Wnt Signaling Research Articles

RETRACTION: PNU-74654 enhances the antiproliferative effects of 5-FU in breast cancer and antagonizes thrombin-induced cell growth via the Wnt pathway.

F. Rahmani, F. Amerizadeh, S. M. Hassanian, M. Hashemzehi, S.-N. Nasiri, H. Fiuji, G. A. Ferns, M. Khazaei, and A. Avan, "PNU-74654 Enhances the Antiproliferative Effects of 5-FU in Breast Cancer and Antagonizes Thrombin-induced Cell Growth via the Wnt Pathway," Journal of Cellular Physiology 234, no. 8 (2019): 14123-14132, https://doi.org/10.1002/jcp.28104. The above article, published online on 11 January 2019 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Robert Heath; and Wiley Periodicals LLC. The retraction has been agreed due to concerns raised by a third party on the data presented in the article. Specifically, image elements in Figure 1c were previously published by the same author group in a different scientific context. Additionally, the panels representing the histological staining corresponding to the "Control" and "5-FU" groups in Figure 3b were found to originate from the same biological sample. Finally, splicing sites have been detected within Figure 6b. The concerns were not satisfactorily addressed by the authors upon request. Accordingly, retraction is warranted as the editors have lost trust in the data presented in the article and in its conclusions. The corresponding author Majid Khazaei disagrees with the decision of retraction. No confirmation was obtained by the remaining co-authors.

The soluble factor milieu in idiopathic pulmonary fibrosis dysregulates epithelial differentiation.

In idiopathic pulmonary fibrosis (IPF), epithelial abnormalities are present including bronchiolization and alveolar cell dysfunction. We hypothesized that the IPF microenvironment disrupts normal epithelial growth and differentiation. We mimicked the soluble factors within an IPF microenvironment using an IPF cocktail (IPFc), composed of nine factors which are increased in IPF lungs (CCL2, IL-1β, IL-4, IL-8, IL-13, IL-33, TGF-β, TNFα, and TSLP). Using IPFc, we asked whether the soluble factor milieu in IPF affects epithelial growth and differentiation and how IPFc compares to TGF-β alone. Epithelial growth and differentiation were studied using mouse lung organoids (primary Epcam+ epithelial cells co-cultured with CCL206 fibroblasts). Organoids exposed to IPFc and TGF-β were re-sorted into epithelial and fibroblast fractions and subjected to RNA sequencing. IPFc did not affect the number of organoids formed. However, pro-surfactant protein C expression was decreased. On these parameters, TGF-β alone had similar effects. However, RNA sequencing of re-sorted organoids revealed that IPFc and TGF-β had distinct effects on both epithelial cells and fibroblasts. IPFc upregulated goblet cell markers, whereas these were inhibited by TGF-β. Although both IPFc and TGF-β increased extracellular matrix gene expression, only TGF-β increased myofibroblast markers. VEGF-C and Wnt signaling were among the most differentially regulated signaling pathways by IPFc versus TGF-β. Interestingly, Wnt pathway activation rescued Sftpc downregulation induced by IPFc. In conclusion, IPFc alters epithelial differentiation in a way that is distinct from TGF-β. Alterations in Wnt signaling contribute to these effects. IPFc may be a more comprehensive representation of the soluble factor microenvironment in IPF.

Autophagy controls neuronal differentiation by regulating the WNT-DVL signaling pathway

ABSTRACT Macroautophagy/autophagy dysregulation is associated with various neurological diseases, including Vici syndrome. We aimed to determine the role of autophagy in early brain development. We generated neurons from human embryonic stem cells and developed a Vici syndrome model by introducing a loss-of-function mutation in the EPG5 gene. Autophagy-related genes were upregulated at the neuronal progenitor cell stage. Inhibition of autolysosome formation with bafilomycin A1 treatment at the neuronal progenitor cell stage delayed neuronal differentiation. Notably, WNT (Wnt family member) signaling may be part of the underlying mechanism, which is negatively regulated by autophagy-mediated DVL2 (disheveled segment polarity protein 2) degradation. Disruption of autolysosome formation may lead to failure in the downregulation of WNT signaling, delaying neuronal differentiation. EPG5 mutations disturbed autolysosome formation, subsequently inducing defects in progenitor cell differentiation and cortical layer generation in organoids. Disrupted autophagy leads to smaller organoids, recapitulating Vici syndrome-associated microcephaly, and validating the disease relevance of our study. Abbreviations: BafA1: bafilomycin A1; co-IP: co-immunoprecipitation; DVL2: dishevelled segment polarity protein 2; EPG5: ectopic P-granules 5 autophagy tethering factor; gRNA, guide RNA; hESC: human embryonic stem cells; KO: knockout; mDA, midbrain dopamine; NIM: neural induction media; NPC: neuronal progenitor cell; qPCR: quantitative polymerase chain reaction; UPS: ubiquitin-proteasome system; WNT: Wnt family member; WT: wild type.

NSUN2 regulates Wnt signaling pathway depending on the m5C RNA modification to promote the progression of hepatocellular carcinoma.

5-Methylcytosine (m5C) RNA modification is a highly abundant and important epigenetic modification in mammals. As an important RNA m5C methyltransferase, NOP2/Sun-domain family member 2 (NSUN2)-mediated m5C RNA modification plays an important role in the regulation of the biological functions in many cancers. However, little is known about the biological role of NSUN2 in hepatocellular carcinoma (HCC). In this study, we found that the expression of NSUN2 was significantly upregulated in HCC, and the HCC patients with higher expression of NSUN2 had a poorer prognosis than those with lower expression of NSUN2. NSUN2 could affect the tumor immune regulation of HCC in several ways. In vitro and in vivo experiments confirmed that NSUN2 knockdown significantly decreased the abilities of proliferation, colony formation, migration and invasion of HCC cells. The methylated RNA immunoprecipitation-sequencing (MeRIP-seq) showed NSUN2 knockdown significantly affected the abundance, distribution, and composition of m5C RNA modification in HCC cells. Functional enrichment analyses and in vitro experiments suggested that NSUN2 could promote the HCC cells to proliferate, migrate and invade by regulating Wnt signaling pathway. SARS2 were identified via the RNA immunoprecipitation-sequencing (RIP-Seq) and MeRIP-seq as downstream target of NSUN2, which may play an important role in tumor-promoting effect of NSUN2-mediated m5C RNA modification in HCC. In conclusion, NSUN2 promotes HCC progression by regulating Wnt signaling pathway and SARS2 in an m5C-dependent manner.

MiRNA-153 attenuates progression of non-small cell lung cancer through targeting positive regulatory/SET domain 2

Background: To explore whether micro ribonucleic acid (miR)-153 regulates positive regulatory/SET domain 2 (PRDM2) in a targeted manner and affects the proliferation and apoptosis of non-small cell lung cancer (NSCLC) A549 cells. Methodology: The expressions of miR-153 and PRDM2 in NSCLC tissues and A549 cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). TargetScan was utilized to predict miR-153 target genes. Methyl thiazolyl tetrazolium (MTT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were carried out to study the cell proliferation and apoptosis. Western blotting was performed to examine the changes in the proteins in the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway following the miR-153 overexpression. Results: The expression of miR-153 was decreased and that of PRDM2 was increased in NSCLC tissues and cells. Target genes regulated by miR-153 participated in self-vascular development, miRNA metabolic process and the Wnt signaling pathway. The overexpression of miR-153 led to an obvious reduction in the proliferation ability of A549 cells, a notable increase in apoptotic cells, and significant decreases in phosphorylated (p)-JAK2 and p-STAT3 proteins. Dual-luciferase reporter gene assay revealed that miR-153 could directly modulate the expression level of PRDM2. Conclusion: MiR-153 directly targets PRDM2 and affects A549 cell proliferation and apoptosis through the JAK/STAT pathway. Keywords: Non-small cell lung cancer; miR-153; positive regulatory/SET domain 2; Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway.

12510 SLC25A11 As A Novel Gene For Carney-Stratakis Syndrome

Abstract Disclosure: F. Freitas-Castro: None. L.S. Santana: None. G.F. Fagundes: None. A.F. Afonso: None. E.C. Lobato: None. F.L. Ledesma: None. I.C. Soares: None. B.B. Mendonca: None. M.C. Fragoso: None. A. Latronico: None. C.A. Stratakis: None. M.Q. Almeida: None. Background: Carney-Stratakis syndrome (CSS, OMIM #606864), also reported as “paraganglioma and gastrointestinal stromal tumor syndrome” or Carney-Stratakis dyad, is an autosomal dominant rare syndrome with incomplete penetrance, characterized by the association of paragangliomas (PGL) and/or pheochromocytomas (PHEO) and gastrointestinal stromal tumors (GIST). CSS is mainly caused by germline heterozygous pathogenic variants (PVs) in the succinate dehydrogenase subunit genes (SDHB, SDHC, SDHD), with SDHB and SDHD being the most frequently affected. Aim: To investigate a novel genetic etiology for CSS not associated with germline SDHx defects. Methods: Genetic investigation using SANGER sequencing and multiplex ligation-dependent probe amplification (MLPA) rule out germline defects for SDHx in a 59-year-old woman diagnosed with a 9 cm left PHEO, 4.8 cm PGL and 9.3 cm GIST. Thus, we performed whole exome sequencing of germline DNA (paired with tumor DNA from PHEO, PGL, and GIST) and applied a targeted analysis with the enrichment of 3,485 potential neuroendocrine tumors susceptibility genes associated with cellular response to hypoxia, mitochondrion, Krebs cycle, and tumorigenesis (MAPK, mTOR, ERK, and Wnt signaling). Additionally, we performed RT-qPCR to determine SLC25A11 expression levels in PHEO and PGL samples, stratified into three groups: tumors from the case harboring the SLC25A11 variant, Cluster 1 (n= 4), and Cluster 2 (n= 8). Tumor DNA methylation status was assessed using immunostaining for 5-hydroxymethylcytosine (5hmC). Results: Exome sequencing revealed a new heterozygous germline variant (c.293G>A / p.Arg98His) in the mitochondrial 2-oxoglutarate/malate carrier gene (SLC25A11). This variant, located in a highly conserved residue of the SLC25A11 mitochondrial carrier domain, is predicted to be deleterious in silico (REVEL score= 0.81). Exome sequencing of the PHEO and PGL did not reveal somatic PVs in genes previously associated with PHEO and PGL. Moreover, somatic c-KIT and PDGFRA PVs were not identified in GIST. Notably, a significant downregulation of SLC25A11 expression was observed in tumor samples harboring the SLC25A11 p.Arg98His variant (0.57 ± 0.13) compared with tumors from cluster 1 (1.39 ± 0.45; p = 0.025) and cluster 2 (1.79 ± 0.71; p < 0.001). Interestingly, immunostaining for 5hmC was negative in all tumors (PHEO, PGL and GIST), indicating tumor hypermethylation. Conclusion: A rare germline deleterious variant in SLC25A11 was identified in a patient with CSS. Moreover, the absence of somatic drivers, hypermethylation status and loss of SLC25A11 expression in the CSS tumors support the involvement of this gene with CSS. Support: Sao Paulo Research Foundation (FAPESP) grant 2019/15873-6 (to M.Q.A.), and FAPESP post-doctoral fellowship 2021/11240-9 (to F.F-C.). Presentation: 6/1/2024

12510 SLC25A11 As A Novel Gene For Carney-stratakis Syndrome

Abstract Disclosure: F. Freitas-Castro: None. L.S. Santana: None. G.F. Fagundes: None. A.F. Afonso: None. E.C. Lobato: None. F.L. Ledesma: None. I.C. Soares: None. B.B. Mendonca: None. M.C. Fragoso: None. A. Latronico: None. C.A. Stratakis: None. M.Q. Almeida: None. Background: Carney-Stratakis syndrome (CSS, OMIM #606864), also reported as “paraganglioma and gastrointestinal stromal tumor syndrome” or Carney-Stratakis dyad, is an autosomal dominant rare syndrome with incomplete penetrance, characterized by the association of paragangliomas (PGL) and/or pheochromocytomas (PHEO) and gastrointestinal stromal tumors (GIST). CSS is mainly caused by germline heterozygous pathogenic variants (PVs) in the succinate dehydrogenase subunit genes (SDHB, SDHC, SDHD), with SDHB and SDHD being the most frequently affected. Aim: To investigate a novel genetic etiology for CSS not associated with germline SDHx defects. Methods: Genetic investigation using SANGER sequencing and multiplex ligation-dependent probe amplification (MLPA) rule out germline defects for SDHx in a 59-year-old woman diagnosed with a 9 cm left PHEO, 4.8 cm PGL and 9.3 cm GIST. Thus, we performed whole exome sequencing of germline DNA (paired with tumor DNA from PHEO, PGL, and GIST) and applied a targeted analysis with the enrichment of 3,485 potential neuroendocrine tumors susceptibility genes associated with cellular response to hypoxia, mitochondrion, Krebs cycle, and tumorigenesis (MAPK, mTOR, ERK, and Wnt signaling). Additionally, we performed RT-qPCR to determine SLC25A11 expression levels in PHEO and PGL samples, stratified into three groups: tumors from the case harboring the SLC25A11 variant, Cluster 1 (n= 4), and Cluster 2 (n= 8). Tumor DNA methylation status was assessed using immunostaining for 5-hydroxymethylcytosine (5hmC). Results: Exome sequencing revealed a new heterozygous germline variant (c.293G>A / p.Arg98His) in the mitochondrial 2-oxoglutarate/malate carrier gene (SLC25A11). This variant, located in a highly conserved residue of the SLC25A11 mitochondrial carrier domain, is predicted to be deleterious in silico (REVEL score= 0.81). Exome sequencing of the PHEO and PGL did not reveal somatic PVs in genes previously associated with PHEO and PGL. Moreover, somatic c-KIT and PDGFRA PVs were not identified in GIST. Notably, a significant downregulation of SLC25A11 expression was observed in tumor samples harboring the SLC25A11 p.Arg98His variant (0.57 ± 0.13) compared with tumors from cluster 1 (1.39 ± 0.45; p = 0.025) and cluster 2 (1.79 ± 0.71; p < 0.001). Interestingly, immunostaining for 5hmC was negative in all tumors (PHEO, PGL and GIST), indicating tumor hypermethylation. Conclusion: A rare germline deleterious variant in SLC25A11 was identified in a patient with CSS. Moreover, the absence of somatic drivers, hypermethylation status and loss of SLC25A11 expression in the CSS tumors support the involvement of this gene with CSS. Support: Sao Paulo Research Foundation (FAPESP) grant 2019/15873-6 (to M.Q.A.), and FAPESP post-doctoral fellowship 2021/11240-9 (to F.F-C.). Presentation: 6/1/2024

8030 Sexually Dimorphic Metabolic Consequences Of CCN5/WISP2 Gene Deletion Revealed A Male-Specific Pattern Of Gene Expression

Abstract Disclosure: M. Yang: None. V. Xega: None. M. Zakikhani: None. J. Liu: None. Matricellular protein CCN5/WISP2 is a novel target of IGF-1 and WNT signaling within the pancreatic islets. Our previous research suggests CCN5 promotes β-cell proliferation and survival through Akt/PKB and focal adhesion kinase signaling pathways. More recently, we have reported sexual dimorphic features of CCN5 gene deletion and overexpression. Specifically, male knockout mice demonstrated improved insulin sensitivity and glucose tolerance upon high-fat diet (HFD)-induced obesity, while females were unaffected (ENDO 2022). On the other hand, male aP2-CCN5 transgenic mice that overexpress CCN5 in adipose tissues were partially protected from HFD-streptozotocin induced diabetes, with improved glucose tolerance; females exhibited impaired glucose tolerance and exacerbated diabetes (ENDO 2023). Our findings indicate that an increased level of CCN5 protects β-cells, but only in male mice. This protection cannot be solely attributed to the pro-islet effect and suggests the involvement of extra-pancreatic and sex-specific roles. This study indirectly screened normal CCN5 gene expression by detecting β-galactosidase activity, measured in X-gal-stained tissues before fixation and sectioning. In order to create the gene deletion through homologous recombination, LacZ was inserted between exons 2 and 5 of the CCN5 gene. As a result, male knockout mice exhibited a distinct blue staining in the pseudostratified columnar epithelium with stereocilia of the epididymis and vas deferens. This type of epithelium comprises a single layer of cells but appears to have multiple layers due to the nuclei being at different levels. The epithelium possesses long projections made of actin filaments and plays a crucial role in absorbing, transporting, and maturing sperm cells. The staining was also clear in Leydig cells of the testis, which produce testosterone, which was confirmed using immunohistochemistry and authentic CCN5 antibody. In female mice, however, only a few cells in the ovary and scattered staining was observed in the endometrial glands of the uterus. Since androgens promote energy expenditure, lipolysis, and insulin sensitivity, knockout or overexpression of CCN5 may potentially affect androgen and/or sperm production, leading to metabolic consequences in males. Our results from in vitro, knockout, and overexpression models support the notion that CCN5 promotes β-cell growth and survival against diabetes and further suggest that it possesses sex-specific, metabolic effects. Presentation: 6/2/2024

Bifunctional mesoporous HMUiO-66-NH2 nanoparticles for bone remodeling and ROS scavenging in periodontitis therapy

Periodontal bone defects represent an irreversible consequence of periodontitis associated with reactive oxygen species (ROS). However, indiscriminate removal of ROS proves to be counterproductive for tissue repair and insufficient for addressing existing bone defects. In the treatment of periodontitis, it is crucial to rationally alleviate local ROS while simultaneously promoting bone regeneration. In this study, Zr-based large-pore hierarchical mesoporous metal-organic framework (MOF) nanoparticles (NPs) HMUiO-66-NH2 were successfully proposed as bifunctional nanomaterials for bone regeneration and ROS scavenging in periodontitis therapy. HMUiO-66-NH2 NPs demonstrated outstanding biocompatibility both in vitro and in vivo. Significantly, these NPs enhanced the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) under normal and high ROS conditions, upregulating osteogenic gene expression and mitigating oxidative stress. Furthermore, in vivo imaging revealed a gradual degradation of HMUiO-66-NH2 NPs in periodontal tissues. Local injection of HMUiO-66-NH2 effectively reduced bone defects and ROS levels in periodontitis-induced C57BL/6 mice. RNA sequencing highlighted that differentially expressed genes (DEGs) are predominantly involved in bone tissue development, with notable upregulation in Wnt and TGF-β signaling pathways. In conclusion, HMUiO-66-NH2 exhibits dual functionality in alleviating oxidative stress and promoting bone repair, positioning it as an effective strategy against bone resorption in oxidative stress-related periodontitis.

9263 Characterization Of A Novel Oral Small Molecule PTH1R Agonist: Proof Of Concept For An Alternative To Injectable Peptide-Based Therapy For Hypoparathyroidism

Abstract Disclosure: J. Zhang: Employee; Self; Septerna Inc.. I. Gomez: None. C. He: Employee; Self; Septerna Inc. J. Wang: Employee; Self; Septerna Inc. X. Du: Employee; Self; Septerna Inc. K. Nguyen: Employee; Self; Septerna Inc. K. Alvarez: Employee; Self; Septerna Inc. A.M. Mirza: Employee; Self; Septerna Inc.. Hypoparathyroidism (HP), a rare endocrine disorder characterized by insufficient levels of parathyroid hormone (PTH), leads to hypocalcemia and hyperphosphatemia. Most HP cases arise due to damage of the parathyroid glands during thyroid or neck surgery. HP symptoms include severe muscle cramps, tingling, burning and numbness, memory loss, and headaches, all leading to a decreased quality of life. Calcium and vitamin D supplementation do not fully ameliorate the disease and may contribute to renal disease. Chronic HP increases the risk of major complications, such as calcium depositions in the brain, eye, and kidneys. Similarly, it can lead to low bone turnover and increased bone mineral density with associated increased bone mineralization. In comparison to standard of care and traditional injectable peptide-based therapies, oral small molecules may have multiple advantages clinically. However, there are no oral small molecules currently in development. PTH functions through activation of the parathyroid hormone 1 receptor (PTH1R), a class B G protein-coupled receptor. The main target tissues for PTH1R activation include bone and kidney. In the kidney, PTH increases tubular reabsorption of calcium and promotes renal excretion of phosphate. In bone, PTH stimulates bone turnover and mobilizes calcium into the circulation to maintain serum calcium. Here we describe the characterization of SP-1462, a novel, potent, and selective oral small molecule, and show that, like the PTH peptides, it acts on PTH1R as an agonist and elicits similar downstream functions in vitro and in vivo. To assess PTH1R engagement in the major targeted organs, kidney and bone, primary human renal proximal epithelial cells (RPTEC) and a human osteoblast cell line (Saos-2) were used, respectively. RPTECs treated with PTH and SP-1462 showed significant changes in transporter genes (SLC22A2 and SLC13A1), immune related genes (CCL2 & DPP4), and matrix reorganization genes (ITGA5 and COL1A1). Saos-2 cells treated with PTH and SP-1462 both significantly affected the expression of Wnt signaling genes (DKK1 and TCF7) and the expression of coupling genes that bridge anabolic/catabolic activities (M-CSF, RANKL, HDAC4, and OPG). Importantly, oral administration of a single dose of SP-1805 in a rat surgical thyroparathyroidectomy (TPTx) model resulted in significant upregulation of serum calcium levels for a period of 24 hours in a dose dependent manner at 3, 10 and 30 mg/kg. The window of calcium upregulation is comparable to injectable PTH peptides currently in development to treat hypoparathyroidism. These data suggest that oral small molecule PTH1R agonists engage PTH pathways similar to native PTH and have the potential to replace injectable PTH peptides to treat PTH-related disorders, including hypoparathyroidism. Presentation: 6/3/2024

8401 Genetic Characterization of a Case of Primary Aldosteronism in an 11-Year-Old Girl carrying a Germline VUS of CACNA1H and a Somatic Mutation of CTNNB1

Abstract Disclosure: S. Parisien-La Salle: None. G. Van Vliet: None. G. Corbeil: None. M. Labrecque: None. M. Tétreault: None. I. Bourdeau: None. Introduction: Primary aldosteronism is a frequent cause of secondary hypertension in adults but is rare in children. We report the case and genetic studies of an 11 yo girl presenting with primary aldosteronism at puberty. Case presentation: An 11-year-old female patient was referred to the pediatric endocrinology clinic for hypertension (166/118). Her past medical history and family history were unremarkable. She reported polyuria, polydipsia and occasional headaches and abdominal pain. Her Tanner stage indicated that puberty was initiated (P2M2). An abdominal CT revealed a 9x10x11cm right adrenal mass with calcifications and a necrotic zone. On [1]8F-FDG PET/CT, the mass had an uptake of 2.9 SUVmax. Laboratory results revealed hypokalemia (K: 2.6 mmol/L [N: 3.5-4.5]) and elevated aldosterone (1712.8 pmol/L [N: 110-1330]) with suppressed renin (0.2 ng/ml/h [N: <5.3]). Her urinary aldosterone level was elevated (114 nmol/day [N: <67]). The rest of the biochemical adrenal panel was normal. Patient underwent a right adrenalectomy by laparotomy. The pathology report showed an adrenal tumor without capsular or vascular invasion. The only malignancy criterion was an area of necrosis and the ki-67% proliferation index was < 1%. Genetic investigations: After informed consent, the patient underwent germline analysis for the chimeric gene CYP11B2/CYP11B1 (Ruhr University Bochum, Germany) and a multigene panel including KCNJ5, CACNA1D, CACNA1H and CLCN2 genes (Fulgent, CA), which were both negative except for a variant of unknown significance (VUS) that was identified in CACNA1H (c.3868G>A, p.Val1290Ile) with a minor allele frequency of 3.291E-5 (GnomAD). Tumor DNA was extracted after adrenalectomy from fresh frozen tissues. Somatic genetic analysis of GNAS, CTNNB1, KCNJ5, ATP1A1, ATP2B3 and CACNA1D was performed using direct Sanger Sequencing (Genome Quebec Innovation Centre) and revealed a heterozygous missence pathogenic variant in the CTNNB1 gene (c.121 A>G p.Thr41Ala). This mutation was absent in leukocyte DNA of the patient. Immunohistochemistry of the adrenal tumor disclosed strong cytoplasmic β-catenin staining in most tumor cells (monoclonal anti-β-catenin antibody; BD Transduction Laboratories). RNA-sequencing of patient tumor RNA was performed (Research Center CHU Québec) and compared to human adrenal total RNA (Clontech, Mountain View, CA). Pathway enrichment analysis showed activation of the Wnt signaling pathway. No variant was identified in GNAQ or GNA11 genes. However, GNRHR was upregulated compared to the control sample with a log2 fold change of 3.96 (p=0.000074). Conclusion: We present a rare pediatric case of an aldosterone producing adenoma harbouring a somatic β-catenin pathogenic variant with upregulation of GNRHR. The implication of the germline CACNA1H VUS still needs to be clarified. Presentation: 6/3/2024

6821 Characterization Of A Novel Oral Small Molecule PTH1R Agonist: Proof Of Concept For An Alternative To Injectable Peptide-Based Therapy For Hypoparathyroidism

Abstract Disclosure: J. Zhang: None. I. Gomez: None. C. He: None. J. Wang: None. X. Du: None. K. Nguyen: None. K. Alvarez: None. A.M. Mirza: None. Hypoparathyroidism (HP), a rare endocrine disorder characterized by insufficient levels of parathyroid hormone (PTH), leads to hypocalcemia and hyperphosphatemia. Most HP cases arise due to damage of the parathyroid glands during thyroid or neck surgery. HP symptoms include severe muscle cramps, tingling, burning and numbness, memory loss, and headaches, all leading to a decreased quality of life. Calcium and vitamin D supplementation do not fully ameliorate the disease and may contribute to renal disease. Chronic HP increases the risk of major complications, such as calcium depositions in the brain, eye, and kidneys. Similarly, it can lead to low bone turnover and increased bone mineral density with associated increased bone mineralization. In comparison to standard of care and traditional injectable peptide-based therapies, oral small molecules may have multiple advantages clinically. However, there are no oral small molecules currently in development. PTH functions through activation of the parathyroid hormone 1 receptor (PTH1R), a class B G protein-coupled receptor. The main target tissues for PTH1R activation include bone and kidney. In the kidney, PTH increases tubular reabsorption of calcium and promotes renal excretion of phosphate. In bone, PTH stimulates bone turnover and mobilizes calcium into the circulation to maintain serum calcium. Here we describe the characterization of SP-1462, a novel, potent, and selective oral small molecule, and show that, like the PTH peptides, it acts on PTH1R as an agonist and elicits similar downstream functions in vitro and in vivo. To assess PTH1R engagement in the major targeted organs, kidney and bone, primary human renal proximal epithelial cells (RPTEC) and a human osteoblast cell line (Saos-2) were used, respectively. RPTECs treated with PTH and SP-1462 showed significant changes in transporter genes (SLC22A2 and SLC13A1), immune related genes (CCL2 & DPP4), and matrix reorganization genes (ITGA5 and COL1A1). Saos-2 cells treated with PTH and SP-1462 both significantly affected the expression of Wnt signaling genes (DKK1 and TCF7) and the expression of coupling genes that bridge anabolic/catabolic activities (M-CSF, RANKL, HDAC4, and OPG). Importantly, oral administration of a single dose of SP-1805 in a rat surgical thyroparathyroidectomy (TPTx) model resulted in significant upregulation of serum calcium levels for a period of 24 hours in a dose dependent manner at 3, 10 and 30 mg/kg. The window of calcium upregulation is comparable to injectable PTH peptides currently in development to treat hypoparathyroidism. These data suggest that oral small molecule PTH1R agonists engage PTH pathways similar to native PTH and have the potential to replace injectable PTH peptides to treat PTH-related disorders, including hypoparathyroidism. Presentation: 6/3/2024

8773 CD44 Variant Isoforms Expressed Within PAX7/SOX2-Positive Pituitary Neuroendocrine Tumors Attribute To Therapy Resistance And Recurrence Of Cushing’s Disease

Abstract Disclosure: J. Chakrabarti: None. J. Eschbacher: None. S. Mallick: None. E. Ferris: None. B. Hermes: None. A. Little: None. Y. Zavros: None. Background: Cushing’s disease (CD) is a serious rare endocrine disease caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that subsequently stimulates the adrenal glands to overproduce cortisol. Approximately 56% of patients will experience disease recurrence during the 2 to 10-year follow-up period. Surgical failures and late recurrences of CD are common, and despite multiple treatments, biochemical control is achieved in only 50% of patients. Therefore, there is a need to advance targeted therapies that prevent recurrence and therapy resistance. The Cluster of Differentiation (CD) 44 transmembrane glycoprotein (CD44) is a prominent WNT signaling target, and deregulation of the Wnt pathway is associated with constitutively active βcatenin in SOX2-positive cells. Although a similar mechanism has been proposed in PitNETs, it has not been fully tested. The alternative mRNA splice variant, CD44v9 is expressed on cancer stem cells promotes resistance to ROS through the stabilization of the SLC7A11 (xCT) cystine glutamate transporter. Proteolytic cleavage of CD44 releases the CD44 intracellular domain which translocates to the nucleus activating cell stemness genes including SOX2. Although CD44 is a known cancer stem cell marker that plays multiple key roles including drug resistance and tumor cell invasiveness, the mechanisms underlying human PitNET tumorigenesis are unclear. Hypothesis: CD44 expressed on PAX7/SOX2-positive corticotroph PitNET cells define a cell population that attributes to therapy resistance and disease recurrence. Methods: CD patient PitNET tissues and matched PitNET-generated organoids (PitNETOs) were used for CosMx™ Spatial Molecular Imaging (SMI) and scRNAseq analyses respectively. Resistance to drug- and radiation-induced apoptosis was analyzed using PitNETOs generated. Results: SMI and scRNAseq data revealed a cell population co-expressing PAX7 and SOX2, CD44 and EpCAM that was abundant in invasive and silent corticotroph PitNET subtypes with Knosp grades 3-4. Invasive and silent corticotroph PitNETs also expressed an increased cell population of CD163+ macrophages, myofibroblast cancer associated fibroblasts (myCAFs), inflammatory CAFs. While cells isolated from the dura revealed growth of PitNETOs, adherent cells expressed increased proliferation (Ki67), and expression of somatostatin receptor subtype 2, cancer stem markers (CD44, SOX2, NESTIN, FUT4, PROM1), PAX7 progenitor cell population expressing stem cell markers NESTIN, SOX2, CD44 and EpCAM. Increased CD44/SLCA7A11 expression in CD44/PAX7/SOX2-positive PitNETOs rendered tumor cells resistant to radiation-induced apoptosis. Conclusions: Drugs targeting the CD44 variant isoforms may increase the efficacy of medical therapies and stereotactic radiosurgery to prevent residual and recurrent PitNETs in CD patients. Presentation: 6/3/2024

Comparative proteomic analysis of regenerative mechanisms in mouse retina to identify markers for neuro-regeneration in glaucoma

The retina is part of the central nervous system (CNS). Neurons in the CNS and retinal ganglion cells lack the ability to regenerate axons spontaneously after injury. The intrinsic axonal growth regulators, their interaction and roles that enable or inhibit axon growth are still largely unknown. This study endeavored to characterize the molecular characteristics under neurodegenerative and regenerative conditions. Data-Independent Acquisition Mass Spectrometry was used to map the comprehensive proteome of the regenerative retina from 14-day-old mice (Reg-P14) and adult mice after lens injury (Reg-LI) both showing regrowing axons in vitro, untreated adult mice, and retina from adult mice subjected to two weeks of elevated intraocular pressure showing degeneration. A total of 5750 proteins were identified (false discovery rate < 1%). Proteins identified in both Reg-P14 and Reg-LI groups were correlated to thyroid hormone, Notch, Wnt, and VEGF signaling pathways. Common interactors comprising E1A binding protein P300 (EP300), CREB binding protein (CBP), calcium/calmodulin dependent protein kinase II alpha (CaMKIIα) and sirtuin 1 (SIRT1) were found in both Reg-P14 and Reg-LI retinas. Proteins identified in both regenerating and degenerative groups were correlated to thyroid hormone, Notch, mRNA surveillance and measles signaling pathways, along with PD-L1 expression and the PD-1 checkpoint pathway. Common interactors across regenerative and degenerative retinas comprising NF-kappa-B p65 subunit (RELA), RNA-binding protein with serine-rich domain 1 (RNPS1), EP300 and SIN3 transcription regulator family member A (SIN3A). The findings from our study provide the first mapping of regenerative mechanisms across postnatal, mature and degenerative mouse retinas, revealing potential biomarkers that could facilitate neuro-regeneration in glaucoma.

An Exogenous Ketone Ester Slows Tumor Progression in Murine Breast and Renal Cancer Models.

Ketone esters (KEs) exhibit promise as anti-cancer agents but their impact on spontaneous metastases remains poorly understood. Although consumption of a ketogenic diet (KD) that is low in carbohydrates and high in fats can lead to KE production in vivo, the restrictive composition of KDs may diminish adherence in cancer patients. We investigated the effects of an exogenous ketone ester-supplemented (eKET), carbohydrate-replete diet on tumor growth, metastasis, and underlying mechanisms in orthotopic models of metastatic breast (4T1-Luc) and renal (Renca-Luc) carcinomas. Mice were randomized to diet after tumor challenge. Administration of KEs did not alter tumor cell growth in vitro. However, in mice, our eKET diet increased circulating β-hydroxybutyrate and inhibited primary tumor growth and lung metastasis in both models. Body composition analysis illustrated the overall safety of eKET diet use, although it was associated with a loss of fat mass in mice with renal tumors. Immunogenetic profiling revealed divergent intratumoral eKET-related changes by tumor type. In mammary tumors, Wnt and TGFβ pathways were downregulated, whereas in renal tumors, genes related to hypoxia and DNA damage repair were downregulated. Thus, our eKET diet exerts potent antitumor and antimetastatic effects in both breast and renal cancer models, albeit with different modes of action and physiologic effects. Its potential as an adjuvant dietary approach for patients with diverse cancer types should be explored further.

Whole-genome resequencing reveals genetic diversity and selection signatures in five populations of Mandarin Fish (Siniperca chuatsi)

Mandarin fish is an important freshwater economic fish in China, and the genome of mandarin fish population may have specific selection signatures after long-term selection, and studying these selection signatures is one of the important strategies for screening functional genes. In this study, the whole genome was resequenced from five mandarin fish populations (HLJ, HN, AH, FJ, and BJ), and a total of high-quality SNPs with 1659,318 single nucleotide polymorphisms were obtained, and the genetic differentiation index and nucleotide polymorphism ratio between populations were used to detect the selected genomic regions. The results showed that all individuals were divided into two branches, with the HLJ population forming an independent cluster, and the HN, AH, FJ, and BJ populations clustering together. Selection signal analysis on the HLJ mandarin fish population and other mandarin fish populations using the FST and π HLJ/other methods. Finally, we identified 26 important candidate genes based on the enrichment pathways related to fat traits such as Glycerolipid metabolism, acyl-CoA oxidase activity, Wnt signaling pathway, and Notch signaling pathway, including notch3, pla2g12a and acox1. This study explores the genetic diversity and genetic structure of mandarin fish from a whole-genome perspective, identifies candidate genes related to fat traits, and provides references for the protection and utilization of mandarin fish genetic resources.

Eniluracil blocks AREG signalling-induced pro-inflammatory fibroblasts of melanoma in heart failure.

Heart failure (HF) is characterized by a heightened risk of melanoma, which often metastasizes to the heart. The overlap pathology between HF and melanoma includes chronic low-grade inflammation and dysregulation of inflammatory cancer-associated fibroblasts (iCAFs). The impact of HF on iCAF-driven tumour inflammation remains obscure. To identify critical genes for HF development, transcriptomic data (GSE57338) containing 313 clinical HF samples [136 healthy controls, 95 ischaemia (ISCH) and 82 dilated cardiomyopathy (DCM)] were analysed to screen differentially expressed genes (DEGs) and perform enrichment analysis. Fifty-one DEGs in ISCH and 62 DEGs in DCM were identified with log2|fold change (FC)|≥1 and P value ≤0.05. All these genes are involved in extracellular matrix organization, immune/inflammatory responses and Wnt signalling pathways. Then, the overall survival curves and prognostic models of DEGs in melanoma were evaluated. The correlation of gene expression with lymphocyte infiltration levels was assessed. Only aldehyde oxidase 1 (AOX1) and amphiregulin (AREG) maintained the same trend in melanoma as in HF, negatively affecting prognosis by regulating lymphocyte infiltration (log-rank P value=0.0017 and 0.0019). The potential drug molecules were screened, and the binding energies were calculated via molecular docking. Eniluracil, a known AOX1 targeting drug, was found to stably bind with AREG (hydrogen bond binding energies: -65.633, -63.592 and -62.813kcal/mol). The increased prevalence of melanoma in HF patients and its propensity for cardiac metastasis may be due to AREG-mediated systemic low-grade inflammation. Eniluracil holds promise as a therapeutic agent that may block AREG signalling, inhibiting the activation of iCAF mediated by regulatory T cell (Treg) and neutrophil.

Exploring DIX-DIX Homo- and Hetero-Oligomers in Wnt Signaling with AlphaFold2.

Wnt signaling is involved in embryo development and cancer. The binding between the DIX domains of Axin1/2, Dishevelled1/2/3, and Coiled-coil-DIX1 is essential for Wnt/β-catenin signaling. Structural and biological studies have revealed that DIX domains are polymerized through head-to-tail interface interactions, which are indispensable for activating β-catenin Wnt signaling. Although different isoforms of Dvl and Axin proteins display both redundant and specific functions in Wnt signaling, the specificity of DIX-mediated interactions remains unclear due to technical challenges. Using AlphaFold2(AF2), we predict the structures of 6 homodimers and 22 heterodimers of DIX domains without templates and compare them with the reported X-ray complex structures. PRODIGY is used to calculate the binding affinities of these DIX complexes. Our results show that the Axin2 DIX homodimer has a stronger binding affinity than the Axin1 DIX homodimer. Among Dishevelled (Dvl) proteins, the binding affinity of the Dvl1 DIX homodimer is stronger than that of Dvl2 and Dvl3. The Coiled-coil-DIX1(Ccd1) DIX homodimer shows weaker binding than the Axin1 DIX homodimer. Generally, heterodimer interactions tend to be stronger than those of homodimers. Our findings provide insights into the mechanism of the Wnt signaling pathway and highlight the potential of AF2 and PRODIGY for studying protein-protein interactions in signaling pathways.

Downregulating LKB1 in bone marrow mesenchymal stem cells could inhibit CD4+ T cell proliferation via the PD-1/PD-L1 signaling pathway

BackgroundOur previous research has shown that LKB1 in amniotic mesenchymal stem cells (MSCs) serves as a vital regulator of regulatory T cell differentiation and T cell proliferation, which may have a similar role in bone marrow MSCs (BMMSCs). Therefore, we investigated the role of LKB1 in BMMSCs for regulating CD4+ T cell proliferation in the bone micro-environment of AML. MethodsRT-PCR was used to assessed LKB1 expression in BMMSCs derived from AML patients and healthy controls. Subsequently, LKB1 was knocked down in the BMMSCs line HS-5 (HS-5-LKB1KD). Co-cultures in vitro were established to analyze the effect of HS-5-LKB1KD on CD4+ T cell. Flow cytometry was employed to measure PD-L1 and CD4+ T cell proliferation levels. Western blot was utilized to detect related proteins. ResultsThe expression of LKB1 in BMMSCs derived from AML patients was decreased. Knockdown of LKB1 in HS-5 resulted in upregulation of PD-L1 expression. Co-culture of peripheral blood CD4+ T cell with HS-5-LKB1KD exhibited reduced CD4+ T cell proliferation compared to co-culture with HS-5-LKB1con. Furthermore, blocking PD-L1 in the co-culture conditions could restore the reduced CD4+ T cell proliferation. Additionally, it was found that upregulation of the Wnt signaling pathway-related proteins following LKB1 knockdown in HS-5, indicating that downregulating LKB1 could promote PD-L1 expression through activation of the Wnt signaling pathway. ConclusionsThe decreased expression of LKB1 in BMMSCs may activate the Wnt signaling pathway, leading to increased PD-L1 expression. This inhibited CD4+ T cell proliferation, which might lead to impaired anti-tumor immunity in AML patients and promote AML progression.

Isoform-specific C-terminal phosphorylation drives autoinhibition of Casein kinase 1

Casein kinase 1δ (CK1δ) controls essential biological processes including circadian rhythms and wingless-related integration site (Wnt) signaling, but how its activity is regulated is not well understood. CK1δ is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, δ1 and δ2, are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C termini (XCT), but with marked changes in potential phosphorylation sites. Here, we test whether the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and hydrogen/deuterium exchange mass spectrometry, we show that the δ1 XCT is preferentially phosphorylated by the kinase and the δ1 tail makes more extensive interactions across the kinase domain. Mutation of δ1-specific XCT phosphorylation sites increases kinase activity both in vitro and in cells and leads to changes in the circadian period, similar to what is reported in vivo. Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. δ1 autoinhibition relies on conserved anion-binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1δ. These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase.