Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC50 of ~10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

Insulinoma associated 1 (Insm1) plays an important role in regulating the development of cells in the central and peripheral nervous systems, olfactory epithelium and endocrine pancreas. To better define the role of Insm1 in pancreatic endocrine cell development we generated mice with an Insm1GFPCre reporter allele and used them to study Insm1-expressing and null populations. Endocrine progenitor cells lacking Insm1 were less differentiated and exhibited broad defects in hormone production, cell proliferation and cell migration. Embryos lacking Insm1 contained greater amounts of a non-coding Neurog3 mRNA splice variant and had fewer Neurog3/Insm1 co-expressing progenitor cells, suggesting that Insm1 positively regulates Neurog3. Moreover, endocrine progenitor cells that express either high or low levels of Pdx1, and thus may be biased towards the formation of specific cell lineages, exhibited cell type-specific differences in the genes regulated by Insm1. Analysis of the function of Ripply3, an Insm1-regulated gene enriched in the Pdx1-high cell population, revealed that it negatively regulates the proliferation of early endocrine cells. Taken together, these findings indicate that in developing pancreatic endocrine cells Insm1 promotes the transition from a ductal progenitor to a committed endocrine cell by repressing a progenitor cell program and activating genes essential for RNA splicing, cell migration, controlled cellular proliferation, vasculogenesis, extracellular matrix and hormone secretion.

SUMMARYCoordinated movement depends on the creation of synapses between specific neurons in the motor circuit. In C. elegans, this important decision is regulated by the UNC-4 homeodomain protein. unc-4 mutants are unable to execute backward locomotion because VA motor neurons are mis-wired with inputs normally reserved for their VB sisters. We have proposed that UNC-4 functions in VAs to block expression of VB genes. This model is substantiated by the finding that ectopic expression of the VB gene ceh-12 (encoding a homolog of the homeodomain protein HB9) in unc-4 mutants results in the mis-wiring of posterior VA motor neurons with VB-like connections. Here, we show that VA expression of CEH-12 depends on a nearby source of the Wnt protein EGL-20. Our results indicate that UNC-4 prevents VAs from responding to a local EGL-20 cue by disabling a canonical Wnt signaling cascade involving the Frizzled receptors MIG-1 and MOM-5. CEH-12 expression in VA motor neurons is also opposed by a separate pathway that includes the Wnt ligand LIN-44. This work has revealed a transcriptional mechanism for modulating the sensitivity of specific neurons to diffusible Wnt ligands and thereby defines distinct patterns of synaptic connectivity. The existence of comparable Wnt gradients in the vertebrate spinal cord could reflect similar roles for Wnt signaling in vertebrate motor circuit assembly.

Summary Using a transgenic mouse model to express MafA, Pdx1, and Neurog3 (3TF) in a pancreatic acinar cell- and doxycycline-dependent manner, we discovered that the outcome of transcription factor-mediated acinar to β-like cellular reprogramming is dependent on both the magnitude of 3TF expression and on reprogramming-induced inflammation. Overly robust 3TF expression causes acinar cell necrosis resulting in marked inflammation and acinar-to-ductal metaplasia. Generation of new β-like cells requires limiting reprogramming-induced inflammation, either by reducing 3TF expression or by eliminating macrophages. The new β-like cells were able to reverse streptozotocin-induced diabetes 6 days after inducing 3TF expression but failed to sustain their function after removal of the reprogramming factors.

e14047 Background: There is currently a lack of easily-accessible data regarding the volume and type of cancer patients seen at VUMC, despite extensive use of an electronic medical record (EMR) with a dedicated tumor registry. This leads to errors in estimating metrics that depend on subject accrual to clinical trials. We aimed to develop a tool to determine the number of head and neck squamous cell carcinoma (HNSCC) patients, stratified by desired subsets, seen yearly at the VUMC in order to achieve a more accurate and efficacious approach to estimating future patient accrual to clinical trials. Methods: Working with Nashville Biosciences, we identified patients with HNSCC using ICD codes in combination with VUMC’s Tumor Registry data (collected for patients diagnosed and treated at VUMC). We estimated the rate of accrual of HNSCC patient blood samples (a proxy for ICD code) based on the number of specimens acquired over the past 3 years. Each patient was counted only once based on their first blood sample acquired during this period, based on a comprehensive list of laboratories and clinical procedures that require a specimen to be drawn. We then evaluated the distribution of patients by primary site, standardized to SEER registry terminology. We also investigated what fraction of patients received treatment with surgery, radiation, and/or chemotherapy (we expect that this includes treatment with biologics) using a combination of ICD9/10 and CPT coding. Results: We identified a total of 1,131 HNSCC patients. SEER summary staging information was available for 603 patients - 432 (72%) had Stage 1-3 disease and 171 (28%) had Stage 4 disease. Primary site data was available for 717 patients - 648 (90%) HNSCC of the oral cavity, 20 (3%) had HNSCC of the oropharynx, 29 (4%) HNSCC of the larynx, 20 (3%) HNSCC of the hypopharynx. Of all patients, we found that 598 had at least 1 code for chemotherapy, 540 for radiotherapy, and 825 for surgery such that the majority (96%) received cancer treatment. We have not evaluated the timing of the treatment relative to diagnosis. Conclusions: By using ICD codes and tumor registry data extracted from VUMC’s EMR, we determined the site distribution and type of treatment for HNSCC over a 3-year period. The disease site distribution did not match prior experience Refinements are ongoing. However, once improved, these methods may be used to estimate future clinical trial accrual, thus reducing start-up time, cost and poor trial site performance, while increasing accrual.