MELK Research Articles

Identification of Crucial Cancer Stem Cell Genes Linked to Immune Cell Infiltration and Survival in Hepatocellular Carcinoma.

Hepatocellular carcinoma is characterized by high recurrence rates and poor prognosis. Cancer stem cells contribute to tumor heterogeneity, treatment resistance, and recurrence. This study aims to identify key genes associated with stemness and immune cell infiltration in HCC. We analyzed RNA sequencing data from The Cancer Genome Atlas to calculate mRNA expression-based stemness index in HCC. A weighted gene co-expression network analysis was performed to identify stemness-related gene modules. A single-sample gene set enrichment analysis was used to evaluate immune cell infiltration. Key genes were validated using RT-qPCR. The mRNAsi was significantly higher in HCC tissues compared to adjacent normal tissues and correlated with poor overall survival. WGCNA and subsequent analyses identified 10 key genes, including minichromosome maintenance complex component 2, cell division cycle 6, forkhead box M1, NIMA-related kinase 2, Holliday junction recognition protein, DNA topoisomerase II alpha, denticleless E3 ubiquitin protein ligase homolog, maternal embryonic leucine zipper kinase, protein regulator of cytokinesis 1, and kinesin family member C1, associated with stemness and low immune cell infiltration. These genes were significantly upregulated in HCC tissues. A functional enrichment analysis revealed their involvement in cell cycle regulation. This study identified 10 key genes related to stemness and immune cell infiltration in HCC. These genes, primarily involved in cell cycle regulation, may serve as potential targets for developing more effective treatments to reduce HCC recurrence and improve patient outcomes.

Spatiotemporal regulation of MELK during mitosis.

Maternal Embryonic Leucine Zipper Kinase (MELK) has been studied intensively in recent years due to its overexpression in multiple cancers. However, the cell biology of MELK remains less characterized despite its well-documented association with mitosis. Here we report a distinctive pattern of human MELK that translocates from the cytoplasm to cell cortex within 3min of anaphase onset. The cortex association lasts about 30min till telophase. The spatiotemporal specific localization of MELK depends on the interaction between its Threonine-Proline (TP) rich domain and kinase associated 1 (KA1) domain, which is regulated by CDK1 kinase and PP4 protein phosphatase. KA1 domains are known to regulate kinase activities through various intramolecular interactions. Our results revealed a new role for KA1 domain to control subcellular localization of a protein kinase.

Targeting MELK in tumor cells and tumor microenvironment: from function and mechanism to therapeutic application.

Maternal embryonic leucine zipper kinase (MELK), a member of the adenosine monophosphate-activated protein kinase (AMPK) protein family, has been reported to be involved in the regulation of many cellular events. The aberrant expression of MELK is associated with tumorigenesis and malignant progression of various tumors. Moreover, MELK plays an essential role in the regulation of tumor microenvironment (TME), which affects the function of immune cells and the responsiveness to immunotherapy. Currently, small molecule inhibitors targeting MELK have been developed and evaluated in clinical trials. A comprehensive understanding of MELK may provide clues and confidence for subsequent basic research and scientific transformation. In this review, we provide a comprehensive overview of the structural features, molecular biological functions, and critical roles of MELK in tumors and TME, as well as the targeted agents under development for the treatment of tumors and discuss the perspective for MELK-targeted therapies for tumors.

Upregulation of MELK promotes chemoresistance and induces macrophage M2 polarization via CSF-1/JAK2/STAT3 pathway in gastric cancer

BackgroundGastric cancer (GC) stands out as one of the most prevalent malignancies affecting the digestive system, characterized by a substantial incidence rate and mortality. Maternal embryonic leucine zipper kinase (MELK) has been implicated in the advancement of various cancer types and the modulation of the tumor microenvironment. This study aims to delve into the involvement of MELK in chemoresistance and the tumor microenvironment of GC.MethodsThe MELK expression was detected using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry. Lentiviral transfection was employed to establish stable cell lines with either overexpressed or silenced MELK. The impact of MELK on the chemoresistance of GC cells and the polarization of macrophages was investigated through in vitro and in vivo functional assays. Additionally, the correlation between MELK and the cytokines colony-stimulating factor 1 (CSF-1), as well as stromal macrophages, was analysed. The prognostic significance of MELK, CSF-1, and CD206 expression levels in clinical samples was further investigated.ResultsMELK was found to be highly expressed in chemoresistant GC cells and tissues. Furthermore, both in vitro and in vivo assays indicated that MELK overexpression conferred chemoresistance in GC cells. Additionally, MELK overexpression was observed to induce M2 macrophage polarization via the CSF-1/JAK2/STAT3 pathway, thereby contributing to chemoresistance within the tumor microenvironment. The expression of MELK in GC tissues from neoadjuvant chemotherapy patients correlated positively with CSF-1 and CD206. Moreover, patients with higher expression levels of MELK, CSF-1, or CD206 exhibited significantly shorter OS and DFS rates.ConclusionsOur investigation underscores the critical role of MELK in promoting chemoresistance and inducing M2 macrophage polarization in GC. It proposes novel targets and methods for the treatment of GC, as well as prognostic factors for neoadjuvant chemotherapy.

An insight into the therapeutic effects of isoliquiritigenin in breast cancer.

Breast cancer ranks as the most widespread malignant condition in women, emerging as a primary contributor to mortality. The primary challenges in cancer treatments involve undesirable side effects. Therefore, exploring natural compounds as additional therapy could provide valuable insights. Isoliquiritigenin (ILN), an isoflavonoid featuring a chalcone moiety primarily sourced from Glycyrrhiza species, has garnered increasing interest in breast cancer research. This review aims to provide a comprehensive understanding of ILN's mechanisms of action in breast cancer, drawing from a range of in vitro and in vivo studies. ILN primarily acts by inhibiting angiogenesis, aromatase, inflammation, and cell proliferation, and preventing invasion and metastasis. Mechanistically, it downregulates miR-374a, phosphoinositide-3-kinase-protein kinase B/Akt, maternal embryonic leucine zipper kinase, vascular endothelial growth factor, and estrogen receptor protein levels, and causes enhancement of Wnt inhibitory factor-1, and Unc-51-like kinase 1 expression to treat breast cancer. ILN emerges as a promising natural option, offering therapeutic advantages with minimal side effects. However, it is important to note that current research on ILN is primarily limited to preclinical models, underscoring the need for further investigation to validate its potential efficacy.

Tumor cell-intrinsic MELK enhanced CCL2-dependent immunosuppression to exacerbate hepatocarcinogenesis and confer resistance of HCC to radiotherapy

BackgroundThe outcome of hepatocellular carcinoma (HCC) is limited by its complex molecular characteristics and changeable tumor microenvironment (TME). Here we focused on elucidating the functional consequences of Maternal embryonic leucine zipper kinase (MELK) in the tumorigenesis, progression and metastasis of HCC, and exploring the effect of MELK on immune cell regulation in the TME, meanwhile clarifying the corresponding signaling networks.MethodsBioinformatic analysis was used to validate the prognostic value of MELK for HCC. Murine xenograft assays and HCC lung metastasis mouse model confirmed the role of MELK in tumorigenesis and metastasis in HCC. Luciferase assays, RNA sequencing, immunopurification–mass spectrometry (IP-MS) and coimmunoprecipitation (CoIP) were applied to explore the upstream regulators, downstream essential molecules and corresponding mechanisms of MELK in HCC.ResultsWe confirmed MELK to be a reliable prognostic factor of HCC and identified MELK as an effective candidate in facilitating the tumorigenesis, progression, and metastasis of HCC; the effects of MELK depended on the targeted regulation of the upstream factor miR-505-3p and interaction with STAT3, which induced STAT3 phosphorylation and increased the expression of its target gene CCL2 in HCC. In addition, we confirmed that tumor cell-intrinsic MELK inhibition is beneficial in stimulating M1 macrophage polarization, hindering M2 macrophage polarization and inducing CD8 + T-cell recruitment, which are dependent on the alteration of CCL2 expression. Importantly, MELK inhibition amplified RT-related immune effects, thereby synergizing with RT to exert substantial antitumor effects. OTS167, an inhibitor of MELK, was also proven to effectively impair the growth and progression of HCC and exert a superior antitumor effect in combination with radiotherapy (RT).ConclusionsAltogether, our findings highlight the functional role of MELK as a promising target in molecular therapy and in the combination of RT therapy to improve antitumor effect for HCC.

Transcriptome analysis reveals hepatic disordered lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses due to maternal undernutrition during late pregnancy

Although lipid metabolism in fetal livers under intrauterine growth restriction (IUGR) conditions has been widely studied, the implications of maternal undernutrition on fetal hepatic lipid metabolism, lipotoxic injury, and abnormal development remain largely unknown. Therefore, this study investigated the effects of maternal undernutrition on disordered hepatic lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses using transcriptome analysis. Seventeen singleton ewes were randomly divided into three groups on day 90 of pregnancy: a control group (CG; 0.63 MJ metabolic energy/body weight (ME/BW)0.75/day, n = 5), maternal undernutrition group 1 (MU1; 0.33 MJ ME/BW0.75/day, n = 6), and maternal undernutrition group 2 (MU2; 0.20 MJ ME/BW0.75/day, n = 6). The fetuses were euthanized and recovered on day 130 of pregnancy. The levels of free fatty acids (FFA) in maternal blood (P < 0.01), fetal blood (P < 0.01), and fetal livers (P < 0.05) were increased in the MU1 and MU2 groups, but fetal hepatic triglyceride (TG) levels in the MU2 group (P < 0.01) and β-hydroxybutyrate levels in the MU1 and MU2 groups (P < 0.01) were decreased compared to the CG. Severe inflammatory cell infiltration and increased non-alcoholic fatty liver disease activity scores were observed in MU1 and MU2 fetuses (P < 0.01). Progressive deposition of fetal hepatic reticular fibers and collagen fibers in the fetal livers of the MU1 and MU2 groups and significant hepatic fibrosis were observed in the MU2 fetuses (P < 0.05). Gene set enrichment analysis showed that genes involved in lipid accumulation and FFA beta oxidation were downregulated in both MU groups compared to those in the controls. The fetal liver mRNA expression of the β-oxidation regulator, acetyl-CoA acetyltransferase 1, and the TCA regulator, isocitrate dehydrogenase were reduced in MU1 (P < 0.05) and MU2 (P < 0.01) fetuses, and downregulated mRNA expression of long chain fatty acid CoA ligase 1 (P < 0.05) and glycerol-3-phosphate acyltransferase (P < 0.01) was observed in MU2 fetuses. Differentially expressed genes (DEGs) in MU1 versus CG (360 DEGs) and MU2 versus CG (746 DEGs) were identified using RNA sequencing. Bioinformatics analyses of the 231 intersecting DEGs between MU1 versus CG and MU2 versus CG indicated that neutrophil extracellular traps (NETs) were induced and played a central role in fetal hepatic injury in IUGR sheep. Increased maternal blood myeloperoxidase (MPO) levels (P < 0.01), NE (Elane)-positive areas in fetal liver sections (P < 0.05), and fetal liver MPO protein expression (P < 0.01) were found in the MU1 and MU2 groups; however, MPO levels were reduced in the fetal membrane (P < 0.01) and fetal blood (P < 0.05) in the MU1 group, and in the maternal-fetal placenta and fetal blood in the MU2 group (P < 0.01). Analysis of gene expression trends in the intersecting DEGs between MU1 versus CG (129 DEGs) and MU2 versus CG (515 DEGs) further revealed that 30 hub genes were essential regulators of the G2/M cell cycle, all of which were associated with hepatocellular carcinoma. G0/G1 phase cells of the fetal liver were reduced in the MU1 (P < 0.05) and MU2 (P < 0.01) groups, whereas G2/M phase cells were elevated in the MU1 and MU2 groups (P < 0.01). The representatives of upregulated hub genes and fetal liver protein expression of maternal embryonic leucine zipper kinase and protein regulator of cytokinesis 1 were progressively enhanced in the MU1 and MU2 groups (P < 0.01), and topoisomerase II alpha protein expression in the MU2 group (P < 0.05), as expected. These results indicate that FFA overload, severe lipotoxic injury, and NETs were induced, and disease-promoting regulators of the G2/M cell cycle were upregulated in the fetal liver of IUGR sheep. These findings provide new insights into the pathogenesis of impaired hepatic lipid metabolism and abnormal development and the molecular origin of post-natal liver disease in IUGR due to maternal undernutrition. This information can support the development of new therapeutic strategies.

Identification and validation of the TRHDE-AS1/hsa-miR-449a/ADAMTS5 axis as a novel prognostic biomarker in prostate cancer.

Despite advancements in cancer research, the prognostic implications of competing endogenous RNA (ceRNA) networks in prostate cancer (PCa) remain incompletely understood. This study aimed to elucidate the prognostic relevance of ceRNA networks in PCa, utilizing a comprehensive bioinformatics approach alongside experimental validation. After searching The Cancer Genome Atlas (TCGA) database, RNA sequencing (RNA-Seq) data were extracted to identify differentially expressed RNAs (DERs) between 491 PCa samples and 51 normal prostate tissues, following which a comprehensive bioinformatics strategy was implemented to construct a ceRNA network. An optimal prognostic signature comprising these DERs was then established and validated using TCGA data. In addition, functional validation was performed through RNA pull-down, dual-luciferase reporter assays, quantitative real-time PCR, and western blot analysis conducted in PC-3 and DU145 cell lines, thereby complementing the bioinformatics analysis. A total of 613 DERs, comprising 103 long noncoding RNAs (lncRNAs), 60 microRNAs (miRNAs), and 450 messenger RNAs (mRNAs), were identified and utilized in constructing a ceRNA network, which encompassed 23 lncRNAs, 9 miRNAs, and 52 mRNAs. An optimal prognostic signature was established, including VPS9D1 antisense RNA 1 (VPS9D1-AS1), miR-449a, cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1), targeting protein for Xklp2 (TPX2), solute carrier family 7 member 11 (SLC7A11), copine7 (CPNE7), and maternal embryonic leucine zipper kinase (MELK), yielding area under the curve (AUC) values exceeding 0.8 across training, validation, and entire datasets. Our experiments results revealed an interaction between lncRNA TRHDE antisense RNA 1 (TRHDE-AS1) and miR-449a and that miR-449a could target the ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) mRNA. Knockdown of miR-449a significantly impeded cell proliferation, G1/S transition, migration and invasion, and promoted apoptosis in PC-3 and DU145 cells. Furthermore, knockdown of miR-449a notably downregulated protein expression of CDK4, cyclin D1, N-cadherin and vimentin, while upregulating protein expression of cleaved caspase-3 and E-cadherin. This study contributes to a deeper understanding of the prognostic-linked ceRNA network in PCa, providing fundamental insights that could improve diagnostic and therapeutic approaches for PCa management.

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.

Molecular docking and dynamics studies to identify novel active compounds targeting potential breast cancer receptor proteins from an indigenous herb Euphorbia thymifolia Linn

Background Breast cancer has become the most prevalent disease and its incidence has almost doubled in the Indian population. This increased burden demands new targeted therapies with novel compounds either synthetically produced or derived from indigenous plants, which could be a promising approach for the development of drugs. Euphorbia thymifolia L is a widely growing tropical herb that has been reported to have various ethnopharmacological properties, including anticancer properties. Therefore, the aim of the present study was to screen the phytoconstituents and identify the active compounds present in the methanolic extract of E. thymifolia (ME.ET) as ligands to inhibit potential protein targets implicated in breast cancer using an In-silico approach. Methods ME.ET was subjected to GC-MS analysis to screen the phytoconstituents, and the identified compounds were docked with protein targets such as extracellular signal-regulated kinases (ERK1), a serine/threonine kinase-1(AKT1), human epidermal growth factor 2 (HER2), estrogen receptor (ER), maternal embryonic leucine zipper kinase (MELK), polo-like kinase-1(PLK1), and protein tyrosine kinase (PTK6). Compounds with good docking scores were further subjected to dynamic studies to understand the protein ligand binding stability, ligand pathway calculation, and molecular mechanics energies combined with Poisson-Boltzmann (MM/PBSA) calculations using the Schrodinger suite. Results GC-MS analysis revealed the presence of 245 phytoconstituents, 219 of which were unique. When subjected to docking, these phytocompounds, namely 3,6,9,12-tetraoxatetradecane-1,14-diyl dibenzoate (TTDB) and succinic acid, 2-(dimethylamino) ethyl 4-isopropylphenyl ester (SADPE), showed good docking scores. Molecular dynamics studies showed a high affinity and low binding energy for TTDB with HER2, ERK1, and SADPE with ER. Conclusions Hence, in this study, we identified two lead compounds in E.thymifolia linn. Further invitro and invivo anticancer studies can be performed to confirm these results and to understand the molecular mechanism by which they exhibit anticancer activity against breast cancer.

Synthesis and evaluation of tryptanthrin derivatives as promising anticancer agents: In vitro, in silico, and SAR insights

In this study, with the aim of identifying new anticancer agents, we synthesized a series of tryptanthrin derivatives. All the synthesized compounds were investigated for their anticancer potential in-vitro, using an MTT-based assay against a panel of three different cancer cell lines (A-549, MCF-7, and MDA-MB-231). Amongst the synthesized derivatives, compound 3 showed excellent anticancer activity, with GI50 values of 5.03 ± 0.77 µM, 3.76 ± 0.55 µM, and 4.20 ± 0.50 µM against A-549, MDA-MB-231 and MCF-7 cells, respectively. Furthermore, these molecules did not show any cytotoxic effect against normal mouse embryonic fibroblast (NIH/3T3) cell lines suggesting no intrinsic cytotoxicity. In this study, ADME and toxicity profiles of the synthesized compounds were also predicted using in silico techniques. According to the findings, all of the synthesized derivatives are nontoxic and non-carcinogenic in the biological system and showed good ADME properties. Finally, potent compounds were subjected to in silico molecular docking and simulation studies were carried out against maternal embryonic leucine zipper kinase (MELK) to uncover potential protein-ligand interactions and affinity at the binding site. This study will help in the future development of novel anticancer agents.

Hsa_circ_0101050 accelerates the progression of Colon cancer by targeting the miR-140–3 p/MELK axis

BackgroundCircular RNAs (circRNAs) are involved in the progression of colon cancer (CC). This study aimed to examine the role of a new circRNA circ_0101050 in CC. MethodsDual-luciferase reporter and RNA immunoprecipitation analyses were performed to validate the target relationships among maternal embryonic leucine zipper kinase (MELK), microRNA (miR)-140–3 p, and circ_0101050. Expression levels were calculated using western blotting and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blotting was performed to evaluate the relative expression of Bcl-2 and Bax proteins to determine cell death. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to determine the proliferative potential of CC cells. The migration rate of CC cells was evaluated using wound healing assays. Tumor formation tests were performed to determine the effect of circ_0101050 on tumor development in vivo. ResultsElevated levels of circ_0101050 and MELK were observed in CC. By inhibiting circ 0,101,050 or MELK, CC cell proliferation and migration were inhibited, but CC cell apoptosis was promoted. Silencing circ_0101050 also inhibited CC growth in vivo. We also found that miR-140–3 p was downregulated, which alleviated the repressive effects of circ_0101050 knockdown on proliferating and migrating CC cells, as well as the stimulating effect on apoptosis. In addition, the absence of MELK alleviated the effects of miR-140–3 p downregulation, which enhanced CC cell malignancy. ConclusionsCirc_0101050 exacerbates malignant phenotypes in CC by targeting the miR-140–3 p/MELK axis. These findings suggested that the circ_0101050/miR-140–3 p/MELK network may be a prospective target for CC treatment.

Abstract 5978: Therapeutic targeting of MELK using a drug repurposing approach to combat TNBC cells

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer linked to high chemo-resistant metastatic cases and limited therapeutic options due to the absence of conventional bioreceptors. Therefore, to treat TNBC, it is imperative to identify promising druggable biological targets. Experimental Procedures: An integrated bioinformatic analysis of microarray datasets was performed to identify significantly differentially expressed genes (DEGs) in TNBC. For selecting a potent therapeutic target, the function and significance of the upregulated DEGs in TNBC was investigated by an in silico study employing the UALCAN portal, String database, KM plotter, and Enrichr tools. This analysis included gene expression validation, insights into protein-protein interactions, survival analysis, and pathway mapping of the genes. To obtain potent drugs binding to the selected target MELK (maternal embryonic leucine zipper kinase), a drug repurposing approach was employed starting with virtual screening followed by molecular dynamic simulation (MDS) using GROMACS. Further, the anti-cancer activity of the top two drugs was validated by live-dead imaging, gene expression analyses, MTT-based cell viability and ROS detection assays. Results: Based on a stringent criterion for significant DEG identification (log2|FC|&amp;gt;1 &amp; adjust P-value &amp;lt; 0.05), 25 overlapping upregulated genes were obtained from three selected datasets. Further, in silico analysis revealed that MELK is significantly upregulated across the four stages and subtypes of breast cancer, including TNBC. Higher MELK expression and its oncogenic interactions correlated with poor overall survival of TNBC patients. Evidence suggests that the existing inhibitor for MELK (OTSSP167) has shown poor specificity. Therefore, to block its activity, a library of 1293 FDA-approved drugs was screened against the kinase domain of MELK. MDS study and analysis using parameters like RMSD, RMSF and pair distance revealed that the top 10 drugs were strongly stabilized by H-bonds in the binding pocket of MELK, with minimal deviation and reduced fluctuations of MELK upon drug binding. Binding free energy calculation of drug-MELK interaction using the MM-PBSA method revealed that nine drugs possessed higher binding energy than the inhibitor. The top two drugs obtained from the study (originally kinase and reductase inhibitors respectively) were further validated for their in vitro anti-cancer efficacy in MDA-MB-231 and MDA-MB-468 cells. Treatment with these drugs exhibited (a) a dose-dependent decrease in proliferation, (b) significant cell death, (c) a marked increase in the cellular ROS levels, and (d) a decrease in the expression of MELK and its downstream targets in TNBC cell lines. Conclusion: The present study opens the avenue for using repurposed drugs as potential therapeutic molecules against MELK activity to combat TNBC progression. Citation Format: Arisha Arora, Shilpi Sarkar, Siddhartha S. Ghosh. Therapeutic targeting of MELK using a drug repurposing approach to combat TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5978.

Exploration of MELK as a downstream of Del-1 and druggable targets in triple-negative breast cancer.

In our previous study, Developmental endothelial locus-1 (Del-1) was a promising predictive marker for breast cancer. However, the downstream targets of Del-1 remain unknown. Here, we sought to discover a druggable target downstream of Del-1 and investigate the mechanism by which it regulates the course of breast cancer. To investigate Del-1 downregulation effect on breast cancer, we performed transcriptome analysis using RNA sequencing of Del-1 knockdowned MDA-MB-231 cell line Plus, to investigate the expression of Del-1 and Maternal embryonic leucine zipper kinase (MELK), mRNA levels in eight different triple-Negative Breast Cancer (TNBC) cell lines were analyzed. High-throughput sequencing was performed on total RNA isolated. OTS167 was used for MELK inhibition. The effects of MELK on cell proliferation and invasion were determined using the MTT and Matrigel transwell assays. Furthermore, we examined MELK expression in breast cancer tissue. Del-1 and MELK mRNA expression levels were significantly higher in the TNBC cell lines, MDA-MB-468, HCC-1806, and MBA-MB-231. Knocking down Del-1 with siRNA in HCC-1806 and MBA-MB-231 cells significantly decreased MELK expression and thus suggested a possible relationship between Del-1 and MELK. In MDA-MB-468 cells, a basal-like 1 TNBC cell line, OTS167 significantly inhibited breast cancer cell proliferation and promoted cell apoptosis. To further investigate the relationship between Del-1 and MELK, dual inhibition of both Del-1 and MELK was performed, which significantly reduced the viability of MDA-MB-468 and MBA-MB-231 cells. We found that MELK acts downstream of Del-1 and is a promising druggable target, especially in basal-like and mesenchymal stem-like subtype.

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.

Discovery of first-in-class PROTACs targeting maternal embryonic leucine zipper kinase (MELK) for the treatment of Burkitt lymphoma.

Maternal embryonic leucine zipper kinase (MELK) is a novel target for the treatment of various kinds of B-cell malignancies. However, the toxicity of inhibitors of MELK has led to clinical failures in cancer treatments. Moreover, inactivation of MELK catalytic domain is insufficient for achieving cancer cell apoptosis. To further confirm the role of MELK in Burkitt lymphoma treatment, we describe herein a structure-guided design of PROTACs targeting MELK. Through design, computer-assisted optimization and SAR studies, we developed the first-in-class MELK-targeting PROTAC MGP-39, which promoted a rapid and potent degradation of MELK in RAMOS cells. Additionally, the newly designed MELK degrader induced significant cell cycle arrest and apoptosis in cancer cells. Notably, compared to MELK inhibitors, MGP-39 has better anti-cancer activity and lower toxicity, indicating the practical role of PROTACs in avoiding the side effects of traditional inhibitors. More importantly, our results show that the use of a PROTAC can be adopted as a general and effective strategy for targeted cancer therapy.

Small Molecule Inhibitor Targeting Nemo-like Kinase Improves Erythropoiesis in Human and Mouse Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies, and an increased risk of developing cancer. Approximately 80% of patients with DBA have a mutation in one of 20 different genes that encode ribosomal proteins, resulting in abnormal protein translation. The current standard of care for patients with DBA includes steroids, chronic red cell transfusions, or stem cell transplantation, but these are all associated with significant side effects including infections, iron overload, and the risk of graft versus host disease. Thus, the development of more effective and less-toxic therapies is needed to treat the anemia seen in patients with DBA. Our previous work demonstrated that Nemo-Like Kinase (NLK) is overly active in red cell precursors in models of DBA as well as in patient samples (Wilkes, et al., 2020). We hypothesize that NLK plays an important role in the pathogenesis of DBA, and is a potential target for DBA therapy. Human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) were transduced with lentivirus co-expressing GFP and shRNA against RPS19/RPL11 or luciferase (control), and were differentiated in erythroid media in the presence of small molecule NLK inhibitors. After screening small molecule compounds that inhibit NLK as an off-target from previously approved or clinically advanced drugs, we found that a known MELK (maternal embryonic leucine zipper kinase) inhibitor, OTS167, improved erythropoiesis (CD71+ cell expansion) by 31% and 27% at a concentration of 50nM in RPS19- and RPL11-knockdown HSPCs, respectively. We also investigated the effect of OTS167 on the erythropoiesis in Rpl11-haploinsufficient mouse model of DBA ( Rpl11 wt/LoxpCre-ERT2 +, Morgado-Palacin, et al., 2015 and Rpl11 wt/LoxpMx-Cre + Liu,et al.,2023). Lineage-negative (Lin-) hematopoietic progenitors isolated from the mouse bone marrow were cultured in erythroid differentiation medium with OTS167. Our data showed that the erythroid progenitors (CD71+ cells) expanded from the cultured Lin- cells were increased by 20% (n=2, p=0.065) at an OTS167 concentration of 30nM. We confirmed that NLK activity was increased in erythroid progenitors from the Rpl11-haploinsufficient mice for in vitro phosphorylation of the substrate compared with tamoxifen-treated wild type mice. Moreover, we performed colony formation assay with OTS167 in the mouse bone marrow cells. OTS167 increased BFU-E colony numbers in a dose-dependent manner with the highest effect at a concentration of 50nM (n=3, p=0.003). OTS167 did not increased CFU-E colony number, suggesting that OTS167 affected the BFU-E stage and/or earlier stages of erythroid development. Also, OTS167 did not affected CFU-GM colony formation as myeloid lineage. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to minimize the need for chronic red cell transfusions or treatment with steroids. Our in vitro data suggest that pharmacologic inhibition of NLK with OTS167 may achieve this goal. We have initiated studies to test the efficacy of OTS167 in vivo using Rpl11-haploinsufficient mouse models. Given that DBA is associated with a number of mutations in the ribosome, using one drug that targets NLK as a common therapeutic target could be a more practical treatment option for patients in contrast to gene therapy or gene editing followed by autologous stem cell rescue. Even if the drug targeting NLK does not completely rescue the anemia in ribosome-insufficient cell models of DBA, the improvement could be enough to help patients especially in combination with other drugs that are currently being used or studied .

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.