Target LEF1 Research Articles

Recurrent UBE3C-LRP5 translocations in head and neck cancer with therapeutic implications

Head and neck cancer is a major cause of morbidity and mortality worldwide. The identification of genetic alterations in head and neck cancer may improve diagnosis and treatment outcomes. In this study, we report the identification and functional characterization of UBE3C-LRP5 translocation in head and neck cancer. Our whole transcriptome sequencing and RT-PCR analysis of 151 head and neck cancer tumor samples identified the LRP5-UBE3C and UBE3C-LRP5 fusion transcripts in 5.3% of patients of Indian origin (n = 151), and UBE3C-LRP5 fusion transcripts in 1.2% of TCGA-HNSC patients (n = 502). Further, whole genome sequencing identified the breakpoint of UBE3C-LRP5 translocation. We demonstrate that UBE3C-LRP5 fusion is activating in vitro and in vivo, and promotes the proliferation, migration, and invasion of head and neck cancer cells. In contrast, depletion of UBE3C-LRP5 fusion suppresses the clonogenic, migratory, and invasive potential of the cells. The UBE3C-LRP5 fusion activates the Wnt/β-catenin signaling by promoting nuclear accumulation of β-catenin, leading to upregulation of Wnt/β-catenin target genes, MYC, CCND1, TCF4, and LEF1. Consistently, treatment with the FDA-approved drug, pyrvinium pamoate, significantly reduced the transforming ability of cells expressing the fusion protein and improved survival in mice bearing tumors of fusion-overexpressing cells. Interestingly, fusion-expressing cells upon knockdown of CTNNB1, or LEF1 show reduced proliferation, clonogenic abilities, and reduced sensitivity to pyrvinium pamoate. Overall, our study suggests that the UBE3C-LRP5 fusion is a promising therapeutic target for head and neck cancer and that pyrvinium pamoate may be a potential drug candidate for treating head and neck cancer harboring this translocation.

Repurposing DPP4 Inhibition to Improve Hair Follicle Activation and Regeneration

Skin injury and several diseases elicit fibrosis and induce hair follicle (HF) growth arrest and loss. The resulting alopecia and disfiguration represent a severe burden for patients, both physically and psychologically. Reduction of profibrotic factors such as dipeptidyl peptidase 4 (DPP4) might be a strategy to tackle this issue. We show DPP4 overrepresentation in settings with HF growth arrest (telogen), HF loss, and nonregenerative wound areas in mouse skin and human scalp. Topical DPP4 inhibition with Food and Drug Administration/European Medicines Agency-approved sitagliptin on preclinical models of murine HF activation/regeneration results in accelerated anagen progress, whereas treatment of wounds with sitagliptin results in reduced expression of fibrosis markers, increased induction of anagen around wounds, and HF regeneration in the wound center. These effects are associated with higher expression of Wnt target Lef1, known to be required for HF anagen/HF-activation and regeneration. Sitagliptin treatment decreases profibrotic signaling in the skin, induces a differentiation trajectory of HF cells, and activates Wnt targets related to HF activation/growth but not those supporting fibrosis. Taken together, our study shows a role for DPP4 in HF biology and shows how DPP4 inhibition, currently used as oral medication to treat diabetes, could be repurposed into a topical treatment agent to potentially reverse HF loss in alopecia and after injury.

Systems Drug Design for Muscle Invasive Bladder Cancer and Advanced Bladder Cancer by Genome-Wide Microarray Data and Deep Learning Method with Drug Design Specifications.

Bladder cancer is the 10th most common cancer worldwide. Due to the lack of understanding of the oncogenic mechanisms between muscle-invasive bladder cancer (MIBC) and advanced bladder cancer (ABC) and the limitations of current treatments, novel therapeutic approaches are urgently needed. In this study, we utilized the systems biology method via genome-wide microarray data to explore the oncogenic mechanisms of MIBC and ABC to identify their respective drug targets for systems drug discovery. First, we constructed the candidate genome-wide genetic and epigenetic networks (GWGEN) through big data mining. Second, we applied the system identification and system order detection method to delete false positives in candidate GWGENs to obtain the real GWGENs of MIBC and ABC from their genome-wide microarray data. Third, we extracted the core GWGENs from the real GWGENs by selecting the significant proteins, genes and epigenetics via the principal network projection (PNP) method. Finally, we obtained the core signaling pathways from the corresponding core GWGEN through the annotations of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway to investigate the carcinogenic mechanisms of MIBC and ABC. Based on the carcinogenic mechanisms, we selected the significant drug targets NFKB1, LEF1 and MYC for MIBC, and LEF1, MYC, NOTCH1 and FOXO1 for ABC. To design molecular drug combinations for MIBC and ABC, we employed a deep neural network (DNN)-based drug-target interaction (DTI) model with drug specifications. The DNN-based DTI model was trained by drug-target interaction databases to predict the candidate drugs for MIBC and ABC, respectively. Subsequently, the drug design specifications based on regulation ability, sensitivity and toxicity were employed as filter criteria for screening the potential drug combinations of Embelin and Obatoclax for MIBC, and Obatoclax, Entinostat and Imiquimod for ABC from their candidate drugs. In conclusion, we not only investigated the oncogenic mechanisms of MIBC and ABC, but also provided promising therapeutic options for MIBC and ABC, respectively.

Investigation of the Role of Wnt/Delta‐Catenin Signaling in Regulating Opioid Pain Relief After Heat Shock Protein 90 Inhibition

Opioid receptors are important regulators of pain, and opioid drugs like morphine are used in a wide variety of clinical settings throughout the world. In spite of their clear utility and efficacy concerning the treatment of pain, the addictive nature of opioids limits their use as an option for the treatment of chronic pain. This illustrates the great medical and social need to improve opioid therapy, both enhancing analgesia and reducing side effects like addiction. More recently, we showed that Heat shock protein 90 (HSP90), a molecular chaperone that participates in a vast array of critical molecular processes in the cell, has been implicated as an important modulator of opioid‐induced pain relief within the spinal cord. We found that inhibition of HSP90 improved the therapeutic index of opioids, boosting pain relief and reducing side effects. In an effort to elucidate the molecular pathways responsible for these benefits, we performed proteomic analysis of spinal cord tissue from male and female CD‐1 mice treated with intrathecal Vehicle or 17‐AAG (0.5 nmol, 24 hrs), an HSP90 inhibitor. We found that the signaling molecule delta‐catenin2 was reduced by 27.6% by 17‐AAG treatment in spinal tissue. Since delta‐catenin2 stabilizes and enables Wnt signaling, this suggests that Wnt signaling is also reduced by HSP90 inhibition. As Wnt has been shown to promote pain, this fits with our model that suppressed Wnt/Catenin signaling could enhance opioid pain relief after HSP90 inhibition. We thus report here our efforts to test this hypothesis by using a Wnt activator (AMBMP) and Wnt inhibitor (JW74) combined with 17‐AAG and morphine in a mouse tail flick pain model; we also report our efforts to confirm molecular Wnt signaling changes in spinal cord by Western Blot and qPCR for Wnt itself along with established Wnt signaling targets VEGF‐A and LEF1. Together these studies will establish a new mechanistic node linking HSP90 to the regulation of opioid pain relief in the spinal cord. This will enhance our basic science understanding of this system and could lead to novel therapeutic targets.

Intervention Mechanism of Hunag-Lian Jie-Du Decoction on Canonical Wnt/β-Catenin Signaling Pathway in Psoriasis Mouse Model.

Background Psoriasis is a common chronic inflammatory skin disease with multifactor etiology, characterized by abnormal proliferation and differentiation of keratinocytes. Huang-Lian Jie-Du decoction (HLJDD) is a traditional Chinese medicine prescription with good clinical curative effect on psoriasis. However, its therapeutic mechanisms are still unclear. Methods The psoriasis model of SKH-1 nude mice was established by imiquimod-induced and HLJDD gavage was given. Hematoxylin and eosin staining were used to evaluate pathological morphologies, and immunohistochemistry was used to detect the expressions of Wnt1, β-catenin, and c-Myc in psoriasis mice. Western blot was used to examine the expressions of Frizzled-2, LRP5/6, GSK-3β, APC, Axin2, TCF4, LEF1, cyclin D1, TBX3, EPHB2, and NOTUM enzyme. Results In this study, HLJDD reduced skin erythema and lesions, decreased the thickness of epidermal and downregulated the expressions of Wnt1, β-catenin, and c-Myc. Western blot results showed that HLJDD reduced the expressions of Wnt receptors Frizzled-2 and LRP5/6, and Wnt downstream target genes TCF4, LEF1, cyclin D1, TBX3, and EPHB2, while upregulated destruction complex proteins GSK-3β, APC, and Axin2. Conclusions HLJDD can effectively treat psoriasis and inhibit the Wnt/β-catenin signaling pathway at multiple stages.

CHARGE syndrome-associated proteins FAM172A and CHD7 influence male sex determination and differentiation through transcriptional and alternative splicing mechanisms.

To gain further insight into chromatin‐mediated regulation of mammalian sex determination, we analyzed the role of the CHARGE syndrome‐associated proteins FAM172A and CHD7. This study is based on our prior discoveries that a subset of corresponding mutant mice display complete male‐to‐female sex reversal, and that both of these proteins regulate co‐transcriptional alternative splicing in neural crest cells. Here, we report that FAM172A and CHD7 are present in the developing gonads when sex determination normally occurs in mice. The interactome of FAM172A in pre‐Sertoli cells again suggests a role at the chromatin‐spliceosome interface, like in neural crest cells. Accordingly, analysis of Fam172a‐mutant pre‐Sertoli cells revealed transcriptional and splicing dysregulation of hundreds of genes. Many of these genes are similarly affected in Chd7‐mutant pre‐Sertoli cells, including several known key regulators of sex determination and subsequent formation of testis cords. Among them, we notably identified Sry as a direct transcriptional target and WNT pathway‐associated Lef1 and Tcf7l2 as direct splicing targets. The identified molecular defects are also associated with the abnormal morphology of seminiferous tubules in mutant postnatal testes. Altogether, our results thus identify FAM172A and CHD7 as new players in the regulation of male sex determination and differentiation in mice, and further highlight the importance of chromatin‐mediated regulatory mechanisms in these processes.

Transmembrane protein 97 exhibits oncogenic properties via enhancing LRP6-mediated Wnt signaling in breast cancer

Upregulation of transmembrane protein 97 (TMEM97) has been associated with progression and poor outcome in multiple human cancers, including breast cancer. Recent studies suggest that TMEM97 may be involved in the activation of the Wnt/β-catenin pathway. However, the molecular mechanism of TMEM97 action on Wnt/β-catenin signaling is completely unclear. In the current study, TMEM97 was identified as an LRP6-interacting protein. TMEM97 could interact with LRP6 intracellular domain and enhance LRP6-mediated Wnt signaling in a CK1δ/ε-dependent manner. The binding of TMEM97 to LRP6 facilitated the recruitment of CK1δ/ε to LRP6 complex, resulting in LRP6 phosphorylation at Ser 1490 and the stabilization of β-catenin. In breast cancer cells, knockout of TMEM97 attenuated the Wnt/β-catenin signaling cascade via regulating LRP6 phosphorylation, leading to a decrease in the expression of Wnt target genes AXIN2, LEF1, and survivin. TMEM97 deficiency also suppressed cell viability, proliferation, colony formation, migration, invasion, and stemness properties in breast cancer cells. Importantly, TMEM97 knockout suppressed tumor growth through downregulating the Wnt/β-catenin signaling pathway in a breast cancer xenograft model. Taken together, our results revealed that TMEM97 is a positive modulator of canonical Wnt signaling. TMEM97-mediated Wnt signaling is implicated in the tumorigenesis of breast cancer, and its targeted inhibition may be a promising therapeutic strategy for breast cancer.

Human fibroblast‐derived extracellular vesicles promote hair growth in cultured human hair follicles

Hair loss is a prevalent medical condition affecting both genders. In this study, we investigate the effects of a specific class of extracellular vesicles (EVs), namely human normal fibroblast-derived EVs (hFB-EVs), on human dermal papilla (DP) and outer root sheath (ORS) cells and examine the molecular mechanisms responsible for hair growth in hair follicles (HFs). We find that Wnt3a, which maintains the hair-generating activity of DP cells, is enriched and more strongly associated with hFB-EVs than with fibroblasts. Furthermore, hFB-EV-associated Wnt3a mediated receptor activation in cultured DP cells, leading to an increase in β-catenin in the cytoplasm and its translocation into the nucleus, thereby elevating expression of the target genes Axin2 and Lef1. Additionally, hFB-EVs promoted the migration, proliferation, and differentiation of ORS cells and elongation of the hair shaft in human HFs. These findings revealed a novel mechanism by which hFB-EVs influence hair growth.

Deciphering the role of Wnt signaling in acute myeloid leukemia prognosis: how alterations in DNA methylation come into play in patients' prognosis.

Acute myeloid leukemia (AML) is a malignant clonal disorder affecting myeloid differentiation through mechanisms that include epigenetic dysregulation. Abnormal changes in DNA methylation and gene expression profiles of pathways involved in hematopoietic development, such as Wnt/β-catenin, contribute to the transformation, development, and maintenance of leukemic cells. This review summarizes the alterations of Wnt signaling-related genes at the epigenetic and transcriptional level and their implications for AML prognosis. Among the implications of epigenetic alterations in AML, methylation of Wnt antagonists is related to poor prognosis, whereas their upregulation has been associated with a better clinical outcome. Furthermore, Wnt target genes c-Myc and LEF-1 present distinct implications. LEF-1 expression positively influences the patient overall survival. c-Myc upregulation has been associated with treatment resistance in AML, although c-Myc expression is not exclusively dependent of Wnt signaling. Understanding the signaling abnormalities could help us to further understand leukemogenesis, improve the current risk stratification for AML patients, and even serve to propose novel therapeutic targets.

Identification of differentially expressed miRNAs in differentiating benign from malignant pleural effusion

BackgroundTuberculosis pleural effusion (TPE) and malignant pleural effusion (MPE) are very common clinical complications. Considering the totally different prognosis and clinical treatment of TPE and MPE, the accurate and non-invasive diagnosis are very critical for patients with pleural effusion to initiate efficient management and treatment. However, effective clinical biomarkers were rarely explored to distinguish benign from MPE. The purpose of this study is to identify potential miRNAs which can probably be used to differentiate malignant pleural effusion from TPE.ResultsA total of 23 significantly differentially expressed miRNAs were identified in MPE, with 18 up-expressed and 5 down-expressed. And the target genes of the miRNAs mainly involved in the biology process of nervous system, cancer, immune system and metabolic process etc. Three high confident target genes, AGO4, FGF9 and LEF1 can be regulated by miR-195-5p, miR-182-5p and miR-34a-5p respectively. And these genes participate in the canonical pathway of regulation of the Epithelial-Mesenchymal and the biological functions of apoptosis, growth of tumor and cell proliferation of tumor cell lines. Further, RT-PCR validation results based on 64 collected individuals showed that the expression levels of the three miRNAs were 2–5 times higher in MPE samples, which were consistent with the microarray results. In addition, ROC curve analysis demonstrated that the combination of the three miRNAs can achieve higher AUC of 0.93 (p-value< 0.0001) to differentiate MPE from TPE.ConclusionsThe identified miR-195-5p, miR-182-5p and miR-34a-5p can become potential diagnostic biomarkers for MPE with further evidences.

Deciphering the molecular morphology of the human hair cycle: Wnt signalling during the telogen-anagen transformation.

The signals that induce anagen (growth) in 'quiescent' human telogen hair follicles (HFs) are as yet unknown. Their identification promises better targeted therapeutic hair growth interventions. Recognizing the central role of Wnt signalling in hair biology, the aim was to delineate the differential expression of key agonists, antagonists and target genes of this pathway during the telogen-to-anagen transformation of human scalp HFs. This differential expression was studied by insitu hybridization in human telogen and early-anagen scalp HF sections. On anagen induction, gene expression of the Wnt ligands WNT3, WNT4 and WNT10B, the Wnt ligand secretion regulator WLS, and the Wnt target genes AXIN2 and LEF1, is significantly increased within the secondary hair germ and the dermal papilla. Conversely, expression of the secreted Wnt inhibitor SFRP1 (secreted frizzled-related protein 1) is reduced. Human epithelial HF stem cells upregulate WNT4 and WNT10A expression, suggesting that these Wnt agonists are important for stem cell activation. We provide the first evidence that key changes in Wnt signalling that drive murine anagen induction also occur in human scalp HFs, yet with notable differences. This provides a rational basis for Wnt-targeting therapeutic interventions to manipulate human hair growth disorders. What's already known about this topic? Upregulation of Wnt agonists and downregulation of Wnt antagonists in the secondary hair germ and/or dermal papilla drives hair growth (anagen) induction in mice. Autocrine Wnt signalling in murine epithelial hair follicle stem cells is required to maintain their stem cell function. Reduction of Wnt ligands or increased expression of Wnt antagonists induces dysregulation of the murine hair follicle cycle and causes alopecia. What does this study add? This study demonstrates for the first time that key Wnt pathway regulatory agonists, antagonists and target genes, are expressed in the human telogen-to-early-anagen transformation. On human anagen induction the Wnt ligands WNT3, WNT4 and WNT10B are increased in the regenerating epithelium, whereas the Wnt antagonist, SFRP1 (secreted frizzled-related protein 1), is reduced. Human anagen induction has fundamental differences in the expression of Wnt ligands compared with the murine system. What is the translational message? Regulation of these Wnt ligands permits targeted therapeutic interventions in human hair growth disorders and informs development of new drugs that promote or suppress anagen induction.

The Wnt/β-Catenin/LEF1 Pathway Promotes Cell Proliferation at Least in Part Through Direct Upregulation of miR-17-92 Cluster

The miR-17-92 cluster is involved in animal development and homeostasis, and its dysregulation leads to human diseases such as cancer. In the present study, we investigated the functional link between miR-17-92 cluster and Wnt/β-catenin signaling pathway in ICP2 and DF1 cells. We demonstrated that ectopic expression of either LEF1 or β-catenin increased the promoter activity of the miR-17-92 cluster host gene (MIR17HG) and combined ectopic expression of LEF1 and β-catenin further enhanced the promoter activity; while knockdown of either LEF1 or β-catenin reduced the MIR17HG promoter activity. Both LEF1 and β-catenin could directly bind to the MIR17HG promoter. Furthermore, we demonstrated that low doses of lithium chloride (LiCl), an activator of Wnt/β-catenin signaling pathway, increased MIR17HG promoter activity and the endogenous expression of the miR-17-92 cluster, while high doses of LiCl had the opposite effects. Treatment with XAV-939, an inactivator of the Wnt/β-catenin pathway, reduced the endogenous expression of miR-17-92 cluster. Finally, we found that low doses of LiCl promoted the proliferation of ICP2 and DF1 cells, while high doses of LiCl inhibited the proliferation of ICP2 and DF1 cells. Taken together, our results reveal that MIR17HG is a target of LEF1 and the Wnt/β-catenin pathway and suggest that the miR-17-92 cluster may, at least in part, mediate the proliferation-promoting effect of the Wnt/β-catenin pathway in cell proliferation.

Mutant Lef1 controls Gata6 in sebaceous gland development and cancer

Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a β‐catenin‐independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9‐positive Lef1‐negative hair follicle progenitors that give rise to the upper SG. Overexpression of Gata6 by in utero lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutant Lef1, Gata6 ablation increases the total number of skin tumors yet decreases the proportion of SG tumors. The increased tumor burden correlates with impaired DNA mismatch repair and decreased expression of Mlh1 and Msh2 genes, defects frequently observed in human sebaceous neoplasia. Gata6 specifically marks human SG tumors and also defines tumors with elements of sebaceous differentiation, including a subset of basal cell carcinomas. Our findings reveal that Gata6 controls sebaceous gland development and cancer.

Longdaysin inhibits Wnt/β-catenin signaling and exhibits antitumor activity against breast cancer.

BackgroundCK1 is involved in regulating Wnt/β-catenin signaling and represents a promising target for the treatment of breast cancer. A purine derivative longdaysin has recently been identified as a novel modulator of cellular circadian rhythms through targeting the protein kinases CK1δ, CK1α, and ERK2. However, the antitumor activity of longdaysin and its underlying mechanisms remain unclear.MethodsThe inhibitory effect of longdaysin on Wnt/β-catenin signaling was investigated using the SuperTOPFlash reporter system. The levels of phosphorylated LRP6, total LRP6, DVL2, active β-catenin, and total β-catenin were examined by Western blot. The expression of Wnt target genes was determined using real-time PCR. The ability of colony formation of breast cancer cells was measured by colony formation assay. The effects of longdaysin on cancer cell migration and invasion were assessed using transwell assays. The effect of longdaysin on cancer stem cells was tested by sphere formation assay. The in vivo antitumor effect of longdaysin was evaluated using MDA-MB-231 breast cancer xenografts.ResultsLongdaysin suppressed Wnt/β-catenin signaling through inhibition of CK1δ and CK1ε in HEK293T cells. In breast cancer Hs578T and MDA-MB-231 cells, micromolar concentrations of longdaysin attenuated the phosphorylation of LRP6 and DVL2 and reduced the expression of active β-catenin and total β-catenin, leading to the downregulation of Wnt target genes Axin2, DKK1, LEF1, and Survivin. Furthermore, longdaysin inhibited the colony formation, migration, invasion, and sphere formation of breast cancer cells. In MDA-MB-231 breast cancer xenografts, treatment with longdaysin suppressed tumor growth in association with inhibition of Wnt/β-catenin signaling.ConclusionLongdaysin is a novel inhibitor of the Wnt/β-catenin signaling pathway. It exerts antitumor effect through blocking CK1δ/ε-dependent Wnt signaling.

Flavonoid Silibinin Increases Hair-Inductive Property Via Akt and Wnt/β-Catenin Signaling Activation in 3-Dimensional-Spheroid Cultured Human Dermal Papilla Cells.

Hair loss, also known as alopecia, is a common dermatological condition of psychosocial significance; development of therapeutic candidates for the treatment of this condition is, hence, important. Silibinin, a secondary metabolite from Silybum marianum, is an effective antioxidant that also prevents various cutaneous problems. In this study, we have investigated the effect of silibinin on hair induction using three-dimensional (3D) cultured, human dermal papilla (DP) spheroids. Silibinin was found to significantly increase viability through AKT serine/threonine kinase (AKT) activation in 3D DP spheroids. This was correlated with an increase in the diameter of the 3D DP spheroids. The activation of the wingless and INT-1 (Wnt)/β-catenin signaling pathway, which is associated with hair growth induction in the DP, was evaluated using the T cell-specific transcription factor and lymphoid enhancer-binding factor (TCF/LEF) transcription factor reporter assay; results indicated significantly increased luciferase activity. In addition, we were able to demonstrate increased expression of the target genes, WNT5a and LEF1, using quantitative real-time PCR assay. Lastly, significantly elevated expression of signature genes associated with hair induction was demonstrated in the 3D DP spheroids treated with silibinin. These results suggest that silibinin promotes proliferation and hair induction through the AKT and Wnt/β-catenin signaling pathways in 3D DP spheroids. Silibinin can be a potential candidate to promote hair proliferation.

Pharmacological Targeting of Osteoblast-Induced MDS and AML

The bone marrow hematopoietic stem cell niche has emerged as a regulator and instigator of hematological myeloid malignancies. Activation of b-catenin signaling in osteoblasts, by deletion of exon3 or by inactivating mutations in APC, induces myelodysplasia (MDS), rapidly progressing to acute myeloid leukemia (AML) in mice. Activated b-catenin signaling is present in osteoblasts of one third of MDS and AML patients and it is the most active pathway in stromal cells of MDS patients suggesting that it may sustain dysplastic hematopoiesis and progression to MDS and AML in humans. Therefore, targeting this pathway may provide a new therapeutic approach for this subgroup of MDS/AML patients. To examine this hypothesis we searched for an FDA-approved compound with the ability to inhibit b-catenin signaling and identified all-trans-retinoic acid (ATRA) as a potential candidate. We show here that treatment of osteoblastic cells with ATRA inhibits the induction of b-catenin target genes by Wnt3a and abrogates nuclear localization of b-catenin, as shown by immunohistochemistry and western blot analysis. These effects are at least in part independent of its phosphorylation by GSK3b on exon3 and are mediated by the retinoic acid receptor a (RARa) since silencing RARa, but not RARb or RARg, in osteoblasts abolishes the inhibitory effect of ATRA on Wnt3a-induced b-catenin signaling. Similarly, treatment of leukemic mice expressing constitutively active b-catenin in their osteoblasts with ATRA, inhibited b-catenin signaling as measured by expression of its target genes Axin2 and Lef1 in bone. At the same time, ATRA improved anemia and thrombocytopenia, decreased the percentage of blasts in bone marrow and blood and prolonged overall survival in these leukemic mice as compared to vehicle-treated mice. The response to ATRA treatment of two MDS patients with active b-catenin in their osteoblasts, and not responding to other treatments, was monitored. The hematologic phenotype of the patients improved and disease status stabilized so that patients remain transfusion independent and disease-related symptom-free with a good quality of life thereafter. The improvement in disease status coincided with absence of b-catenin in the nucleus as flow cytometry analysis showed a high percentage of patient osteoblasts expressing activated b-catenin in pre-therapy samples but not in on-therapy samples. These results suggest that ATRA may find a new use in the treatment of the specific portion of MDS/AML patients with activated b-catenin in their osteoblasts. DisclosuresRaza:Geoptix: Speakers Bureau; Onconova: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Kura Oncology: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Syros: Research Funding.

Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development

AbstractNotch1 signaling must elevate to high levels in order to drive the proliferation of CD4−CD8− double-negative (DN) thymocytes and progression to the CD4+CD8+ double-positive (DP) stage through β-selection. During this critical phase of pre–T-cell development, which is also known as the DN-DP transition, it is unclear whether the Notch1 transcriptional complex strengthens its signal output as a discrete unit or through cofactors. We previously showed that the protein inhibitor of activated STAT–like coactivator Zmiz1 is a context-dependent cofactor of Notch1 in T-cell leukemia. We also showed that withdrawal of Zmiz1 generated an early T-lineage progenitor (ETP) defect. Here, we show that this early defect seems inconsistent with loss-of-Notch1 function. In contrast, at the later pre–T-cell stage, withdrawal of Zmiz1 impaired the DN-DP transition by inhibiting proliferation, like withdrawal of Notch. In pre–T cells, but not ETPs, Zmiz1 cooperatively regulated Notch1 target genes Hes1, Lef1, and Myc. Enforced expression of either activated Notch1 or Myc partially rescued the Zmiz1-deficient DN-DP defect. We identified residues in the tetratricopeptide repeat (TPR) domain of Zmiz1 that bind Notch1. Mutating only a single residue impaired the Zmiz1-Notch1 interaction, Myc induction, the DN-DP transition, and leukemic proliferation. Similar effects were seen using a dominant-negative TPR protein. Our studies identify stage-specific roles of Zmiz1. Zmiz1 is a context-specific cofactor for Notch1 during Notch/Myc-dependent thymocyte proliferation, whether normal or malignant. Finally, we highlight a vulnerability in leukemic cells that originated from a developmentally important Zmiz1-Notch1 interaction that is hijacked during transformation from normal pre–T cells.

Abstract 5897: Single cell-derived analysis of desmoid tumors for studying tumor-stroma interactions

Abstract Cancer associated fibroblasts play an important role in the maintenance and remodeling of the tumor microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. Desmoid tumors (DT), also called aggressive fibromatosis, are rare, locally invasive soft tissue tumors that consist of fibroblastic cells embedded in extracellular matrix. Identification of the stromal cells to study tumor-stroma interactions is difficult due to both populations displaying a fibroblastic phenotype, and no cell marker exists that reliably differentiates between the two populations. Majority of DT arise sporadically due to somatic activating mutations in beta-catenin (CTNNB1), a major effector molecule of canonical Wnt signaling. We established single cell derived colonies from multiple DT samples and characterized the beta-catenin mutation status of each clone by Sanger sequencing. Indeed, we were able to establish both mutant and non-mutant colonies from DT samples. Quantitative PCR for beta-catenin targets AXIN2 and LEF1 confirmed differential activity between the mutant and non-mutant colonies. The specific CTNNB1 codon mutation had no difference on beta-catenin transcriptional activity. We next performed a high throughput surface antigen screen to identify cell markers that can distinguish between the two subpopulations. Our screen found CD142 to be uniquely expressed by the mutant colonies, while the non-mutant colonies uniquely expressed Podoplanin. Quantitative PCR confirmed the differential expression of these markers. Furthermore, the CD142-positive population in heterogeneous DT samples correlated with their mutation frequency. Importantly, CD142-based cell sorting allowed the isolation of the mutant subpopulation even in samples that appeared as wild-type by Sanger sequencing. We also studied the expression of secreted factors in our mutant and non-mutant populations. We observed that CTHRC1, a ligand related to the Wnt/PCP pathway, is highly elevated exclusively in the mutant subpopulations. Recombinant CTHRC1 increased the proliferation rate of DT primary cultures, as measured by BrdU incorporation, while neutralizing antibodies against CTHRC1 decreased cell proliferation. The importance of tumor-stroma interactions cannot be studied without first identifying and characterizing the two populations. This has been especially difficult in soft tissue sarcomas where both the neoplastic and stromal cells exhibit a mesenchymal phenotype. Our study offers a novel method for identifying the mutant and non-mutant subpopulations within desmoid tumors to study how they may interact. Rapidly quantifying tumor composition will also support efforts to understand the natural progression of disease and how it responds to therapy. Citation Format: Mushriq Al-Jazrawe, Steven Xu, Qingxia Wei, Raymond Poon, Benjamin Alman. Single cell-derived analysis of desmoid tumors for studying tumor-stroma interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5897. doi:10.1158/1538-7445.AM2017-5897

Activation of Wnt Signaling Increases Numbers of Enteric Neurons Derived From Neonatal Mouse and Human Progenitor Cells

Neural stem and progenitor cells from the enteric nervous system (ENS) might serve as a source of cells for treatment of neurogastrointestinal disorders. Before we can use these cells, we must increase our understanding of the signaling mechanisms that regulate proliferation and differentiation. We systematically evaluated the effects of canonical Wnt signaling on proliferation and differentiation of cultured ENS progenitor cells from neonatal mice and humans. We isolated ENS progenitors from tunica muscularis of the small intestine of newborn (postnatal day 0) wild-type C57BL/6 mice as well as from Wnt1-Cre2 reporter mice. We also obtained intestinal tissue samples from infants (2 and 7 months old) undergoing surgery for imperforate anus or focal intestinal perforation and isolated ENS cells. ENS cells were cultured under proliferation conditions leading to formation of 3-dimensional spheres, which we activated with Wnt3a and SB216763 in order to activate the β-catenin-dependent canonical Wnt pathway. We used immunoblot and quantitative polymerase chain reaction to evaluate the molecular response to Wnt stimuli and immunohistochemistry, proliferation, and cell death assays to identify new neurons. In proliferating enterospheres derived from ENS progenitor cells, we verified the expression of Wnt receptors frizzled 1-10 and the co-receptors low-density lipoprotein receptor-related proteins 5 and 6. Pharmacologic stimulation with Wnt agonists led to intracellular accumulation of Wnt-dependent β-catenin and up-regulated expression of known Wnt target genes axin2, lef1, and lgr5. Activation of the canonical Wnt pathway promoted growth of ENS cell spheres during cell expansion and increased the number of newborn neurons derived from mouse and human progenitor cells. In studies of human and mouse ENS progenitors, we found activation of the Wnt signaling pathway to promote neurogenesis of the ENS invitro. The neurogenic effect of Wnt agonists on ENS progenitors supports their use in generation of cell pools for autologous cell replacement therapies.

OR14 HLA class I antibodies increased WNT/β-catenin pathway component protein lymphoid-binding factor 1 (LEF1) in endothelial cells in foxo1 protein dependent fashion

Introduction and aim Forkhead box (FOX) proteins are a family of important transcription factors that plays important role in maintaining endothelial cells functions. The protective role of FOXO1 in multiple cellular functions is supported by several studies. In many cellular systems, the forkhead transcription factors such as FOXO1 activity is regulated by post-translational modification through phosphorylation and acetylation. The purpose of the current study was to investigate the effect of HLA class I on the phosphorylation/inactivation of the FOXO1 protein. We also investigated the impact of the HLA antibodies on the mRNA levels of the fibrosis protein LEF1. Methods Western blot analysis was used to examine the phosphorylation status of FOXO1 in human umbilical cord endothelial cells (HUVECs) after treatment with monoclonal antibodies directed against HLA class I. Real-time PCR assay was used to measure the mRNA levels of LEF1 in endothelial cells. Results Coupling of endothelial cells with monoclonal antibodies directed to HLA class I but not class II resulted in increased phosphorylation (inactivation) of the FOXO1 protein at Ser256 and Th24. In addition, the mRNA levels of FOXO1 down-stream target LEF1 were significantly up-regulated in endothelial cells treated with HLA class I antibodies compared to control treated cells. shRNAi mediated knock-down of FOXO1 protein in endothelial cells resulted in increased expression of LEF1 in endothelial cells. Conclusion This data identifies a new role of HLA antibodies in regulating endothelial cells functions by the inactivation of FOXO1 and increased its down-stream target LEF1. This data in addition to the previously published data indicating the involvement of the LEF1 in fibrosis might suggest a novel mechanism for allograft fibrosis mediated by HLA antibodies. Further studies to evaluate the in vitro findings in human allograft tissues are required.