Role Of Maternal Embryonic Leucine Zipper Kinase Research Articles

Maternal embryonic leucine zipper kinase in cancer progress and therapeutic potentials

This review delves into the investigation of Maternal Embryonic Leucine Zipper Kinase (MELK) and its significance in different cancer types. The primary emphasis is placed on understanding its role in cancer cell proliferation, migration, invasion, and resistance to therapeutic interventions. The intricate mechanisms of action exhibited by MELK are of significant importance in the context of cancer progression. These mechanisms encompass a wide range of biological processes, including gene regulation, cellular activities, and interactions at the tissue level. This highlights the multifaceted nature of MELKs involvement in cancer development and underscores its significance in this context. This essay explores the potential of MELK as a diagnostic and prognostic biomarker, with a focus on its altered expression patterns in various cancer types. Furthermore, this study delves into the investigation of MELK as a highly promising target for cancer therapy. It provides an in-depth analysis of the progress made in the development of MELK inhibitors and explores their potential clinical applications. The research endeavours in this study focus on addressing challenges related to therapeutic resistance and biomarker validation. Additionally, the investigation explores potential opportunities for combination therapies and personalised medicine approaches. The future directions of research on maternal embryonic leucine zipper kinase (MELK) encompass an in-depth investigation into its underlying molecular mechanisms, the validation of clinical biomarkers associated with MELK, and the exploration of effective combination strategies involving MELK. The role of MELK in cancer and the possibility to utilize it as a target for therapy have garnered significant attention due to their potential to advance precision cancer care and improve patient outcomes.

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.

MELK promotes HCC carcinogenesis through modulating cuproptosis-related gene DLAT-mediated mitochondrial function

Cuproptosis caused by copper overload is mediated by a novel regulatory mechanism that differs from previously documented mechanisms regulating cell death. Cells dependent on mitochondrial respiration showed increased sensitivity to a copper ionophore elesclomol that induced cuproptosis. Maternal embryonic leucine zipper kinase(MELK) promotes tumorigenesis and tumor progression through the PI3K/mTOR pathway, which exerts its effects partly by targeting the pyruvate dehydrogenase complex(PDHc) and reprogramming the morphology and function of mitochondria. However, the role of MELK in cuproptosis remains unclear. Here, we validated that elevated MELK expression enhanced the activity of PI3K/mTOR signaling and subsequently promoted Dihydrolipoamide S-Acetyltransferase (DLAT) expression and stabilized mitochondrial function. This regulatory effect helped to improve mitochondrial respiration, eliminate excessive intracellular reactive oxygen species (ROS), reduce intracellular oxidative stress/damage and the possibility of mitochondria-induced cell fate alternations, and ultimately promote the progression of HCC. Meanwhile, elesclomol reduced translocase of outer mitochondrial membrane 20(TOM 20) expression and increased DLAT oligomers. Moreover, the above changes of MELK to HCC were abolished by elesclomol. In conclusion, MELK enhanced the levels of the cuproptosis-related signature(CRS) gene DLAT (especially the proportion of DLAT monomer) by activating the PI3K/mTOR pathway, thereby promoting elesclomol drug resistance, altering mitochondrial function, and ultimately promoting HCC progression.

Abstract P6-10-08: Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers

Abstract Background Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are aggressive breast cancer subtypes with poor clinical outcomes due to the lack of well-validated and actionable targets and the onset of chemo-resistant metastasis. TNBC and IBC account for 15-20% and 2-4% of all breast cancer diagnoses but result in 30% and 8%–10% of breast cancer-related deaths, respectively. MELK (maternal embryonic leucine zipper kinase) is a potential therapeutic target in both TNBC and triple-negative IBC (TN-IBC). We have shown that 1) MELK expression is higher in basal tumors (mostly TNBC) and in IBC than in non-IBC, 2) high MELK expression is an independent prognostic factor for poor overall survival (P=0.0002) and poor distant metastasis-free survival (P=0.008) in breast cancer, and 3) MELK knockout leads to significantly lower metastatic burden and prolonged survival in a xenograft model of TNBC (unpublished data). MELK inhibitor (MELKi) OTS167 is in clinical trials but it lacks specificity and cross-reacts with other essential kinases. It is critical to discover novel, selective inhibitors with good bioavailability that target MELK and minimize adverse effects. We have developed a second-generation small-molecule MELK inhibitor 30e that is a highly potent and slow-binding ATP-competitive inhibitor of MELK. 30e exhibits potent cellular inhibition of MELK (IC50< 10 nM), as assessed using a live cell NanoBRET assay, and is highly selective as assessed by live-cell KiNativ profiling experiments in MDA-MB-231 TNBC cells. Methods We screened a panel of TNBC and IBC cell lines for MELK expression by western blot and qPCR analysis. We assessed the effects of MELK inhibition with the 30e on migration and invasion. The role of MELK in regulating cancer stem cell phenotypes was assessed using the mammosphere assay and flow cytometry (CD44+/CD24-/low surface marker). MELK’s protumorigenic role was determined in vitro using a soft agar assay, and the anti-tumor activity of 30e was assessed in vivo using a TN-IBC orthotopic xenograft mouse model. Results We found that TNBC had high mRNA levels of MELK (62%) compared to non-TNBC cell lines (25%), which correlated with MELK protein levels. 30e inhibited cell viability in TNBC and IBC cells in a dose-dependent manner and with IC50s ranging from 0.45 to 1.76 μM (P≤0.05). However, no significant effect was observed when normal MCF-10A breast cells were treated with 30e (IC50>20 μM). 30e also significantly reduced colony formation ability. Further, we observed that 30e reduced mammosphere formation efficiency and the CD44+/CD24-/low subpopulation in both TNBC and IBC cells in a dose-dependent manner (P≤0.05), suggesting the potential of 30e to inhibit the stem cell population in vitro. The soft agar assay also showed a significant decrease in colony formation in the TNBC and IBC cells after treatment with 30e, an indirect indicator of in vivo tumorigenicity. To assess the efficacy of 30e treatment on tumor growth, we evaluated this inhibitor as a single agent in a SUM-149 (TN-IBC) orthotopic mouse model. The mice were treated with intraperitoneal injections daily with 3 doses (2.5, 5, and 10 mg/kg) of 30e. Our data show that 30e suppressed tumor growth in a dose-dependent manner (P< 0.007). Conclusions and Future Directions Our data demonstrate that a novel, potent and specific MELK inhibitor inhibits tumor growth by suppressing the cancer stem cell phenotype. MELK is a promising target for aggressive cancers such as TNBC and IBC. We are evaluating how MELK inhibition modulates the tumor microenvironment, which is a critical component of breast cancer response to treatment. We performed a cytokine antibody array and are validating the top targets. Further, we will evaluate 30e in combination with standard-of-care treatment for toxicity and inhibition of metastasis. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Hector Gonzalez, Emilly S. Villodre, Bisrat Debeb, Debu Tripathy, Geoffrey Bartholomeusz, Juhyeon Lee, Kevin Dalby, Chandra Bartholomeusz. Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-08.

Abstract P5-08-14: Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has high rates of relapse and metastasis. It has a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, plays a critical role in promoting CSC maintenance, cell proliferation, and malignant transformation. Here, we assessed the role of MELK in TNBC aggressiveness. Methods: The clinical relevance of MELK in TNBC was analyzed using the World IBC Consortium dataset (n = 314). The effects of MELK knockdown (siRNA), knockout (CRISPR-Cas9), and MELK inhibition with a novel selective small molecule inhibitor MELK-In-7 on migration and invasion were determined using the migration and invasion assays, respectively. MELK’s role in regulating CSC phenotypes was assessed using the mammosphere assay and flow cytometry. The impact of MELK knockdown on epithelial-to-mesenchymal transition (EMT) was determined using Western blotting. MELK’s protumorigenic role was determined in vitro using soft agar assay and in vivo using TNBC xenograft mouse models. Results: MELK mRNA expression level was higher in TNBC tumors than in tumors expressing hormone receptor, HER2, or both (P < 0.0001). In univariate analysis, breast cancer patients with high-MELK-expressing tumors had worse overall survival (OS, P < 0.001) and distant metastasis-free survival (DMFS, P < 0.01) than patients with low-MELK-expressing tumors, suggesting a prognostic impact of MELK in breast cancer. MELK knockdown using siRNA or MELK inhibition using MELK-In-7 significantly reduced migration, invasion, mammosphere formation, CD24-/CD44+ subpopulations, and ALDH activity, and reversed EMT in TNBC cells. This result suggests a role for MELK in regulating CSCs and EMT, which play crucial roles in tumor invasion and metastasis. Nude mice injected with MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and significantly improved OS compared with the mice injected with parental MDA-MB-231 cells (P < 0.05). Further, MELK-selective inhibitor (MELK-In-7) suppressed the growth of 4T1 mouse TNBC tumors in a dose-dependent manner in syngeneic BALB/c mice (P < 0.001). These results indicate an essential role for MELK in promoting TNBC tumor growth and metastasis. To uncover the underlying molecular mechanism of MELK’s regulation of TNBC metastasis, we performed microarray analysis and identified SERPINE1, a serine protease inhibitor known to regulate metastasis, as a downstream target of MELK. We validated that mRNA levels of SERPINE1 were reduced in MELK-knockout MDA-MB-231 cells and MELK-In-7-treated MDA-MB-231 cells. Moreover, SERPINE1 knockdown using siRNA reduced migration and invasion of TNBC cells. These results suggest that MELK regulates TNBC cell motility through SERPINE1. Conclusion: This work confirms MELK as a driver of aggressiveness and metastasis in TNBC. The mechanisms underlying MELK’s regulation of TNBC metastasis via SERPINE1 require further studies. Citation Format: Xuemei Xie, Gaurav B. Chauhan, Ramakrishna Edupuganti, Takahiro Kogawa, Jihyun Park, Moises T Tacam, Fnu Vidhu, Diane D. Liu, Juliana M. Taliaferro, Mary Kathryn Pitner, Yu Shen, Naoto T. Ueno, Savitri Krishnamurthy, Gabriel N. Hortobagyi, Debu Tripathy, Steven J. Van Laere, Geoffrey Bartholomeusz, Kevin Dalby, Chandra Bartholomeusz. Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-14.

OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in osteosarcoma.

Osteosarcoma is the most common primary malignancy of bones and frequently affects young children and adolescents. There are several challenges associated with treating osteosarcoma owing to the aggressiveness of the disease, as well as the risk of chemoresistance. Numerous studies are being performed with the aim of identifying improved prognostic and therapeutic markers for this malignancy. Maternal embryonic leucine zipper kinase (MELK) is an oncogene that has been studied in several types of cancer in recent years. In the present study, the expression of MELK in osteosarcoma and normal tissue samples was examined, and the effects of MELK expression on osteosarcoma cellular proliferation, metastasis, the cell cycle and apoptosis were demonstrated using CCK-8, wound healing, migration and invasion and apoptosis assays. The role of MELK in cancer progression in osteosarcoma was determined, revealing the association between MELK expression and prognosis of osteosarcoma. It was demonstrated that knockdown of MELK resulted in reduced proliferation, migration and invasion in vitro along with potentiation of apoptosis and cell cycle arrest. Furthermore, the effect of the targeted MELK inhibitor, OTSSP167, on tumor progression of osteosarcoma in vitro and in vivo was assessed. Mechanistically, it was demonstrated that MELK promoted osteosarcoma proliferation and metastasis by regulating PCNA and MMP9 expression via the PI3K/Akt/mTOR signaling pathway. Thus, the present study revealed the oncogenic role played by MELK, and established MELK as a valuable prognostic and therapeutic marker in osteosarcoma.

Maternal embryonic leucine zipper kinase is a novel target for diffuse large B cell lymphoma and mantle cell lymphoma

Diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are among the most aggressive B cell non-Hodgkin lymphomas. Maternal embryonic leucine zipper kinase (MELK) plays a role in cancer cell cycle progression and is associated with poor prognosis in several cancer cell types. In this study, the role of MELK in DLBCL and MCL and the therapeutic potential of MELK targeting is evaluated. MELK is highly expressed in DLBCL and MCL patient samples, correlating with a worse clinical outcome in DLBCL. Targeting MELK, using the small molecule OTSSP167, impaired cell growth and survival and induced caspase-mediated apoptosis in the lymphoma cells. Western blot analysis revealed that MELK targeting decreased the phosphorylation of FOXM1 and the protein levels of EZH2 and several mitotic regulators, such as Cdc25B, cyclin B1, Plk-1, and Aurora kinases. In addition, OTSSP167 also sensitized the lymphoma cells to the clinically relevant Bcl-2 inhibitor venetoclax by strongly reducing Mcl1 levels. Finally, OTSSP167 treatment of A20-inoculated mice resulted in a significant prolonged survival. In conclusion, targeting MELK with OTSSP167 induced strong anti-lymphoma activity both in vitro and in vivo. These findings suggest that MELK could be a potential new target in these aggressive B cell malignancies.

MELK is a novel therapeutic target in high-risk neuroblastoma.

Maternal embryonic leucine zipper kinase (MELK) is known to modulate intracellular signaling and control cellular processes. However, the role of MELK in oncogenesis is not well defined. In this study, using two microarray datasets of neuroblastoma (NB) patients, we identified that MELK expression is significantly correlated to poor overall survival, unfavorable prognosis, and high-risk status. We found that MELK is a direct transcription target of MYCN and MYC in NB, and MYCN increases MELK expression via direct promoter binding. Interestingly, knockdown of MELK expression significantly reduced the phosphorylation of target protein Retinoblastoma (pRb) and inhibited NB cell growth. Furthermore, pharmacological inhibition of MELK activity by small-molecule inhibitor OTSSP167 significantly inhibited cell proliferation, anchorage-independent colony formation, blocked cell cycle progression, and induced apoptosis in different NB cell lines including a drug-resistant cell line. Additionally, OTSSP167 suppressed NB tumor growth in an orthotopic xenograft mouse model. Overall, our data suggest that MELK is a novel therapeutic target for NB and its inhibitor OTSSP167 is a promising drug for further clinical development.

Abstract 952: Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia

Abstract Maternal embryonic leucine-zipper kinase (MELK), a member of the serine-threonine kinases snf1/AMP-activated protein family is involved in mammalian embryonic development. MELK is aberrantly upregulated in several types of solid cancer including glioblastoma and breast cancer, and implicated in formation and maintenance of cancer stem cells. Little is known about the relevance of this kinase in hematological malignancies. Our study aimed to explore the role of MELK in acute myeloid leukemia (AML) and identify whether targeting this kinase in leukemia stem cells may have therapeutic relevance. In order to characterize the expression of MELK in AML, we examined the protein levels in AML cell lines and primary blasts by western blot. MELK protein was expressed at variable levels in 10 out of 11 AML cell lines and primary blasts obtained from AML patients (n=5), representing diverse molecular and cytogenetic abnormalities of the disease. Importantly, we also found that MELK protein levels were higher in CD34+ cells compared with the more differentiated CD34- primary blasts from AML patient. To determine the role of MELK in AML, we utilized a loss of function approach in three AML cell lines (KG1, MV4-11 and U937 cells) and assessed cell viability by MTS. Cells transfected with MELK-siRNA showed significant decrease in cell viability (40-60%, P<0.05) compared with cells transfected with control-siRNA. In addition, knockdown of MELK expression resulted in increased apoptosis in MV4-11 and U937 cells assessed by AnnexinV staining. OTS167, a small molecule MELK kinase inhibitor is currently undergoing phase I clinical testing in patients with advanced solid malignancies. To examine the anti-leukemia activity of OTS167 in AML, we treated nine AML cell lines with this compound and assessed cells viability by MTS assay. Decrease in cell viability following treatment with increasing doses of OTS167 was time and dose dependent. Variable sensitivities among AML cell lines (IC50∼10-40nM at 48hrs) were observed. In addition, the compound promoted cell differentiation as assessed by CD11b staining in U937 cells. Treatment with (25 and 50nM) of OTS167 induced apoptosis by ∼30 and 40% in MV4-11 and MOLM13 cells at 48 hours post treatment (P=0.01 and 0.05, respectively). Similarly, OTS167 cytotoxicity (IC50 ∼ 10-40nM; n=3) and induced apoptosis was observed in primary AML blasts. In conclusion, MELK is expressed in AML cell lines and AML blasts with higher levels observed in less differentiated cells. MELK knockdown results in growth inhibition, apoptosis, and cell death suggesting that this kinase may act as a potential therapeutic target. MELK kinase inhibitor (OTS167) causes cytotoxicity in AML cell lines and primary blasts. These results warrant further preclinical evaluation of this compound as a novel targeted therapy for patients with AML. Citation Format: Houda Alachkar, Martin Mutonga, Suyoun Chung, Yo Matsuo, Wendy Stock, Yusuke Nakamura. Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia. [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 952. doi:10.1158/1538-7445.AM2014-952

Abstract 4607: Stathmin is involved in the maternal embryonic leucine zipper kinase pathway and impacts in the outcome of glioblastoma

Abstract INTRODUCTION: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in several cell processes, including the cell cycle, differentiation, proliferation, and apoptosis. MELK has also shown multiple features consistent with the potential utility as an anticancer target, including in astrocytomas. However, the detailed downstream signaling pathway of MELK in cancer cells is still not fully understood and the putative role of MELK in astrocytomas remains unclear. The discovery of proteins involved in the MELK pathway might be important to develop additional small molecular compound to improve effectiveness. PURPOSE: This study aim to analyze proteins and/or genes involved in the MELK pathway in the tumorigenesis of gliomas. MATERIAL AND METHODS: Analysis of the expression profile of a panel of protein relevant to the process of tumorigenesis that MELK is involved was performed by Proteomic analysis by two dimensional electrophoresis and mass spectrometry for human glioma cell line U87MG transfected with siRNA for the MELK compared to U87MG transfected with non-target control (NTC) cells. Analysis and validation of gene expression was performed by Quantitative real time PCR (qRT-PCR) using SYBR Green method in a series of 153 astrocytomas of different malignant grade (23 AGI, 26 AGII, 18 AGIII and 86 AGIV or glioblastoma - GBM) and 22 non neoplastic (NN) brain samples. RESULTS: We identified Stathmin/oncoprotein 18 (STMN1) involved in the cell cycle, as one of twelve proteins differentially expressed, with lower expression when MELK was silenced. A significant higher expression of STMN1 in malignant diffusely infiltrative astrocytomas when compared to non-invasive pilocytic astrocytoma was found (p<0.0001). Strong correlation for MELK and STMN1 associated expression was observed (r=0.741, p<0.0001) in GBM. STMN1 hyperexpression alone impacted at GBM outcome with an overall survival of 5.4 months in contrast to 13.4 months when both genes were hypoexpressed (log rank=0.002). CONCLUSIONS: Thus, our results showed that STMN1 is downstream in the MELK pathway and might play an important role in astrocytoma tumorigenesis. Citation Format: Miyuki Uno, Sueli Mieko Oba-Shinjo, Roseli Silva, Marcela Gimenez, Jose Cesar Rosa, Suely Kazue Nagahashi Marie. Stathmin is involved in the maternal embryonic leucine zipper kinase pathway and impacts in the outcome of glioblastoma. [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 4607. doi:10.1158/1538-7445.AM2014-4607

Abstract 3299: Maternal embryonic leucine zipper kinase is critical for the growth and migration of triple negative breast cancer cells

Abstract Background: Breast cancer constitutes 30% of all new cancer cases in women. Despite steady decrease in breast cancer mortality, the lack of therapeutic targets is still a major problem. While the hormone-receptor positive (ER+) and human epidermal growth factor receptor 2-positive (HER2+) breast cancers respond to current targeted therapies, no targeted therapy is available for the treatment of triple negative breast cancer (TNBC), which lacks the expression of ER alpha (ER), progesterone receptor (PR), and HER2-receptor. Hence, there is an urgent need for effective targets against this sub-type of breast cancer. Previous studies in our laboratory used gene expression profiling of human breast cancers to identify kinases overexpressed in ER-negative breast cancers. One of these highly expressed kinases is the maternal embryonic leucine-zipper kinase (MELK). MELK is a serine/threonine protein kinase known to have a role in cell cycle progression, apoptosis and DNA repair. The purpose of this study was to test the hypothesis that MELK is required for the growth and migration of TNBC. Methods: RNA and protein was isolated from a panel of TNBC and ER-positive breast cancer cell lines and MELK expression was quantified by qPCR and immunoblotting. To determine whether MELK regulates cell growth, ER-positive and TNBC cell lines were transfected with siRNA targeting MELK and cell number was measured by manual counting. Anchorage-independent growth was measured using soft agar assays. Effect on migration and invasion was determined using Boyden Chamber assays. Immunostaining with actin-phalloidin was performed on MELK knockdown cells to determine effect of MELK loss on the cytoskeleton. Results: MELK mRNA and protein levels were significantly higher in TNBC cell lines compared to ER-positive breast cancers. Knockdown of MELK suppressed growth (≥50% growth inhibition) in six TNBC cell lines but had no effect on growth of six ER positive cell lines. Colony formation was also greatly reduced in TNBC cell lines but was not affected in ER positive cell lines upon siRNA knockdown of MELK. In addition, knockdown of MELK reduced migration of three TNBC cell lines (MDAMB231, MDAMB468 and Hcc70) but had no effect on the ER-positive cell line (MCF7). Decreased staining of actin filaments was observed in cell lines where migration was reduced upon MELK knockdown suggesting a role of MELK in formation of actin cytoskeleton. Current studies are focused on understanding how MELK regulates the cell growth and migration in TNBC. Conclusions: MELK is an important growth regulator of TNBC, but not of ER positive breast cancers. Our results indicate that MELK promotes cell migration in TNBC cells. These findings suggest that MELK is a promising target for the treatment of TNBC. Supported by a Susan G. Komen for the Cure Promise Grant (KG081694), and the John Charles Cain Award. Citation Format: Nidhi Batra, Corey Speers, Ivan Uray, Abhijit Mazumdar, Anna Tsimelzon, Susan Hilsenbeck, Gordon Mills, Powel Brown. Maternal embryonic leucine zipper kinase is critical for the growth and migration of triple negative breast cancer cells. [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 3299. doi:10.1158/1538-7445.AM2014-3299

Maternal embryonic leucine zipper kinase regulates pancreatic ductal, but not β-cell, regeneration.

The maternal embryonic leucine zipper kinase (MELK) is expressed in stem/progenitor cells in some adult tissues, where it has been implicated in diverse biological processes, including the control of cell proliferation. Here, we described studies on its role in adult pancreatic regeneration in response to injury induced by duct ligation and β‐cell ablation. MELK expression was studied using transgenic mice expressing GFP under the control of the MELK promoter, and the role of MELK was studied using transgenic mice deleted in the MELK kinase domain. Pancreatic damage was initiated using duct ligation and chemical beta‐cell ablation. By tracing MELK expression using a MELK promoter‐GFP transgene, we determined that expression was extremely low in the normal pancreas. However, following duct ligation and β‐cell ablation, it became highly expressed in pancreatic ductal cells while remaining weakly expressed in α‐cells and β‐ cells. In a mutant mouse in which the MELK kinase domain was deleted, there was no effect on pancreatic development. There was no apparent effect on islet regeneration, either. However, following duct ligation there was a dramatic increase in the number of small ducts, but no change in the total number of duct cells or duct cell proliferation. In vitro studies indicated that this was likely due to a defect in cell migration. These results implicate MELK in the control of the response of the pancreas to injury, specifically controlling cell migration in normal and transformed pancreatic duct cells.

Multi-kinase inhibitor C1 triggers mitotic catastrophe of glioma stem cells mainly through MELK kinase inhibition.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.

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