eEF2K Expression Research Articles

Abstract 461: Dual-kinase targeted miRNA nanotherapy for the treatment of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represents 20% of breast cancer (BC) patients (51,000 cases/year in the US). It is associated with a highly aggressive course, early metastasis and relapses, and drug-resistant phenotype. About 40% of the patients die within 5 years despite available therapies. If the disease is metastatic, patients survive only 13 months. Since TNBC does not express targetable receptors such as ER, PR, and HER2, and it is a highly heterogeneous cancer with six genetically defined sub-types, currently, there are no common actionable molecular targets and no effective targeted therapies for TNBC. microRNAs (miRNAs) are 18-20 nt long non-coding RNAs and are often dysregulated in cancer cells. Tumor-suppressive miRNAs specifically silence their protooncogenic target genes (mRNAs) by binding specifically to their 3’-UTRs located in their target mRNAs, leading to inhibition of oncogenes that play critical roles in cell proliferation, cell cycle, migration, invasion, angiogenesis, drug resistance, tumor growth, and progression. We have previously demonstrated that Eukaryotic Elongation factor-2 kinase (eEF2K) and AXL are oncogenic kinases, and their higher expression is associated with poor survival in patients with TNBC. However, currently, there is no FDA-approved eEF2K and AXL-targeted therapeutics. To effectively co-target these kinases, we extensively analyzed miRNA databases using miRNA target prediction algorithms and recently, we discovered that miR-329-3p has a specific binding site at both 3’-UTRs of eEF2K and AXL mRNAs. Analyzing the TCGA TNBC database, we found that low expression of miR-329-3p is correlated with shorter overall survival in TNBC patients, and the expression of miR-329-3p is commonly reduced or lost in TNBC patient tumors. Since the successful clinical application of miRNA-based therapeutics requires safe and effective nano-delivery systems, we have developed magnetic iron-oxide-based nanoparticles (MNPs) for the delivery of miR-329-3p. We showed that miR-329-3p significantly suppresses cell proliferation, invasion, and migration by targeting both eEF2K and AXL and their downstream mediators such as SRC/FAK and Cyclin D1 in TNBC cell lines. In vivo systemic treatment with MNPs-miR-329 completely blocks tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC xenograft models in mice. Analysis of tumors shows remarkable inhibition in eEF2K and AXL expression, and clinically significant downstream targets including SRC, FAK, and Cyclin D1. We also demonstrated that miR-329 nanotherapy induces apoptosis and inhibits intratumoral proliferation in TNBC tumors in mice. Toxicity markers in mouse blood samples show that miR-329 nanotherapy is safe and not toxic to mice. Taken together, our study suggests that miR-329-3p nanotherapy may be used as a novel potential therapeutic approach in TNBC patients. Citation Format: Goknur Kara, Pinar Atalay Dundar, Nermin Kahraman, Emir Baki Denkbas, Bulent Ozpolat. Dual-kinase targeted miRNA nanotherapy for the treatment of triple-negative breast cancer [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 461.

Enhanced NK cell activation via eEF2K-mediated potentiation of the cGAS–STING pathway in hepatocellular carcinoma

BackgroundLiver cancer, particularly hepatocellular carcinoma (HCC), is characterized by a high mortality rate, attributed primarily to the establishment of an immunosuppressive microenvironment. Within this context, we aimed to elucidate the pivotal role of eukaryotic elongation factor 2 kinase (eEF2K) in orchestrating the infiltration and activation of natural killer (NK) cells within the HCC tumor microenvironment. By shedding light on the immunomodulatory mechanisms at play, our findings should clarify HCC pathogenesis and help identify potential therapeutic intervention venues. MethodsWe performed a comprehensive bioinformatics analysis to determine the functions of eEF2K in the context of HCC. We initially used paired tumor and adjacent normal tissue samples from patients with HCC to measure eEF2K expression and its correlation with prognosis. Subsequently, we enrolled a cohort of patients with HCC undergoing immunotherapy to examine the ability of eEF2K to predict treatment efficacy. To delve deeper into the mechanistic aspects, we established an eEF2K-knockout cell line using CRISPR/Cas9 gene editing. This step was crucial for verifying activation of the cGAS–STING pathway and the subsequent secretion of cytokines. To further elucidate the role of eEF2K in NK cell function, we applied siRNA-based techniques to effectively suppress eEF2K expression in vitro. For in vivo validation, we developed a tumor-bearing mouse model that enabled us to compare the infiltration and activation of NK cells within the tumor microenvironment following various treatment strategies. ResultsWe detected elevated eEF2K expression within HCC tissues, and this was correlated with an unfavorable prognosis (30.84 vs. 20.99 months, P = 0.033). In addition, co-culturing eEF2K-knockout HepG2 cells with dendritic cells led to activation of the cGAS–STING pathway and a subsequent increase in the secretion of IL-2 and CXCL9. Moreover, inhibiting eEF2K resulted in notable NK cell proliferation along with apoptosis reduction. Remarkably, after combining NH125 and PD-1 treatments, we found a significant increase in NK cell infiltration within HCC tumors in our murine model. Our flow cytometry analysis revealed reduced NKG2A expression and elevated NKG2D expression and secretion of granzyme B, TNF-α, and IFN-γ in NK cells. Immunohistochemical examination confirmed no evidence of damage to vital organs in the mice treated with the combination therapy. Additionally, we noted higher levels of glutathione peroxidase and lipid peroxidation in the peripheral blood serum of the treated mice. ConclusionTargeted eEF2K blockade may result in cGAS–STING pathway activation, leading to enhanced infiltration and activity of NK cells within HCC tumors. The synergistic effect achieved by combining an eEF2K inhibitor with PD-1 antibody therapy represents a novel and promising approach for the treatment of HCC.

Design and Characterization of a Novel eEF2K Degrader with Potent Therapeutic Efficacy Against Triple-Negative Breast Cancer.

Dysregulated eEF2K expression is implicated in the pathogenesis of many human cancers, including triple-negative breast cancer (TNBC), making it a plausible therapeutic target. However, specific eEF2K inhibitors with potent anti-cancer activity have not been available so far. Targeted protein degradation has emerged as a new strategy for drug discovery. In this study, a novel small molecule chemical is designed and synthesized, named as compound C1, which shows potent activity in degrading eEF2K. C1 selectively binds to F8, L10, R144, C146, E229, and Y236 of the eEF2K protein and promotes its proteasomal degradation by increasing the interaction between eEF2K and the ubiquitin E3 ligase βTRCP in the form of molecular glue. C1 significantly inhibits the proliferation and metastasis of TNBC cells both in vitro and in vivo and in TNBC patient-derived organoids, and these antitumor effects are attributed to the degradation of eEF2K by C1. Additionally, combination treatment of C1 with paclitaxel, a commonly used chemotherapeutic drug, exhibits synergistic anti-tumor effects against TNBC. This study not only generates a powerful research tool to investigate the therapeutic potential of targeting eEF2K, but also provides a promising lead compound for developing novel drugs for the treatment of TNBC and other cancers.

Abstract 3786: In vivo miR-449b mimic-based nanotherapy suppresses growth and progression of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) comprises 15-20% of breast cancers and is associated with an aggressive course and the highest mortality rate in all breast cancer subtypes owing to high rates of early metastasis recurrence, and a low response to adjuvant chemotherapy. Currently, there is no effective targeted therapy strategy for TNBC due to significant genetic heterogeneity and a lack of defined molecular targets. Recent evidence from our and other laboratories revealed that non-coding RNAs such as tumor suppressor microRNAs (miRNAs) are dysregulated in cancer cells by regulating the expression of target genes (mRNAs) that are involved in critical cellular processes such as cell cycle, proliferation, differentiation, and migration/invasion, drug resistance, and tumor growth. Analyzing the TCGA-patient database, we recently found that reduced miR-449b expression was associated with significantly shorter survival in TNBC patients (n=56 patients with high expression, n=41 patients with low expression, p=0.0011). Our studies revealed that miR449b expression is reduced in TNBC cell lines and its ectopic expression using commercially available transfection reagent inhibited TNBC cell proliferation, migration, and invasion. Since in vivo delivery of miRNA therapeutics has been challenging, we developed magnetic nanoparticles for miR-449b delivery which inhibited in vitro cell proliferation, invasion, and migration. To elucidate molecular mechanisms underlying miR-449b-induced tumor suppressive effects, we investigated potential targets of miR449b using several target prediction algorithms and found that miR-449b has binding sites in the 3'-UTR of the proto-oncogene, eukaryotic elongation factor-2 kinase (eEF2K) gene. We demonstrated eEF2K expression is highly upregulated in TNBC cell lines and eEF2K expression correlates with shorter overall survival in patients with TNBC. More importantly, eEF2K expression was markedly inhibited after ectopic expression of miR-449b in TNBC cells. TNBC cells were also treated with siRNAs specific for eEF2K which led to reduced cell growth and invasion/migration, recapitulating the effects of miR-449b expression Furthermore, in vivo administration of miR-449b-loaded magnetic nanoparticles (0.6 mg/kg once a week in TNBC orthotopic xenograft models (MDA-MB-231) in mice resulted in significant inhibition of eEF2K and tumor growth. Additionally, magnetic nanoparticles-miR-449b treatment in mice did not cause detectable side effects. These findings suggest that miR-449b functions as a tumor suppressor by targeting eEF2K-mediated oncogenic signaling leading to the tumor growth and progression of TNBC. Taken together, our study suggests that the miR-449 nanotherapy using magnetic nanoparticles may be a promising therapeutic strategy that provides a safe and effective antitumor efficacy against TNBC. Citation Format: Goknur Kara, Bulent Ozpolat. In vivo miR-449b mimic-based nanotherapy suppresses growth and progression of triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3786.

Impact of silencing eEF2K expression on the malignant properties of chordoma.

Eukaryotic elongation factor 2 kinase (eukaryotic elongation factor 2 kinase, eEF2K) is a calcium calmodulin dependent protein kinase that keeps the highest energy consuming cellular process of protein synthesis under check through negative regulation. eEF2K pauses global protein synthesis rates at the translational elongation step by phosphorylating its only kown substrate elongation factor 2 (eEF2), a unique translocase activity in ekaryotic cells enabling the polypeptide chain elongation. Therefore, eEF2K is thought to preserve cellular energy pools particularly upon acute development of cellular stress conditions such as nutrient deprivation, hypoxia, or infections. Recently, high expression of this enzyme has been associated with poor prognosis in an array of solid tumor types. Therefore, in a growing number of studies tremendous effort is being directed to the development of treatment methods aiming to suppress eEF2K as a novel therapeutic approach in the fight against cancer. In our study, we aimed to investigate the changes in the tumorigenicity of chordoma cells in presence of gene silencing for eEF2K. Taking a transient gene silencing approach using siRNA particles, eEF2K gene expression was suppressed in chordoma cells. Silencing eEF2K expression was associated with a slight increase in cellular proliferation and a decrease in death rates. Furthermore, no alteration in the sensitivity of chordoma cells to chemotherapy was detected in response to the decrease in eEF2K expression which intriguingly promoted suppression of cell migratory and invasion related properties. Our findings indicate that the loss of eEF2K expression in chordoma cell lines results in the reduction of metastatic capacity.

Abstract A088: Targeting endoplasmic reticulum stress-induced apoptosis to reduce health disparity in high-risk B-cell acute lymphoblastic leukemia in Hispanic/Latino children

Abstract B-cell Acute Lymphoblastic Leukemia (ALL) is the most common childhood malignancy. The death rate from B-ALL in Hispanic/Latino (H/L) is 40% higher than in non-Hispanic whites (NHW) after correcting socioeconomic factors. Deletion of one IKZF1 allele is highly increased H/L children, which provides a biological rationale for the worse prognosis of B-ALL in this population. The very high incidence (31% in all children and 65% in children ≥10 yrs) makes the deletion of one IKZF1 allele the most frequent genetic alteration that confers adverse prognosis in B-ALL in H/L children. The IKZF1 gene encodes a DNA-binding protein, IKAROS, which functions as a transcriptional regulator. Identification of IKAROS target genes in B-ALL of H/L children is essential to understand the specificity of B-ALL pathogenesis in those children and develop the targeted treatment to reduce the health disparity in survival for H/L children with B-ALL. Global DNA-binding analysis of primary B-ALL cells from H/L children showed that IKAROS binds to the promoter of eukaryotic elongation factor 2 kinase (eEF2K). eEF2K regulates protein synthesis, cell cycle progression and malignant transformation. The activity of eEF2K is increased in human malignancies, including B-ALL, and eEF2K is a critical regulator of endoplasmic reticulum (ER) stress-induced autophagy and apoptosis in tumor cells. The role of IKAROS in the regulation of eEF2K expression in B-ALL of H/L children was established using gain-of-function and loss-of-function experiments. Overexpression of IKZF1 in H/L B-ALL cells results in reduced expression of eEF2K, while IKZF1 knock-down results in increased transcription of eEF2K in H/L B-ALL. Since IKAROS function in leukemia is regulated by pro-oncogenic Casein Kinase 2 (CK2), we tested whether CK2 can control the ability of IKAROS to regulate the expression of eEF2K in H/L B-ALL. Increased expression of CK2 in H/L B-ALL results in increased expression of the eEF2K gene due to the loss of IKAROS binding to the promoter of the eEF2K genes. Inhibition of CK2 with shRNA and/or a specific CK2 inhibitor, CX-4945, restores IKAROS binding to promoter of eEF2K in high-risk B-ALL with deletion of one IKZF1 allele in H/L children. Restoration of IKAROS binding to promoter of eEF2K in high-risk B-ALL in H/L children results in reduced expression of this gene. This was associated with increased sensitivity to Akt inhibition by MK-2206. Combination treatment with CK2 inhibitor and MK-2206 showed a synergistic effect on B-ALL cells from H/L children. In summary, presented data demonstrate that IKAROS and CK2 regulate ER stress-induced apoptosis via regulation of eEF2K expression in high-risk B-ALL in Hispanic/Latino children. Results suggest that targeting ER stress-induced apoptosis in combination with CK2 inhibitors can be an efficient treatment to reduce the health disparity in survival for Hispanic/Latino children with B-ALL. Citation Format: Sinisa Dovat, Yali Ding, Dhimant Desai, Arati Sharma. Targeting endoplasmic reticulum stress-induced apoptosis to reduce health disparity in high-risk B-cell acute lymphoblastic leukemia in Hispanic/Latino children [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A088.

A functional eEF2K-eEF2 pathway in the NAc is critical for the expression of cocaine-induced psychomotor sensitisation and conditioned place preference

Recent evidence links synaptic plasticity and mRNA translation, via the eukaryotic elongation factor 2 kinase (eEF2K) and its only known substrate, eEF2. However, the involvement of the eEF2 pathway in cocaine-induced neuroadaptations and cocaine-induced behaviours is not known. Knock-in (KI) mice and shRNA were used to globally and specifically reduce eEF2K expression. Cocaine psychomotor sensitization and conditioned place preference were used to evaluate behavioural outcome. Changes in eEF2 phosphorylation were determined by western blot analyses. No effect was observed on the AMPA/NMDA receptor current ratio in the ventral tegmental area, 24 h after cocaine injection in eEF2K-KI mice compared with WT. However, development and expression of cocaine psychomotor sensitization were decreased in KI mice. Phosphorylated eEF2 was decreased one day after psychomotor sensitization and returned to baseline at seven days in the nucleus accumbens (NAc) of WT mice, but not in eEF2K-KI mice. However, one day following cocaine challenge, phosphorylated eEF2 decreased in WT but not KI mice. Importantly, specific targeting of eEF2K expression by shRNA in the NAc decreased cocaine condition place preference. These results suggest that the eEF2 pathway play a role in cocaine-induced locomotor sensitization and conditioned place preference.

MiRNA-193b-5p Suppresses Pancreatic Cancer Cell Proliferation, Invasion, Epithelial Mesenchymal Transition, and Tumor Growth by Inhibiting eEF2K.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer deaths in the US due to the lack of effective targeted therapeutics and extremely poor prognosis. The study aims to investigate the role of miR-193b and related signaling mechanisms in PDAC cell proliferation, invasion, and tumor growth. Using PDAC cell lines, we performed cell viability, colony formation, in vitro wound healing, and matrigel invasion assays following transfection with miR-193b mimic or control-miR. To identify potential downstream targets of miR-193b, we utilized miRNA-target prediction algorithms and investigated the regulation of eukaryotic elongation factor-2 kinase (eEF2K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways and mediators of epithelial mesenchymal transition (EMT). The role of miR-193b in PDAC tumorigenesis was evaluated in in vivo tumor growth of Panc-1 xenograft model in nude mice. We found that miR-193b is under expressed in PDAC cells compared to corresponding normal pancreatic epithelial cells and demonstrated that ectopic expression of miR-193b reduced cell proliferation, migration, invasion, and EMT through downregulation of eEF2K signaling in PDAC cells. miR-193b expression led to increased expression of E-Cadherin and Claudin-1 while decreasing Snail and TCF8/ZEB1 expressions via eEF2K and MAPK/ERK axis. In vivo systemic injection of miR-193b using lipid-nanoparticles twice a week reduced tumor growth of Panc-1 xenografts and eEF2K expression in nude mice. Our findings suggest that miR-193b expression suppresses PDAC cell proliferation, migration, invasion, and EMT through inhibition of eEF2K/MAPK-ERK oncogenic axis and that miR-193b-based RNA therapy might be an effective therapeutic strategy to control the growth of PDAC.

Eukaryotic Extension Factor 2 Kinase may Affect the Occurrence and Development of Glioblastoma Through Immune Cell Infiltration.

Glioblastoma (GBM) is one of the most common malignancies among primary brain tumors in adults, featuring a poor prognosis and a high recurrence rate. Eukaryotic elongation factor 2 kinase (eEF2K) is a calcium/calmodulin-dependent protein kinase that is involved in promoting tumor cell proliferation, migration, invasion, and survival. However, its expression level in GBM, its prognostic impact and correlation with immune infiltration are not yet known. In this study, we used The Cancer Genome Atlas (TCGA) database to explore the potential molecular mechanisms of eEF2K in GBM development and clinical prognosis in terms of gene expression, survival status, immune infiltration, and associated cellular pathways. We found that eEF2K expression levels were elevated in GBM, but eEF2K was not associated with the prognosis of GBM patients; eEF2K expression in GBM was associated with multiple immune cell infiltrations. These results show a statistical correlation between eEF2K expression and the development of GBM and immune cell infiltration, which helps us to understand the roles of eEF2K in GBM from different perspectives.

EEF2K promotes PD-L1 stabilization through inactivating GSK3β in melanoma

BackgroundImmune checkpoint blockade (ICB) targeting programmed death ligand-1 (PD-L1)/programmed cell death protein-1 (PD-1) pathway has become an attractive strategy for cancer treatment; however, unsatisfactory efficacy has limited its clinical benefits....

Novel inhibitors of eukaryotic elongation factor 2 kinase: In silico, synthesis and in vitro studies

Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual alpha kinase whose expression is highly upregulated in various cancers and contributes to tumor growth, metastasis, and progression. More importantly, eEF2K expression is associated with poor clinical outcome and shorter patient survival in breast, lung and ovarian cancers. Therefore, eEF2K is an emerging molecular target for development of novel targeted therapeutics and precision medicine in solid cancers. Currently, there are not any available potent and specific eEF2K inhibitors for clinical translation. In this study, we designed and synthesized a series of novel compounds with coumarin scaffold with various substitutions and investigated their effects in inhibiting eEF2K activity using in silico approaches and in vitro studies in breast cancer cells. We utilized an amide substitution at position 3 on the coumarin ring with their pharmacologically active groups containing pyrrolidine, piperidine, morpholine and piperazine groups with (CH2)2 bridged for aliphatic amides. Due to their ability to form covalent binding to the target enzyme, we also investigated the effects of boron containing groups on functionalized coumarin ring (3 compounds) and designed novel aliphatic and aromatic derivatives of coumarin scaffolds (10 compounds) and phenyl ring with boron groups (4 compounds). The Glide/SP module of the Maestro molecular modeling package was used to perform in silico analysis and molecular docking studies. According to our combined results, structure activity relationship (SAR) was performed in detail. Among the newly designed, synthesized, and tested compounds, our in vitro findings revealed that several compounds displayed a highly effective eEF2K inhibition at submicromolar concentrations in in vitro breast cancer cells. In conclusion, we identified novel compounds that can be used as eEF2K inhibitors and that they should be further evaluated by in vivo preclinical tumor models studies for antitumor efficacy and clinical translation.

EEF2K as a novel metastatic and prognostic biomarker in gastric cancer patients

BackgroundAlthough eukaryotic elongation factor 2 kinase (eEF2K) has been reported to be a potential oncogenic factor in many human cancers, its usefulness as a clinical prognostic biomarker for gastric cancer has not been investigated. MethodsIn this study, data about 540 patients with stomach adenocarcinoma (STAD) were analyzed from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases to determine the expression of eEF2K. Immunohistochemistry (IHC), western blots, and real-time polymerase chain reaction (RT-PCR) were also performed to determine the clinical significance of eEF2K expression in 96 postoperative patients with gastric cancer. Among the 96 patients, 36 had low expression of eEF2K and 60 had high expression. ResultsAnalysis of the TCGA and GEO datasets revealed that eEF2K expression was significantly higher in the STAD tissue samples than in the non-tumorous gastric tissues. IHC, western blots, and RT-PCR confirmed these findings. The high expression level of eEF2K was found to be related to the presence of lymph node metastasis (p = 0.002). Moreover, multivariate analysis showed that eEF2K was an independent indicator of prognosis for overall survival (OS) (hazard ratio [HR] = 1.72, 95% confidence interval [CI] = 1.06–2.79; p = 0.03) and disease-free survival (DFS) (HR = 1.66, 95% CI = 0.997–2.765; p = 0.052) in patients with surgically resected STAD. ConclusionCollectively, our findings suggest that eEF2K is a clinical indicator of metastatic and prognostic significance for STAD survival and could serve as a potential therapeutic target.

NcRNA therapy with miRNA-22-3p suppresses the growth of triple-negative breast cancer

Deregulation of noncoding RNAs, including microRNAs (miRs), is implicated in the pathogenesis of many human cancers, including breast cancer. Through extensive analysis of The Cancer Genome Atlas, we found that expression of miR-22-3p is markedly lower in triple-negative breast cancer (TNBC) than in normal breast tissue. The restoration of miR-22-3p expression led to significant inhibition of TNBC cell proliferation, colony formation, migration, and invasion. We demonstrated that miR-22-3p reduces eukaryotic elongation factor 2 kinase (eEF2K) expression by directly binding to the 3′ untranslated region of eEF2K mRNA. Inhibition of EF2K expression recapitulated the effects of miR-22-3p on TNBC cell proliferation, motility, invasion, and suppression of phosphatidylinositol 3-kinase/Akt and Src signaling. Systemic administration of miR-22-3p in single-lipid nanoparticles significantly suppressed tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC models. Evaluation of the tumor response, following miR-22-3p therapy in these models using a novel mathematical model factoring in various in vivo parameters, demonstrated that the therapy is highly effective against TNBC. These findings suggest that miR-22-3p functions as a tumor suppressor by targeting clinically significant oncogenic pathways and that miR-22-3p loss contributes to TNBC growth and progression. The restoration of miR-22-3p expression is a potential novel noncoding RNA-based therapy for TNBC.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 lowered blood pressure in spontaneously hypertensive rats via inducing vasorelaxation

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) suppresses protein translation. We previously reported eEF2K expression was upregulated in mesenteric arteries (MA) from spontaneously hypertensive rats (SHR). We have recently revealed A484954, an eEF2K inhibitor, acutely suppressed vasopressor agonists-induced increase of blood pressure (BP) in normal Wistar rats. In this study, we examined the acute effects of A484954 on BP in SHR and explored underlying mechanisms. BP was measured by a carotid cannulation method in SHR. Isometric contraction in MA from SHR was measured. Endothelial nitric oxide synthase (eNOS) dimerization was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A484954 lowered BP in 15-week-old SHR. A484954 induced relaxation in MA from both 4- and 7-9-week-old SHR. In MA from 4-week-old SHR, A484954-induced relaxation was inhibited almost completely by a NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME) and significantly by a β blocker, propranolol. In MA from 7-9-week-old SHR, on the other hand, A484954-induced relaxation was inhibited partly either by l-NAME, indomethacin, a cyclooxygenase inhibitor, or l-NAME + indomethacin. A484954 promoted the dimerization of eNOS in human endothelial cells. In summary, we have revealed A484954 lowers BP in SHR perhaps through the vasorelaxation via the production of endothelium-derived relaxing factors.

Abstract B27: A link between small noncoding RNAs and mRNA translation elongation: The let7-eEF2K axis in pediatric tumor adaptation to nutrient deprivation

Abstract Background: Medulloblastoma (MB) and neuroblastoma (NB) are the most common pediatric solid cancers and the leading cause of childhood cancer-related deaths. Genetic amplifications of the MYC and MYCN oncogenes are the strongest independent predictor of adverse outcomes in MB and NB, underscoring a dire need to improve therapeutic approaches. Tumors continually weather stress from acute microenvironmental changes, including limited nutrient availability. The let-7 family of small noncoding RNAs (miRNAs) inhibits tumor progression and regulates metabolism by selectively targeting and degrading mRNAs, including MYC and MYCN. Indeed, let-7 miRNAs are frequently repressed in several cancers, including in MYC-driven MB and NB. We previously reported that the mRNA translation elongation regulator eukaryotic eEF2K is a pivotal mediator of cancer cell adaptation to nutrient deprivation. Our preliminary data indicate that the eEF2K 3´ untranslated region (UTR) harbors a potential binding site for let-7 miRNAs. In addition, eEF2K mRNA and let-7 miRNA expressions negatively correlate in MB and NB. We therefore hypothesized that let-7 downregulation induces eEF2K expression, thereby supporting MYC-driven MB and NB adaptation to nutrient deprivation and tumor progression. Methods: Effects of eEF2K pharmacologic and genetic inhibition on MB and NB cell death were evaluated in vitro by flow cytometry, while cellular mRNA translation rates were studied by polysome fractionation and Click chemistry. The ability of let-7 to degrade eEF2K mRNA was assessed by let-7 miRNAs transfection into MB cells, followed by RT-PCR and Western blotting for eEF2K. Binding of let-7 to the eEF2K 3´UTR was validated by luciferase reporter assay. Finally, NB xenograft mouse models confirmed in vitro observations. Results: Inhibition of eEF2K significantly reduces survival of MYC/MYCN-amplified NB and MB cell lines under nutrient deprivation, altering their mRNA translation rates. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels by 40-50%. Downregulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3´UTR. eEF2K knockdown decreases growth twofold in MYCN-amplified NB xenografts in mice under calorie-restriction diets. Conclusions: Let-7 miRNAs degrade the eEF2K mRNA by binding to its 3´UTR, indicating that let-7 repression in MYC-driven NB and MB is partially responsible for increased eEF2K levels. Moreover, the let-7-eEF2K axis constitutes a critical mechanism for MYC-driven MB and NB adaptation to acute metabolic stress, representing a promising therapeutic target. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs, as eEF2K activity appears critical under metabolic stress conditions. Citation Format: Alberto Delaidelli, Gian Luca Negri, Xue Qi Wang, Simran Sidhu, Brian Cho, Gabriel Leprivier, Volker Hovestadt, Stefan Pfister, Michael Taylor, Marcel Kool, Poul Sorensen. A link between small noncoding RNAs and mRNA translation elongation: The let7-eEF2K axis in pediatric tumor adaptation to nutrient deprivation [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B27.

Abstract B55: MYCN promotes m6a-dependent translation of eEF2K mRNA during adaptation to ER stress

Abstract MYCN-amplified neuroblastoma is one of the most common and malignant pediatric tumors with low survival rates. Attempts to develop specific inhibitors to target MYCN have not been successful, prioritizing research into MYCN downstream targets. It was previously shown that eukaryotic translation elongation factor 2 (eEF2K) is upregulated in MYCN-amplified neuroblastoma and facilitates its adaptation to the nutrient deprivation (ND). However, the mechanism of eEF2K overexpression and its role in such adaptation is unknown. Using SUnSET assays, we showed that MYCN overexpression increases protein synthesis rates, demonstrating that MYCN-amplified cells depend on protein biosynthesis to cover the needs of fast proliferation. Enhanced protein biosynthesis increases the susceptibility for protein aggregates and subsequent unfolded protein response (UPR) activation and ER stress. ER stress also occurs under ND due to accumulation of unglycosylated proteins in the ER. To check how MYCN-amplified tumors might adapt to ER stress, we performed gene set enrichment analyses (GSEA) of genes that are overexpressed in MYCN amplified neuroblastoma and found “UPR” in the top 10 gene sets. As eEF2K regulates protein synthesis rates by slowing down the elongation step of mRNA translation, we examined whether eEF2K is part of the ER stress adaptation mechanism in MYCN-amplified cells. We found that during ND, eEF2K KO cells are more sensitive to thapsigargin (Tg) and tunicamycin (Tm), ER stress-inducing compounds, and show higher levels of ATF4, a major ER stress marker. Notably, low doses of cycloheximide, which blocks translation elongation, rescued the phenotype in eEF2K KO cells, confirming that the observed effect of eEF2K KO is linked to translation elongation. Finally, GSEA of RNAseq data obtained from TET21N WT vs. eEF2K KO after ND showed upregulation of genes responsible for the UPR and ER stress in eEF2K KO cells. Therefore, eEF2K appears to be linked to ER stress adaptation. To find potential mechanisms of MYCN-dependent regulation of eEF2K expression, we performed bioinformatic analyses of the eEF2K mRNA and found potential binding sites for METTL3, an enzyme that functions in the methylation of adenosine residues in mRNAs, in the eEF2K 3´-UTR. METTL3 knockdown resulted in a marked decrease of eEF2K protein without significant changes at the mRNA level. We applied polysome fractionation to study the effect of m6a modification on eEF2K mRNA translation. We discovered that METTL3 KD brings eEF2K mRNA to lighter polysome fractions compared with WT cells. As YTHDF1 is the only known reader of m6a modification that increases translation, we performed YTHDF1 knockdown and observed a decrease in eEF2K protein levels. Our findings indicate that MYCN amplified neuroblastoma cells increase translation of eEF2K mRNAs via a METTL3-YTHDF1 axis, and that this potentially functions as part of an adaptation mechanism under increased protein synthesis and subsequent ER stress. Citation Format: Andrii Vislovukh, Cristopher Hughes, Michael Lizardo, Poul H. Sorensen. MYCN promotes m6a-dependent translation of eEF2K mRNA during adaptation to ER stress [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B55.

A Novel Micropeptide Encoded by Y-Linked LINC00278 Links Cigarette Smoking and AR Signaling in Male Esophageal Squamous Cell Carcinoma.

Long noncoding RNAs (lncRNA) have been shown to play critical roles in many diseases, including esophageal squamous cell carcinoma (ESCC). Recent studies have reported that some lncRNA encode functional micropeptides. However, the association between ESCC and micropeptides encoded by lncRNA remains largely unknown. In this study, we characterized a Y-linked lncRNA, LINC00278, which was downregulated in male ESCC. LINC00278 encoded a Yin Yang 1 (YY1)-binding micropeptide, designated YY1BM. YY1BM was involved in the ESCC progression and inhibited the interaction between YY1 and androgen receptor (AR), which in turn decreased expression of eEF2K through the AR signaling pathway. Downregulation of YY1BM significantly upregulated eEF2K expression and inhibited apoptosis, thus conferring ESCC cells more adaptive to nutrient deprivation. Cigarette smoking decreased m6A modification of LINC00278 and YY1BM translation. In conclusion, these results provide a novel mechanistic link between cigarette smoking and AR signaling in male ESCC progression. SIGNIFICANCE: Posttranscriptional modification of a micropeptide-encoding lncRNA is negatively impacted by cigarette smoking, disrupting negative regulation of the AR signaling pathway in male ESCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2790/F1.large.jpg.See related commentary by Banday et al., p. 2718.

EEF2 kinase mediated autophagy as a potential therapeutic target for paclitaxel-resistant triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) are initially responsive to chemotherapy, but most recurrent TNBCs develop resistance. Autophagy is believed to play dual roles in cancer and might contribute to chemoresistance. In this study, we aimed to investigate the role of autophagy and its regulator, eukaryotic elongation factor 2 kinase (eEF2K), in determining the biological nature of TNBC. We used in vitro models of TNBC, namely, paclitaxel-resistant cell lines derived from sensitive cell lines. Various approaches to measuring autophagy flux were applied. We assessed the effects of inhibiting autophagy and silencing eEF2K on cell viability, tumor formation and invasion. We also collected residual tumor samples from 222 breast cancer patients who underwent neoadjuvant chemotherapy and measured eEF2K and LC3 expression levels by immunohistochemistry (IHC). Multivariate survival analysis was used to determine prognostic variables. Compared to the parental lines, the chemoresistant lines exhibited enhanced starvation-stimulated autophagy and showed significant decreases in cell viability, growth and invasion upon treatment with autophagy inhibitors. eEF2K silencing also resulted in the suppression of autophagic activity and in aggressive biological behavior. In the survival analysis, residual tumor LC3 (P=0.001) and eEF2K (P=0.027) expression levels were independent prognostic factors for patients who underwent neoadjuvant chemotherapy, especially in those with TNBC. Our study indicated that eEF2K and autophagy play key roles in the maintenance of aggressive tumor behavior and chemoresistance in resistant TNBC. eEF2K silencing may be a novel strategy for the treatment of TNBC.

Abstract 3660: A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation

Abstract BACKGROUND/OBJECTIVES: MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN and MYC amplification in childhood neuroblastoma (NB) and medulloblastoma (MB) are associated with aggressive disease and high mortality, underscoring a dire need for novel therapies. Let-7 microRNAs (miRNAs) inhibit tumor progression and regulate metabolism by degrading several mRNAs, including MYC. Let-7 miRNAs are therefore frequently repressed in cancer, including MYC-driven NB and MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cells adaptation to nutrient deprivation (ND). Publicly available transcriptomic database analyses indicate that eEF2K expression significantly correlate with MYCN and MYC expression in multiple tumor cohorts. Our preliminary data also indicate that the eEF2K 3’ untranslated region (UTR) harbors a potential binding site for let-7 miRNAs. In addition, eEF2K mRNA and let-7 miRNA expression negatively correlates in NB and MB, suggesting a potential regulation of the former by the latter. We therefore hypothesized that let-7 down-regulation induces eEF2K expression, thereby supporting MYC-driven NB and MB adaptation to ND and tumor progression. METHODS: Immunohistochemistry for eEF2K substrate (p-eEF2) was performed on NB and MB tissue microarrays to link results with MYC expression and outcome. Effects of eEF2K pharmacological and genetic inhibition on NB and MB cell survival were evaluated in vitro by MTT assay and PI staining. The ability of let-7 to degrade eEF2K mRNA was assessed by let-7 miRNAs transfection into MB cells, followed by RT-PCR and Western Blotting for eEF2K. Binding of let-7 to the eEF2K 3’UTR was validated by luciferase reporter assay. Finally, NB xenograft mouse models were used to confirm in vitro observations. RESULTS: High eEF2K activity is linked to MYC over-expression and reduced survival in NB and MB (p<0.05). Pharmacological and genetic inhibition of eEF2K significantly reduces survival of MYC/MYCN-amplified NB and MB cell lines under ND. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40-50%), and down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3’UTR. Knockdown of eEF2K determines a twofold growth decrease of MYCN-amplified NB xenografts when mice are kept under caloric restriction diet. CONCLUSIONS: Let-7 miRNAs degrade eEF2K mRNA by binding to its 3'UTR, indicating that let-7 repression in MYC-driven NB and MB is partially responsible for eEF2K increased levels and activity. Moreover, the let-7-eEF2K axis represents a critical mechanism for MYC-driven NB and MB adaptation to ND, constituting a promising therapeutic target. Citation Format: Alberto Delaidelli, Gian Luca Negri, Brian Cho, Simran Sidhu, Stefan Pfister, Michael Taylor, Gabriel Leprivier, Marcel Kool, Poul Soresnsen. A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3660.

Effects of cellular energy homeostasis modulation through AMPK on regulation of protein translation and response to hypoxia

Abstract Objective AMP-activated-protein-kinase (AMPK) regulates cellular energy-homeostasis. Eukaryotic-elongation-factor-2-kinase (eEF2K) plays important roles in regulating protein-synthesis, functions similarly to AMPK to protect cancer-cells from metabolic stress. Tumor-hypoxia induces angiogenesis with activation of hypoxia-inducible-factor-1-alpha (HIF-1α). AMPK might be implicated both in regulation of protein-translation and response-to-hypoxia. We aimed to investigate the effects of AMPK modulation on regulation of protein translation and response-to-hypoxia by evaluating eEF2K and HIF-1α proteins in breast and hepatocellular cancers. Materials and methods Hepatocellular (Huh-7, HepG2) and breast (SKBR-3, MDA-MB-453) cancer-cells were incubated with AMPK-activator (A769662) or inhibitor (dorsomorphin) for 8/24 h. Alterations in eEF2K/HIF-1α protein expressions were examined. Results Cancer-cells slightly increased eEF2K expression after 24-h of AMPK-activation. Significant decreases in eEF2K expressions were observed with AMPK inhibition in all cancer-cells except Huh-7. Slight transient decrease in HIF-1α expression was observed after 8-h of AMPK-activation in all cancer-cells except MDA-MB-453. AMPK-inhibition decreased HIF-1α expression, especially in HepG2-cells. Conclusion The effects of AMPK modulation on eEF2K/HIF-1α protein expressions were investigated. Cells with varying molecular-expression-profiles demonstrate different metabolic activities. AMPK-activation may provide adaptive advantage to cancers and such an advantage may be reverted with an AMPK-inhibitor. The current study contributes to the literature in determining the effects of therapeutic strategies targeting AMPK on cancer-cell metabolic-pathway regulation.