Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells.

Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFßSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax- and SMAD-mediated transactivation of CDKN1C and CDKN1A. Notably, combined DYRK1A and TGFß inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both in vitro and in vivo.

Insights into beta cell regeneration for diabetes via integration of molecular landscapes in human insulinomas.

Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the "genomic recipe" for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells. Here, we show at the pathway level that the majority of the insulinomas display mutations, copy number variants and/or dysregulation of epigenetic modifying genes, most prominently in the polycomb and trithorax families. Importantly, these processes are coupled to co-expression network modules associated with cell proliferation, revealing candidates for inducing beta cell regeneration. Validation of key computational predictions supports the concept that understanding the molecular complexity of insulinoma may be a valuable approach to diabetes drug discovery.Diabetes results in part from a deficiency of functional pancreatic beta cells. Here, the authors study the genomic and epigenetic landscapes of human insulinomas to gain insight into possible pathways for therapeutic beta cell regeneration, highlighting epigenetic genes and pathways.

Effects of N-Substitutions on the Tetrahydroquinoline (THQ) Core of Mixed-Efficacy µ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Ligands.

N-Acetylation of the tetrahydroquinoline (THQ) core of a series of µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist ligands increases DOR affinity, resulting in ligands with balanced MOR and DOR affinities. We report a series of N-substituted THQ analogues that incorporate various carbonyl-containing moieties to maintain DOR affinity and define the steric and electronic requirements of the binding pocket across the opioid receptors. 4h produced in vivo antinociception (ip) for 1 h at 10 mg/kg.

4E-BP2/SH2B1/IRS2 Are Part of a Novel Feedback Loop That Controls ß-Cell Mass.

The mammalian target of rapamycin complex 1 (mTORC1) regulates several biological processes, although the key downstream mechanisms responsible for these effects are poorly defined. Using mice with deletion of eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), we determine that this downstream target is a major regulator of glucose homeostasis and ß-cell mass, proliferation, and survival by increasing insulin receptor substrate 2 (IRS2) levels and identify a novel feedback mechanism by which mTORC1 signaling increases IRS2 levels. In this feedback loop, we show that 4E-BP2 deletion induces translation of the adaptor protein SH2B1 and promotes the formation of a complex with IRS2 and Janus kinase 2, preventing IRS2 ubiquitination. The changes in IRS2 levels result in increases in cell cycle progression, cell survival, and ß-cell mass by increasing Akt signaling and reducing p27 levels. Importantly, 4E-BP2 deletion confers resistance to cytokine treatment in vitro. Our data identify SH2B1 as a major regulator of IRS2 stability, demonstrate a novel feedback mechanism linking mTORC1 signaling with IRS2, and identify 4E-BP2 as a major regulator of proliferation and survival of ß-cells.

A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.

Types 1 and 2 diabetes affect some 380 million people worldwide. Both ultimately result from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with a peak percentage (∼2%) engaged in the cell cycle in the first year of life. In embryonic life and after early childhood, beta cell replication is barely detectable. Whereas beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts. Hence, there remains an urgent need for antidiabetic therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, using a high-throughput small-molecule screen (HTS), we find that analogs of the small molecule harmine function as a new class of human beta cell mitogenic compounds. We also define dual-specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine and the nuclear factors of activated T cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation and differentiation. Using three different mouse and human islet in vivo-based models, we show that harmine is able to induce beta cell proliferation, increase islet mass and improve glycemic control. These observations suggest that harmine analogs may have unique therapeutic promise for human diabetes therapy. Enhancing the potency and beta cell specificity of these compounds are important future challenges.

Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA.

A cell line, WE-spleen6, has been developed from the stromal layer of primary spleen cell cultures. On conventional plastic, WE-spleen6 cells had a spindle-shaped morphology at low cell density but grew to become epithelial-like at confluency. On the commercial extracellular matrix (ECM), Matrigel, the cells remained spindle-shaped and formed lumen-like structures. WE-spleen6 cells had intermediate filament protein, vimentin and the ECM protein, collagen I, but not smooth muscle α-actin (SMA) and von Willebrand factor (vWF) and lacked alkaline phosphatase and phagocytic activities. WE-spleen6 was more susceptible to infection with VHSV IVb than a fibroblast and epithelial cell lines from the walleye caudal fin, WE-cfin11f and WE-cfin11e, respectively. Viral transcripts and proteins appeared earlier in WE-spleen6 cultures as did cytopathic effect (CPE) and significant virus production. The synthetic double-stranded RNA (dsRNA), polyinosinic: polycytidylic acid (pIC), induced the antiviral protein Mx in both cell lines. Treating WE-spleen6 cultures with pIC prior to infection with VHSV IVb inhibited the early accumulation of viral transcripts and proteins and delayed the appearance of CPE and significant viral production. Of particular note, pIC caused the disappearance of viral P protein 2 days post infection. WE-spleen6 should be useful for investigating the impact of VHSV IVb on hematopoietic organs and the actions of pIC on the rhabdovirus life cycle.

Quantity and three-dimensional position of the recurrent and superior laryngeal nerve lower motor neurons in a rat model.

OBJECTIVES: We sought to elucidate the 3-dimensional position and quantify the lower motor neurons (LMNs) of the recurrent laryngeal nerve (RLN) and the superior laryngeal nerve (SLN) in a rat model. Quantification and mapping of these neurons will enhance the usefulness of the rat model in the study of reinnervation following trauma to these nerves. METHODS: Female Sprague-Dawley rats underwent microsurgical transection of the RLN, the SLN, or both the RLN and SLN or sham surgery. After transection, either Fluoro-Ruby (FR) or Fluoro-Gold (FG) was applied to the proximal nerve stumps. The brain stems were harvested, sectioned, and examined for fluorolabeling. The LMNs were quantified, and their 3-dimensional position within the nucleus ambiguus was mapped. RESULTS: Labeling of the RLN was consistent regardless of the labeling agent used. A mean of 243 LMNs was documented for the RLN. The SLN labeling with FR was consistent and showed a mean of 117 LMNs; however, FG proved to be highly variable in labeling the SLN. The SLN LMNs lie rostral and ventral to those of the RLN. In the sham surgical condition, FG was noted to contaminate adjacent tissues--in particular, in the region of the SLN. CONCLUSIONS: Fluorolabeling is an effective tool to locate and quantify the LMNs of the RLN and SLN. The LMN positions and counts were consistent when FR was used in labeling of either the RLN or the SLN. Fluoro-Gold, however, because of its tendency to contaminate surrounding structures, can only be used to label the RLN. Also, as previously reported, the SLN LMNs lie rostral and ventral to those of the RLN. This information results in further clarification of a rat model of RLN injury that may be used to investigate the effects of neurotrophic factors on RLN reinnervation.

Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas.

The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors.

Whole-body sleeping beauty mutagenesis can cause penetrant leukemia/lymphoma and rare high-grade glioma without associated embryonic lethality.

The Sleeping Beauty (SB) transposon system has been used as a somatic mutagen to identify candidate cancer genes. In previous studies, efficient leukemia/lymphoma formation on an otherwise wild-type genetic background occurred in mice undergoing whole-body mobilization of transposons, but was accompanied by high levels of embryonic lethality. To explore the utility of SB for large-scale cancer gene discovery projects, we have generated mice that carry combinations of different transposon and transposase transgenes. We have identified a transposon/transposase combination that promotes highly penetrant leukemia/lymphoma formation on an otherwise wild-type genetic background, yet does not cause embryonic lethality. Infiltrating gliomas also occurred at lower penetrance in these mice. SB-induced or accelerated tumors do not harbor large numbers of chromosomal amplifications or deletions, indicating that transposon mobilization likely promotes tumor formation by insertional mutagenesis of cancer genes, and not by promoting wide-scale genomic instability. Cloning of transposon insertions from lymphomas/leukemias identified common insertion sites at known and candidate novel cancer genes. These data indicate that a high mutagenesis rate can be achieved using SB without high levels of embryonic lethality or genomic instability. Furthermore, the SB system could be used to identify new genes involved in lymphomagenesis/leukemogenesis.

lin-35/Rb and the CoREST ortholog spr-1 coordinately regulate vulval morphogenesis and gonad development in C. elegans.

Using a genetic screen to identify genes that carry out redundant functions during development with lin-35/Rb, the C. elegans Retinoblastoma family ortholog, we have identified a mutation in spr-1. spr-1 encodes the C. elegans ortholog of human CoREST, a protein containing Myb-like SANT and ELM2 domains, which functions as part of a transcriptional regulatory complex. CoREST recruits mediators of transcriptional repression, including histone deacetylase, and demethylase, and interacts with the tumor suppression protein REST. spr-1/CoREST was previously shown in C. elegans to suppress defects associated with loss of the presenilin sel-12, which functions in the proteolytic processing of LIN-12/Notch. Here we show that lin-35 and spr-1 coordinately regulate several developmental processes in C. elegans including the ingression of vulval cells as well as germline proliferation. We also show that loss of lin-35 and spr-1 hypersensitizes animals to a reduction in LIN-12/Notch activity, leading to the generation of proximal germline tumors. This defect, which is observed in lin-35; spr-1; lin-12(RNAi) and lin-35; spr-1; hop-1(RNAi) triple mutants is likely due to a delay in the entry of germ cells into meiosis.

lin-35/Rb and xnp-1/ATR-X function redundantly to control somatic gonad development in C. elegans.

In screens for genetic modifiers of lin-35/Rb, the C. elegans retinoblastoma protein (Rb) homolog, we have identified a mutation in xnp-1. Mutations in xnp-1, including a presumed null allele, are viable and, in general, appear indistinguishable from the wild type. In contrast, xnp-1 lin-35 double mutants are typically sterile and exhibit severe defects in gonadal development. Analyses of the abnormal gonads indicate a defect in the lineages that generate cells of the sheath and spermatheca. xnp-1 encodes the C. elegans homolog of ATR-X, a human disease gene associated with severe forms of mental retardation and urogenital developmental defects. xnp-1/ATR-X is a member of the Swi2/Snf2 family of ATP-dependent DEAD/DEAH box helicases, which function in nucleosome remodeling and transcriptional regulation. Expression of an xnp-1 Colon, two colons GFP promoter fusion is detected throughout C. elegans development in several cell types including neurons and cells of the somatic gonad. Our findings demonstrate a new biological role for Rb family members in somatic gonad development and implicate lin-35 in the execution of multiple cell fates in C. elegans. In addition, our results suggest a possible conserved function for xnp-1/ATR-X in gonadal development across species.