Person:

Rubin, Lee

Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smo’s actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies.

The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK–PI3K–AKT–GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA.

Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. Here, we identify five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons that recapitulate the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibit TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we show that the technique can reveal novel aspects of human disease phenotypes in vitro.

Purpose: Inappropriate activation of the Hedgehog (Hh) signaling pathway in skin is critical for the development of basal cell carcinomas (BCC). We have investigated the anti-BCC efficacy of topically-applied CUR61414, an inhibitor of the Hh signal transduction molecule Smoothened. Experimental Design: In preclinical studies, we used a depilatory model to evaluate the ability of topical formulations of CUR61414 to repress Hh responsive cells found at the base of hair follicles in normal skin. We also tested the in vivo effects of topical CUR61414 on murine BCCs developed in (Ptch1^{ +/−}) K14-CreER2 p53 fl/fl mice. In a phase I clinical study, we evaluated the safety, tolerability, and efficacy of a multidose regimen of CUR61414 (0.09%, 0.35%, 1.1%, and 3.1%) applied topically to human superficial or nodular BCCs for up to 28 days. Results: In mice, topical CUR61414 significantly inhibited skin Hh signaling, blocked the induction of hair follicle anagen, and shrank existing BCCs. However, we observed no clinical activity of this formulation in human superficial or nodular BCCs in a phase I clinical study. Conclusions: Our data highlight some of the challenges of translating preclinical experience into successful human results for a topical anticancer agent.

The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by Western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration.

The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA.

Dysfunction or death of pancreatic β cells underlies both types of diabetes. This functional decline begins with β cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate β cell stress nor prevent, let alone reverse, β cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature β cells, is down-regulated in the early stages of T2D in mice and when β cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature β cell state, we screen for factors that reverse β cell de-differentiation. We find that a small molecule inhibitor of TGFβ receptor I (Alk5) protects cells from the loss of key β cell transcription factors and restores a mature β cell identity even after exposure to prolonged and severe diabetes. DOI: http://dx.doi.org/10.7554/eLife.02809.001

Stepwise differentiation from embryonic stem cells (ESCs) to functional insulin-secreting beta cells will identify key steps in beta-cell development and may yet prove useful for transplantation therapy for diabetics. An essential step in this schema is the generation of pancreatic progenitors—cells that express Pdx1 and produce all the cell types of the pancreas. High-content chemical screening identified a small molecule, (-)-indolactam V, that induces differentiation of a substantial number of Pdx1-expressing cells from human ESCs. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine, exocrine and duct cells, in vitro and in vivo. Further analyses showed that (-)-indolactam V works specifically at one stage of pancreatic development, inducing pancreatic progenitors from definitive endoderm. This study describes a chemical screening platform to investigate human ESC differentiation and demonstrates the generation of a cell population that is a key milepost on the path to making beta cells.

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.