Maternal Embryonic Leucine Zipper Kinase Inhibitor Research Articles

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.

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.

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 .

Integrated bioinformatics analysis and experimental validation reveals hub genes of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by systemic inflammation, especially synovitis, leading to joint damage. It is important to explore potential biomarkers and therapeutic targets to improve the clinical treatment of RA. However, the potential underlying mechanisms of action of available treatments for RA have not yet been fully elucidated. The present study investigated the potential biomarkers of RA and identified specific targets for therapeutic intervention. A comprehensive analysis was performed using mRNA files downloaded from the Gene Expression Omnibus. Differences in gene expression were analyzed and compared between the normal and RA groups. In addition, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed genes (DEGs). A protein-protein interaction network, Molecular Complex Detection and cytoHubba network were evaluated to identify hub genes. Finally, using an experimental RA rat model induced by Freund's complete adjuvant (FCA), the expression of potential biomarkers or target genes in RA were verified through reverse transcription-quantitative PCR. The results of the mRNA dataset processing revealed 195 DEGs in patients with RA when compared with the healthy controls. Moreover, 10 hub genes were identified in patients with RA and four candidate mRNAs were identified, as follows: Discs large homolog-associated protein 5 (DLGAP5), kinesin family member 20A (KIF20A), maternal embryonic leucine zipper kinase (MELK) and nuclear division cycle 80 (NDC80). Finally, the bioinformatics analysis results were validated by quantifying the expression of the DLGAP5, KIF20A, MELK and NDC80 genes in the FCA-induced experimental RA rat model. The findings of the present study suggested that the treatment of RA may be successful through the inhibition of DLGAP5, KIF20A, MELK and NDC80 expression. Therefore, the targeting of these genes may result in more effective treatments for patients with RA.

Structure-based virtual screening of chemical libraries as potential MELK inhibitors and their therapeutic evaluation against breast cancer

New targeted therapy for triple negative breast cancer (TNBC) is an urgent need, as advanced disease responds poorly to conventional chemotherapy. Genomic and proteomic studies are currently investigating new genes and proteins as promising therapeutic targets. One of such therapeutic targets is a cell cycle regulatory kinase; Maternal Embryonic Leucine Zipper Kinase (MELK), overexpressed in TNBC and correlated with cancer development. We performed molecular docking for virtual screening of chemical libraries (phytochemicals/synthetic drugs) against MELK protein structure and identified 8 phytoconstituents (isoxanthorin, emodin, gamma-coniceine, quercetin, tenuazonic acid, isoliquiritigenin, kaempferol, and Nobiletin) and 8 synthetic drugs (tetrahydrofolic acid, alfuzosin, lansoprazole, ketorolac, ketoprofen, variolin B, orantinib, and firestein) as potential hits interacting with the active site residues of MELK based on bound poses, hydrogen bond, hydrophobic interactions and MM/GBSA binding free energies. ADME and drug-likeness prediction further identified few hits with high drug-likeness properties and were further tested for anti-tumorigenic potential. Two phytochemicals isoliquiritigenin and emodin demonstrated growth inhibitory effects on TNBC MDA-MB-231 cells while much lower effect was observed on non-tumorigenic MCF-10A mammary epithelial cells. Treatment with both molecules downregulated MELK expression, induced cell cycle arrest, accumulated DNA damage and enhanced apoptosis. The study identified isoliquiritigenin and emodin as potential MELK inhibitors and provides a basis for subsequent experimental validation and drug development against cancer.

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.

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.

MELK predicts poor prognosis and promotes metastasis in esophageal squamous cell carcinoma via activating the NF‑κB pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide with a low 5-year survival rate due to the lack of effective therapeutic strategies. Accumulating evidence has indicated that maternal embryonic leucine zipper kinase (MELK) is highly expressed in several tumors and associated with tumor development. However, the biological effects of MELK in ESCC remain unknown. In the present study, cell phenotypical experiments and animal metastasis assays were performed to detect the influence of MELK knockdown in vitro and in vivo. The potential molecular mechanism of MELK-mediated ESCC metastasis was further investigated by western blotting and immunofluorescence staining. The results revealed that the expression of MELK in human ESCC tissues was higher than that in adjacent normal tissues and was positively associated with the poor prognosis of patients. Reducing MELK expression resulted in growth inhibition and suppression of the invasive ability of ESCC cells in vitro and in vivo. MELK inhibition induced alterations of epithelial-mesenchymal transition-associated proteins. Mechanistically, MELK interacted with IκB kinase (IKK) and promoted the phosphorylation of IKK, by which MELK regulated activation of the NF-κB pathway. Collectively, the present study revealed the function and mechanism of MELK in the cell metastasis of ESCC, which may be a potential therapeutic target for ESCC.

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.

Consensus Virtual Screening Identified [1,2,4]Triazolo[1,5-b]isoquinolines As MELK Inhibitor Chemotypes.

Maternal Embryonic Leucine‐zipper Kinase (MELK) is a current oncotarget involved in a diverse range of human cancers, with the usage of MELK inhibitors being explored clinically. Here, we aimed to discover new MELK inhibitor chemotypes from our in‐house compound library with a consensus‐based virtual screening workflow, employing three screening concepts. After careful retrospective validation, prospective screening and in vitro enzyme inhibition testing revealed a series of [1,2,4]triazolo[1,5‐b]isoquinolines as a new structural class of MELK inhibitors, with the lead compound of the series exhibiting a sub‐micromolar inhibitory activity. The structure‐activity relationship of the series was explored by testing further analogs based on a structure‐guided selection process. Importantly, the present work marks the first disclosure of the synthesis and bioactivity of this class of compounds.

Inhibition of FLT3-ITD and Melk By Small Molecule Inhibitor OTS167 in FLT3 mutant Acute Myeloid Leukemia (AML)

Background and Rationale: Maternal embryonic leucine zipper kinase (MELK) is a Ser/Thr protein kinase that is aberrantly expressed in many tumor types and demonstrated to be important in formation and maintenance of cancer stem cells. We have previously reported that MELK is also aberrantly expressed in AML cell lines and primary patient leukemia cells and is associated with a poor prognosis (Alachkar et. al., Oncotarget, 2014). MELK knockdown resulted in leukemia cell growth inhibition and apoptosis. Based on these preclinical insights, a small molecule inhibitor of MELK kinase activity, OTS167, (OncoTherapy Sciences, Tokyo, Japan) has been developed and is being tested in an early phase clinical trial in advanced myeloid malignancies at the University of Chicago (IRB#15-1373). Here, we report the activity of MELK signaling blockade in AML with FLT3 mutations, which are associated with drug resistance and poor clinical outcomes, and demonstrate synergy with known FLT3 kinase inhibitors.Methods: Both MELK and FLT3-ITD activity were targeted by treating the human AML cell lines MV4:11, MOLM-13, KOCL-48 and KG-1 which aberrantly express MELK as well as a FLT3-ITD, FLT3-TKD or FLT3-WT with the small molecule MELK inhibitor OTS167 alone or in combination with FLT3 kinase inhibitors Crenolanib or Gilteritinib.Results: OTS167 induced cell death of FLT3 mutant cell lines in a dose dependent manner and with IC50 similar to known FLT3 kinase inhibitors (OTS167 IC50 1.8-18.2nM; Crenolanib IC50 2.31-13.8nM; Gilteritinib IC50 4.9-36.3nM). Using isobologram analysis, OTS167 in combination with either Gilteritinib or Crenolanib induced synergistic cell death in FLT3 mutant leukemia cells at 48hrs (Figure 1A), but not in FLT3-WT leukemia cells. We found that OTS167 alone had the effect of inhibiting auto-phosphorylation of FLT3-ITD, as well as inhibiting MELK activity resulting in downregulation of MELK protein expression. Treatment of cells with 50nM OTS167 resulted in loss of FLT3-ITD (Y591) phosphorylation within 1hr, and total FLT3-ITD protein expression within 24 hours (Figure 1B). Loss of FLT3-ITD protein expression occurred in the presence of FLT3 mRNA and was not due to decreased stability of the protein, as treatment with OTS167 in the presence of cycloheximide did not decrease FLT3-ITD lifespan. Notably, the loss of FLT3-ITD protein expression observed with OTS167 treatment was not observed with the same concentration of FLT3 inhibitors Crenolanib or Gilteritinib. In vitro kinase assays using recombinant FLT3-ITD confirmed that OTS167 could act directly to inhibit FLT3-ITD kinase activity at an IC50 of 4.9nM, compared to a reported MELK IC50 of 0.11-1.1nM (Chung, et. al., Oncotarget, 2012). In MV4:11 cells, the constitutive activation of the MAPK, PI3K, and STAT5 signaling pathways by FLT3-ITD is inhibited by 50nM OTS167, similar to inhibition by 50nM FLT3 inhibitors Crenolanib and Gilteritinib (Figure 1C).In addition, treatment with either OTS167 or FLT3 inhibitor resulted in loss of MYC mRNA and protein expression at 3hr, suggesting that more effective downregulation of MYC expression by the combination may increase the susceptibility of FLT3 mutant AML to apoptosis. The inhibition of FLT3-ITD activation and expression after OTS167 treatment was not observed when MELK protein activity was downregulated using siRNA, suggesting the inhibitor may simultaneously target two independent signaling pathways active in cellular transformation. These results indicate that OTS167 may have a dual specificity in FLT3 mutant AML through targeting both aberrant MELK and FLT3 activity.Conclusion: In summary, the MELK inhibitor, OTS167, may be an attractive therapeutic agent for FLT3 mutant AML, with dual targeting resulting in decreased levels of FLT3-ITD protein and kinase activity. These data provide a rationale for testing dual MELK and FLT3 kinase inhibitors in patients with FLT3 mutant AML. [Display omitted] DisclosuresOdenike:CTI/Baxalta: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria. Stock:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Maternal Embryonic Leucine Zipper Kinase Inhibitor OTSSP167 Inhibits Osteoclast Activity and Restores Osteoblast Activity in a Murine Model of Multiple Myeloma Bone Disease

Multiple myeloma bone disease (MMBD) is characterized by an increased osteoclast activity and a decreased osteoblast differentiation in the multiple myeloma (MM) microenvironment, resulting in the development of lytic bone lesions. A large majority of MM patients suffer from MMBD, which not only causes morbidity but also negatively impacts patient survival. The development of novel therapies, ideally with a combined anti-resorptive and bone-anabolic effect, is of great interest because lytic lesions persist with the current standard of care, even in patients in complete remission. We have previously shown that maternal embryonic leucine zipper kinase (MELK) drives a high-risk gene network in MM and that MELK inhibition with OTSSP167 reduces tumor development in the 5TGM.1 model. In the current study, we assessed the effect of OTSSP167 treatment on bone cell activity and the development of MMBD.OTSSP167 inhibited osteoclast activity in vitro by decreasing monocytic progenitor cell viability as well as via a direct anti-resorptive effect on mature osteoclasts in both human (derived from PBMCs) and murine (RAW264.7) cell cultures. In addition, OTSSP167 stimulated matrix deposition and mineralization by human BMSC-TERT osteoblasts in vitro, which coincided with an increased expression of osterix . This combined anti-resorptive and osteoblast-stimulating effect of OTSSP167 resulted in the complete prevention of lytic lesions and bone loss in MM-bearing 5TGM.1 mice, as assessed by micro-computed tomography. OTSSP167 reduced the number of cortical perforations in MM-bearing mice, without affecting cortical thickness. The loss of trabecular bone volume that occurs in MM-bearing mice compared to healthy controls, was completely prevented, due to an increase in the number of trabeculae and a decrease in trabecular separation. Immunohistomorphometric analyses on the femurs of these mice showed that the increase in osteoclast surface observed in MM-bearing mice was completely prevented with OTSSP167 treatment. In addition, OTSSP167 treatment resulted in a recovery of osteoblast and osteoid surface levels.In conclusion, we show that OTSSP167 has a direct anti-MMBD effect in addition to its anti-MM activity, which warrants further clinical development of MELK inhibition in MM. [Display omitted] DisclosuresLudwig:Celgene: Speakers Bureau; Bristol-Meyers: Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Speakers Bureau; Takeda: Research Funding, Speakers Bureau.

KLF5-induced BBOX1-AS1 contributes to cell malignant phenotypes in non-small cell lung cancer via sponging miR-27a-5p to up-regulate MELK and activate FAK signaling pathway

BackgroundNon-small cell lung cancer (NSCLC) is a major histological subtype of lung cancer with high mortality and morbidity. A substantial amount of evidence demonstrates long non-coding RNAs (lncRNA) as critical regulators in tumorigeneis and malignant progression of human cancers. The oncogenic role of BBOX1 anti-sense RNA 1 (BBOX1-AS1) has been reported in several tumors. As yet, the potential functions and mechanisms of BBOX1-AS1 in NSCLC are obscure.MethodsThe gene and protein expression was detected by qRT-PCR and western blot. Cell function was determined by CCK-8, colony forming, would healing and transwell assays. Bioinformatics tools, ChIP assays, dual luciferase reporters system and RNA pull-down experiments were used to examine the interaction between molecules. Subcutaneous tumor models in nude mice were established to investigate in vivo NSCLC cell behavior.ResultsBBOX1-AS1 was highly expressed in NSCLC tissues and cells. High BBOX1-AS1 expression was associated with worse clinical parameters and poor prognosis. BBOX1-AS1 up-regulation was induced by transcription factor KLF5. BBOX1-AS1 deficiency resulted in an inhibition of cell proliferation, migration, invasion and EMT in vitro. Also, knockdown of BBOX1-AS1 suppressed NSCLC xenograft tumor growth in mice in vivo. Mechanistically, BBOX1-AS1 acted act as a competetive “sponge” of miR-27a-5p to promote maternal embryonic leucine zipper kinase (MELK) expression and activate FAK signaling. miR-27a-5p was confirmed as a tumor suppressor in NSCLC. Moreover, BBOX1-AS1-induced increase of cell proliferation, migration, invasion and EMT was greatly reversed due to the overexpression of miR-27a-5p. In addition, the suppressive effect of NSCLC progression owing to BBOX1-AS1 depletion was abated by the up-regulation of MELK. Consistently, BBOX1-AS1-mediated carcinogenicity was attenuated in NSCLC after treatment with a specific MELK inhibitor OTSSP167.ConclusionsKLF5-induced BBOX1-AS1 exerts tumor-promotive roles in NSCLC via sponging miR-27a-5p to activate MELK/FAK signaling, providing the possibility of employing BBOX1-AS1 as a therapeutic target for NSCLC patients.

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.

A novel carbon-11 radiolabeled maternal embryonic leucine zipper kinase inhibitor for PET imaging of triple-negative breast cancer

Maternal embryonic leucine zipper kinase (MELK) plays an important role in the regulation of tumor cell growth. It is abundant in triple-negative breast cancers (TNBC), making it a promising target for molecular imaging and therapy. Based on the structure of a potent MELK inhibitor (OTSSP167) with high affinity, we developed a novel carbon-11 radiolabeled molecular probe 11C-methoxy-OTSSP167, and evaluated its application in positron emission tomography (PET) imaging of TNBC. 11C-methoxy-OTSSP167 was successfully synthesized and was identical to its non-radiolabeled compound methoxy-OTSSP167 in high-pressure liquid chromatography (HPLC) chromatogram. The obtained tracer had 10 ± 2% radiolabeling yield with a total synthesis time of 40 min. The radiochemical purity of the tracer was more than 95%. The maximum uptake (9.97 ± 0.70%) of 11C-methoxy-OTSSP167 in MELK-overexpressing MDA-MB-231 cells was at 60 min in vitro. On PET, MDA-MB-231 tumors were clearly visible at 30, 60, and 90 min after injection of 11C-methoxy-OTSSP167, while no obvious radioactivity accumulation was found in the low-MELK MCF-7 tumors. In vivo biodistribution data were consistent with the findings of the PET images. However, the radioactive tracer showed high uptake in normal organs such as liver and intestine, which may limit the application of the tracer. In addition, a markedly different MELK expression level in MDA-MBA-231 and MCF-7 tumors was verified via IHC staining. In conclusion, 11C-methoxy-OTSSP167 was successfully developed and exhibited elevated uptake in MELK overexpressed tumor, indicating its potential for noninvasively imaging of MELK overexpressed TNBC.

An electrophilic warhead library for mapping the reactivity and accessibility of tractable cysteines in protein kinases.

Targeted covalent inhibitors represent a viable strategy to block protein kinases involved in different disease pathologies. Although a number of computational protocols have been published for identifying druggable cysteines, experimental approaches are limited for mapping the reactivity and accessibility of these residues. Here, we present a ligand based approach using a toolbox of fragment-sized molecules with identical scaffold but equipped with diverse covalent warheads. Our library represents a unique opportunity for the efficient integration of warhead-optimization and target-validation into the covalent drug development process. Screening this probe kit against multiple kinases could experimentally characterize the accessibility and reactivity of the targeted cysteines and helped to identify suitable warheads for designed covalent inhibitors. The usefulness of this approach has been confirmed retrospectively on Janus kinase 3 (JAK3). Furthermore, representing a prospective validation, we identified Maternal embryonic leucine zipper kinase (MELK), as a tractable covalent target. Covalently labelling and biochemical inhibition of MELK would suggest an alternative covalent strategy for MELK inhibitor programs.

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.

Mass spectrometry–based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.