breast cancer-initiating stem cells more than those transfected with mock vector or kinase-dead MELK mutant plasmid. Although several substrates of MELK were reported, we are still not fully understood how MELK contributes to breast cancer development or progression. To elucidate the MELK signaling pathway in breast cancer cells, we examined 2D-PAGE and mass spectrometric analysis. Finally, we successfully identified two novel substrates of MELK. We confirmed the phosphorylation of these substrates by MELK via in vitro kinase assay and identified phosphorylation sites on each substrate by mass spectrometry. These results provide new information for better understanding of the biological role of MELK. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 75. doi:1538-7445.AM2012-75

Abstract A38: Maternal embryonic leucine zipper kinase (MELK) is upregulated in high-grade prostate cancer

Abstract Loss of cell cycle control is a prerequisite for cancer onset and progression. In prostate cancer, increased activity of cell cycle proliferating genes has been associated with prognostic parameters such as biochemical relapse and survival. The identification of novel oncogenic and druggable targets in patient subgroups with poor prognosis may help to develop targeted therapy approaches. We analyzed prostate cancer and corresponding benign tissues (n = 98) using microarrays. The comparison of high and low grade tumors (Gleason score ≥ 4+3 vs. ≤ 3+4) revealed 144 differentially expressed genes (p &amp;lt; 0.05), which are associated with a severe disease course. Out of these, 15 genes are assigned to cell cycle process. The gene maternal embryonic leucine zipper kinase (MELK) was identified to be highly correlated with cell cycle and prostate cancer associated genes like UBE2C, TOP2A, CCNB2 and AURKB, and was therefore further included into validation and functional experiments. The association between MELK gene expression and high-risk prostate cancer was validated in an independent patient cohort (p &amp;lt; 0.005, n = 79). Furthermore, our finding was supported by a public microarray dataset showing a higher MELK expression in prostate cancer patients with biochemical relapse. Immunohistochemical analysis using a tissue microarray (n = 94) revealed increased MELK protein expression in prostate cancer tissues of high Gleason scores. Knockdown of MELK decreased cell viability in PC3 and LNCaP cells. Microarray analysis upon MELK knockdown experiments revealed downstream processes like chromatin modification, embryonic development and cell migration. Our findings indicate that the protein serine/threonine kinase MELK represents a promising marker for advanced prostate cancer disease. Citation Format: Ruprecht Kuner, Holger Sültmann, Maria Fälth, Nicole Chui Pressinotti, Jan Christoph Brase, Sabrina Balaguer-Puig, Georg Schäfer, Georg Bartsch, Eberhard Steiner, Helmut Klocker. Maternal embryonic leucine zipper kinase (MELK) is upregulated in highgrade prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A38.

Phosphorylation of microtubule‐associated protein tau by AMPK‐related kinases

Microtubule-associated protein tau is abnormally hyperphosphorylated in the intracellular filamentous inclusions seen in neurodegenerative disorders with dementia, such as Alzheimer's disease and other tauopathies. Microtubule-associated protein/microtubule-affinity regulating kinases (MARKs) have previously been identified as kinases which phosphorylate KxGS motifs in the tandem repeats of tau. They are members of the 5'-AMP-activated protein kinase (AMPK)-related kinases in the Ca(2+)/calmodulin-dependent protein kinase group. In this study, we examined the ability of AMPK-related kinases, brain-specific kinases 1 and 2, maternal embryonic leucine-zipper kinase, MARK1, and salt-inducible kinase (SIK), to phosphorylate tau. We found that they phosphorylated S262 and S356 in KxGS motifs in the repeats of tau, thus resulting in immunoreactivity with antibody 12E8. MARK1 and SIK most effectively phosphorylated tau, and their down-regulation resulted in a reduction of 12E8-labelling. BX 795, an inhibitor of MARK1 and SIK, reduced 12E8-immunolabelling of tau in rat cortical neurons. These findings reveal a significant contribution of AMPK-related kinases to the phosphorylation of tau at S262/S356.

Resistance of colorectal cancer cells to radiation and 5-FU is associated with MELK expression

It was reported that the local recurrence would be caused by cancer stem cells acquiring chemo- and radio-resistance. Recently, one of the potential therapeutic targets for colorectal and other cancers has been identified, which is maternal embryonic leucine zipper kinase (MELK). MELK is known as an embryonic and neural stem cell marker, and associated with the cell survival, cell proliferation, and apoptosis. In this study, SNU-503, which is a rectal cancer cell line, was treated with radiation or 5-fluorouracil (5-FU), and elevation of the MELK expression level was observed. Furthermore, the cell line was pre-treated with small interfering RNA (siRNA) against MELK mRNA before treatment of radiation or 5-FU and its effects on cell cycle and proliferation were observed. We demonstrated that knockdown of MELK reduced the proliferation of cells with radiation or 5-FU treatment. In addition, MELK suppression caused changes in cell cycle. In conclusion, MELK could be associated with increased resistance of colorectal cancer cells against radiation and 5-FU.

Cortical localization of maternal embryonic leucine zipper kinase (MELK) implicated in cytokinesis in early xenopus embryos.

MELK has been implicated in a large variety of functions. Because its level is elevated in cancer tissues and it is involved in cell proliferation, MELK is considered as a potential therapeutic target for cancers. In a recent, study we have shown that MELK is involved in cytokinesis in early Xenopus laevis embryos. MELK dynamically accumulates at the cell cortex including a narrow band corresponding to the presumptive division furrow shortly before cytokinesis onset. MELK co-localizes and interacts with anillin an important regulator of cytokinesis. In addition, MELK overexpression interferes with accumulation at the cleavage furrow of activated Rho GTPase another crucial regulator of cytokinesis. Interestingly, our study also revealed that a transition implying a change in the direction of asymmetric furrow ingression occurs during early development. After this transition, MELK, as well as other proteins involved in cytokinesis, do not localize anymore as a band at the equatorial cortex but still localizes at the cell cortex. Our results indicate that cortical localization is an important feature of MELK in X. laevis embryos.

Cortical localization of Maternal Embryonic Leucine zipper Kinase (MELK) implicated in cytokinesis in early Xenopus embryos

MELK has been implicated in a large variety of functions. Because its level is elevated in cancer tissues and it is involved in cell proliferation, MELK is considered as a potential therapeutic target for cancers. In a recent, study we have shown that MELK is involved in cytokinesis in early Xenopus laevis embryos. MELK dynamically accumulates at the cell cortex including a narrow band corresponding to the presumptive division furrow shortly before cytokinesis onset. MELK co-localizes and interacts with anillin an important regulator of cytokinesis. In addition, MELK overexpression interferes with accumulation at the cleavage furrow of activated Rho GTPase another crucial regulator of cytokinesis. Interestingly, our study also revealed that a transition implying a change in the direction of asymmetric furrow ingression occurs during early development. After this transition, MELK, as well as other proteins involved in cytokinesis, do not localize anymore as a band at the equatorial cortex but still localizes at the cell cortex. Our results indicate that cortical localization is an important feature of MELK in X. laevis embryos.

Expression of stem cell marker and receptor kinase genes in glioblastoma tissue quantified by real-time RT-PCR

Glioblastoma is dependent on a specific signaling pathway to maintain its tumor phenotype. The receptor tyrosine kinase (RTK) family mediates the multiple oncogenic growth factor receptor signaling and contributes to the pathogenesis of glioblastoma. Recently, many studies have shown that the expression of stem cell marker in glioblastoma tissue has prognostic significance, which indicates that the quantification of stem cell markers and RTK genes yields biological information about glioblastoma. In this study, we quantified RNA expression levels of stem cell markers [CD133, Nestin, BMI-1, maternal embryonic leucine zipper kinase (MELK), and Notch1–4] as well as RTKs (EGFR, ErbB4, VEGFR1-3, FGFR1, -2, PDGFRΑ, and PDGFRΒ) in 42 clinical samples of glioblastoma by the real-time RT-PCR method. We demonstrated that the expression of MELK is exclusively upregulated in glioblastoma tissue. Notch receptor expression is moderately upregulated and is correlated with that of VEGFR2, VEGFR3, and PDGFRβ. Unsupervised clustering identified one unique sample group that showed high expression of most of the genes analyzed. Our results suggest that quantification of these stem cell markers and RTK genes can stratify patients based on the expression profile, which might provide insight into the glioma biology in each cluster.

Siomycin A targets brain tumor stem cells partially through a MELK-mediated pathway

Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.