Tyrosine Kinase Research Articles

Tumor-intrinsic kinome landscape of pancreatic cancer reveals new therapeutic approaches.

Effective therapies for pancreatic ductal adenocarcinoma (PDAC) have been largely elusive. Here, we perform Multiplexed kinase Inhibitor Bead Mass Spectrometry on 102 patient derived xenografts derived from 14 unique primary PDAC to define the tumor-intrinsic kinome landscape. Our findings uncover three kinome subgroups making up two tumor-intrinsic kinome subtypes that we call kinotypes. The kinotypes show enrichment of different kinase classes and recapitulate previously described molecular subtypes, basal-like and classical. The kinotype characterizing basal-like tumors shows enrichment of receptor tyrosine kinases, whereas the kinotype characterizing classical tumors is enriched in understudied kinases involved in Wnt signaling and immune pathways. We validate our findings in two clinical trials and show that only patients with basal-like tumors derive significant benefit from EGFR inhibitors. Our results provide a comprehensive tumor-intrinsic kinome landscape of PDAC that strongly supports actionable kinotype specific kinase targets and provides a roadmap for kinase inhibitor therapy in PDAC.

Comprehensive Analysis of TEK Variants in Patients With Vascular Malformations.

Pathogenic variants in the receptor tyrosine kinase TIE2, encoded by TEK, are known to cause vascular malformations (VMs). In this study, we retrospectively reviewed the deidentified data generated through clinical NGS testing in our laboratory and found 88 VM cases with a total of 107 clinically significant TEK variants. Among those, 23 unique variants at the amino acid level were identified, including five novel (p.Cys1040Arg, p.Arg1099PhefsTer12, p.Glu1109Ter, p.Phe1111LeufsTer7, p.Phe1111ValfsTer7) and 18 previously published variants. Missense variants were identified more often in the tyrosine kinase domain, while all nonsense/frameshift variants were clustered in the C-terminal tail (CTT). In addition, most variants occurred as solitary alterations, whereas certain variants always co-occurred with a second TEK variant. Five patterns of TEK variants (P1-P5) were identified: (P1) Arg849 + another variant; (P2) Tyr897 + another variant; (P3) Leu914 single variants; (P4) Arg915/918 single variants; and (P5) CTT single /co-occurring variants. This study provides the most comprehensive view of pathogenic TEK variants in VMs to date.

Clinicopathological characteristics of Japanese patients with breast cancer and MET exon 14 skipping alterations.

In non-small cell lung cancer, alterations in mesenchymal-epithelial transition (MET) have been recognized as novel therapeutic targets. In particular, the MET exon 14 skipping mutation (METex14s) is a rare oncogenic driver. Targeted therapy with MET tyrosine kinase inhibitors has recently been approved for this mutation. However, c-MET expression and METex14s frequency and their clinicopathological effects in Japanese patients with breast cancer (BC) remain unknown. Tissue microarray-based immunohistochemistry (IHC) was performed to measure c-MET expression in 930 patients with BC (808 with invasive and 122 with noninvasive BC). Reverse transcription polymerase chain reaction was performed to analyse METex14s in patients exhibiting c-MET expression. Clinicopathological characteristics, including patient prognosis based on c-MET expression and METex14s, were elucidated. IHC staining revealed c-MET expression in 91/930 (9.7%) patients. Notably, IHC expression was frequently observed in apocrine carcinomas (11/26 cases). Among the c-MET IHC-positive cases, METex14s frequency was 25.9% (14/54 cases) in invasive BC and 54.1% (20/37 cases) in noninvasive BC. Furthermore, 4/11 informative noninvasive and invasive BC cases with apocrine differentiation carried METex14s. The nuclear grade was significantly higher in the METex14s-positive group among invasive BC with c-met IHC expression. Furthermore, patients' age and negative rate for PgR IHC was significantly lower in the METex14s-positive group among noninvasive BC. Regarding the factors associated with patient outcomes, both c-MET IHC staining and METex14s expression did not affect survival, regardless of the hormone receptor status. High c-MET expression and METex14s are common in apocrine carcinoma.

Lack of TYK2 signaling enhances host resistance to Candida albicans skin infection

Candida albicans is the most common human fungal pathogen, causing diseases ranging from local to life-threating systemic infections. Tyrosine kinase 2 (TYK2), a crucial mediator in several cytokine signaling pathways, has been associated with protective functions in various microbial infections. However, its specific contribution in the immune response to fungal infections has remained elusive. In this study, we show that mice lacking TYK2 or its enzymatic activity exhibit enhanced resistance to C. albicans skin infections, limiting fungal spread and accelerating wound healing. Impaired TYK2-signaling prompted the formation of a distinctive layer of necrotic neutrophils around the fungal pathogens. Transcriptomic analysis revealed TYK2’s pivotal role in regulating interferon-inducible genes in neutrophils, thereby impacting their antifungal capacity during infection. Furthermore, we show that TYK2-dependent interferon-gamma (IFNγ) production contributes to fungal dissemination from the skin to the kidneys. Our study uncovers a hitherto unrecognized detrimental role of TYK2 in cutaneous C. albicans infections.

Discovery of a molecular glue for EGFR degradation.

Aberrant expression of epidermal growth factor receptor (EGFR) plays a critical role in the pathogenesis of various tumors, potentially representing a target for therapeutic intervention. Nonetheless, EGFR remains a challenging protein to target pharmacologically in triple-negative breast cancer (TNBC). An emerging approach to address the removal of such proteins is the application of molecular glue (MG) degraders. These compounds facilitate protein-protein interactions between a target protein and an E3-ubiquitin ligase, subsequently leading to protein degradation. Herein, we identified a new MG (CDDO-Me, C-28 methyl ester of 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acid), which orchestrated binding between EGFR and KEAP1 (an E3-ubiquitin ligase adapter), thereby initiating the ubiquitination and degradation of EGFR. CDDO-Me directly interacted with the tyrosine kinase (TK) domain of EGFR, resulting in its degradation via an autophagy-dependent lysosomal pathway. Knockdown of KEAP1 decreased the degradation of EGFR by reducing its K63-linked ubiquitination, leading to diminished EGFR colocalization in autophagosomes and lysosomes. Notably, CDDO-Me attenuates TNBC progression by accelerating EGFR degradation in cell-derived xenografts and patient-derived organoid models, highlighting its clinical application potential. Consequently, induction of EGFR degradation through MG degraders represents a viable therapeutic strategy for TNBC.

Design and Synthesis of Novel Spiro [Chromane‐2,4′‐Piperidin]‐4‐One Derivatives: Anti‐Proliferative Investigation and Molecular Docking Studies

AbstractChemists around the globe are extensively working for remedial solutions to cancer, one of the greatest health hazards. We have synthesized twenty‐three novel spiro [chromane‐2,4′‐piperidin]‐ 4 ‐one derivatives (KBS and KMS series of analogues) as part of our ongoing research to combat this deadly disease, and confirmed their structures using 1H NMR, 13C NMR, HRMS, and FT‐IR. Furthermore, we employed single‐crystal XRD to identify the compound structures of KBS4 and KMS10. We have tested the compounds on the cell line such as MCF‐7; U87‐MG; SCC‐25; and HEK‐293T, via WST‐1 assay. Eight compounds showed IC50 values ranging 3.9–10 μM; against the cell line MCF‐7. The best compounds of all were KMS9 (IC50=3.83 μM), KMS5 (IC50=4.14 μM), and KBS8 (IC50=8.24 μM), which promoted apoptosis in MCF‐7 cells. KMS5 and KMS9 compounds showed G1 cell cycle arrest, while compound KBS8 showed G2 cell cycle arrest. Insilco ADME studies were carried out. Molecular docking and dynamics experiments showed how KMS5, KMS9, and KBS8 bind to the active region of the EGFR family – a group of receptor tyrosine kinase (RTK) proteins (PDB ID: 7JXP, 2.16 Å). Further structural modifications of the KMS5, KMS9, and KBS8 may improve their activity against breast cancer.

Transcriptomic analysis delineates potential regulatory network as therapeutic alternatives in chronic myeloid leukemia

BackgroundChronic myeloid leukemia (CML) relapse and progression after discontinuation of tyrosine kinase inhibitors (TKI) therapy in the chronic phase (CP) are attributed to primitive quiescent leukemic progenitor cells (LPCs). This study aimed to identify key differentially expressed genes (DEGs) involved in molecular pathways alternative to BCR-ABL, which could lead to the eradication of leukemic stem/progenitor cells.MethodsmRNA expression profiling dataset comprising newly diagnosed untreated CP CML leukemic progenitor cell samples n=5 and normal hematopoietic progenitor cell samples n=5 were selected for DEGs identification. A weighted gene co-expression network was constructed to identify significant hub modules. Protein–protein interaction (PPI) network construction and pathway enrichment analysis provided additional information on the biological pathways involved. The genes involved in the resultant PPI network were subjected to a gene transcriptional regulatory network based on transcription factors (TFs) and microRNAs (miRNAs) involved in the target genes.ResultsA total of 709 genes that were differentially expressed among those corresponding to p<0.05 and log2FC>0.5 0.5$$\\end{document}]]>. The WGCNA showed a significant hub module comprising 267 DEGs. The major pathways of the hub genes involved in the PPI network at the highest confidence score >0.9 0.9$$\\end{document}]]> were related to mitochondrial translational elongation. The integrated gene transcriptional regulatory network revealed a TF (ETS1), two miRNAs (miR-34a-5p and miR-205-5p) targeting a hub gene (GFM1) as the highest-order motif or subnetwork in the three-node miRNA feed-forward loop.ConclusionsThe study suggests that the regulatory feed-forward loop may provide a potential therapeutic target in CML. Consequently, the mitochondrial translation pathway may be explored as an alternative to BCR-ABL pathways, which could aid in eliminating or increasing sensitization of CML progenitor/stem cells to TKI treatment.

Lung Cancer with Brain Metastasis—Treatment Strategies and Molecular Characteristics

Lung cancer is a leading cause of brain metastases (BMs), with 10–20% of patients with non-small cell lung cancer (NSCLC) presenting with BMs at diagnosis and 25–50% developing them over the course of their disease. Historically, BMs have posed significant therapeutic challenges, partly due to the blood brain barrier (BBB), which restricts drug penetration to the central nervous system. Consequently, BMs were initially managed with local treatments, including surgical resection, stereotactic radiosurgery, and whole brain radiation therapy. In recent years, however, systemic treatments for BMs have advanced significantly, particularly with the development of molecularly-targeted therapies and immunotherapies. The discovery of driver mutations and the development of novel tyrosine kinase inhibitors (TKIs) have yielded encouraging intracranial responses in NSCLC patients with actionable genetic alterations (e.g., EGFR, ALK, ROS1). Genomic profiling has also suggested genetic heterogeneity between BMs and primary sites. Immunotherapies, alone or in combination with other treatments, have demonstrated promising results in NSCLC with BMs, although most clinical trials have included only selected patients with asymptomatic or previously treated BMs. In this review, we discuss the molecular and immune characteristics of NSCLC with BMs, analyze intracranial efficacy findings from clinical trials, and explore treatment strategies for lung cancer patients with BMs.

Ivacaftor, a CFTR potentiator, synergizes with osimertinib against acquired resistance to osimertinib in NSCLC by regulating CFTR-PTEN-AKT axis.

Osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has demonstrated significant clinical benefits in the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). However, inevitable acquired resistance to osimertinib limits its clinical utility, and there is a lack of effective countermeasures. Here, we established osimertinib-resistant cell lines and performed drug library screening. This screening identified ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, as a synergistic enhancer of osimertinib-induced anti-tumor activity both in vitro and in vivo. Mechanistically, ivacaftor facilitated the colocalization of CFTR and PTEN on the plasma membrane to promote the function of PTEN, subsequently inhibiting the PI3K/AKT signaling pathway and suppressing tumor growth. In summary, our study suggests that activating CFTR enhances osimertinib-induced anti-tumor activity by regulating the PTEN-AKT axis. Furthermore, ivacaftor and osimertinib constitute a potential combination strategy for treating osimertinib-resistant EGFR-mutated NSCLC patients.

Peripheral Neuropathy in the Phase 3 ASPEN Study of Bruton Tyrosine Kinase Inhibitors for Waldenström Macroglobulinemia.

Peripheral neuropathy (PN) is a significant cause of morbidity associated with Waldenström macroglobulinemia (WM). The phase 3 ASPEN study compared the efficacy and safety of zanubrutinib with ibrutinib in patients with WM. This ad hoc analysis examined treatment outcomes with zanubrutinib or ibrutinib on PN symptoms associated with WM in patients enrolled in ASPEN. Logistic regression was performed between PN symptom resolution and several predictors. Health-related quality of life (HRQOL) was assessed using the validated European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30. Forty-nine patients with PN symptoms were included (zanubrutinib treated, n=27; ibrutinib treated, n=22). Overall, 35 patients (71.4%) experienced resolution of PN symptoms, with a median time to resolution of 10.1 months (range, 1-46.8). In cohort 1 (MYD88 mutation), the median time to PN symptom resolution was 4.6 months (range, 1.1-46.8) with zanubrutinib and 14.1 months (range, 1-44) with ibrutinib. Logistic regression demonstrated a significant relationship between PN symptom resolution and both major response (hazard ratio [HR], 10.67 [95% CI,2.20-51.81]; P=.0033) and lower baseline anti-MAG antibody levels (HR, 0.72 [95% CI, 0.52-1.00]; P=.0486). Patients with PN symptom resolution had greater improvement in HRQOL. Physical functioning improved in patients with PN symptom resolution and was unchanged in patients without resolution. Improvements observed in PN symptoms may be in response to a reduction in IgM. While further investigation is required, this analysis supports the potential use and further exploration of Bruton tyrosine kinase inhibitors to treat PN symptoms in patients with WM. ClinicalTrials.gov: NCT03053440.

Fostamatinib for Hospitalized Adults With COVID-19 and Hypoxemia: A Randomized Clinical Trial.

Fostamatinib, a spleen tyrosine kinase inhibitor, has been reported to improve outcomes of COVID-19. To evaluate the efficacy and safety of fostamatinib in adults hospitalized with COVID-19 and hypoxemia. This multicenter, phase 3, placebo-controlled, double-blinded randomized clinical trial was conducted at 41 US sites and 21 international sites between November 17, 2021, and September 27, 2023; the last follow-up visit was December 31, 2023. Participants were adults aged 18 years or older hospitalized with acute SARS-CoV-2 infection and hypoxemia. Data were analyzed between January 10 and March 8, 2024. Fostamatinib, 150 mg orally twice daily for 14 days, or placebo. The primary outcome was oxygen-free days, an ordinal outcome classifying a participant's status at day 28 based on mortality and duration of supplemental oxygen use. An adjusted odds ratio (AOR) greater than 1.0 was considered to indicate superiority of fostamatinib over placebo. A key secondary outcome was 28-day all-cause mortality. Safety outcomes included elevated transaminase values, neutropenia, and hypertension. Of the 400 participants randomized (median age, 67 years [IQR, 58-76 years]; 210 [52.5%] men), 199 received fostamatinib and 201 received placebo. The mean (SD) number of oxygen-free days was 13.4 (12.4) in the fostamatinib group and 14.2 (12.1) in the placebo group (unadjusted mean difference, -1.26 days [95% CI, -3.52 to 1.00 days]; AOR, 0.82 [95% credible interval (CrI), 0.58-1.17]). Mortality at 28 days occurred in 22 of 195 patients (11.3%) in the fostamatinib group and 16 of 197 (8.1%) in the placebo group (AOR, 1.44; 95% CrI, 0.72-2.90). Aspartate aminotransferase elevation occurred more commonly in the fostamatinib group (23 [11.6%]) than in the placebo group (11 [5.5%]; AOR, 2.28; 95% CrI, 1.07-4.84). Other safety outcomes were similar between groups. In this randomized clinical trial of adults hospitalized with COVID-19 and hypoxemia, fostamatinib did not increase the number of oxygen-free days compared with placebo. These results do not support the hypothesis that fostamatinib improves outcomes among adults hospitalized with hypoxemia during the Omicron era. ClinicalTrials.gov Identifier: NCT04924660.

Pre-pregnancy obesity is associated with an altered maternal metabolome and reduced Flt3L expression in preterm birth

Mechanisms linking pre-pregnancy obesity to increased preterm birth risk are unclear. Here, we examined the impact of pre-pregnancy obesity on metabolites, Fms-related tyrosine kinase 3 ligand (Flt3L), and proinflammatory cytokine profiles in preterm birth. We used cytokine bead array, ELISA and Gas Chromatography-Mass Spectrometry (GC-MS) to determine cytokine and metabolite profiles in maternal and cord blood samples from 124 pregnant women in Australia, who gave birth at term (n = 86) or preterm (n = 38). Besides the expected variations in birth weight and gestational age, all demographic characteristics, including pre-pregnancy body mass index, were similar between the term and preterm birth groups. Mothers in the preterm birth group had reduced Flt3L (P = 0.002) and elevated IL-6 (P = 0.002) compared with term birthing mothers. Among mothers who gave birth preterm, those with pre-pregnancy obesity had lower Flt3L levels (P = 0.02) compared with lean mothers. Flt3L and IL-6 were similar in cord blood across both groups, but TNFα levels (P = 0.02) were reduced in preterm newborns. Metabolomic analysis revealed significant shifts in essential metabolites in women with pre-pregnancy obesity, some of which were linked to preterm births. Our findings suggest that maternal pre-pregnancy obesity alters the metabolome and reduces Flt3L expression, potentially increasing risk of preterm birth.

Clinical Implications of Erb-B2 Receptor Tyrosine Kinase 2 S310 Mutations in Non–Small-Cell Lung Cancer: Two Case Reports

Clinical Implications of Erb-B2 Receptor Tyrosine Kinase 2 S310 Mutations in Non–Small-Cell Lung Cancer: Two Case Reports

ErbB4 precludes the occurrence of PTSD-like fear responses by supporting the bimodal activity of the central amygdala

AbstractPost-traumatic stress disorder (PTSD) often arises after exposure to traumatic events and is characterized by dysregulated fear responses. Although the associations of erb-b2 receptor tyrosine kinase 4 (ErbB4) with various neuropsychiatric diseases, including schizophrenia and bipolar disorder, have been widely examined, the physiological roles of ErbB4 in PTSD and fear responses remain unclear. Using Cre-dependent ErbB4 knockout (KO) mice, we observed that PTSD-like fear behaviors emerged in ErbB4-deficient mice, particularly in inhibitory neurons. Specifically, the loss of ErbB4 in somatostatin-expressing (SST+) neurons was sufficient to induce PTSD-like fear responses. We also adopted the CRISPR/Cas9 system for region-specific KO of ErbB4, which revealed that ErbB4 deletion in SST+ neurons of the lateral division of the amygdala (CeL) caused elevated anxiety and PTSD-like fear generalization. Consistent with its physiological role, ErbB4 expression was diminished in CeLSST neurons from mice that exhibited PTSD-like phenotypes. While fear On and Off cells identified in the CeL displayed distinct responses to conditioned and novel cues, as previously shown, the selectivity of those On and Off cells was compromised in SSTErbB4-/- and stressed mice, which displayed strong fear generalization. Therefore, the bimodal activity that CeL On/Off cells display is likely required for proper discrimination of fearful stimuli from ambient stimuli, which should be sustained by the presence of ErbB4. Taken together, our data substantiate the correlation between PTSD-like fear responses and ErbB4 expression in CeLSST neurons and further underscore the functional effects of ErbB4 in CeLSST neurons, supporting the bimodal responses of CeL neurons.

Would the utilization of sFlt-1/PlGF ratio in clinical practice prevent unnecessary hospital admissions of cases with preeclampsia?

ABSTRACT Objectives Preeclampsia (PET) is a serious pregnancy complication with potential adverse maternal and fetal outcomes. Recent research has examined the soluble fms-like tyrosine kinase 1 (sFlt-1)/placental growth factor (PlGF) ratio for predicting PET. The study aimed to assess the efficacy of the sFlt-1/PlGF ratio in ruling out unnecessary hospital admission and PET. Methods sFlt-1/PlGF ratio was performed in patients as clinically indicated. Results The sFlt-1/PlGF ratio at a cutoff value of 51 predicted hospital admission with AUC, PPV, NPV, sensitivity, and specificity of 85%, 44%, 98%, 88%, and 85%, respectively. Additionally, at a cutoff value of 38, the sFlt-1/PlGF ratio predicted the short-term development of PET with PPV, NPV, sensitivity, and specificity of 42%, 100%, 100%, and 81%, respectively. The PlGF alone at a cutoff value of 193.36 predicted hospital admission with AUC, PPV, NPV, sensitivity, and specificity of 87%, 80%, 97%, 67%, and 98%, respectively. Moreover, at a cutoff value of 51.35, the PlGF alone predicted the development of PET with PPV, NPV, sensitivity, and specificity of 54%, 98%, 88%, and 90%, respectively. There was a significant association between a high sFlt-1/PlGF ratio and developing PET. Conclusion The sFlt-1/PlGF ratio and PlGF are promising for ruling out PET and hospital admission in pregnant women.

Cardiovascular toxicity risk assessment of tyrosine kinase inhibitors: a pharmacovigilance study using the VigiBase database

IntroductionAdvances in the early detection and treatment of cancer have significantly improved the prognosis of patients with cancer. Tyrosine kinase inhibitors (TKIs) are effective targeted treatments for various malignancies that act by inhibiting kinase activity. Although these drugs share a common mechanism of action, they differ in their targeted kinases, pharmacokinetics, and side effects. TKIs can cause cardiovascular side effects, which adversely affect the prognosis of cancer survivors. This study aimed to assess the risk of cardiac toxicity associated with TKIs using the World Health Organization Global Database, VigiBase.MethodsWe conducted a cross-sectional analysis of data from VigiBase, a comprehensive global database of suspected drug reactions. The dataset included reports up to December 2022. We identified patients treated with Food and Drug Administration-approved TKIs and analyzed their age and sex data. The primary outcome was cardiovascular impairment, defined by 21 preferred terms in the Medical Dictionary for Regulatory Activities Terminology version 25.1. Disproportionality analysis using the reported odds ratio was performed to detect adverse cardiovascular signals. Statistical analyses were conducted using R 3.3.2, with a P-value &amp;lt;0.05 considered significant.ResultsOf the 32, 520, 983 reports in VigiBase, 23, 181, 539 were eligible for the analysis. Significant cardiovascular signals were identified for 17 TKIs, including erlotinib, gefitinib, and imatinib. Stratified analyses revealed potential sex- and age-related differences in the risk of adverse events. Heatmaps indicated significant signals for drugs such as lapatinib in males and gefitinib in younger patients.DiscussionOur findings indicate that some TKIs, particularly those classified as VEGFR, BCR-ABL, and BTK, pose similar risks of cardiotoxicity, while others, including EGFR, HER2, and ALK TKIs, exhibit varied risk profiles. These results underscore the importance of individualized risk assessment and management of TKI-treated patients. In conclusion, this study provides valuable insights into the cardiotoxic risk of TKIs, which is essential for developing tailored treatment plans.

EphA4 mediates ephrinB1-dependent adhesion in retinal ganglion cells

Eph/ephrin signaling is crucial for organizing retinotopic maps in vertebrates. Unlike other EphAs, which are expressed in the embryonic ventral retina, EphA4 is found in the retinal ganglion cell (RGC) layer at perinatal stages, and its role in mammalian visual system development remains unclear. Using classicin vitrostripe assays, we demonstrate that, while RGC axons are repelled by ephrinB2, they grow on ephrinB1 stripes through EphA4-mediated adhesion. In vivo, retinal axons from EphA4-deficient mice from either sex show impaired arborization in the medial, but not lateral, regions of the superior colliculus that express ephrinB1. Gain-of-function experiments further reveal that ephrinB1-mediated adhesion depends on EphA4 tyrosine kinase activity but it is independent of its Sterile Alpha Motif. Together, our findings suggest that EphA4/ephrinB1 forward signaling likely facilitates adhesion between retinal axon terminals and cells in the medial colliculus, contributing to the establishment of proper connectivity within the visual system.Significance statementThe significance of our findings goes beyond unveiling the mechanisms underlying topographic visual map formation. By discovering a dual role for EphA4 in mediating both repulsion and adhesion, which challenges the conventional understanding of EphA4/ephrin interactions, this work opens up new avenues for exploring EphA4 broader implications in other cellular contexts, including cell differentiation, migration and synaptic plasticity.

Global Identification of Anti-Melanoma Cellular Targets by Photochemically Induced Coupling of L-Shikonin Reactions on the Surface of Magnetic Particles

Background: L-Shikonin, an active component of Arnebia euchroma (Royle) Johnst., has remarkable pharmacological effects, particularly in its anti-tumour activity. Nonetheless, the specific targets and mechanisms of action remain to be further explored. Methods: A novel Fe3O4@L-Shikonin was designed and synthesized in this study by linking Fe3O4 and L-Shikonin through benzophenone. Fe3O4@L-Shikonin was characterized using several techniques, including scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and drug removal methods, to determine the content of L-Shikonin on the surface of the magnetic particles. Target hooking technology was utilized to identify the target proteins of the compound in melanoma. The synthesized Fe3O4@L-Shikonin was co-incubated with A375 cell lysate, followed by the target proteins, which were purified by magnetic enrichment using magnetic microspheres and identified by high-resolution mass spectrometry. Results: AutoDock Vina software was employed for molecular docking analysis, where it was found that L-Shikonin targets RPN1, CPEB4, and HNRNPUL1 proteins. Subsequently, A375 cells were treated with L-Shikonin at different concentrations (2.5, 5.0, 10.0 μM) for 48 h, and the expressions of the three proteins were observed. The results showed a significant reduction in the relative expression of CPEB4 in the high-dose group compared to the control group (p &lt; 0.01). Moreover, the relative expression of HNRNPUL1 was decreased in the medium- and high-dose groups (p &lt; 0.05). Conclusions: This study initially revealed from the source that L-Shikonin may regulate melanoma-specific markers, melanosomes, tyrosine kinases related to abnormal tyrosine metabolism, and melanoma through multiple targets such as CPEB4 and HNRNPUL1. Proliferation and metastasis work together to exert an anti-melanoma mechanism, which provides a new idea for the follow-up study of the molecular pharmacological mechanism of the complex system of total naphthoquinones in Arnebia euchroma (Royle) Johns.

Multiomics profiling of DNA methylation, microRNA, and mRNA in skeletal muscle from monozygotic twin pairs discordant for type 2 diabetes identifies dysregulated genes controlling metabolism

BackgroundA large proportion of skeletal muscle insulin resistance in type 2 diabetes (T2D) is caused by environmental factors.MethodsBy applying multiomics mRNA, microRNA (miRNA), and DNA methylation platforms in biopsies from 20 monozygotic twin pairs discordant for T2D, we aimed to delineate the epigenetic and transcriptional machinery underlying non-genetic muscle insulin resistance in T2D.ResultsUsing gene set enrichment analysis (GSEA), we found decreased mRNA expression of genes involved in extracellular matrix organization, branched-chain amino acid catabolism, metabolism of vitamins and cofactors, lipid metabolism, muscle contraction, signaling by receptor tyrosine kinases pathways, and translocation of glucose transporter 4 (GLUT4) to the plasma membrane in muscle from twins with T2D. Differential expression levels of one or more predicted target relevant miRNA(s) were identified for approximately 35% of the dysregulated GSEA pathways. These include miRNAs with a significant overrepresentation of targets involved in GLUT4 translocation (miR-4643 and miR-548z), signaling by receptor tyrosine kinases pathways (miR-607), and muscle contraction (miR-4658). Acquired DNA methylation changes in skeletal muscle were quantitatively small in twins with T2D compared with the co-twins without T2D. Key methylation and expression results were validated in muscle, myotubes, and/or myoblasts from unrelated subjects with T2D and controls. Finally, mimicking T2D-associated changes by overexpressing miR-548 and miR-607 in cultured myotubes decreased expression of target genes, GLUT4 and FGFR4, respectively, and impaired insulin-stimulated phosphorylation of Akt (Ser473) and TBC1D4.ConclusionsTogether, we show that T2D is associated with non- and epigenetically determined differential transcriptional regulation of pathways regulating skeletal muscle metabolism and contraction.

Opposed regulation of endoplasmic reticulum retention under stress by ERp44 and PDIA6.

Conditions of endoplasmic reticulum (ER) stress reduce protein synthesis by provoking translation regulation, governed by the eIF2α kinase PERK. When PERK is inhibited during ER stress, retention of a selective subset of glycoproteins occurs, a phenomenon we termed selective endoplasmic reticulum retention (sERr). sERr clients are enriched with tyrosine kinase receptors (RTKs), which form large molecular weight disulfide bonded complexes in the ER. The protein disulfide isomerase ERp44 promotes sERr and increases the size of sERr complexes. Here we show that sERr is reversible upon washout. Pulse chase analyses show that upon recovery, only a small fraction of the sERr complexes disintegrates and contributes to the matured proteins, while most are newly synthesized. Sequential inductions of sERr and washouts demonstrate an accelerated recovery that is dependent on the unfolded protein response transducer IRE1. Since IRE1 regulates the expression level PDIA6, we analyzed its contribution to sERr. We found that PDIA6 and ERp44 constitutively interact by disulfides and have opposite effects on resumed recovery of trafficking following removal of sERr conditions. Deletion of ERp44 accelerates, while deletion of PDIA6 slows down recovery with a minimal effect on total protein synthesis. ERp44 is a primary interactor with sERr clients. When missing, PDIA6 partitions more into sERr complexes. Deletion of the tumor suppressor PTEN, which induces RTK signaling, promoted sERr formation kinetics, and accelerated the recovery, suggesting feedback between RTKs signaling and sERr. This study suggests that sERr, should develop physiologically or pathologically, is counteracted by adaptation responses that involve IRE1 and PDIA6.