The Inhibitory Properties of a Novel, Selective LMTK3 Kinase Inhibitor.

Recently, the oncogenic role of lemur tyrosine kinase 3 (LMTK3) has been well established in different tumor types, highlighting it as a viable therapeutic target. In the present study, using in vitro and cell-based assays coupled with biophysical analyses, we identify a highly selective small molecule LMTK3 inhibitor, namely C36. Biochemical/biophysical and cellular studies revealed that C36 displays a high in vitro selectivity profile and provides notable therapeutic effect when tested in the National Cancer Institute (NCI)-60 cancer cell line panel. We also report the binding affinity between LMTK3 and C36 as demonstrated via microscale thermophoresis (MST). In addition, C36 exhibits a mixed-type inhibition against LMTK3, consistent with the inhibitor overlapping with both the adenosine 5'-triphosphate (ATP)- and substrate-binding sites. Treatment of different breast cancer cell lines with C36 led to decreased proliferation and increased apoptosis, further reinforcing the prospective value of LMTK3 inhibitors for cancer therapy.

Discovery of cyclic guanidine-linked sulfonamides as inhibitors of LMTK3 kinase.

Lemur tyrosine kinase 3 (LMTK3) is oncogenic in various cancers. In breast cancer, LMTK3 phosphorylates and modulates the activity of estrogen receptor-α (ERα) and is essential for the growth of ER-positive cells. LMTK3 is highly expressed in ER-negative breast cancer cells, where it promotes invasion via integrin ß1. LMTK3 abundance and/or high nuclear expression have been linked to shorter disease free and overall survival time in a variety of cancers, supporting LMTK3 as a potential target for anticancer drug development. We sought to identify small molecule inhibitors of LMTK3 with the ultimate goal to pharmacologically validate this kinase as a novel target in cancer. We used a homogeneous time resolve fluorescence (HTRF) assay to screen a collection of mixture-based combinatorial chemical libraries containing over 18 million compounds. We identified several cyclic guanidine-linked sulfonamides with sub-micromolar activity and evaluated their binding mode using a 3D homology model of the LMTK3 KD.

The lncRNA MIR2052HG regulates ERα levels and aromatase inhibitor resistance through LMTK3 by recruiting EGR1.

BACKGROUND: Our previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG's mechanism of action. METHODS: RNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines. RESULTS: MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. CONCLUSIONS: Our findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.

Lemur Tyrosine Kinase-3 Suppresses Growth of Prostate Cancer Via the AKT and MAPK Signaling Pathways.

BACKGROUND/AIMS: Lemur tyrosine kinase (LMTK)-3 is a member of the receptor tyrosine kinase (RTK) family. Abnormal expression of LMTK-3 exists in various types of cancers, especially in endocrine-resistant breast cancers; however, the precise level of expression and the biological function in prostate cancer are poorly understood. METHODS: In the present study, we determined the expression of LMTK-3 in prostate cancer using immunohistochemistry and Western blotting. We infected PC3 and LNCaP cells with lentivirus-LMTK-3 and observed the biologic characteristics of the PC3 and LNCaP cells in vitro with TUNEL, and migration and invasion assays, respectively. We also established a transplant tumor model of human prostate cancer with infected cells in 15 BALB/c-nu/nu nude mice. RESULTS: LMTK-3 was expressed in prostate epithelial cells. There was a significant decline in the level of LMTK-3 expression in prostate cancers compared to normal tissues. LMTK-3 inhibited PC3 and LNCaP cell growth, migration, and invasion, and induced cell apoptosis in vitro. We also observed that LMTK-3 induced PC3 cell apoptosis in vivo. Further study showed that LMTK-3 inhibited phosphorylation of AKT and ERK, and promoted phosphorylation and activation of p38 kinase and Jun kinase (JNK). CONCLUSION: Recombinant lentivirus with enhanced expression of LMTK-3 inhibited prostate cancer cell growth and induced apoptosis in vitro and in vivo. AKT and MAPK signaling pathways may contribute to the process.

Designing and optimization of novel human LMTK3 inhibitors against breast cancer - a computational approach.

Estrogen receptor-α (ERα) is expressed more in patients with breast cancer and its level correlated with endocrine resistance. LMTK3 is reported as breast cancer target with regulation of estrogen receptor-α (ERα) through phosphorylation. In this computational study, structure-based inhibitor screening was performed on human LMTK3 using ZINC database. ATP-binding cavity with critical residues involved in the LMTK3 phosphorylation was used as target site for the screening. From the large ligand library, the best compounds were screen with three-phase virtual screening methods in Dockblaster, AutoDock Vina and AutoDock, respectively. The evaluation of ligands was carried out by binding energy and weak interactions, such as hydrogen bond interactions and hydrophobic contacts, in the target site that favors LMTK3 inhibition. Top compounds were found to be more effective in druglikeness activity by ADME prediction. The stability and binding affinity of ligand complexes were optimized by trajectory analysis such as RMSD, Rg, SASA and interhydrogen bonds from molecular dynamics simulations. The behavior of protein motion after ligand binding was illustrated by eigenvectors from principal component analysis (PCA). In addition, binding free energy of the LMTK3-ligand complexes were calculated by MM/PBSA methods and results supported the strong binding in dynamic system. Thus, the computational studies illustrated the structural insights on LMTK3 inhibition mechanism by ligands ZINC04670539, ZINC05607079 and ZINC04344028, also proposed as potent lead candidates. Our findings step towards developing novel LMTK3 inhibitors and identified lead candidates can be future breast cancer drugs with further experimental studies.

High expression of LMTK3 is an independent factor indicating a poor prognosis in estrogen receptor α-positive breast cancer patients.

OBJECTIVE: Over 70% of breast cancers are estrogen receptor alpha-positive, and endocrine therapy targeting estrogen action decreases mortality from breast cancer. Recently, a novel protein kinase that regulates estrogen receptor alpha activity, lemur tyrosine kinase-3, has been identified. In this study, we investigated whether messenger RNA expression and polymorphisms of the gene encoding the kinase, LMTK3, are associated with prognosis in breast cancer patients during long-term follow-up. METHODS: First, we investigated the relationship between messenger RNA expression of LMTK3 and patient outcome in 219 breast cancers. The effects of several variables on survival were tested by Cox proportional hazards regression analysis. Next, we performed LMTK3 genotyping in 471 breast cancers to clarify the prognostic role of these polymorphisms. RESULTS: Our data showed that LMTK3 expression level was not associated with prognosis in all patients. We then analyzed the impact of LMTK3 mRNA expression on the prognosis of breast cancer according to estrogen receptor alpha status. Both disease-free survival and overall survival were significantly shorter in estrogen receptor alpha-positive patients with high LMTK3 expression receiving adjuvant endocrine therapy than in those patients with low LMTK3 expression. Multivariate Cox regression analysis revealed that high LMTK3 expression was an independent poor prognostic factor in estrogen receptor alpha-positive breast cancer patients. We did not find any correlation between LMTK3 genotypes and prognosis of breast cancer patients in our series. CONCLUSIONS: Our results show that high expression of LMTK3 is an independent prognostic factor in estrogen receptor alpha-positive breast cancer patients receiving adjuvant endocrine therapy.

Chemoinformatics and machine learning techniques to identify novel inhibitors of the lemur tyrosine kinase-3 receptor involved in breast cancer.

Breast cancer is still the largest cause of cancer death in women, and around 70% of primary breast cancer patients are estrogen receptor (ER)-positive, which is the most frequent kind of breast cancer. The lemur tyrosine kinase-3 (LMTK3) receptor has been linked to estrogen responsiveness in breast cancer. However, the function of LMTK3 in reaction to cytotoxic chemotherapy has yet to be studied. Breast cancer therapy research remains tricky due to a paucity of structural investigations on LMTK3. We performed structural investigations on LMTK3 using molecular docking and molecular dynamics (MD) simulations of the LMTK3 receptor in complex with the top three inhibitor molecules along with a control inhibitor. Analysis revealed the top three compounds show the best binding affinities during docking simulations. Interactive analysis of hydrogen bonds inferred hotspot residues Tyr163, Asn138, Asp133, Tyr56, Glu52, Ser132, Asp313, and Asp151. Some other residues in the 5-Å region determined strong alkyl bonds and conventional hydrogen bond linkages. Furthermore, protein dynamics analysis revealed significant modifications among the top complexes and the control system. There was a transition from a loop to a-helix conformation in the protein-top1 complex, and in contrast, in complexes top2 and top3, the formation of a stabilizing sheet in the C chain was observed, which limited significant mobility and increased complex stability. Significant structural alterations were observed in the protein-top complexes, including a shorter helix region and the creation of some loop regions in comparison to the control system. Interestingly, binding free energies, including MMGB/PBSA WaterSwap analysis estimation, reveals that the top1 complex system was more stable than other systems, especially in comparison to the control inhibitor complex system. These results suggest a the plausible mode of action for the novel inhibitors. Therefore, the current investigation contributes to understanding the mechanism of action, serving as a basis for future experimental studies.

Integrated multi-omics approach to distinct molecular characterization and classification of early-onset colorectal cancer.

Incidence of early-onset colorectal cancer (EOCRC), defined by a diagnosed age under 50 years, is increasing, but its heterogeneous etiologies that differ from general CRC remain undetermined. We initially characterize the genome, epigenome, transcriptome, and proteome of tumors from 79 patients in a Chinese CRC cohort. Data for an additional 126 EOCRC subjects are obtained from the International Cancer Genome Consortium Chinese cohort and The Cancer Genome Atlas European cohort. We observe that early-onset tumors have a high tumor mutation burden; increased DNA repair features by mutational signature 3 and multi-layer pathway enrichments; strong perturbations at effects of DNA methylation and somatic copy-number alteration on gene expression; and upregulated immune infiltration as hot tumors underlying immunophenotypes. Notably, LMTK3 exhibits ancestral mutation disparity, potentially being a functional modulator and biomarker that drives molecular alterations in EOCRC development and immunotherapies. This integrative omics study provides valuable knowledge for precision oncology of CRC.

Lemur tyrosine kinase-3 (LMTK3) induces chemoresistance to cetuximab in colorectal cancer via the ERK/MAPK pathway.

As an oncogenic kinase in multiple cancers, LMTK3 was deeply implicated in cancer pathogenesis. Nevertheless, its biological function in colorectal cancer (CRC) is still unclear. In this study, LMTK3 mRNA expression was assessed by RT-qPCR. LMTK3, phospho-ERK1/2 (p-ERK1/2), ERK1/2, and cleaved caspase-3 protein levels were detected by western blotting. Cetuximab (CTX)-resistant CRC cell models were constructed to investigate the mechanism of LMTK3-regulated CTX resistance in CRC. CTX half-maximal inhibitory concentration (IC50), viability, apoptosis, cell cycle, migration, and invasion of CRC cells were analyzed via Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, and transwell assays. We found LMTK3 was distinctly upregulated in CRC tissues and cells, particularly in CTX-resistant CRC tissues and cells. LMTK3 inhibition lowered CTX half-maximal inhibitory concentration (IC50) value, inhibited cell viability, induced cell apoptosis, triggered cell-cycle arrest, and impaired cell metastatic capability in CTX-resistant CRC cells. Moreover, we also demonstrated that LMTK3 induced CTX resistance in CRC via the activation of ERK/MAPK signaling in vitro. These results suggested a novel molecular mechanism by which LMTK3 participates in the development of CTX resistance in CRC.

Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α.

OBJECTIVE: The curative effect of high-efficiency progesterone and other therapeutic drugs for endometrioid adenocarcinoma patients with preservation of reproductive capacity has not been satisfactory so far. Novel therapeutic drugs need to be explored. METHODS: We investigated the cytoplastic and nuclear expression levels of LMTK3 between endometrioid adenocarcinoma tissues and adjacent endometrial tissues by immunohistochemistry. We detected the effects of LMTK3 on cell viability of Ishikawa cells by CCK-8. We detected the effects of LMTK3 on cell cycle and apoptosis of Ishikawa cells by flow cytometry. We also detected the effects of LMTK3 knockdown on mRNA and protein levels of ERα by qRT-PCR and western blotting, respectively. We also used the cBioPortal online database to analyze the coexpression of LMTK3 and ESR1 in 1647 UCEC samples. RESULTS: We used TMAs to identify that LMTK3 was mainly detected in the cytoplasm of endometrioid tissues, and cytoplasmic LMTK3 expression in endometrioid tissues was higher than that in adjacent endometrial tissues (P < 0.05). LMTK3 knockdown decreased the proliferation of Ishikawa cells through decreasing cell viability (P < 0.01), increasing G1 (P < 0.001) arrest, and promoting apoptosis (P < 0.01). There was a positive correlation between the mRNA expression levels of LMTK3 and ESR1 (Spearman: P=2.011e-5, R=0.13; Pearson: P=7.18e-8, R=0.17). Knockdown of LMTK3 also reduced the mRNA (P < 0.001) and protein (P < 0.001) levels of ERα. CONCLUSIONS: Inhibitors of LMTK3 may be a possible future treatment for ERα and LMTK3 highly expressed endometrioid adenocarcinoma following appropriate studies.

The multifaceted role of lemur tyrosine kinase 3 in health and disease.

In the last decade, LMTK3 (lemur tyrosine kinase 3) has emerged as an important player in breast cancer, contributing to the advancement of disease and the acquisition of resistance to therapy through a strikingly complex set of mechanisms. Although the knowledge of its physiological function is largely limited to receptor trafficking in neurons, there is mounting evidence that LMTK3 promotes oncogenesis in a wide variety of cancers. Recent studies have broadened our understanding of LMTK3 and demonstrated its importance in numerous signalling pathways, culminating in the identification of a potent and selective LMTK3 inhibitor. Here, we review the roles of LMTK3 in health and disease and discuss how this research may be used to develop novel therapeutics to advance cancer treatment.

AKT1 Polymorphism (rs10138227) and Risk of Colorectal Cancer in Moroccan Population: A Case Control Study.

BACKGROUND: LMTK3 and AKT1 each have a role in carcinogenesis and tumor progression. The analysis of single nucleotide polymorphisms of AKT1 and LMTK3 could lead to more complete and accurate risk estimates for colorectal cancer. AIM: We evaluated the association between single nucleotide polymorphisms (SNPs) of AKT1 and LMTK3 and the risk of colorectal cancer in a case-control study in Moroccan population. METHODS: Genomic DNA from 70 colorectal cancer patients and 50 healthy control subjects was extracted from whole blood. Genotyping was performed by direct sequencing after polymerase chain reactions for the 7 SNPs (AKT1rs1130214G/T, AKT1rs10138227C/T, AKT1rs3730358C/T, AKT1rs1000559097G/A, AKT1rs2494737A/T, LMTK3rs8108419G/A, and LMTK3rs9989661A/G.). Study subjects provided detailed information during the collection. All P values come from bilateral tests. RESULTS: In the logistic regression analysis, a significantly high risk of colorectal cancer was associated with TC/TT genotypes of rs10138227 with adjusted odds ratio [OR] equal to 2.82 and 95% confidence interval [CI] of 1.15 to 6.91. CONCLUSION: Our results suggest that the SNP AKT1rs10138227 could affect susceptibility to CRC, probably by modulating the transcriptional activity of AKT1. However, larger independent studies are needed to validate our results.

Lmtk3-KO Mice Display a Range of Behavioral Abnormalities and Have an Impairment in GluA1 Trafficking.

Accumulating evidence suggests that glutamatergic signaling and synaptic plasticity underlie one of a number of ways psychiatric disorders appear. The present study reveals a possible mechanism by which this occurs, through highlighting the importance of LMTK3, in the brain. Behavioral analysis of Lmtk3-KO mice revealed a number of abnormalities that have been linked to psychiatric disease such as hyper-sociability, PPI deficits and cognitive dysfunction. Treatment with clozapine suppressed these behavioral changes in Lmtk3-KO mice. As synaptic dysfunction is implicated in human psychiatric disease, we analyzed the LTP of Lmtk3-KO mice and found that induction is severely impaired. Further investigation revealed abnormalities in GluA1 trafficking after AMPA stimulation in Lmtk3-KO neurons, along with a reduction in GluA1 expression in the post-synaptic density. Therefore, we hypothesize that LMTK3 is an important factor involved in the trafficking of GluA1 during LTP, and that disruption of this pathway contributes to the appearance of behavior associated with human psychiatric disease in mice.

Molecular screening and docking analysis of LMTK3and AKT1 combined inhibitors.

The abnormal activation of AKT/mTOR signaling pathway and overexpression of LMTK3, are the main factors involved in the generation of drug resistance. Therefore, the use of computer-aided drug design in the inhibitors discovery offers an advantage to provide new candidates for the treatment of this resistance. We realised the virtual screening and molecular docking of AKT1 and LMTK3 proteins by the Dockblaster server. In addition, with abundance of candidates under development for AKT1 kinase, we have also conducted a Quantitative Structure-Activity Relationship (QSAR) study based on these compounds, in order to design more active compounds and predict their activity for development of a new inhibitor of AKT1. QSAR tests were performed for AKT1 using the Partial Least Squares method with a correlation coefficient of R2=0.8062 and a cross-validation of q2=0.6995. This test has selected five compounds as competitive inhibitors-AKT1-ATP with a better biological activities. In parallel the molecular screening has selected five other compounds as competitive ATP-inhibitors of LMTK3. One of them is a common inhibitor with AKT1, and it is marketed as a moderate to severe pain therapy. The ADME predictions confirmed the inhibitors pharmacological activity of these compounds for potential consideration as drug candidates.

Identification of curcumin derivatives as human LMTK3 inhibitors for breast cancer: a docking, dynamics, and MM/PBSA approach.

Human lemur tyrosine kinase-3 (LMTK3) is primarily involved in regulation of estrogen receptor-α (ERα) by phosphorylation activity. LMTK3 acts as key biomarker for ERα positive breast cancer and identified as novel drug target for breast cancer. Due to the absence of experimental reports, the computational approach has been followed to screen LMTK3 inhibitors from natural product curcumin derivatives based on rational inhibitor design. The initial virtual screening and re-docking resulted in identification of top three leads with favorable binding energy and strong interactions in critical residues of ATP-binding cavity. ADME prediction confirmed the pharmacological activity of the leads with various properties. The stability and binding affinity of leads were well refined in dynamic system from 25 ns MD simulations. The behavior of protein motion towards closure of ATP-binding cavity was evaluated based on eigenvectors by PCA. In addition, MM/PBSA calculations also confirmed the relative binding free energy of LMTK3-lead complexes in favor of the effective binding. From our study, novel LMTK3 inhibitors tetrahydrocurcumin, curcumin 4,4'-diacetate, and demethoxycurcumin have been proposed with inhibition mechanism. Further experimental evaluation on reported lead candidates might prove its role in breast cancer therapeutics.

LMTK3 escapes tumour suppressor miRNAs via sequestration of DDX5.

Lemur tyrosine kinase-3 (LMTK3) plays an important role in cancer progression and is associated with breast, lung, gastric and colorectal cancer. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that typically repress target genes at post-transcriptional level and have an important role in tumorigenesis. By performing a miRNA expression profile, we identified a subset of miRNAs modulated by LMTK3. We show that LMTK3 induces miR-34a, miR-196-a2 and miR-182 levels by interacting with DEAD-box RNA helicase p68 (DDX5). LMTK3 binds via DDX5 to the pri-miRNA of these three mature miRNAs, thereby sequestrating them from further processing. Ectopic expression of miR-34a and miR-182 in LMTK3-overexpressing cell lines (MCF7-LMTK3 and MDA-MB-231-LMTK3) inhibits breast cancer proliferation, invasion and migration. Interestingly, miR-34a and miR-182 directly bind to the 3'UTR of LMTK3 mRNA and consequently inhibit both its stability and translation, acting as tumour suppressor-like miRNAs. In aggregate, we show that LMTK3 is involved in miRNA biogenesis through modulation of the Microprocessor complex, inducing miRNAs that target LMTK3 itself.

Lemur tyrosine kinase-3 is a significant prognostic marker for patients with colorectal cancer.

Lemur tyrosine kinase-3 (LMTK3) belongs to the family of serine-threonine-tyrosine kinases and the aberrant expression of LMTK3 was observed in several human malignancies. However, the association of LMTK3 with clinical outcomes in colorectal cancer patients is unclear. Thus, this present study was to evaluate the association of LMTK3 expression level with clinicopathologic factors and prognosis of patients with colorectal cancer (CRC). The expression level of LMTK3 in 69 archival paraffin-embedded colorectal tumor tissue specimens was examined by immunohistochemistry (IHC). As a result, we found that the LMTK3 expression level was significantly elevated in CRC tissues as compared with Crohn's disease or colorectal polyp tissues (P<0.0001, P<0.0001, respectively). Positive LMTK3 signals in the colorectal cancer cells were observed in about 89.9% (62 of 69) CRC tissue specimens. Additionally, LMTK3 expression was significantly correlated with lymph node metastasis and tumor-node-metastasis (TNM) classification (P=0.003, and P=0.008, respectively), but not with sex, age, tumor location, histological differentiation, tumor size, or depth of tumor invasion (all P>0.05). Kaplan-Meier survival curves showed that the overall survival rate was significantly higher in the patients with low expression of LMTK3 when compared with those patients with high LMTK3 (P=0.010). Moreover, multivariate analysis revealed that LMTK3 expression was an independent prognostic factor for CRC patients (P=0.047). These results suggest that LMTK3 protein could serve as a prognostic marker for CRC patients.