A multiparametric calcium signal screening platform using iPSC-derived cortical neural spheroids.

Discovery of therapeutics for neurological diseases is hampered by the lack of predictive in vitro and in vivo models. Traditionally, in vitro assays rely on engineered cell lines grown two-dimensionally (2D) outside a physiological tissue context, which makes them very amenable for large scale drug screening but reduces their relevance to in vivo neurophysiology. In recent years, three-dimensional (3D) neural cell culture models derived from human induced pluripotent stem cells (iPSCs) have been developed as an in vitro assay platform to investigate brain development, neurological diseases, and for drug screening. iPSC-derived neural spheroids or organoids can be developed to include complex neuronal and glial cell populations and display spontaneous, synchronous activity, which is a hallmark of in vivo neural communication. In this report we present a proof-of-concept study evaluating 3D iPSC-derived cortical neural spheroids as a physiologically- and pharmacologically-relevant high-throughput screening (HTS) platform and investigate their potential for use for therapeutic development. To this end, a library of 687 neuroactive compounds were tested in a phenotypic screening paradigm which measured calcium activity as a functional biomarker for neural modulation through fluctuations in calcium fluorescence. Pharmacological responses of cortical neural spheroids were analyzed using a multi-parametric approach, whereby seven peak characteristics from the calcium activity in each well were quantified and incorporated into principal component analysis and Sammon mapping to measure compound response. Here, we describe the implementation of the 687-compound library screen and data analysis demonstrating that iPSC-derived cortical spheroids are a robust and information-rich assay platform for HTS.

Screening for modulators of neural network activity in 3D human iPSC-derived cortical spheroids.

Human induced Pluripotent Stem Cells (iPSCs) are a powerful tool to dissect the biology of complex human cell types such as those of the central nervous system (CNS). However, robust, high-throughput platforms for reliably measuring activity in human iPSC-derived neuronal cultures are lacking. Here, we assessed 3D cultures of cortical neurons and astrocytes displaying spontaneous, rhythmic, and highly synchronized neural activity that can be visualized as calcium oscillations on standard high-throughput fluorescent readers as a platform for CNS-based discovery efforts. Spontaneous activity and spheroid structure were highly consistent from well-to-well, reference compounds such as TTX, 4-AP, AP5, and NBQX, had expected effects on neural spontaneous activity, demonstrating the presence of functionally integrated neuronal circuitry. Neurospheroid biology was challenged by screening the LOPAC®1280 library, a collection of 1280 pharmacologically active small molecules. The primary screen identified 111 compounds (8.7%) that modulated neural network activity across a wide range of neural and cellular processes and 16 of 17 compounds chosen for follow-up confirmed the primary screen results. Together, these data demonstrate the suitability and utility of human iPSC-derived neurospheroids as a screening platform for CNS-based drug discovery.

Functional and Mechanistic Neurotoxicity Profiling Using Human iPSC-Derived Neural 3D Cultures.

Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity. Here, we employed a human induced pluripotent stem cell (iPSC)-based 3D neural platform composed of mature cortical neurons and astrocytes as a model for this purpose. The iPSC-derived human 3D cortical neuron/astrocyte co-cultures (3D neural cultures) present spontaneous synchronized, readily detectable calcium oscillations. This advanced neural platform was optimized for high-throughput screening in 384-well plates and displays highly consistent, functional performance across different wells and plates. Characterization of oscillation profiles in 3D neural cultures was performed through multi-parametric analysis that included the calcium oscillation rate and peak width, amplitude, and waveform irregularities. Cellular and mitochondrial toxicity were assessed by high-content imaging. For assay characterization, we used a set of neuromodulators with known mechanisms of action. We then explored the neurotoxic profile of a library of 87 compounds that included pharmaceutical drugs, pesticides, flame retardants, and other chemicals. Our results demonstrated that 57% of the tested compounds exhibited effects in the assay. The compounds were then ranked according to their effective concentrations based on in vitro activity. Our results show that a human iPSC-derived 3D neural culture assay platform is a promising biologically relevant tool to assess the neurotoxic potential of drugs and environmental toxicants.

Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.

Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC). We investigated potential bypass mechanisms to PI3K/mTOR inhibition in KRAS-mutant CRC. Using genetically engineered mouse model cells that had acquired resistance to the dual PI3K/mTOR small-molecule inhibitor PF-04691502, we determined with chemical library screens that inhibitors of the ERBB [epidermal growth factor receptor (EGFR)] family restored the sensitivity to PF-04691502. Although EGFR inhibitors alone have limited efficacy in reducing KRAS-mutant tumors, we found that PF-04691502 induced the abundance, phosphorylation, and activity of EGFR, ERBB2, and ERBB3 through activation of FOXO3a (forkhead box O 3a), a transcription factor inhibited by the PI3K to AKT pathway. PF-04691502 also induced a stem cell-like gene expression signature. KRAS-mutant patient-derived xenografts from mice treated with PF-04691502 had a similar gene expression signature and exhibited increased EGFR activation, suggesting that this drug-induced resistance mechanism may occur in patients. Combination therapy with dacomitinib (a pan-ERBB inhibitor) restored sensitivity to PF-04691502 in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Our findings suggest that combining PI3K/mTOR and EGFR inhibitors may improve therapeutic outcome in patients with KRAS-mutant CRC.

Chemogenetic evaluation of the mitotic kinesin CENP-E reveals a critical role in triple-negative breast cancer.

Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E-dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic-pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer.

Potent agonists of the Hedgehog signaling pathway.

A family of biaryl substituted 1,4-diaminocyclohexanamides of 3-chlorobenzothiophene-2-carboxylic acid is reported as picomolar modulators of Hedgehog protein function. SAR for the 1,4-diaminocyclohexane group is shown to be exquisitely sensitive to substitution on the 4-amino group, and SAR for the 3-chlorobenzothiophene group is highly specific. Preliminary SAR studies of the biaryl substituent led to a picomolar compound with in vivo activity.

Potent inhibitors of the hedgehog signaling pathway.

A small family of phenyl quinazolinone ureas is reported as potent modulators of Hedgehog protein function. Preliminary SAR studies of the urea substituent led to a nanomolar Hedgehog antagonist.

IFNalpha induces Fas expression and apoptosis in hedgehog pathway activated BCC cells through inhibiting Ras-Erk signaling.

Basal cell carcinoma (BCC), the most common form of human cancer, is understood to be associated with activation of the sonic hedgehog pathway, through loss-of-function mutations of tumor suppressor PTCH1 or gain-of-function mutations of smoothened. Interferon (IFN)-based therapy is quite effective in BCC treatment, but the molecular basis is not well understood. Here we report a novel mechanism by which IFNalpha mediates apoptosis in BCCs. In the presence of IFNalpha, we observed increased apoptosis in a BCC cell line ASZ001, in which PTC is null, and therefore with constitutive activation of the sonic hedgehog pathway. We demonstrate that SMO agonist Ag-1.4 mediates activation of extracellular signal-regulated kinase (Erk) phosphorylation, which is abrogated by IFNalpha in sonic hedgehog responsive C3H10T1/2 cells. In transient transfection experiments, we demonstrate that IFNalpha inhibits Erk phosphorylation and serum response element activation induced by expression of SMO, Gli1, PDGFRalpha and activated Raf, but not activated mitogen-activated Erk-regulating kinase (Mek), suggesting that IFNalpha targets mainly on Mek function. We further show that IFNalpha induces expression of Fas in BCC cells through interfering with Mek function. The role of the Fas-L/Fas signaling axis in IFNalpha-mediated apoptosis is demonstrated by the fact that addition of Fas-L neutralizing antibodies, just as caspase-8 inhibitor Z-IETD-FMK, effectively prevents IFNalpha-mediated apoptosis. Thus, our data indicate that IFNalpha-based BCC therapy induces Fas expression and apoptosis through interfering with Mek function.

Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions.

The link between basal cell carcinoma (BCC) and aberrant activation of the Hedgehog (Hh) signaling pathway has been well established in humans and in mouse models. Here we report the development of assays, including two novel in vitro BCC models, which allowed us to screen for Hh inhibitors and test their validity as potential treatments for BCC. We identified a novel small molecule Hh inhibitor (CUR61414) that can block elevated Hh signaling activity resulting from oncogenic mutations in Patched-1. Moreover, CUR61414 can suppress proliferation and induce apoptosis of basaloid nests in the BCC model systems, whereas having no effect on normal skin cells. These findings directly demonstrate that the use of Hh inhibitors could be a valid therapeutic approach for treating BCC.

Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists.

BACKGROUND: The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc. RESULTS: We have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro, while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null, but not Smo-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo. CONCLUSIONS: Smo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.

Sonic hedgehog is a potent inducer of rat oligodendrocyte development from cortical precursors in vitro.

Sonic Hedgehog (Shh) induces oligodendrocyte development in the ventral neural tube and telencephalon but its role in oligodendrocyte generation in dorsal telencephalon is debated. Transcripts for Shh and its receptor complex were detected in subventricular zone and neocortex from E17 to birth. As Shh is not yet expressed in E15 neocortex, we grew E15 cortical precursors (CP) into neurospheres in the presence of recombinant Octyl-Shh (O-Shh). After sphere adhesion and removal of O-Shh, enhanced neurite outgrowth and cell migration were already observed at 3 h. Three days after O-Shh treatment, oligodendrocyte progenitors (OP) emerged and continued to increase in number for 7 days while the ratio of neuronal cells decreased compared to control. Shh selectively triggered mitosis of OP but not neuronal progenitors and enhanced growth of neonatal OP. Thus Shh in E15-17 embryonic neocortex can signal CP to adopt an oligodendrocyte fate and favors expansion of this lineage.