Overexpression Of Maternal Embryonic Leucine Zipper Kinase Research Articles

MiR-181a/b-5p negatively regulates keratinocytes proliferation by targeting MELK.

Accumulating evidence indicates that microRNAs (miRNAs) have a vital effect on the pathogenesis of psoriasis. This study is conducted to investigate the potential involvement of miR-181a-5p and miR-181b-5p in the proliferation of HaCaT keratinocytes. Cell viability and proliferation were evaluated respectively in this study using the CCK-8 and the 5-ethynyl-2'-deoxyuridine (EdU) assays. The expression of Maternal Embryonic Leucine Zipper Kinase (MELK) and Keratin 16 (KRT16) mRNA and protein in tissues and cells was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The Luciferase reporter system analyzes the connection between miR-181a-5p/miR-181b-5p and MELK. The results showed that miR-181a/b-5p expression was downregulated in the psoriasis lesions and negatively regulated the proliferation of keratinocytes. MELK was directly targeted by miR-181a-5p/miR-181b-5p. In addition, HaCaT keratinocytes proliferation was inhibited by knockdown of MELK while promoted dramatically by MELK overexpression. Notably, miR-181a/b-5p mimics could attenuate the effects of MELK in keratinocytes. In conclusion, our research findings suggested miR-181a-5p and miR-181b-5p negatively regulate keratinocyte proliferation by targeting MELK, providing potential diagnostic biomarkers and therapeutic targets for psoriasis.

Up-Regulation of MELK Promotes Cell Growth and Invasion by Accelerating G1/S Transition and Indicates Poor Prognosis in Lung Adenocarcinoma.

Maternal embryonic leucine zipper kinase (MELK) is an oncogene in many tumors, although its contribution to lung adenocarcinoma (LUAD) is unclear. We examined MELK expression in patient LUAD tissue and matched healthy lung tissues. We investigated the connection between MELK expression and tumor differentiation, lymph node metastasis, and patient survival. We downregulated MELK expression using small-hairpin RNA to assess its impact on LUAD cell proliferation, clonogenicity, and invasion. We also investigated the molecular mechanism underlying these effects. MELK expression was significantly heightened in LUAD tissue as opposed to the matching healthy lung tissues. LUAD patients who had MELK overexpression had a worse prognosis. Suppression of MELK hinders proliferation, clonogenicity, and invasion of LUAD cells. The MELK suppression led to the arrest of the cell cycle's G1/S phase by reducing the cyclin E1 and cyclin D expression. Our outcomes manifest that MELK can function as a beneficial prognostic indication and a new therapy target for LUAD. MELK has an essential function in progressing LUAD, manifesting potential as a viable target for therapeutic intervention in this disease management.

Targeting MELK improves PD-1 blockade efficiency in cervical cancer via enhancing antitumor immunity

The balance between T helper 1 (Th1)/T helper 2 (Th2) has critical function in determining intratumoral immune response and anti-PD-1 immunotherapy. The level of maternal embryonic leucine zipper kinase (MELK) is reported to correlate with infiltrating of immune cells in cancers, but the underlying molecular mechanism is not clarified. In the present study, we are aimed to elucidate the potential function of MELK in cervical cancer. We found that MELK was upregulated and acted an oncogenic role in cervical cancer. MELK overexpression shifted Th1/Th2 balance towards Th2 predisposition in mouse cervical tumors in vivo and naïve T cells from human PBMCs in vitro, while MELK knockdown exhibited opposite effects. MELK overexpression activated NF-κB signaling and promoted IL-6 secretion by cervical cancer cells. Depletion of IL-6 by neutralization antibodies abrogated the influence of MELK on Th1/2 balance. Besides, MELK modulated the antitumor activity of cytotoxic CD8+ T cells in cervical tumors, but depletion of Th2 cells by IL-4 neutralization abrogated this effect. Finally, MELK overexpression conferred tolerance to PD-1 blockade in cervical tumors, while targeting MELK by OTSSP167 significantly enhanced PD-1 blockade efficiency. Our data elucidated a novel role of MELK in regulating Th1/Th2 balance and anti-PD-1 immunotherapy in cervical cancer.

Preclinical assessment of synergistic efficacy of MELK and CDK inhibitors in adrenocortical cancer

BackgroundAdrenocortical cancer (ACC) is a rare and aggressive cancer with dismal 5-year survival due to a lack of effective treatments. We aimed to identify a new effective combination of drugs and investigated their synergistic efficacy in ACC preclinical models.MethodsA quantitative high-throughput drug screening of 4,991 compounds was performed on two ACC cell lines, SW13 and NCI-H295R, based on antiproliferative effect and caspase-3/7 activity. The top candidate drugs were pairwise combined to identify the most potent combinations. The synergistic efficacy of the selected inhibitors was tested on tumorigenic phenotypes, such as cell proliferation, migration, invasion, spheroid formation, and clonogenicity, with appropriate mechanistic validation by cell cycle and apoptotic assays and protein expression of the involved molecules. We tested the efficacy of the drug combination in mice with luciferase-tagged human ACC xenografts. To study the mRNA expression of target molecules in ACC and their clinical correlations, we analyzed the Gene Expression Omnibus and The Cancer Genome Atlas.ResultsWe chose the maternal embryonic leucine zipper kinase (MELK) inhibitor (OTS167) and cyclin-dependent kinase (CDK) inhibitor (RGB-286638) because of their potent synergy from the pairwise drug combination matrices derived from the top 30 single drugs. Multiple publicly available databases demonstrated overexpression of MELK, CDK1/2, and partnering cyclins mRNA in ACC, which were independently associated with mortality and other adverse clinical features. The drug combination demonstrated a synergistic antiproliferative effect on ACC cells. Compared to the single-agent treatment groups, the combination treatment increased G2/M arrest, caspase-dependent apoptosis, reduced cyclins A2, B1, B2, and E2 expression, and decreased cell migration and invasion with reduced vimentin. Moreover, the combination effectively decreased Foxhead Box M1, Axin2, glycogen synthase kinase 3-beta, and β-catenin. A reduction in p-stathmin from the combination treatment destabilized microtubule assembly by tubulin depolymerization. The drug combination treatment in mice with human ACC xenografts resulted in a significantly lower tumor burden than those treated with single-agents and vehicle control groups.ConclusionsOur preclinical study revealed a novel synergistic combination of OTS167 and RGB-286638 in ACC that effectively targets multiple molecules associated with ACC aggressiveness. A phase Ib/II clinical trial in patients with advanced ACC is therefore warranted.

Ectopic expression of MELK in oral squamous cell carcinoma and its correlation with epithelial mesenchymal transition.

Epithelial–mesenchymal transition (EMT) is closely correlated to metastasis formation generation and maintenance of cancer stem cells, nevertheless, the underlying mechanisms are unclear. The aim of this study is to investigate the role of maternal embryonic leucine-zipper kinase (MELK) in EMT regulation in oral squamous cell carcinoma (OSCC). We found that there was overexpression of MELK in human OSCC tissues, and high MELK expression was correlated with lymphatic metastasis and led to poor prognosis in patients with OSCC. We also confirmed that MELK is closely correlated to the EMT process using a human OSCC tissue microarray. Additionally, MELK expression was observed to be regulated in several OSCC cell lines, and knockdown of MELK genes inhibited cell proliferation, migration, invasion and EMT of OSCC cells in vitro. Furthermore, silencing of MELK suppressed tumour growth in vivo, and experimental research verified that MELK may augment OSCC development via mediating the Wnt/Notch signalling pathway. Our findings suggest that MELK serves as an oncogene to improve malignant development of OSCC via enhancing EMT, and MELK might be a potential target for anticancer therapeutic.

Phillygenin, a MELK Inhibitor, Inhibits Cell Survival and Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells.

IntroductionPancreatic cancer (PC) is one of the leading causes of cancer, with the lowest 5-year survival rate of all cancer types. Given the fast metastasis of PC and its resistance to surgery, radiotherapy, chemotherapy, and combinations thereof, it is imperative to develop more effective anti-PC drugs. Phillygenin (PHI) has been reported to exert anti-cancer, anti-bacterial, and anti‐inflammatory properties. However, the mechanism of PHI in the development of PC is still unclear.MethodsThe cytotoxicity of PHI in pancreatic cancer cells was evaluated by MTT assay, and clonogenic assay was used to test the anti-proliferation of PHI. The pro-apoptotic effect of PHI was detected by flow cytometry analysis. The changes of epithelial–mesenchymal transition (EMT) in pancreatic cancer cells treated with PHI were determined by Western blot. Transwell assay was used to test the migration and invasion of PC cells after treatment with PHI. Molecular docking was used to predict the potential binding site of candidate target with PHI.ResultsPHI could inhibit the proliferation, migration, and EMT of PC cells (PANC-1 and SW1990) and induce its apoptosis. Analysis of the Cancer Genome Atlas database indicated that elevated MELK levels correlated with poor overall survival (OS) and disease-free survival (DFS) of PC patients. In addition, molecular modeling showed that PHI may potentially target the catalytic domain of maternal embryonic leucine zipper kinase (MELK). Overexpression of MELK muted the anti-PC effects of PHI.ConclusionPHI holds promise as a potent candidate drug for the treatment of PC via targeted MELK.

Maternal Embryonic Leucine Zipper Kinase Promotes Tumor Growth and Metastasis via Stimulating FOXM1 Signaling in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignancy and is one of the most important cause of cancer related mortalities in the world. However, there is no clinically effective targeted therapeutic drugs for ESCC due to lack of valuable molecular therapeutic targets. In the present study, we investigated the biological function and molecular mechanisms of maternal embryonic leucine zipper kinase (MELK) in ESCC. The expression of MELK mRNA and protein was determined in cell lines and clinical samples of ESCC. MTT, focus formation and soft agar assays were carried out to measure cell proliferation and colony formation. Wound healing and transwell assays were used to assess the capacity of tumor cell migration and invasion. Nude mice models of subcutaneous tumor growth and lung metastasis were performed to examine the function of MELK in tumorigenecity and metastasis of ESCC cells. High expression of MELK was observed in ESCC cell line and human samples, especially in the metastatic tumor tissues. Moreover, overexpression of MELK promoted cell proliferation, colony formation, migration and invasion, and increased the expression and enzyme activity of MMP-2 and MMP-9 in ESCC cells. More importantly, enhanced expression of MELK greatly accelerated tumor growth and lung metastasis of ESCC cells in vivo. In contrast, knockdown of MELK by lentiviral shRNA resulted in an opposite effect both in vitro and in animal models. Mechanistically, MELK facilitated the phosphorylation of FOXM1, leading to activation of its downstream targets (PLK1, Cyclin B1, and Aurora B), and thereby promoted tumorigenesis and metastasis of ESCC cells. In conclusion, MELK enhances tumorigenesis, migration, invasion and metastasis of ESCC cells via activation of FOXM1 signaling pathway, suggesting MELK is a potential therapeutic target for ESCC patients, even those in an advanced stage.

Abstract 837A: Combining CRISPR/Cas9 mutagenesis and a small-molecule inhibitor to probe the function of MELK in cancer

Abstract The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of cytotoxic chemotherapies, and in vivo. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. By combining our MELK-knockout clones with a new highly-specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cancer patients identifies MELK expression as a correlate of mitotic activity, explaining its association with poor prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies. Citation Format: Christopher J. Giuliano, Ann Lin, Joan C. Smith, Ann C. Palladino, Jason M. Sheltzer. Combining CRISPR/Cas9 mutagenesis and a small-molecule inhibitor to probe the function of MELK in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 837A.

Inhibition of maternal embryonic leucine zipper kinase with OTSSP167 displays potent anti-leukemic effects in chronic lymphocytic leukemia.

TP53 pathway defects contributed to therapy resistance and adverse clinical outcome in chronic lymphocytic leukemia (CLL), which represents an unmet clinical need with few therapeutic options. Maternal embryonic leucine zipper kinase (MELK) is a novel oncogene, which plays crucial roles in mitotic progression and stem cell maintenance. OTSSP167, an orally administrated inhibitor targeting MELK, is currently in a phase I/II clinical trial in patients with advanced breast cancer and acute myeloid leukemia. Yet, no investigation has been elucidated to date regarding the oncogenic role of MELK and effects of OTSSP167 in chronic lymphocytic leukemia (CLL). Previous studies confirmed MELK inhibition abrogated cancer cell survival via p53 signaling pathway. Thus, we aimed to determine the biological function of MELK and therapeutic potential of OTSSP167 in CLL. Herein, MELK over-expression was observed in CLL cells, and correlated with higher WBC count, advanced stage, elevated LDH, increased β2-MG level, unmutated IGHV, positive ZAP-70, deletion of 17p13 and inferior prognosis of CLL patients. In accordance with functional enrichment analyses in gene expression profiling, CLL cells with depletion or inhibition of MELK exhibited impaired cell proliferation, enhanced fast-onset apoptosis, induced G2/M arrest, attenuated cell chemotaxis and promoted sensitivity to fludarabine and ibrutinib. However, gain-of-function assay showed increased cell proliferation and cell chemotaxis. In addition, OTSSP167 treatment reduced phosphorylation of AKT and ERK1/2. It decreased FoxM1 phosphorylation, expression of FoxM1, cyclin B1 and CDK1, while up-regulating p53 and p21 expression. Taken together, MELK served as a candidate of therapeutic target in CLL. OTSSP167 exhibits potent anti-tumor activities in CLL cells, highlighting a novel molecule-based strategy for leukemic interventions.

Sanguinarine triggers intrinsic apoptosis to suppress colorectal cancer growth through disassociation between STRAP and MELK

BackgroundPrevious studies showed sanguinarine induced apoptosis in CRC cells but did not define the underlying mechanisms. The purpose of this work was to determine the in vivo and in vitro effects of sanguinarine on CRC tumors and to elucidate the mechanism in regulating the intrinsic apoptosis.MethodsCell viability of CRC cell lines treated with sanguinarine was measured by MTT assay. Apoptotic cells stained with Annexin V and 7-AAD were detected by flow cytometry. Mitochondrial membrane potential and reactive oxygen species (ROS) were analyzed by JC-1 and DCFH-DA staining, respectively. The in vitro kinase activity of MELK was analyzed by using HTRF® KinEASE™-STK kit. The expression of proteins were determined using Western blotting and immunohistochemistry. Co-immunoprecipitation and immunofluorecence were used to study the interaction between STRAP and MELK. The anti-neoplastic effect of sanguinarine was observed in vivo in an orthotopic CRC model.ResultsSanguinarine decreased the tumor size in a dose-dependent manner in orthotopical colorectal carcinomas through intrinsic apoptosis pathway in BALB/c-nu mice. It significantly increased cleavage of caspase 3 and PARP in implanted colorectal tissues. Sanguinarine increased mitochondrial ROS and triggered mitochondrial outer membrane permeabilization in multiple colorectal cancer (CRC) cell lines. NAC pretreatment lowered ROS level and downregulated apoptosis induced by sanguinarine. The intrinsic apoptosis induced by sanguinarine was Bax-dependent. The elevated expression and association between serine-threonine kinase receptor-associated protein (STRAP) and maternal embryonic leucine zipper kinase (MELK) were observed in Bax positive cells but not in Bax negative cells. Sanguinarine dephosphorylated STRAP and MELK and disrupted the association between them in HCT116 and SW480 cells. The expression and association between STRAP and MELK were also attenuated by sanguinarine in the tumor tissues. Importantly, we found that STRAP and MELK were overexpressed and highly phosphorylated in colorectal adenocarcinomas and their expression were significantly correlated with tumor stages. Furthermore, the expression of MELK, but not STRAP, was associated with lymph node metastasis.ConclusionsSanguinarine dephosphorelates STRAP and MELK and disassociates the interaction between them to trigger intrinsic apoptosis. Overexpression of STRAP and MELK may be markers of CRC and their disassociation may be a determinant of therapeutic efficacy.

Elucidating the Role of the Maternal Embryonic Leucine Zipper Kinase in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate <35%. Mortality remains high due to lack of targeted therapies. Using bioinformatic analyses, we identified maternal embryonic leucine zipper kinase (MELK) as 4.1-fold overexpressed in ACC compared with normal adrenal samples. High MELK expression in human tumors correlated with shorter survival and with increased expression of genes involved in cell division and growth. We investigated the functional effects of MELK inhibition using newly developed ACC cell lines with variable MELK expression, CU-ACC1 and CU-ACC2, compared with H295R cells. In vitro treatment with the MELK inhibitor, OTSSP167, resulted in a dose-dependent decrease in rates of cell proliferation, colony formation, and cell survival, with relative sensitivity of each ACC cell line based upon the level of MELK overexpression. To confirm a MELK-specific antitumorigenic effect, MELK was inhibited in H295R cells via multiple short hairpin RNAs. MELK silencing resulted in 1.9-fold decrease in proliferation, and 3- to 10-fold decrease in colony formation in soft agar and clonogenicity assays, respectively. In addition, although MELK silencing had no effect on survival in normoxia, exposure to a hypoxia resulted in a sixfold and eightfold increase in apoptosis as assessed by caspase-3 activation and TUNEL, respectively. Together these data suggest that MELK is a modulator of tumor cell growth and survival in a hypoxic microenvironment in adrenal cancer cells and support future investigation of its role as a therapeutic kinase target in patients with ACC.

MELK expression correlates with tumor mitotic activity but is not required for cancer growth.

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Previously, we established that triple-negative breast cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK proliferate at wild-type levels in vitro (<xref ref-type="bibr" rid="bib34">Lin et al., 2017</xref>). Here, we generate several additional knockout clones of MELK and demonstrate that across cancer types, cells lacking MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of cytotoxic chemotherapies, and in vivo. By combining our MELK-knockout clones with a recently described, highly specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cohorts of cancer patients identifies MELK expression as a correlate of tumor mitotic activity, explaining its association with poor clinical prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies.

MELK serves as a potent independent biomarker to predict survival of diffuse large B‐cell lymphoma patients treated with rituximab‐CHOP regimen

Introduction: Maternal embryonic leucine zipper kinase (MELK) is a novel oncogene, which plays pivotal roles in maintenance of cancer stem cells and cancer cell survival. However, the clinical significance of MELK in the progression of diffuse large B-cell lymphoma (DLBCL) remains poorly understood. Currently, 30% to 40% DLBCL patients remain incurable with standard therapy (cyclophosphamide, doxorubicin, vincristine, prednisone combined with rituximab, and R-CHOP), resulting in multiple studies on identifying effective prognostic biomarker. Herein, we aimed to assess prognostic significance of MELK in DLBCL patients with R-CHOP regimen. Methods: MELK expression pattern in DLBCL and normal lymph node tissues was characterized in microarray from Gene Expression Ominous (GEO) GSE56315. Prognostic value of MELK expression was evaluated in 471 de novo DLBCL patients treated with R-CHOP, collected as part of the International DLBCL R-CHOP Consortium Program Study, on tissue microarray from GSE31312 using Affymetrix GeneChip Human Genome HG-U133 Plus 2.0 chips. Optimal cutoff value of MELK expression was determined by calculating Youden's index from receiver operating characteristic (ROC) curve. Kaplan-Meier survival curve analyses with log-rank test, univariate and multivariate Cox regression model analyses of overall survival (OS), and progression-free survival (PFS) were performed. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis in whole-genome profiling were conduct to predict potential functions of MELK in DLBCL. Results: Markedly increased expression of MELK in DLBCL tissues (9.299 ± 0.081, n = 89) compared with B cells from normal tonsil tissues (7.218 ± 0.243, n = 33) were observed (Figure A). Furthermore, in the cohort of 471 DLBCL patients with R-CHOP treatment, stratified high MELK expression is closely associated with advanced Ann Arbor stage (P = .026), high IPI score (P = .036), activated B-cell–like (ABC) DLBCL subtype (P = .013), and patients' non-CR rates (P = .001). Kaplan-Meier survival curve analyses indicated MELK overexpression was of significant correlation with inferior OS (P = .001) and PFS (P = .018) (Figure B, C). Multivariate Cox regression analysis adjusting for validated significant parameters in univariate analysis confirmed independent prognostic value of MELK in OS of DLBCL patients (HR: 1.600; 95% CI, 1.096-2.335, P = .015). Functional enrichment assays indicate that MELK is tightly associated with biological processes including cell division, apoptosis process and cell cycle. Besides, MELK might contribute to cancer progression of DLBCL via regulating PI3K/Akt, Ras, and p53 signaling pathways. Keywords: diffuse large B-cell lymphoma (DLBCL); gene expression profile (GEP); R-CHOP

MELK is an oncogenic kinase essential for early hepatocellular carcinoma recurrence

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Many kinases have been found to be intimately involved in oncogenesis and the deregulation of kinase function has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth and survival. Previously, we have performed gene expression profile analysis on HCC samples and have identified a host of kinases that are remarkably overexpressed in HCC. Among these, the Maternal Embryonic Leucine Zipper Kinase (MELK) is highly overexpressed in HCC and its overexpression strongly correlates with early recurrence and poor patients' survival. Silencing MELK inhibited cell growth, invasion, stemness and tumorigenicity of HCC cells by inducing apoptosis and mitosis. We further showed that the overexpression of MELK in HCC samples strongly correlated with the cell cycle- and mitosis-related genes which are directly regulated as part of the forkhead transcription factor FoxM1-related cell division program. Together, our data establish MELK as an oncogenic kinase involved in the pathogenesis and recurrence of HCC and could provide a promising molecular target to develop therapeutic strategies for patients with advanced HCC.

Abstract C26: Development of selective MELK kinase inhibitors for breast cancer treatment

Abstract Breast cancer is the second most common cancer in the world and the most frequent cancer among women. Despite the progress in developing breast cancer therapies, approximately, 15% of all breast cancers are diagnosed as triple negative breast cancer (TNBC) and due to the lack of estrogen and progesterone receptors this subgroup of patients remains difficult to treat with hormonal therapies. Additionally, therapies targeting HER2, such as Herceptin, are also inefficient against TNBC. In recent years, maternal embryonic leucine zipper kinase (MELK) has been identified as a novel oncogenic target that is highly expressed in several types of solid cancers: breast (especially triple negative breast cancer), colon, ovary, lung, and brain and present at low levels in normal tissues. MELK overexpression in patient tumors strongly correlates with poor prognosis in glioblastoma and breast cancer. siRNA mediated knockdown of MELK kinase significantly inhibits growth of tumor cell lines both in vitro and in vivo. Therefore, MELK kinase is emerging as a novel and interesting target with significant potential for therapeutic intervention in cancer. MELK is an atypical member of the AMPK family of serine-threonine kinases that been implicated has been implicated in stem cell renewal, cell cycle progression, cytokinesis, mRNA splicing and apoptosis. Its activity is correlated with its phosphorylation level, is cell cycle dependent, and maximal during mitosis although direct upstream regulators of MELK kinase activity are unknown. Despite the fact that the exact function is currently under investigation, selective targeting of MELK may be an effective cancer treatment strategy in a wide range of solid tumors. In this study, we are reporting development of a series of selective MELK kinase inhibitors. Synthesized compounds exert excellent selectivity and potency in MELK inhibition in a low nanomolar range. Therapeutic effect of the compounds was investigated in the panel of breast cancer cell lines with different genetic background as well as with different MELK kinase levels; it was shown that for some cell lines compounds induced cell death with nanomolar ED50 values. The compound's effect on the proliferation and in the colony formation assay was also investigated. Taken altogether, the presented data supports our rationale of using MELK kinase inhibitors as a novel approach for the cancer therapy. Citation Format: Piotr Kowalczyk, Paulina Węgrzyn, Monika Prokopowicz, Martyna Knop, Karolina Mazur, Katarzyna Dziedzic, Karolina Gluza, Martyna Knop, Katarzyna Dziedzic, Karolina Mazur, Adam Radzimierski, Claude Commandeur, Magdalena Zawadzka, Kristjan Bloudoff, Fred Vaillancourt, Nick Larsen, John Wang, Dom Reynolds, Daisuke Ito, Jian Zou, Michelle Aicher, Pete Smith, Ping Zhu, Krzysztof Brzózka. Development of selective MELK kinase inhibitors for breast cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C26.

Abstract 2386: Molecular-cytogenetic analysis of the maternal embryonic leucine-zipper kinase (MELK) oncogene in cancer

Abstract Introduction: The MELK gene is located on chromosome 9p13.2 and encodes a serine/threonine kinase that is involved in cell cycle regulation and apoptosis. The MELK protein is abundantly expressed in various tumors including breast cancer, and is associated with poor patient survival. MELK is an attractive molecular target due to its critical roles in cancer stem cell maintenance. However, the function and mechanism of MELK overexpression remain elusive. Gene amplification/copy number gain is one of the potential mechanisms underlying MELK overexpression. In this pilot study, we used fluorescence in situ hybridization (FISH) to detect MELK gene amplification and analyzed other MELK gene copy number alterations (CNA) in endometrial, ovarian, and breast cancer cell lines. Methods: To date, 3 endometrial, 1 ovarian and 13 breast cancer cell lines of different subtypes were screened for MELK CNA. Of these, six breast cancer cell lines (BT20, MCF7, MDAMB231, SKBR3, BT549, and T47D) had known status of MELK: all carried high levels of protein expression by Western Blot. Cell harvest and metaphase slides were prepared according to standard protocols. MELK FISH probe (BAC RP11-450B8) directly labeled with SpectrumGreen was developed and validated in our laboratory. The chromosome 9 centromere enumeration probe (CEP9) labeled with SpectrumOrange (Abbott Molecular) was used to distinguish gene amplification from gene polysomy (gene and chromosome copy number gain ≥ 3). Mean copies of each signal per cell and copy number ratios per cell were calculated. Ratio of MELK to CEP9 ≥ 2.0 was a cut off point for MELK amplification. Results: Across all cell lines, the ratios of MELK to CEP9 ranged between 0.5 to 1.7, showing no amplification. However, 10 cell lines (59%) displayed 3-8 copies of MELK due to polysomy for chromosome 9, 4 (24%) harbored both gene polysomy and structural alterations (duplications and translocations); only two cell lines exhibited normal MELK and CEP9 copies and one presented with heterozygous deletion of MELK. Interestingly, all 6 MELK- overexpressed cell lines showed either gene polysomy or complex chromosomal alterations or both. Conclusions: Our pilot study suggests that amplification of MELK gene does not occur or is a rare event in human cancer cells in vitro. However, recurrent MELK structural alterations (duplications and translocations) and gene polysomy can cause elevated protein expression in breast cancer cell lines. MELK FISH study in primary tumors from breast cancer patients will be presented. Supported by: American Cancer Society Citation Format: Ashley Hardeman, Tatyana A. Grushko, Maria J. Gomez, Mariann Coyle, Yusuke Nakamura, Olufunmilayo I. Olopade. Molecular-cytogenetic analysis of the maternal embryonic leucine-zipper kinase (MELK) oncogene in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2386. doi:10.1158/1538-7445.AM2014-2386

The structures of the kinase domain and UBA domain of MPK38 suggest the activation mechanism for kinase activity.

Murine protein serine/threonine kinase 38 (MPK38) is the murine orthologue of human maternal embryonic leucine-zipper kinase (MELK), which belongs to the SNF1/AMPK family. MELK is considered to be a promising drug target for anticancer therapy because overexpression and hyperactivation of MELK is correlated with several human cancers. Activation of MPK38 requires the extended sequence (ExS) containing the ubiquitin-associated (UBA) linker and UBA domain and phosphorylation of the activation loop. However, the activation mechanism of MPK38 is unknown. This paper reports the crystal structure of MPK38 (T167E), which mimics a phosphorylated state of the activation loop, in complex with AMP-PNP. In the MPK38 structure, the UBA linker forces an inward movement of the αC helix. Phosphorylation of the activation loop then induces movement of the activation loop towards the C-lobe and results in interlobar cleft closure. These processes generate a fully active state of MPK38. This structure suggests that MPK38 has a similar molecular mechanism regulating activation as in other kinases of the SNF1/AMPK family.

Abstract P6-04-04: Maternal embryonic leucine zipper kinase (MELK) is a novel radiosensitizing and therapeutic target and is independently prognostic in triple-negative breast cancer

Abstract Background: While effective targeted therapies exist for estrogen receptor (ER)-positive and HER2/neu-positive breast cancer, no such effective therapies exist for ER-negative, PR-negative, and HER2-negative (“triple negative”) cancers. Given the lack of targeted agents for triple negative (TN) disease and their relative radiation insensitivity, it is clear that additional targets for treatment are critically needed. Our previous work identified one such novel molecular target as maternal embryonic leucine zipper kinase (MELK), and we sought to investigate the impact of MELK expression on radiation response and patient outcomes. Methods: Using gene expression arrays, we interrogated the expression of MELK in 2,061 breast tumor samples as well as a panel of 51 breast cancer cell lines. We measured protein expression in TN cancers with western blotting and used clonogenic survival assays to quantitate radiosensitivity of BCC lines at baseline and after MELK inhibition. Multiple datasets were used to evaluate the prognostic import of MELK. Kaplan-Meier analysis using local control and survival data was performed. Chi squared scores were calculated to determine significance and hazard ratios (HR) and 95% confidence intervals (CI) were calculated. A Cox proportional hazards model was constructed to identify potential factors of survival. Results: We demonstrate that MELK expression is significantly elevated in human TN breast cancers, including chemoradiation resistant tumors (305 tumors compared to 1756 non-TN breast tumors; p-value 7.5 e-21). MELK protein and RNA expression is induced by ionizing radiation (5.6-7.5 fold at 72 hours, p-value &amp;lt;0.01). We characterized the radiation sensitivity of BCC lines and demonstrated that MELK expression is significantly correlated with radioresistance (as measured by clonogenic survival) in 21 breast cancer cell lines (R: 0.62, p-value 0.003). Inhibition of MELK using both siRNA and small molecule inhibitors induces radiation sensitivity in vitro with and enhancement ratio (ER) of 1.5-1.6. We demonstrate that high MELK expression is strongly correlated with p53 mutation positive status (p-value &amp;lt;0.001). Finally, local control and survival analyses of patients with BC showed that those patients whose tumors have high expression of MELK have significantly higher rates of LR after radiation and an overall poorer prognosis than patients with low expression of MELK (HR for LR 1.89-2.23, p-value 0.001; HR for overall survival 1.46-3.3; p-value &amp;lt;0.001 in 3 independent datasets). In multivariate analysis of all patients, only MELK expression and grade were significantly associated with worse local recurrence free (LRF) survival with a HR of 1.35 (95% CI 1.05-1.72, p-value &amp;lt; 0.01). Conclusion: Here, we identify MELK as a potential biomarker of radioresistance and target for radiosensitization in triple negative breast cancers. MELK overexpression was associated with local failure across multiple data sets. MVA identified MELK as the strongest factor associated with poor local control. Our results support the rationale for developing clinical strategies to inhibit MELK as a novel target in triple-negative breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-04.

Maternal Embryonic Leucine Zipper Kinase (MELK): A Novel Regulator in Cell Cycle Control, Embryonic Development, and Cancer

Maternal embryonic leucine zipper kinase (MELK) functions as a modulator of intracellular signaling and affects various cellular and biological processes, including cell cycle, cell proliferation, apoptosis, spliceosome assembly, gene expression, embryonic development, hematopoiesis, and oncogenesis. In these cellular processes, MELK functions by binding to numerous proteins. In general, the effects of multiple protein interactions with MELK are oncogenic in nature, and the overexpression of MELK in kinds of cancer provides some evidence that it may be involved in tumorigenic process. In this review, our current knowledge of MELK function and recent discoveries in MELK signaling pathway were discussed. The regulation of MELK in cancers and its potential as a therapeutic target were also described.

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