DLK1 Expression Research Articles

Enrichment of trimethyl histone 3 lysine 4 in the Dlk1 and Grb10 genes affects pregnancy outcomes due to dietary manipulation of excess folic acid and low vitamin B12.

The aberrant expression of placental imprinted genes due to epigenetic alterations during pregnancy can impact fetal development. We investigated the impact of dietary modification of low vitamin B12 with varying doses of folic acid on the epigenetic control of imprinted genes and fetal development using a transgenerational model of C57BL/6J mice. The animals were kept on four distinct dietary combinations based on low vitamin B12 levels and modulated folic acid, mated in the F0 generation within each group. In the F1 generation, each group of mice is split into two subgroups; the sustained group was kept on the same diet, while the transient group was fed a regular control diet. After mating, maternal placenta (F1) and fetal tissues (F2) were isolated on day 20 of gestation. We observed a generation-wise opposite promoter CpG methylation and gene expression trend of the two developmental genes Dlk1 and Grb10, with enhanced gene expression in both the sustained and transient experimental groups in F1 placentae. When fetal development characteristics and gene expression were correlated, there was a substantial negative association between placental weight and Dlk1 expression (r = -0.49, p < 0.05) and between crown-rump length and Grb10 expression (r = -0.501, p < 0.05) in fetuses of the F2 generation. Consistent with these results, we also found that H3K4me3 at the promoter level of these genes is negatively associated with all fetal growth parameters. Overall, our findings suggest that balancing vitamin B12 and folic acid levels is important for maintaining the transcriptional status of imprinted genes and fetal development.

DLK1 Is Associated with Stemness Phenotype in Medullary Thyroid Carcinoma Cell Lines

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor, often requiring systemic treatment in advanced or metastatic stages, where drug resistance presents a significant challenge. Given the role of cancer stem cells (CSCs) in cancer recurrence and drug resistance, we aimed to identify CSC subpopulations within two MTC cell lines harboring pathogenic variants in the two most common MEN2-associated codons. We analyzed 15 stemness-associated markers, along with well-established thyroid stem cell markers (CD133, CD44, and ALDH1), a novel candidate (DLK1), and multidrug resistance proteins (MRP1 and MRP3). The ability to efflux the fluorescent dye Hoechst 3342 and form spheroids, representing CSC behavior, was also assessed. MZ-CRC-1 cells (p.M918T) displayed higher expressions of canonical markers, DLK1, and MRP proteins than TT cells (p.C634W). MZ-CRC-1 cells also formed more spheroids and showed less dye accumulation (p &lt; 0.0001). Finally, we observed that DLK1+ cells (those expressing DLK1) in both cell lines exhibited significantly higher levels of stemness markers compared to DLK1− cells (those lacking DLK1 expression). These findings underscore DLK1’s role in enhancing the stemness phenotype, providing valuable insights into MTC progression and resistance and suggesting potential therapeutic implications.

In-utero exposure to real-life environmental chemicals disrupts gene expression within the hypothalamo-pituitary-gonadal axis of prepubertal and adult rams

Environmental chemicals (ECs) have been associated with a broad range of disorders and diseases. Daily exposure to various ECs in the environment, or real-life exposure, has raised significant public health concerns. Utilizing the biosolids-treated pasture (BTP) sheep model, this study demonstrates that in-utero exposure to a real-life EC mixture disrupts hypothalamo-pituitary-gonadal (HPG) axis gene expression and reproductive traits in prepubertal (8-week-old, 8w) and adult (11-month-old) male sheep. Ewes were maintained on either BTP or pastures fertilized with inorganic fertilizer [control (C)] from approximately one month prior to insemination until around parturition. Thereafter, all animals were kept under control conditions. Effects on reproductive parameters including testosterone concentrations and the expression of key genes in the HPG axis were evaluated in eight-week-old and adult male offspring from both C and biosolids-exposed (B) groups. Results showed that, at 8w, relative to C (n = 11), B males (n = 11) had lower body weight, and altered testicular expression of HSD3B1, LHR and HSD17B3, BMP4, ABP, P27kip and CELF1. Principal component analysis (PCA) identified two 8w B subgroups, based on hypothalamic expression of GnRH, ESR1, and AR, and pituitary expression of KISSR. The two subgroups also exhibited different serum testosterone concentrations. The largest biosolids effects were observed in the hypothalamus of adult rams with NKB, ESR1, KISS1, AR, DLK1 and GNRH1 mRNA expression differing between B (n = 10) and C (n = 11) rams. Testicular steroidogenic enzymes CYP11A1 and HSD3B1 mRNA expression also differed between exposure groups. PCA identified two adult B subgroups, with BS1 (n = 6) displaying hypothalamic effects and BS2 (n = 4) both hypothalamic and testicular effects. The subgroups also differed in circulating testosterone concentrations. These findings demonstrate that exposure to a real-life EC mixture may predispose some males to infertility, by disrupting key functional HPG markers before puberty with consequent downstream effects on steroid hormones and spermatogenesis.

Methylation aberrations in partner spermatozoa and impaired expression of imprinted genes in the placentae of early-onset preeclampsia

IntroductionDisturbed paternal epigenetic status of imprinted genes has been observed in infertility and recurrent spontaneous abortions. Shallow placentation has been associated with early-onset preeclampsia. Hence, the present study aimed to investigate the methylation patterns of imprinted genes involved in placental development, in the spermatozoa of partners of women experiencing preeclampsia. MethodsThe study involved recruitment of couples into preeclampsia (n = 14) and control (n = 25) groups. Methylation analysis of imprinted gene differentially methylated regions (DMRs) and LINE1 repetitive element was carried out by pyrosequencing in the spermatozoa and placental villi. Global 5mC levels in the spermatozoa were measured through ELISA. Expression of imprinted genes was quantified in the placental villi by real time qPCR. Association of birth weight with DNA methylation and gene expression was assessed. ResultsKvDMR, PEG3 DMR, PEG10 DMR and DLK1-GTL2 IG-DMR were differentially methylated in the spermatozoa and placental villi of preeclampsia group. Global 5mC content and LINE1 methylation levels did not differ between the spermatozoa of the two groups. Increased transcript levels of PEG3, IGF2, DLK1, PHLDA2 and CDKN1C were observed in the preeclamptic placental villi. Birth weight showed significant association with KvDMR, PEG10 DMR, DLK1-GTL2 IG-DMR and LINE1 methylation levels in the spermatozoa. DLK1 expression levels showed a negative association with birth weight. DiscussionThe study highlighted the paternal contribution to early-onset preeclampsia, in the form of disrupted sperm DNA methylation patterns at imprinted gene loci. These loci, after further evaluation in future studies, could serve as sperm-based preeclampsia predictive markers, for couples planning pregnancy.

DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells

BackgroundC3H10T1/2 is a mesenchymal cell line capable of differentiating into osteoblasts, adipocytes and chondrocytes. The differentiation of these cells into osteoblasts is modulated by various transcription factors, such as RUNX2. Additionally, several interconnected signaling pathways, including the NOTCH pathway, play a crucial role in modulating their differentiation into mature bone cells. We have investigated the roles of DLK1 and DLK2, two non-canonical inhibitory ligands of NOTCH receptors, in the osteogenic differentiation of C3H10T1/2 cells.ResultsOur results corroborate existing evidence that DLK1 acts as an inhibitor of osteogenesis. In contrast, we demonstrate for the first time that DLK2 enhances this differentiation process. Additionally, our data suggest that NOTCH2, 3 and 4 receptors may promote osteogenesis, as indicated by their increased expression during this process, whereas NOTCH1 expression, which decreases during cell differentiation, might inhibit osteogenesis. Moreover, treatment with DAPT, a NOTCH signaling inhibitor, impeded osteogenic differentiation. We have confirmed the increase in ERK1/2 MAPK and p38 MAPK phosphorylation in C3H10T1/2 cells induced to differentiate to osteoblasts. Our new findings reveal increased ERK1/2 MAPK phosphorylation in differentiated C3H10T1/2 cells with a decrease in DLK1 expression or an overexpression of DLK2, which is coincident with the behavior of those transfectants where we have detected an increase in osteogenic differentiation. Additionally, p38 MAPK phosphorylation increases in differentiated C3H10T1/2 cells with reduced DLK1 levels.ConclusionsOur results suggest that DLK1 may inhibit osteogenesis, while DLK2 may promote it, by modulating NOTCH signaling and the phosphorylation of ERK1/2 and p38 MAPK pathways. Given the established inhibitory effect of DLK proteins on NOTCH signaling, these new insights could pave the way for developing future therapeutic strategies aimed at treating bone diseases.

8134 Hypothalamic Dlk1 Expression is Not Essential for Preventing Early Maturation of the Reproductive Axis in Female and Male Mice

Abstract Disclosure: D.B. Macedo: None. H. Kim Kyeol: None. A. Abreu: None. A. Latronico: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Inactivating mutations in Delta-like homolog 1 (DLK1), a maternally imprinted gene on chromosome 14, have been recognized as a genetic cause of central precocious puberty (CPP) in humans. It is well established that the timing of puberty, especially in females, is highly sensitive to energy stores and metabolic cues, with lower body fat negatively correlated with age at puberty and menarche. Mice lacking Dlk1 have pre- and postnatal growth retardation. Despite their considerably lower body weight (BW), we found that Dlk1 deficient females achieved puberty at the same age as controls (‘relative precocious puberty’). Moreover, Dlk1 deficiency led to activation of the reproductive axis despite lower levels of kisspeptin and leptin, an adipose tissue hormone with a permissive/stimulatory effect on metabolic control of reproduction. Dlk1 is a transmembrane protein that plays an essential role in inhibiting adipocyte differentiation and its biologically active form is soluble. Increased hypothalamic expression postnatally suggests a neuroendocrine function, but the precise mechanism by which DLK1 deficiency leads to CPP is yet to be deciphered. In this study, we aimed to investigate the role of hypothalamic Dlk1 expression on pubertal onset without the interference of diminished BW, using a conditional knockout (cKO) mouse model. Methods and Results: We generated a targeted Dlk1 knockout mouse model by crossing mice expressing Cre recombinase under the control of Nestin gene (Nes Cre), expressed primarily in neuro-epithelial cells, with mice harboring the Dlk1 gene flanked by loxP sites (Dlk1fl). Since Dlk1 is imprinted and expressed only by the paternal allele, we bred heterozygous Dlk1fl/+ male mice with Nes Cre females to obtain Dlk1fl/Cre (Dlk1 cKO), Dlk1+/Cre (Nes Cre) and Dlk1+/+ (wild type - WT). We confirmed by RT-qPCR that Dlk1 mRNA was undetectable in the mediobasal hypothalamus of Dlk1 cKO adult male mice, whereas it was present in Nes Cre and WT mice. We noted a lower BW in Nes Cre compared to WT mice, as previously reported. However, no difference in BW was observed between Dlk1 cKO and Nes Cre mice at the timing of puberty (females: Dlk1 cKO 12.6 ± 0.8 g vs. Nes Cre 13.8 ± 0.2 g, P = 0.18; males: Dlk1 cKO 12.4 ± 0.2 g vs. Nes Cre 13.1 ± 0.5 g, P = 0.26). Puberty onset, assessed by age at vaginal opening (females) and at preputial separation (males), was not affected by hypothalamic Dlk1 deletion (Dlk1 cKO: 35.6 ± 2.7 days vs. Nes Cre 34.6 ± 0.7 days, P = 0.73; Dlk1 cKO 29.0 ± 0.6 vs. Nes Cre: 30.3 ± 0.6 days, P = 0.19). Conclusion: In contrast to global Dlk1 deficiency, targeted deletion of Dlk1 from neuronal and glial cells did not affect body weight or timing of pubertal onset. These findings suggest that Dlk1 originates from peripheral tissues, such as adipocytes, to regulate reproduction and metabolism. Presentation: 6/1/2024

Dual effects of ARX poly-alanine mutations in human cortical and interneuron development.

Infantile spasms, with an incidence of 1.6 to 4.5 per 10,000 live births, are a relentless and devastating childhood epilepsy marked by severe seizures but also leads to lifelong intellectual disability. Alarmingly, up to 5% of males with this condition carry a mutation in the Aristaless-related homeobox ( ARX ) gene. Our current lack of human-specific models for developmental epilepsy, coupled with discrepancies between animal studies and human data, underscores the gap in knowledge and urgent need for innovative human models, organoids being one of the best available. Here, we used human neural organoid models, cortical organoids (CO) and ganglionic eminences organoids (GEO) which mimic cortical and interneuron development respectively, to study the consequences of PAE mutations, one of the most prevalent mutation in ARX . ARX PAE produces a decrease expression of ARX in GEOs, and an enhancement in interneuron migration. That accelerated migration is cell autonomously driven, and it can be rescued by inhibiting CXCR4. We also found that PAE mutations result in an early increase in radial glia cells and intermediate progenitor cells, followed by a subsequent loss of cortical neurons at later timepoints. Moreover, ARX expression is upregulated in COs derived from patients at 30 DIV and is associated with alterations in the expression of CDKN1C . Furthermore, ARX PAE assembloids had hyperactivity which were evident at early stages of development. With effective treatments for infantile spasms and developmental epilepsies still elusive, delving into the role of ARX PAE mutations in human brain organoids represents a pivotal step toward uncovering groundbreaking therapeutic strategies.

DLK1 promoted ischemic angiogenesis through notch1 signaling in endothelial progenitor cells.

Delta like non-canonical Notch ligand 1 (DLK1), as a member of epidermal growth factor-like family, plays a critical role in somatic growth, tissue development and possibly tissue renewal. Though previous studies had indicated that DLK1 contributed to adipogenesis and myogenesis, it's still controversial whether DLK1 affects angiogenesis and how it interacts with Notch signaling with numerous conflicting reports from different models. Based on our preliminary finding that DLK1 expression was up-regulated in mice ischemic gastrocnemius and in the border zone of infarcted myocardium, we administered either recombinant DLK1 (rDLK1) or PBS in C57BL/6 mice after establishment of hindlimb ischemia (HLI) and myocardial infarction (MI), respectively. Exogenous rDLK1 administration significantly improved both blood perfusion of mice ischemic hindlimbs and muscle motor function on the 3rd, 7th day after HLI, by promoting neovascularization. Similar effect on neovascularization was verified in mice on the 28th day after MI as well as improvement of cardiac failure. Correspondingly, the number of CD34+KDR+ cells, indicated as endothelial progenitor cells (EPCs), was significantly in mice ischemic gastrocnemius by rDLK1 administration, which was abrogated by DAPT as the specific inhibitor of Notch intracellular domain (NICD). Furthermore, bone marrow mononuclear cells were obtained from C57BL/6 mice and differentiated to EPCs ex vivo. Incubation with rDLK1 triggered Notch1 mRNA and NICD protein expressions in EPCs as exposed to hypoxia and serum deprivation, promoting EPCs proliferation, migration, anti-apoptosis and tube formation. Otherwise, rDLK1 incubation significantly decreased intracellular and mitochondrial reactive oxygen species, increased ATP content andmitochondrial membrane potential, downregulated short isoform of OPA-1 expression whereas upregulated mitofusin (-1, -2) expression in EPCs by Notch1 signaling, which were all abrogated by DAPT. In summary, the present study unveils the pro-angiogenesis and its mechanism of rDLK1 through activation of Notch1 signaling in endothelial progenitor cells.

Nausea-induced suppression of feeding is mediated by central amygdala Dlk1-expressing neurons

The motivation to eat is suppressed by satiety and aversive stimuli such as nausea. The neural circuit mechanisms of appetite suppression by nausea are not well understood. Pkcδ neurons in the lateral subdivision of the central amygdala (CeA) suppress feeding in response to satiety signals and nausea. Here, we characterized neurons enriched in the medial subdivision (CeM) of the CeA marked by expression of Dlk1. CeADlk1 neurons are activated by nausea, but not satiety, and specifically suppress feeding induced by nausea. Artificial activation of CeADlk1 neurons suppresses drinking and social interactions, suggesting a broader function in attenuating motivational behavior. CeADlk1 neurons form projections to many brain regions and exert their anorexigenic activity by inhibition of neurons of the parabrachial nucleus. CeADlk1 neurons are inhibited by appetitive CeA neurons, but also receive long-range monosynaptic inputs from multiple brain regions. Our results illustrate a CeA circuit that regulates nausea-induced feeding suppression.

Pericardial delta like non-canonical NOTCH ligand 1 (Dlk1) augments fibrosis in the heart through epithelial to mesenchymal transition.

Heart failure due to myocardial infarction (MI) involves fibrosis driven by epicardium-derived cells (EPDCs) and cardiac fibroblasts, but strategies to inhibit and provide cardio-protection remains poor. The imprinted gene, non-canonical NOTCH ligand 1 (Dlk1), has previously been shown to mediate fibrosis in the skin, lung and liver, but very little is known on its effect in the heart. Herein, human pericardial fluid/plasma and tissue biopsies were assessed for DLK1, whereas the spatiotemporal expression of Dlk1 was determined in mouse hearts. The Dlk1 heart phenotype in normal and MI hearts was assessed in transgenic mice either lacking or overexpressing Dlk1. Finally, in/ex vivo cell studies provided knowledge on the molecular mechanism. Dlk1 was demonstrated in non-myocytes of the developing human myocardium but exhibited a restricted pericardial expression in adulthood. Soluble DLK1 was twofold higher in pericardial fluid (median 45.7 [34.7 (IQR)) μg/L] from cardiovascular patients (n=127) than in plasma (median 26.1μg/L [11.1 (IQR)]. The spatial and temporal expression pattern of Dlk1 was recapitulated in mouse and rat hearts. Similar to humans lacking Dlk1, adult Dlk1-/- mice exhibited a relatively mild developmental, although consistent cardiac phenotype with some abnormalities in heart size, shape, thorax orientation and non-myocyte number, but were functionally normal. However, after MI, scar size was substantially reduced in Dlk1-/- hearts as compared with Dlk1+/+ littermates. In line, high levels of Dlk1 in transgenic mice Dlk1fl/fl xWT1GFPCre and Dlk1fl/fl xαMHCCre/+Tam increased scar size following MI. Further mechanistic and cellular insight demonstrated that pericardial Dlk1 mediates cardiac fibrosis through epithelial to mesenchymal transition (EMT) of the EPDC lineage by maintaining Integrin β8 (Itgb8), a major activator of transforming growth factor β and EMT. Our results suggest that pericardial Dlk1 embraces a, so far, unnoticed role in the heart augmenting cardiac fibrosis through EMT. Monitoring DLK1 levels as well as targeting pericardial DLK1 may thus offer new venues for cardio-protection.

A Fanca knockout mouse model reveals novel Fancd2 function

Fanconi anemia (FA) is a genetically and clinically heterogenous inherited disorder. Clinically, Fanca subtype patients exhibited milder phenotypes compared to Fancd2 subtypes. Increasing evidence suggests that Fancd2 perform independent functions, but the detailed mechanisms are not well characterized. In this study, we developed a Fanca KO mice model in C57BL/6 background with ATG region deletion, then performed a detailed FA phenotypes characterization and analysis with Fanca KO mice and Fancd2 KO mice in the same congenic background. We found that both the Fanca KO and Fancd2 KO cause severe FA phenotypes in mice. However, Fanca KO mice exhibited milder FA phenotypes comparing to Fancd2 KO mice. Fanca KO mice showed higher embryonic and postnatal survival rate, less congenital eye defects in early development. At adult stage, Fanca KO mice showed increased HSC number and reconstitution function. Furthermore, we did RNA-seq study and identified differential expression of Dlk1 and Dlk1 pathway genes in Fanca KO and Fancd2 KO embryonic cells and adult HSCs. Finally, we revealed that Fancd2 was expressed and physically interact with Dlk1 in Fanca KO cells. Collectively, our findings suggested that Fancd2 has distinct functions in the absence of Fanca.

OR04-06 Mkrn3, Dlk1 And Mecp2 Have Distinct Hypothalamic Expression Patterns Across Pubertal Maturation In Male And Female Mice

Abstract Disclosure: F.R. Tinano: None. S.A. Pereira: None. N.P. Boris: None. D.B. Macedo: None. A.P. Canton: None. A. Abreu: None. R.S. Carroll: None. A. Latronico: None. U.B. Kaiser: None. Context: Central precocious puberty (CPP) results from premature re-activation of the hypothalamic-pituitary-gonadal (HPG) axis. The most prevalent genetic cause of CPP is loss-of-function mutations in makorin ring finger 3 (MKRN3), with loss-of-function mutations in delta-like 1 homolog (DLK1) occurring less frequently. While the mechanisms by which MKRN3 regulates puberty onset have been partially elucidated in recent years, the manner by which DLK1 regulates HPG axis maturation remains unknown. Methyl CpG binding protein 2 (MeCP2) is associated with the neurodevelopmental Rett syndrome, with reports of early pubertal development among affected patients. Recently, rare variants in MeCP2 were identified in children with CPP with or without neurologic abnormalities. The pathways by which MECP2 may influence pubertal timing have not been determined. The aim of this study was to characterize Mkrn3, Dlk1 and Mecp2 expression patterns in the hypothalamus of male and female mice at different pubertal stages.Methods and Results: Male and female wild type C57BL/6 mice were euthanized at postnatal (P) age 10, 15, 22, 30 and 60 days. The mediobasal hypothalamus (MBH) and pre-optic area (POA) were isolated from the brains of each mouse. mRNA levels of Mkrn3, Dlk1 and Mecp2 were quantified by RT-qPCR and compared across the ages studied. Mkrn3 expression in both MBH and POA was highest at P10, then significantly lower at P15 and P22, remaining low until adulthood, with a similar pattern in male and female mice. In contrast, Dlk1 expression was higher at P60 compared to P22 in the MBH of male mice, while in female mice it was higher at P22 and P30 compared to P10. In the POA of male mice, Dlk1 expression was higher at P30 and P60 compared to P10, while no significant difference with age was seen in the POA of female mice. Mecp2 had higher expression in the MBH at P10 compared to P15 and P22 in male mice, and compared to P15, P30 and P60 in female mice. Mecp2 expression in the POA of both male and female mice was higher at P10 compared to all the other ages. No differences were observed in the hypothalamic expression of the three genes between male and female mice. Statistical significance was set at p &amp;lt; 0.05. Conclusions:Mkrn3 and Mecp2 had decreased hypothalamic expression in male and female mice with age, suggesting inhibitory roles on the HPG axis, consistent with the phenotype of CPP in patients with loss-of-function mutations in these genes. In contrast, hypothalamic expression of Dlk1 increased in both male and female mice toward adulthood, which is intriguing considering the association of loss-of-function mutations in this gene with CPP and suggests that the role of Dlk1 in the HPG axis is distinct from those of Mkrn3 and Mecp2. Defining the changes in hypothalamic expression of these three genes across pubertal development is critical to understanding their roles and mechanisms of action in regulating the HPG axis. Presentation: Thursday, June 15, 2023

Identification and validation of core genes for type 2 diabetes mellitus by integrated analysis of single-cell and bulk RNA-sequencing

BackgroundThe exact mechanisms of type 2 diabetes mellitus (T2DM) remain largely unknown. We intended to authenticate critical genes linked to T2DM progression by tandem single-cell sequencing and general transcriptome sequencing data.MethodsT2DM single-cell RNA-sequencing data were submitted by the Gene Expression Omnibus (GEO) database and ArrayExpress (EBI), from which gene expression matrices were retrieved. The common cell clusters and representative marker genes were ascertained by principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), CellMarker, and FindMarkers in two datasets (GSE86469 and GSE81608). T2DM-related differentially expressed marker genes were defined by intersection analysis of marker genes and GSE86468-differentially expressed genes. Receiver operating characteristic (ROC) curves were utilized to assign representative marker genes with diagnostic values by GSE86468, GSE29226 and external validation GSE29221, and their prospective target compounds were forecasted by PubChem. Besides, the R package clusterProfiler-based functional annotation was designed to unveil the intrinsic mechanisms of the target genes. At last, western blot was used to validate the alternation of CDKN1C and DLK1 expression in primary pancreatic islet cells cultured with or without 30mM glucose.ResultsThree common cell clusters were authenticated in two independent T2DM single-cell sequencing data, covering neurons, epithelial cells, and smooth muscle cells. Functional ensemble analysis disclosed an intimate association of these cell clusters with peptide/insulin secretion and pancreatic development. Pseudo-temporal trajectory analysis indicated that almost all epithelial and smooth muscle cells were of neuron origin. We characterized CDKN1C and DLK1, which were notably upregulated in T2DM samples, with satisfactory availability in recognizing three representative marker genes in non-diabetic and T2DM samples, and they were also robustly interlinked with the clinical characteristics of patients. Western blot also demonstrated that, compared with control group, the expression of CDKN1C and DLK1 were increased in primary pancreatic islet cells cultured with 30 mM glucose for 48 h. Additionally, PubChem projected 11 and 21 potential compounds for CDKN1C and DLK1, respectively.ConclusionIt is desirable that the emergence of the 2 critical genes indicated (CDKN1C and DLK1) could be catalysts for the investigation of the mechanisms of T2DM progression and the exploitation of innovative therapies.

Imprinted Dlk1 dosage as a size determinant of the mammalian pituitary gland.

Co-regulated genes of the Imprinted Gene Network are involved in the control of growth and body size, and imprinted gene dysfunction underlies human paediatric disorders involving the endocrine system. Imprinted genes are highly expressed in the pituitary gland, among them, Dlk1, a paternally expressed gene whose membrane-bound and secreted protein products can regulate proliferation and differentiation of multiple stem cell populations. Dosage of circulating DLK1 has been previously implicated in the control of growth through unknown molecular mechanisms. Here we generate a series of mouse genetic models to modify levels of Dlk1 expression in the pituitary gland and demonstrate that the dosage of DLK1 modulates the process of stem cell commitment with lifelong impact on pituitary gland size. We establish that stem cells are a critical source of DLK1, where embryonic disruption alters proliferation in the anterior pituitary, leading to long-lasting consequences on growth hormone secretion later in life.

Abstract P3031: The Cross Talk Between Increased Abdominal Adiposity And Calcification Of Aortic Valve

Calcified aortic valve disease (CAVD) is the most prevalent valvular heart disease in western populations affecting nearly 2.5% of individuals over 65. Currently, there is no viable pharmacological treatment to stop the disease's progression or activate mineral regression. The only effective therapy to treat CAVD is surgical valve replacement or trans-catheterization. Obesity is the major common nutritional disorder worldwide and is one of the important risk factors for CAVD. It has been shown that DLK1-/- mice have increased abdominal adiposity. Indeed Dlk1 plays an essential role in regulating adipocytes differentiation and proliferation. Recently, it has been shown that there is an association between fat tissue accumulation and an increased risk of aortic stenosis. We hypothesize that dysregulation of DLK1 stimulates adiposity, increases circulating lipids, enhances valve lipids retention, and stimulates calcification. Herein, our objective is to examine the expression of DLK1 in calcified aortic valves and its impact on lipid retention. Method: We examined the effect of Dlk1 loss of function on lipid retention in aortic valve tissue in ApoE-/- Dlk1-/- mice fed with a high-fat diet and in human explanted aortic valves using immuno-staining. Results: The complete deletion of DLK-1 in ApoE-/- mice were associated with an increased oxidized-LDL deposit in aortic valve tissue. In addition, circulating cholesterol (420 ± 8 mg/dl vs 250± 12 mg/dl, P=0.002) and triglyceride concentration (200 ± 3 mg/dl vs 50± 10 mg/dl, P&lt;0.001) were higher in ApoE-/- DLK1-/- compared to apoE-/- mice. An increased valvular calcification accompanied these changes, as shown by the over-expression of RUNX2 and the increased aortic jet velocity in ApoE-/- DLK1-/- mice. Interestingly the overexpression of DLK1 in isolated human valve cells decreased calcification and lipid retention in vitro. Conclusion: This study showed the significant role of Dlk1-derived abdominal adiposity in valve-lipid retention and calcification.

Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis

How neuronal signaling affects brain myelination remains poorly understood. We show dysregulated neuronal RHEB-mTORC1-DLK1 axis impairs brain myelination. Neuronal Rheb cKO impairs oligodendrocyte differentiation/myelination, with activated neuronal expression of the imprinted gene Dlk1. Neuronal Dlk1 cKO ameliorates myelination deficit in neuronal Rheb cKO mice, indicating that activated neuronal Dlk1 expression contributes to impaired myelination caused by Rheb cKO. The effect of Rheb cKO on Dlk1 expression is mediated by mTORC1; neuronal mTor cKO and Raptor cKO and pharmacological inhibition of mTORC1 recapitulate elevated neuronal Dlk1 expression. We demonstrate that both a secreted form of DLK1 and a membrane-bound DLK1 inhibit the differentiation of cultured oligodendrocyte precursor cells into oligodendrocytes expressing myelin proteins. Finally, neuronal expression of Dlk1 in transgenic mice reduces the formation of mature oligodendrocytes and myelination. This study identifies Dlk1 as an inhibitor of oligodendrocyte myelination and a mechanism linking altered neuronal signaling with oligodendrocyte dysfunction.

Long chains fatty acids induce differentiation of epicardial progenitor cells into adipocytes through preadipocyte marker DLK1

The atrial fibrillation (AF), the most frequent cardiac arrhythmia is most often the clinical expression of the atrial cardiomyopathy. The epicardial adipose tissue (EAT) is now considered as an important component of the atrial cardiomyopathy notably when AF is associated metabolic disorders such as obesity. We previously showed that the epicardial progenitor cells (EPC) are the main source of EAT. Here we tested the hypothesis that long chain free fatty acids (LCFFA) present in the serum of patients with metabolic disorders can induce the differentiation of EPC into adipocytes. First, characterization of the effects LCFFA oleate (OLE) and palmitate (PAL) on differentiation of EPC; second, to examine the involvement of DLK1/Pref-1, a key actor of adipogenesis in the differentiation of EPC into adipocytes. In a rat model of atrial cardiomyopathy secondary to ischemic heart failure, EPC were harvested and cultured in presence of OLE (500 μM) and PAL (250 μM) for 7 days. EPC-derived adipocytes were characterized with oil red-O staining and Bodipy labelling. The transcriptomic of EPC was performed with single cell RNA sequencing together with a gene expression of EPC-derived adipocytes performed with qPCR at 3 and 7days after treatment. Soluble form of Pref-1 was measured in supernatant of EPC treated by using ELISA assay. The Notch/Pref-1 signaling pathway was investigated with biochemistry approach. After 3 days, the subpopulation of EPC expressing pref1 accumulated lipid droplets evidenced by red oil. At day 7, DLK1 expression was decreased whereas that of the adipogenic marker PPARγ was increased at the transcriptomic level (n = 5; P < 0.05). Furthermore, the expression of short soluble form of Pref-1 accumulated in supernatant of EPC indicated the shedding of the protein. Preliminary data indicated an effect of the short soluble form of Pref-1 on EPC proliferation. Single cell RNA sequencing revealed Notch expression in cluster of EPC engaged in the adipocyte differentiation pathway. Serum long chain fatty acid can trigger the adipogenic differentiation of EPC and DLK1/Notch signaling pathway appears to be an early step during this process. It remains to determine mechanisms of lipidic receptor GPR40 in EPC differentiation and the contribution to the expansion of atrial EAT during metabolic diseases.

Abstract CT177: A phase I, first in human study of CBA-1205, glycoengineered humanized anti-DLK-1 monoclonal antibody in patients with advanced solid tumors

Abstract Background: CBA-1205 is a novel humanized anti-delta-like 1 homolog (DLK-1) antibody. It is a glycoengineered antibody by GlymaxX® technology to potentiate antibody-dependent cellular cytotoxicity activity. DLK-1 is a membrane protein with 6 tandem EGF-like motifs in extracellular region. Overexpression of DLK-1 has been reported in variety of cancer types including hepatocellular carcinoma (HCC), suggesting that DLK-1 could be an ideal target for cancer therapy. Methods: This is a first in human, multicenter, open-label Phase I study. The purpose of part 1 is to evaluate the safety and tolerability in patients with advanced solid tumors who had no standard therapy or were refractory or intolerant to standard therapies and to determine the starting dose for part 2 in HCC patients. The standard 3+3 dose escalation design was utilized in seven cohorts (0.1, 0.3, 1, 3, 10, 20, 30 mg/kg). CBA-1205 was administered at 2-week intervals in a 28-day cycle. If the patient had no Dose Limiting Toxicity (DLT) during 28 days after the first two doses, CBA-1205 administration will be continued for further cycles unless any of the criteria for discontinuation. Serum DLK-1 concentration before the start of CBA-1205 administration was analyzed using the Human Pref-1/DLK1/FA1 DuoSet ELISA (R&amp;D Systems, Inc.). Immunohistochemical analysis of DLK-1 expression in the archival tumor specimen was determined in FFPE tissue sections using mouse anti-hDLK-1 antibody (clone DI-2-20, Chiome Bioscience). Results: 22 patients with advanced solid tumors were enrolled. CBA-1205 was well tolerated in these patients and no DLT was observed even at the highest dose tested. No ≥ Grade 3 treatment related adverse events was observed throughout the study periods. The serum concentration of CBA-1205 increased dose-proportionally. The exposure to CBA-1205 exceeded the effective concentrations observed in a mouse xenograft model. No serum anti-CBA-1205 antibody was detected. The disease control was seen in 8 patients (38.1%, all with SD). Especially, long SD was observed in 3 patients with invasive thymoma, malignant melanoma, and pancreatic carcinoma, where progression free survival (PFS) was 39, 72(ongoing, at data cutoff), and 41 weeks, respectively. The serum concentration of soluble DLK-1 was detected in 6 out of 22 patients. Patients with SD by the best overall response had a higher rate of detectable serum DLK-1 concentration and a higher mean concentration than patients with PD. No DLK-1 expression in tumor was detected by IHC method, in 14 patients who consented to use the archival tumor specimens. Conclusions: CBA-1205 was well tolerated without any severe toxicity in patients with advanced solid tumors.Part 2 of this Phase I study with two cohorts (20, 30 mg/kg) in patients with unresectable advanced/recurrent HCC refractory or intolerable to the standard therapy has been initiated. Citation Format: Masafumi Ikeda, Nobuaki Matsubara, Chihiro Kondoh, shose taoka, Yuki Katsuya, Takafumi Koyama, Noboru Yamamoto. A phase I, first in human study of CBA-1205, glycoengineered humanized anti-DLK-1 monoclonal antibody in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT177.

Abstract 1862: Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models

Abstract Delta-like 1 homolog (DLK-1), a non-canonical notch ligand, is a membrane protein with 6 tandem EGF-like motifs in extracellular region. While the expression of DLK-1 is restricted in some neuroendocrine tissues such as adrenal gland and hypophysis in normal adult tissues, its over-expression in cancers have been reported in a variety of cancer types, such as hepatocellular carcinoma (HCC), small cell lung carcinoma, ovarian carcinoma, adrenal cortical carcinoma, pancreatic carcinoma, gastrointestinal stromal tumor, etc. Interestingly, it has been suggested that DLK-1 is a marker of cancer stem cells in HCC. CBA-1205 is a humanized antibody (IgG1/κ) targeting DLK-1, which is glycoengineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. CBA-1205 showed no toxicology issues in cynomolgus monkey GLP studies and the Phase 1 clinical trial in patients with solid tumors is under way (JapicCTI-205384). Previously, we demonstrated potent anti-tumor efficacy of CBA-1205 as a monotherapy in multiple human DLK-1 positive cancer xenograft mouse models including HCC (AACR 2019 annual meeting). Lenvatinib is an oral multi-kinase inhibitor which has been used for 1st line therapy of unresectable advanced HCC globally. The purpose of present study is to evaluate the combinatorial efficacy of CBA-1205 with lenvatinib in two HCC xenograft models (Hep3B and HepG2). Hep3B model was sensitive to lenvatinib monotherapy; oral daily dosing of lenvatinib at 3 mg/kg treatment showed 50% tumor growth inhibition (TGI) and at 10 mg/kg treatment showed more than 80% TGI. In contrast HepG2 model was less sensitive to Lenvatinib; TGI at 3 mg/kg treatment group was 20 % and that for 10 mg/kg treatment group was 50 %. When CBA-1205 (1 mg/kg, twice a week, total 4 times) was intraperitoneally administered in combination with 3 mg/kg of lenvatinib (daily, total 10 times) in Hep3B xenograft mice, synergistic and long-lasting anti-tumor efficacy was observed compared either with CBA-1205 treatment alone or lenvatinib treatment alone. Tumor growth of all mice (N=8) treated with CBA-1205 and lenvatinib showed almost complete inhibition even 2 weeks after the last dosing. More importantly, 4 out of 8 mice in CBA-1205 and lenvatinib treatment group showed tumor regression. Similar results were obtained with CBA-1205 at 1 mg/kg dosage in combination with lenvatinib at 10 mg/kg dosage in HepG2 xenograft model. In conclusion, we demonstrated synergistic and long-lasting anti-tumor efficacy of CBA-1205 in combination with lenvatinib in two different HCC pre-clinical models suggesting that the combination could be a new treatment option for unresectable advanced HCC. Citation Format: Koji Nakamura, Izumi Sakaguchi, Kota Takahashi. Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models [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 1862.

Delta-Like Factor 1 Negatively Regulates Angiogenesis as a Target Gene of MIR-126-5P after Indirect Revascularization Surgery in Patients with Moyamoya Disease

Introduction: Moyamoya disease (MMD) is a chronic cerebral hypoperfusion state with increased narrowing of the intracranial internal carotid artery. Objectives: The main objective of the study is to analyses the delta-like factor 1 negatively regulates angiogenesis as a target gene of miR-126-5p after indirect revascularization surgery in patients with moyamoya disease. Material and methods: This retrospective study was conducted in Akhtar Saeed Medical College Lahore during 2020 to 2021. First, we compared the DLK1 expression in DM tissues from patient with moyamoya disease (n = 8) and unruptured aneurysms (n = 8, control group). DM samples (1.0 × 0.5 cm) were collected from patients with moyamoya disease during revascularization procedures. DM samples were harvested from the temporal part of the head and stored for further use. Results: EPC markers CD31, VE-cadherin, and VEGFR-2 were strongly expressed in cultured cells, while hematopoietic marker CD133 did not display remarkable expression, suggesting highly purified EPC isolation. Practical implication: This study will be helpful in EC proliferation and angiogenesis. Conclusion: It is concluded that DLK1, a target gene of miR-126-5p, negatively regulates EC proliferation and angiogenesis and that downregulating DLK1 expression potentially promotes angiogenesis in chronically ischaemic brains. Keywords: DLK1, EC, Proliferation, Angiogenesis