Expression Of Wnt-related Genes Research Articles

Abstract 6946: Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids

Abstract Colorectal cancer (CRC) is one of the most diagnosed cancers worldwide. Though several treatment strategies are available today, around a million people die of this disease each year worldwide. Thus, there is still a pivotal need for new and more effective treatments. Genetic and non-genetic intra-tumor heterogeneity has emerged lately as an intrinsic feature of malignancies and as one of the factors responsible for therapy resistance or tolerance. In particular, the nature and role that phenotypically distinct, yet genetically indistinguishable, subpopulations have on tumor biology is far from being understood. Here we investigate phenotypic heterogeneity in CRC by identifying specific transcriptionally well defined cell subpopulations. We aim at deciphering the interactions between populations in terms of differentiation and plasticity, tumor growth and potentially exposed targetable vulnerabilities. To this end, we used patient-derived metastatic CRC (mCRC) organoids, including KRAS wild type (wt) and mutant (kras) models. We performed in total scRNAseq for 7 mCRC samples (4 wt; 3 kras) in different growth media: normal growth medium (supplemented with EGF) (n=1); medium without EGF (noEGF) (n=7); or treated with Cetuximab (CTX, a clinically approved monoclonal antibody against EGFR) (n=7). We found that the wt sample grown in EGF presents 2 distinct subpopulations: one with stemness characteristics (LGR5 high) and the other with a more differentiated phenotype (KRT20 high), in agreement with the current bibliography. Interestingly, in EGF deprived cultures new subpopulations appear. In particular, we were able to identify a subpopulation with distinct expression of WNT-related genes (APCDD1, WNT6, WIF1). This subpopulation is also present, and in cases increasing in fraction, when organoids are treated with CTX (in 3 out of 7 samples). Interestingly, a fraction of the WNT positive subpopulation seems to be able to proliferate under CTX treatment (Ki67 positive). We identified the negative regulator of the WNT pathway, APCDD1, as a potential marker to track this subpopulation. We took one wt and one kras sample as models to validate these findings by flow cytometry. Furthermore, sorting of APCDD1 positive and negative cells, and further RT-qPCR analysis, confirmed good correlation between antibody staining and gene expression. Moreover, we found that both types of cells are able to re-grow as organoids after re-culturing. This suggests that this WNT-associated subpopulation could be contributing, at least in part, to CTX tolerance or resistance. Our results show promising potential to help unravel basic features of intra-tumor heterogeneity both in basal conditions and upon treatment, and possibly to find new therapeutic targets. Citation Format: Sabrina J. Fletcher, Irene Catalano, Elena Grassi, Sofia Borgato, Barbara Peracino, Elena Piretto, Luca Alessandri, Raffaele Calogero, Luca Primo, Livio Trusolino, Andrea Bertotti, Alberto Puliafito. Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6946.

Effect of estradiol after bacterial infection on the Wnt/β-catenin pathway in bovine endometrium epithelial cells and organoids

Endometritis is a disease caused by a postpartum bacterial infection with a poor prognosis that primarily affects dairy cows. Three-dimensional organoids have been used as a model for endometritis, because they exhibit a structure comparable to that of the endometrium, demonstrating both expansibility and hormone responsiveness. These characteristics render them an ideal platform for in vitro investigations of endometrial diseases. Estradiol (E2) is an endogenous steroid hormone with demonstrated anti-inflammatory properties, and the objective of this study was to determine the mechanism by which E2 modulates the inflammatory response and the Wnt signal transduction pathway in bovine endometrial epithelial cells and organoids following E. coli infection. We present the techniques for isolating and culturing primary bovine endometrial epithelial cells (BEECs), and producing endometrial organoids. For the experiments, the endometrial epithelial cells and organoids were infected with E. coli for 1 h, followed by incubation with E2 for 12 h. The mRNA and protein expressions of the inflammation-related genes, IL-1β, IL-6, TLR4, and NF-κB, as well as the Wnt pathway-related genes, Wnt4, β-catenin, c-Myc, and CyclinD1, were assessed using real-time quantitative-PCR and western blotting, respectively. The CCK8 viable cell counting assay was utilized to determine the optimal concentration of the Wnt inhibitor, IWR-1. The mRNA and protein expression of Wnt pathway-related genes was assessed following IWR-1 treatment, while the expression levels of proliferation-associated genes (Ki67, PCNA) and barrier repair genes (occludin, claudin, and Zo-1) in BEECs and organoids were evaluated after E2 treatment. The results of this study show that mRNA expression of the inflammatory genes, IL-1β, TLR4, and NF-κB (P < 0.05) decreased in BEECs following E2 treatment compared to the E. coli group. The protein expression of the IL-1β, IL-6, TLR4 and NF-κB genes was also inhibited (P < 0.05). Similar results were observed in tests on the organoids. Our findings demonstrate that E2 significantly upregulates the expression of Wnt-related genes, including β-catenin and c-Myc, while concurrently downregulating the expression of GSK3β (P < 0.05). Next, we treated E. coli-infected BEECs and organoids with the Wnt inhibitor, IWR-1. Compared with E. coli and E. coli + E2, the expression of mRNA and protein from Wnt 4, β-catenin, and CyclinD1 in E. coli + E2 and E. coli + IWR-1 was down-regulated (P < 0.05). The expression of the proliferation genes, Ki67, PCNA, and the tight junction genes, occludin, claudin1, and Zo-1, in organoids was significantly higher than that in BEECs (P < 0.05). In summary, we found strong potential for E2 mitigation of the E. coli-induced inflammatory response in BEECs and organoids, through activation of the Wnt pathway. In addition, the proliferation and repair capacity of organoids was much higher than that of BEECs.

SFRP1 Expression Regulates Wnt Signaling in Chronic Myeloid Leukemia K562 Cells.

Wnt signaling cascades play important roles in cell fate decisions and their deregulation has been documented in many diseases, including malignant tumors and leukemia. One mechanism of aberrant Wnt signaling is the silencing of Wnt inhibitors through epigenetic mechanisms. The sFRPs are one of the most studied Wnt inhibitors; and the sFRP1 loss is known in many hematological malignancies. Therefore, we aimed to compare the expression of Wnt related genes in the presence and absence of sFRP1 in a chronic myeloid leukemia (CML) cell line. It is important to understand how sFRP1 and sFRP1 perform their effects on CML to design new agents and strategies for resistant and advanced forms of CML. We used K562 cells, which normally do not express sFRP1 and its sFRP1 expressing subclone K562s. Total RNA was isolated from K562 and K562s cell lines and converted to cDNA. PCR Array experiments were performed using Human Wnt Signaling Pathway Plus RT2 Profiler™ kit. Wnt signaling pathway activation was studied by western blot for downstream signaling targets. The WNT3, LRP6, PRICKLE1 and BTRC expressions were significantly decreased in the presence of sFRP1; while WNT5B increased. The sFRP1 expression inhibited stabilization of total β-catenin protein and downstream effector phosphorylation of noncanonical Wnt/PCP signaling; whereas Ca2+/PKC signaling remained active. The results suggest that sFRP1 could be a promising therapeutic anticancer agent. Defining these pathway interactions is crucial for designing new agents resistant and advanced forms of CML.

Dehydrated human amniotic membrane modulates canonical Wnt signaling in multiple cell types in vitro

Canonical Wnt signaling is a major pathway known to regulate diverse physiological processes in multicellular organisms. Signaling is tightly regulated by feedback mechanisms; however, persistent dysregulation of this pathway is implicated in the progression of multiple disease states. In this study, proteomic analysis identified endogenous Wnt antagonists in micronized dehydrated human amnion/chorion membrane (μdHACM); thereby, prompting a study to further characterize the intrinsic properties of μdHACM as it relates to Wnt activity, in vitro. A TCF/LEF reporter cell line demonstrated the general ability of μdHACM to inhibit β-catenin induced transcription activity. Furthermore, in vitro systems, modeling elevated Wnt signaling, were developed in relevant cell types including tenocytes, synoviocytes, and human dermal fibroblasts (HDFs). Stimulation of these cells with Wnt3A resulted in translocation of β-catenin to the nucleus and increased expression of Wnt related genes. The subsequent addition of μdHACM, in the continued presence of Wnt-stimulus, mitigated the downstream effects of Wnt3A in tenocytes, synoviocytes, and HDFs. Nuclear localization of β-catenin was abated with corresponding reduction of Wnt related gene expression. These data demonstrate the in vitro regulation of canonical Wnt signaling as an inherent property of μdHACM and a novel mechanism of action.

Abstract 4861: Loss of YAP1 activates Wnt/beta-Catenin signaling in prostate cancer

Abstract Prostate cancer (PCa) is the most common non-cutaneous cancer among American men. In 2019, 174,650 new patients will be diagnosed from this disease. Most patients with localized PCa could be treated successfully. However, when PCa develops into castrate-resistant prostate cancer (CRPCa), the five-year survival rate drops below 30%. Neuroendocrine prostate cancer (NEPCa) is an aggressive subtype of PCa observed in 20%-30% CRPCa patients. Understanding how NEPCa progressed from prostate adenocarcinoma could reveal novel therapeutic targets. Previous studies indicate that activation of Wnt/beta-Catenin signaling promotes the progression to CRPCa with an increased NE differentiation. Additionally, animal studies suggest that Wnt/beta-Catenin signaling is active in NEPCa. Here we explored the mechanisms that promote Wnt/beta-Catenin signaling in NEPCa. Using TRAMP mice, which develop NEPCa after castration, we assessed the expression of Wnt-related genes and found that after androgen deprivation, the expression of Wnt7A is increased in prostate derived from both wild type and TRAMP mice but the expression of DKK1, a Wnt inhibitor, is decreased in wild type prostate but not in TRAMP prostate. The elevated expression of Wnt7a in combination with the lack of induction of Dkk1 in TRAMP tumors provides a mechanism to activate Wnt/beta-Catenin signaling in these tumors. Furthermore, we found the expression of YES-associated Protein (YAP1), a key transcriptional coactivator of Hippo pathway, is lost in NEPCa, in contrast to the elevated expression of YAP1 in high-grade prostate adenocarcinoma. YAP1 knockdown activated Wnt/beta-Catenin activity in PCa PC3 and 22Rv1 cells, indicating that the loss of YAP1 expression provides another mechanism to activate Wnt/beta-Catenin signaling, promoting NEPCa phenotype in PCa cells. Funding: This study is supported by: FWCC/Foundation Legacy Funds, DOD New Investigator Award, The Office of Research SEED awards, NIH R01 AND R03. Citation Format: Siyuan Cheng, Shu Yang, Nestor Prieto-Dominguez, Zachary M. Connelly, Yingli Shi, Xiuping Yu. Loss of YAP1 activates Wnt/beta-Catenin signaling in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4861.

Abstract B77: PORCN inhibition prolongs survival, decreases tumor burden, and alters the immune microenvironment in ovarian cancer

Abstract Background: Wnt/β-catenin pathway upregulation has been correlated with immune evasion in multiple cancers, including epithelial ovarian cancer (EOC). Porcupine (PORCN) is an enzyme necessary for cells to produce WNT ligand, which is necessary for the activation of the pathway. Our hypothesis was that the administration of a PORCN inhibitor (CGX1321) would decrease Wnt signaling and thereby lead to a decrease in immune evasion, creating a “hot” tumor microenvironment (TME), which would result in increased survival and a decrease tumor burden. Methods: RNA sequencing was performed in a cohort of primary ovarian cancer samples. CGX1321 was administered to C57Bl6 mice injected intraperitoneally with ID8 or ID8p53−/− cells. Tumor growth was measured by bioluminescence and omental weight. H&amp;E slides were analyzed for amount of tumor in the omentum. Amount of tumor-infiltrating lymphocytes (TILs), Tregs, dendritic cells (DCs), macrophages, and monocytes in the TME were quantified via flow cytometry. CD11c-cre x β-catenin-flox C57Bl6 mice, a mouse model with no DC-intrinsic β-catenin, and the administration of anti-CD8β antibody were used to evaluate tumor burden with and without CGX1321. Results: Increased Wnt activity was correlated with a decreased T-cell signature in our human ovarian cancer samples. Treatment with CGX1321 prolonged survival (P=0.0001) and decreased omentum weight (P=0.0056) in mice injected with ID8 cells. H&amp;E slides revealed decreased amount of tumor in the omentum with treatment. There was an increase of DCs (P=0.0159), macrophages (P=0.0079), and monocytes (P value=0.0079) with treatment, which was only significant for mice injected with ID8 cells, not ID8p53−/− mice. Tumor burden was decreased in CD11c-cre x β-catenin-flox mice (P=0.1014) without treatment, and with CGX 1321 treatment (P=0.0450). Tumor burden was not decreased with CGX1321 treatment in the absence of CD8+ T-cells (P=0.3175). Conclusions: Consistent with EOC TCGA data, we identified a correlation between increased Wnt signaling and decreased T-cell signature (i.e., “cold” tumor). ID8 cells have a higher level of total β-catenin and expression of WNT related genes compared with ID8p53−/−, which may explain the prolonged survival, decreased tumor burden, and more profound increase of DCs, macrophages, and monocytes in the TME. This may suggest a reliance on these cells for tumor recognition with PORCN inhibition. Furthermore, tumor burden was decreased in a DC-intrinsic β-catenin absent model, suggesting the presence of β-catenin in this antigen-presenting cell contributes to immune evasion in the TME. The lack of effect of CGX1321 on tumor burden in the absence of CD8+ T-cells indicates a partial reliance on CD8+ T-cells for tumor recognition. These data warrant further investigation of Wnt inhibition in both preclinical models and clinical trials in EOC. Citation Format: Whitney N. Goldsberry, Selene Meza-Perez, Ashwini A. Katre, Angelina Londoño, Bryan T. Mott, Jaclyn M. Arquiette, Troy D. Randall, Sara J. Cooper, Lyse A. Norian, Rebecca C. Arend. PORCN inhibition prolongs survival, decreases tumor burden, and alters the immune microenvironment in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B77.

Abstract A09: SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates strong inhibition of the Wnt signaling pathway and antitumor effects in diverse ovarian cancer models

Abstract Aberrant activation of the Wnt signaling pathway is observed in ovarian cancer (OC) and is associated with chemoresistance, immune evasion, and poor prognosis. SM08502 is a novel, oral, small-molecule pan-CLK inhibitor that has been shown to potently inhibit the Wnt signaling pathway in preclinical colorectal cancer models. The purpose of these studies was to test the in vitro and in vivo activity of SM08502 in preclinical models of OC. The effect of SM08502 on cell viability was tested in 10 OC cell lines of various histotypes, including high-grade serous (HGS), serous, endometrioid, clear-cell, and teratocarcinoma lines in vitro. Cell proliferation was impaired in all cell lines by SM08502 regardless of histotype and mutation profile (average EC50=0.123 μM [0.034 – 0.275]). Relative to DMSO, SM08502 (1 μM) also potently inhibited Wnt-related gene expression (TCF7, DVL2, LRP5, and ERBB2), which correlated with inhibition of protein expression in HGSOC, endometrioid, and teratocarcinoma cell lines. Additionally, in cell lines derived from these histotypes, SM08502 strongly inhibited cyclin E1 gene and protein expression, the amplification/overexpression of which has been associated with treatment resistance and poor overall survival in HGSOC. In vivo antitumor effects and tolerability of oral SM08502 (6.25, 12.5 and 25 mg/kg QD for 15-22 days) were assessed in mice bearing OVCAR-3 (HGSOC; TP53mut), PA-1 (teratocarcinoma; N-Ras mut), and TOV-112D (endometrioid; CTNNB1mut) xenografts (n=5 mice per group). In OVCAR-3 xenografts, significant tumor growth inhibition (TGI) vs. vehicle occurred in mice treated with SM08502 12.5 mg/kg (66%, p&amp;lt;0.01) and 25 mg/kg (89%, p&amp;lt;0.001). SM08502 25 mg/kg also induced tumor regression in all mice. Similarly, in PA-1 xenografts, SM08502 induced TGI of 64% (p&amp;lt;0.01) at 12.5 mg/kg and 93% (p&amp;lt;0.01) with 4/5 tumors regressing at 25 mg/kg. SM08502 at 25 mg/kg also induced TGI of 64% (p&amp;lt;0.01) in TOV-112D xenografts. In addition, SM08502 (25 mg/kg QD) was assessed in 5 patient-derived xenograft (PDX) models of metastatic OC with double-hit TP53 and BRCA1/2 mutations (Crown Biosciences). Inhibition of tumor growth was observed in all tested PDXs (average TGI=61% [47-73], p&amp;lt;0.001). SM08502 was well tolerated in all xenograft models tested based on body weight measurements. In summary, SM08502 potently inhibited cell proliferation and expression of Wnt-related genes and cyclin E1 in OC cell lines. SM08502 also demonstrated strong in vivo antitumor effects, including tumor regressions, in different subtypes of OC xenografts and PDX models. These data suggest that SM08502 may provide clinical benefit for OC patients regardless of histotype, BRCA mutation status, or cyclin E1 expression. A phase 1 study assessing safety, tolerability, and pharmacokinetics of SM08502 in advanced solid tumors is ongoing (NCT03355066). Citation Format: Heekyung Chung, Lauren Sitts, Emily Creger, Erica Liao, Brian Eastman, Chi-Ching Mak, K.C. Sunil, Betty Tam, Gail Bucci, Josh Stewart, Carine Bossard, Timothy Phalen, Steven Cha. SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates strong inhibition of the Wnt signaling pathway and antitumor effects in diverse ovarian cancer models [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A09.

Loss of Rnf43 to accelerate KRAS-mediated neoplasia in a clinically relevant genetically engineered mouse model of pancreatic adenocarcinoma.

733 Background: The genetic heterogeneity of pancreatic ductal adenocarcinoma (PDAC) demands a personalized molecular-targeted treatment approach. While activating KRAS mutations are a near ubiquitous event in PDAC pathogenesis, 5-10% of cases display deleterious driver mutations in the Wnt-signaling negative regulator, ring finger 43 (RNF43). Despite this characterization there are no personalized treatment options for this subset of patients. Methods: We have developed a genetically engineered mouse model (GEMM) of PDAC, driven by an activating mutation in Kras and deletion of Rnf43 under control of a pancreas specific promoter (KRC). Mice were followed for survival outcomes and histological changes which were compared to a Kras driven PDAC GEMM (KC). Mice underwent serial magnetic resonance imaging (MRI), with and without dynamic contrast enhanced (DCE) imaging, to evaluate cystic tumor morphology and contrast enhancement during tumor progression. Single cell RNA sequencing (scRNAseq) was also performed to assess changes in single cell populations during tumor progression. Lastly, we established ex vivo cultures from KRC and KC tumors and performed bulk RNA-sequencing (RNAseq) and in vitro pharmacology studies. Results: KRC mice displayed a decrease in overall survival and higher incidence of both high grade pre-neoplastic lesions and invasive PDAC compared to KC mice. Serial MRI revealed increased cystic morphology of KRC mice during tumor progression with increasing DCE intensity. scRNAseq of KRC tumors from moribund mice displayed two distinct populations of both macrophages and fibroblasts, similar to our previous report of Kras/Tp53 and Kras/Ink4a driven GEMMs. Lastly, primary cultures from KRC tumors demonstrated increased expression of Wnt-related genes by RNAseq and increased sensitivity to small molecule porcupine inhibition relative to KC cell lines, demonstrating functional Wnt dependence of the KRC system. Conclusions: Rnf43 is a bona fide tumor suppressor gene in PDAC. This GEMM is a novel platform for drug discovery in RNF43-mutated PDAC with the eventual goal of implementing a precision oncology approach in these patients.

Annexin A8 regulates Wnt signaling to maintain the phenotypic plasticity of retinal pigment epithelial cells

Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Annexin A8 (AnxA8), a calcium-dependent phospholipid-binding protein, and canonical Wnt signalling mechanisms have both been implicated in retinal pigment epithelial (RPE) cell differentiation. The aim here was to examine the possibility of cross-talk between AnxA8 and Wnt signalling, as both are down-regulated upon fenretinide (FR)-mediated RPE transdifferentiation. AnxA8 suppression in RPE cells via siRNA or administration of FR induced neuronal-like cell transdifferentiation and reduced expression of Wnt-related genes, as measured by real-time PCR and western blotting. AnxA8 gene expression, on the other hand, remained unaltered upon manipulating Wnt signalling, suggesting Wnt-related genes to be downstream effectors of AnxA8. Co-immunoprecipitation revealed an interaction between AnxA8 and β-catenin, which was reduced in the presence of activated TGF-β1. TGF-β1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced β-catenin translocation and GSK-3β phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active β-catenin and GSK-3β phosphorylation. These data demonstrate an important role for AnxA8 as a regulator of Wnt signalling and a determinant of RPE phenotype, with implications for regenerative medicine approaches that utilise stem cell-derived RPE cells to treat conditions such as age-related macular degeneration.

Inhibition of Wnt signalling pathway by XAV939 enhances radiosensitivity in human cervical cancer HeLa cells

Cervical cancer is the second most common malignant tumour threatening women’s health. In recent years, heavy-ion beam therapy is becoming a newly emerging therapeutic mean of cancer; however, radio-resistance and radiation-induced damage constitute the main obstacles for curative treatment of cervical cancer. Therefore, to identify the radiosensitizers is essential. Here, we investigated the effects of Wnt signalling pathway on the response of 12C6+ radiation in HeLa cells. XAV939, an inhibitor of Wnt signalling pathway, was added two hours before 12C6+ radiation.12C6+ radiation inhibited the viability of HeLa cells in a time-dependent manner, and inhibiting Wnt signalling using XAV939 significantly intensified this stress. Meanwhile, 12C6+ radiation induced a significant increased cell apoptosis, G2/M phase arrest, and the number of γ-H2AX foci. Supplementation with XAV939 significantly increased the effects induced by 12C6+ radiation alone. Combining XAV939 with 12C6+ irradiation, the expression of apoptotic genes (p53, Bax, Bcl-2) was significantly increased, while the expression of Wnt-related genes (Wnt3a, Wnt5a, β-catenin, cyclin D1 and c-Myc) was significantly decreased. Overall, these findings suggested that blockage of the Wnt/β-catenin pathway effectively sensitizes HeLa cells to 12C6+ irradiation, and it may be a potential therapeutic approach in terms of increasing the clinical efficacy of 12C6+ beams.

SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in Mantle Cell Lymphoma Models

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) that accounts for ~7% of all NHL in the U.S. MCL is associated with aberrant activation of the Wnt signaling pathway, which plays a key role in the survival and maintenance of MCL-initiating cells. Many MCL patients experience relapse and subsequent disease progression due to chemoresistance following initial therapy; hence, novel therapies are needed. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. The purpose of these studies was to examine the antitumor activity of SM09419 in preclinical models of MCL. SM09419 potently inhibited both CLK1-CLK4 (IC50 for all &lt;0.02 µM) and Wnt signaling pathway (average EC50=0.068 µM) activities. In REC-1 and GRANTA-519 MCL cells, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt-related genes (CCND1, LEF1, TCF7) and proteins vs. vehicle. In tests on 5 MCL cell lines, cell proliferation was strongly impaired by SM09419 across all lines (average EC50=0.102 µM [0.021-0.236]). SM09419 also induced apoptosis in REC-1 and GRANTA-519 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression vs. vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD 20-21 days) were assessed in mice bearing REC-1 and JeKo-1 flank xenografts (n=5/group). In REC-1 xenografts, strong tumor growth inhibition (TGI) vs. vehicle occurred in mice treated with 12.5, 25, and 50 mg/kg SM09419 (TGI 88% [p&lt;0.01], 100%, and 100% [p&lt;0.001], respectively), and the two highest doses induced complete tumor regression in all mice from D14. Similarly, in JeKo-1 xenografts, SM09419 (12.5 and 25 mg/kg) induced significant TGI vs. vehicle (71% and 100%, respectively; p&lt;0.0001) with complete tumor regression at 25 mg/kg, whereas acalabrutinib (50 mg/kg BID) was not efficacious (27% TGI) when tested in parallel. SM09419 25mg/kg induced reversible suppression of phospho-SRSF6 protein and inhibited Wnt pathway-related gene expression (TCF7 and DVL2) in JeKo-1 tumors in a single-dose PD study, demonstrating downstream target engagement in vivo. SM09419 was also assessed in 2 patient-derived xenograft (PDX) mouse models of MCL. PDX cells were injected intravenously and treatment was initiated upon 8-12% engraftment of human CD45+CD19+ cells in peripheral blood. In the first model, derived from a patient who was progressive after 8 modalities including ibrutinib, SM09419 (25 mg/kg QD) increased survival vs. vehicle (100% through D26 vs. 0% by D12, respectively; n=6/group) and suppressed MCL engraftment in the blood (12% at D26 vs. 69% at D8 and D12, respectively; p=0.002) and bone marrow (30% at D26 vs. 91% at D8 and D12, respectively; p=0.002). In the second model, derived from a patient refractory after ibrutinib and anti-PDL1 treatment, SM09419 (25 mg/kg QD) significantly suppressed MCL engraftment vs. vehicle in the blood (8% vs. 72%), bone marrow (20% vs. 57%), and spleen (15% vs. 96%) at D28 (study end; p&lt;0.001 for all; n=4/group). In addition, SM09419 greatly inhibited splenomegaly vs. vehicle (0.04 g vs. 0.4 g, respectively; p&lt;0.001). In a subsequent experiment in the same model, mice (n=7/group) were treated with 12.5 or 25 mg/kg SM09419 or vehicle for 12 weeks (to D85). Blood MCL engraftment at D41 was significantly lower in mice treated with SM09419 (40% at 12.5 mg/kg and 23% at 25 mg/kg) vs. vehicle (88%; p&lt;0.01 and p&lt;0.001, respectively). SM09419 dose-dependently increased survival (28.6% at 12.5 mg/kg and 85.7% at 25 mg/kg at D85) vs. vehicle (0% at D63); survival was maintained in both dose groups during post-treatment monitoring (to D99). SM09419 was well tolerated in all tested mouse models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation Wnt signaling pathway activity, and cell proliferation and induced apoptosis in MCL cell lines. The strong in vivo antitumor effects observed as a single agent suggest that SM09419 may provide a clinical benefit for patients with treatment-resistant or refractory MCL. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in Tumor Protein p53 (TP53)-Mutant Acute Myeloid Leukemia Models

Acute myeloid leukemia (AML) with TP53 mutation makes up ~13% of AML cases and is an aggressive, treatment-resistant subtype with dismal prognosis and limited therapeutic options. Aberrant activation of the Wnt signaling pathway is associated with AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells, making Wnt signaling inhibition a potential therapeutic modality for adverse AML. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway in a Wnt signaling reporter assay. The purpose of these studies was to examine the antitumor activity of SM09419 as a single agent and in combination with standard therapies in preclinical models of TP53 mutant (TP53mut) AML. In TF-1a and KG-1a AML cells with TP53 mutations, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt-related genes (LEF1, MYC, DVL2) and proteins vs. vehicle. The effect of SM09419 on cell proliferation was tested in 6 TP53mut AML cell lines. Proliferation was strongly impaired by SM09419 across all cell lines (EC50=0.23 + 0.056 µM). SM09419 significantly induced apoptosis in TF-1a and KG-1a cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In addition, SM09419 potently inhibited cell proliferation when tested in 27 leukapheresis-derived human primary AML cell lines (EC50=0.046 + 0.0061 µM) regardless of TP53 status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with cytarabine (Ara-C), venetoclax (VEN), or azacytidine (AZA) were assessed in mice bearing TP53mut flank xenografts (n=5-15/group). In TF-1a xenografts, SM09419 (12.5 and 25 mg/kg) induced significant tumor growth inhibition (TGI) vs. vehicle at D20 (TGI 55-56% [p&lt;0.01]). VEN (50mg/kg) was not effective (3% TGI) and combining VEN with SM09419 had no additional benefit (52%-60% TGI). In Kasumi-1 xenografts, SM09419 (12.5 and 25 mg/kg), AZA (0.8 mg/kg), and VEN (25 mg/kg) induced TGI vs. vehicle of 87%, 95% (both p&lt;0.0001), 72% (p&lt;0.001), and 48% (NS), respectively at D18. SM09419 25 mg/kg alone induced tumor regression in 40% (2/5) of the mice. SM09419 (12.5 mg/kg) + VEN induced greater TGI vs. vehicle (96%, p&lt;0.0001) with tumor regression in 80% (4/5) of the mice, while AZA + VEN induced 79% TGI (p&lt;0.001) with no tumor regression. In KG-1a xenografts, single-agent SM09419 (12.5 and 25 mg/kg) and Ara-C (10mg/kg) induced significant TGI vs. vehicle (53%, 98%, and 80% [p&lt;0.001], respectively) at D28 but VEN (12.5mg/kg) did not (35% TGI). The combination of SM09419 (12.5 mg/kg) + VEN (12.5 mg/kg) improved TGI (98%) vs. vehicle. Tumor regression was seen in all mice with single-agent SM09419 (25 mg/kg) and 12.5 mg/kg + VEN. In another KG-1a xenograft study, mice were treated with combinations of SM09419 (12.5 mg/kg), AZA (0.8 mg/kg), and VEN (25 mg/kg) for 20 days followed by 21 days of SM09419 (25 mg/kg) or vehicle maintenance in some groups. SM09419 + VEN, SM09419 + AZA, and AZA + VEN induced TGI of 95%, 64%, and 58%, respectively (all p&lt;0.0001), with 80% (12/15) regression in SM09419 + VEN. The triplet induced 91% TGI but was not well tolerated due to GI toxicity. In the maintenance phase, SM09419 given QD or QOD greatly slowed tumor regrowth vs. vehicle at D41 in mice previously treated with SM09419 + VEN (80% and 72% TGI [p&lt;0.001], respectively). SM09419 QD maintenance therapy also slowed tumor regrowth following AZA + VEN (p&lt;0.0001). SM09419 alone and in combination (except with AZA + VEN) was well tolerated in all tested xenografts. In summary, SM09419 potently inhibited SRSF phosphorylation and Wnt pathway signaling and induced apoptosis in TP53mut AML cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of TP53 status. Strong in vivo antileukemic effects were observed with SM09419 as a single agent or in combination with other AML therapies, suggesting that it is a potential treatment for hard-to-treat AML subtypes such as TP53mut AML. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

SAT-520 The Effects Of AKAP13 On Osteogenesis By Modulation Of RhoA And Wnt Signaling

[Introduction] AKAP13 (Protein Kinase A Anchoring Protein 13) is a guanine nucleotides exchange factor (GEF) that activates RhoA as well as a scaffold protein integrating multiple signaling cascades. Previous study has shown that Akap13 hetero knockout mice develop osteoporotic changes and result in decreased osteogenesis and we believe AKAP13 is involved in the early stages of osteogenesis. On the other hand, the involvement of Wnt signaling in bone metabolism has been extensively studied, however the detailed mechanism has not been elucidated. Recent study showed that focal adhesion kinase (FAK)-integrin contributes to bone formation via Wnt signaling pathway. Thus, it is suggested that Akap13 may influence osteogenesis via Wnt signaling through FAK mediated integrin pathway that is linked to Rho GTPase. In this study, we investigated the involvement of AKAP13 in bone formation and the relationship with Wnt signaling. [Methods] uCT analyses of femurs of Akap13 heterozygous-conventional knock-out mice were performed. We transfected the Akap13 or control siRNA into osteoblastic cells and examined the Wnt-related genes expression and osteogenic markers by real time RT-PCR. Osteogenic marker expression was examined after application of RhoA expression vector with or without Akap13 siRNA to osteoblastic cells. [Results] Mice haploinsufficient for Akap13 (Akap13+/-) displayed reduced bone mineral density resembling the changes observed in osteoporotic bone. Colony forming unit-fibroblast numbers were diminished in Akap13+/- mice. Osteoblast numbers in periosteum and extracellular matrix production in Akap13+/- mice was reduced compared to control littermates. Runx2 and Alp mRNAs were both reduced in femora of Akap13+/- mice. Akap13 knockdown showed a significant decrease of Lef1 mRNA expression in MC3T3-E1 cells as well as a reduction of Runx2 and ALP mRNA expression in immortalized bone marrow stem cells (BMSCs). RhoA overexpression in BMSCs showed Alp mRNA increment in RhoA induction dose dependent manner. Reduction of Alp mRNA by AKAP13 knockdown was amplified in higher RhoA overexpression. [Discussion] The Akap13 knockdown showed decreased expression of Wnt-related genes in osteoblastic cells, suggesting the possibility that AKAP13 plays an important role in regulating osteogenesis through Wnt signaling. AKAP13 may be involved in or contribute directly to Wnt signaling through interaction with RhoA by mechanical stress or stimulation of extracellular matrix.<!--EndFragment-->

Effects of ionizing radiation and HLY78 on the zebrafish embryonic developmental toxicity

The health-related effects of ionizing radiation on embryonic development and their underlying mechanisms are still unclear. The aim of this study was to investigate the role of Wnt signaling in mediating the developmental toxicity induced by heavy ion and proton radiation using zebrafish embryos. Zebrafish embryos were radiated with carbon ions or protons. HLY78, an activator of the Wnt signaling pathway, was added immediately after radiation. Carbon ion radiation induced a significant increase of mortality, and activating Wnt signaling using HLY78 after radiation significantly alleviated this stress. Both carbon ion and proton radiation significantly increased malformation rates and decreased hatching rates. Supplementation with HLY78 significantly reduced the effects induced by carbon ion radiation alone. After irradiation with carbon ions, embryos showed a significant decrease in heart rate, spontaneous movement, and locomotive behavior. The expression of apoptotic genes was significantly increased, while the expression of anti-apoptotic and Wnt-related genes was significantly decreased. Supplementation with HLY78 was able to reduce these effects. However, embryos irradiated with proton radiation did not show significant changes in the expression of Wnt-related genes. The results of this study improve our understanding of the mechanisms of carbon ion radiation-induced developmental toxicity, which potentially involves the inhibition of Wnt signaling.

Exploring the Wnt signaling pathway in schizophrenia and bipolar disorder

The Wnt signaling pathway plays a crucial role in neurodevelopment and in regulating the function and structure of the adult nervous system. Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders with evidence of subtle neurodevelopmental, structural and functional neuronal abnormalities. We aimed to elucidate the role of aberrant regulation of the Wnt system in these disorders by evaluating plasma levels of secreted Wnt modulators in patients (SCZ = 551 and BD = 246) and healthy controls (HCs = 639) using enzyme immune-assay. We also investigated the expression of 141 Wnt-related genes in whole blood in a subsample (SCZ = 338, BD = 241, and HCs = 263) using microarray analysis. Both SCZ and BD had dysregulated mRNA expression of Wnt-related genes favoring attenuated canonical (beta-catenin-dependent) signaling, and there were also indices of enhanced non-canonical Wnt signaling. In particular, FZD7, which may activate all Wnt pathways, but favors non-canonical signaling, and NFATc3, a downstream transcription factor and readout of the non-canonical Wnt/Ca2+ pathway, were significantly increased in SCZ and BD (p < 3 × 10−4). Furthermore, patients had lower plasma levels of soluble dickkopf 1 and sclerostin (p < 0.01) compared with HC. Our findings suggest that SCZ and BD are characterized by abnormal Wnt gene expression and plasma protein levels, and we propose that drugs targeting the Wnt pathway may have a role in the treatment of severe mental disorders.

Silencing of the Drosophila ortholog of SOX5 leads to abnormal neuronal development and behavioral impairment.

SOX5 encodes a transcription factor that is expressed in multiple tissues including heart, lung and brain. Mutations in SOX5 have been previously found in patients with amyotrophic lateral sclerosis (ALS) and developmental delay, intellectual disability and dysmorphic features. To characterize the neuronal role of SOX5, we silenced the Drosophila ortholog of SOX5, Sox102F, by RNAi in various neuronal subtypes in Drosophila. Silencing of Sox102F led to misorientated and disorganized michrochaetes, neurons with shorter dendritic arborization (DA) and reduced complexity, diminished larval peristaltic contractions, loss of neuromuscular junction bouton structures, impaired olfactory perception, and severe neurodegeneration in brain. Silencing of SOX5 in human SH-SY5Y neuroblastoma cells resulted in a significant repression of WNT signaling activity and altered expression of WNT-related genes. Genetic association and meta-analyses of the results in several large family-based and case-control late-onset familial Alzheimer's disease (LOAD) samples of SOX5 variants revealed several variants that show significant association with AD disease status. In addition, analysis for rare and highly penetrate functional variants revealed four novel variants/mutations in SOX5, which taken together with functional prediction analysis, suggests a strong role of SOX5 causing AD in the carrier families. Collectively, these findings indicate that SOX5 is a novel candidate gene for LOAD with an important role in neuronal function. The genetic findings warrant further studies to identify and characterize SOX5 variants that confer risk for AD, ALS and intellectual disability.

Andrographolide Exerts Pro-Osteogenic Effect by Activation of Wnt/β-Catenin Signaling Pathway in Vitro

Background/Aims: Osteoporosis is a metabolic bone disorders that tortures about millions of people worldwide. Recent studies showed that Andrographolide (AP) is a promising natural compound for the treatment of osteoclast-related bone diseases. However, its potential in treatment of osteoporosis has not been fully explored. Methods: In this study, the effect of AP on osteoblasts metabolism was investigated via the detection of cell proliferation, cell viability, ALP activity, the expression of osteogenic specific genes including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), osteocalcin (OCN), Bone morphogenic protein-2 (BMP2) and Alkaline phosphatase(ALP) for 3, 5 and 7 days respectively. Further exploration of the association of AP with WNT/β-catenin signaling pathway was performed by examination of the expression of WNT related genes and proteins. Results: Results showed that AP of 4.46 and 8.92 µM, especially 8.92 µM was beneficial to osteogenic differentiation by upregulating ALP activity and expression of osteogenic related genes (P<0.05). Pathway analyses identify canonical WNT/β-catenin pathway as an important mediator in AP-induced osteogenesis. Conclusion: This study indicates that AP exerts its pro-osteogenic potential via activation of the WNT/β-catenin in osteoblasts and thus may represent a candidate of therapeutic agent for osteoporosis.

MiR-23a/-24-induced gene silencing results in mesothelial cell integration of pancreatic cancer.

Background:Invasion of the surrounding tissue is part of the metastatic cascade. Here, we examined the invasion of pancreatic ductal adenocarcinoma (PDAC) cells into the mesothelial barrier and identified the related microRNA (miRNA) expression profiles.Methods:The interactions between PDAC cells and mesothelial monolayers were characterised and quantified using a specific time-lapse videomicroscopy assay. Pancreatic ductal adenocarcinoma cells were further evaluated using the adhesion assay, and miRNA, mRNA and protein expressions were determined using microarray, q-RT–PCR and western blots, respectively. These data were correlated with in vivo dissemination scores.Results:Two groups of PDAC cell lines were distinguished by their integration capacity into the mesothelial monolayer using mean elongation factors (MEFs). Adhesion assays showed a concordant relation between adhesive properties and integration capacity. The distant metastases scores were reverse correlated with MEFs. Microarray analysis of these groups revealed that miR-23a and/or miR-24 target for FZD5, HNF1B and/or TMEM92, respectively, and that they are significantly deregulated.Conclusions:MiR-23a and/or miR-24 overexpression leads to gene silencing of FZD5, TMEM92 and/or HNF1B. Their downregulation induces deregulated expression and degradation of E-cadherin and β-catenin causing destabilisation of the cadherin/catenin complex, and altered the expression of Wnt-related genes. We propose a molecular (epi)genetic mechanism by which increased EMT-like cell shape transformation and integration into mesothelial monolayers of PDAC cells can be observed.

Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders

We reported previously that the expression of Wnt-related genes is lower in osteoporotic hip fractures than in osteoarthritis. We aimed to confirm those results by analyzing β-catenin levels and explored potential genetic and epigenetic mechanisms involved.β-Catenin gene expression and nuclear levels were analyzed by real time PCR and confocal immunofluorescence. Increased nuclear β-catenin was found in osteoblasts isolated from patients with osteoarthritis (99±4 units vs. 76±12, p=0.01, n=10), without differences in gene transcription, which is consistent with a post-translational down-regulation of β-catenin and decreased Wnt pathway activity.Twenty four single nucleotide polymorphisms (SNPs) of genes showing differential expression between fractures and osteoarthritis (WNT4, WNT10A, WNT16 and SFRP1) were analyzed in DNA isolated from blood of 853 patients. The genotypic frequencies were similar in both groups of patients, with no significant differences.Methylation of Wnt pathway genes was analyzed in bone tissue samples (15 with fractures and 15 with osteoarthritis) by interrogating a CpG-based methylation array. Six genes showed significant methylation differences between both groups of patients: FZD10, TBL1X, CSNK1E, WNT8A, CSNK1A1L and SFRP4. The DNA demethylating agent 5-deoxycytidine up-regulated 8 genes, including FZD10, in an osteoblast-like cell line, whereas it down-regulated other 16 genes.In conclusion, Wnt activity is reduced in patients with hip fractures, in comparison with those with osteoarthritis. It does not appear to be related to differences in the allele frequencies of the Wnt genes studied. On the other hand, methylation differences between both groups could contribute to explain the differences in Wnt activity.

Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage.

Objective:It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue.Methods:Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes.Results:Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca2+/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers.Conclusions:WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca2+/WNT pathway was activated in OA cartilage.