HDAC3 Regulates the Transition to the Homeostatic Myelinating Schwann Cell State.

The formation of myelinating Schwann cells (mSCs) involves the remarkable biogenic process, which rapidly generates the myelin sheath. Once formed, the mSC transitions to a stable homeostatic state, with loss of this stability associated with neuropathies. The histone deacetylases histone deacetylase 1 (HDAC1) and HDAC2 are required for the myelination transcriptional program. Here, we show a distinct role for HDAC3, in that, while dispensable for the formation of mSCs, it is essential for the stability of the myelin sheath once formed-with loss resulting in progressive severe neuropathy in adulthood. This is associated with the prior failure to downregulate the biogenic program upon entering the homeostatic state leading to hypertrophy and hypermyelination of the mSCs, progressing to the development of severe myelination defects. Our results highlight distinct roles of HDAC1/2 and HDAC3 in controlling the differentiation and homeostatic states of a cell with broad implications for the understanding of this important cell-state transition.

Design of a small molecule against an oncogenic noncoding RNA.

The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

Therapeutic targeting of casein kinase 1δ in breast cancer.

Identification of specific drivers of human cancer is required to instruct the development of targeted therapeutics. We demonstrate that CSNK1D is amplified and/or overexpressed in human breast tumors and that casein kinase 1δ (CK1δ) is a vulnerability of human breast cancer subtypes overexpressing this kinase. Specifically, selective knockdown of CK1δ, or treatment with a highly selective and potent CK1δ inhibitor, triggers apoptosis of CK1δ-expressing breast tumor cells ex vivo, tumor regression in orthotopic models of triple-negative breast cancer, including patient-derived xenografts, and tumor growth inhibition in human epidermal growth factor receptor 2-positive (HER2(+)) breast cancer models. We also show that Wnt/ß-catenin signaling is a hallmark of human tumors overexpressing CK1δ, that disabling CK1δ blocks nuclear accumulation of ß-catenin and T cell factor transcriptional activity, and that constitutively active ß-catenin overrides the effects of inhibition or silencing of CK1δ. Thus, CK1δ inhibition represents a promising strategy for targeted treatment in human breast cancer with Wnt/ß-catenin involvement.

Identification of an EGFRvIII-JNK2-HGF/c-Met-Signaling Axis Required for Intercellular Crosstalk and Glioblastoma Multiforme Cell Invasion.

Glioblastoma multiforme (GBM) is the most aggressive and common form of adult brain cancer. Current therapeutic strategies include surgical resection, followed by radiotherapy and chemotherapy. Despite such aggressive multimodal therapy, prognosis remains poor, with a median patient survival of 14 months. A proper understanding of the molecular drivers responsible for GBM progression are therefore necessary to instruct the development of novel targeted agents and enable the design of effective treatment strategies. Activation of the c-Jun N-terminal kinase isoform 2 (JNK2) is reported in primary brain cancers, where it associates with the histologic grade and amplification of the epidermal growth factor receptor (EGFR). In this manuscript, we demonstrate an important role for JNK2 in the tumor promoting an invasive capacity of EGFR variant III, a constitutively active mutant form of the receptor commonly found in GBM. Expression of EGFR variant III induces transactivation of JNK2 in GBM cells, which is required for a tumorigenic phenotype in vivo. Furthermore, JNK2 expression and activity is required to promote increased cellular invasion through stimulation of a hepatocyte growth factor-c-Met signaling circuit, whereby secretion of this extracellular ligand activates the receptor tyrosine kinase in both a cell autonomous and nonautonomous manner. Collectively, these findings demonstrate the cooperative and parallel activation of multiple RTKs in GBM and suggest that the development of selective JNK2 inhibitors could be therapeutically beneficial either as single agents or in combination with inhibitors of EGFR and/or c-Met.

Macrophage-Induced Blood Vessels Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves.

The peripheral nervous system has remarkable regenerative capacities in that it can repair a fully cut nerve. This requires Schwann cells to migrate collectively to guide regrowing axons across a 'bridge' of new tissue, which forms to reconnect a severed nerve. Here we show that blood vessels direct the migrating cords of Schwann cells. This multicellular process is initiated by hypoxia, selectively sensed by macrophages within the bridge, which via VEGF-A secretion induce a polarized vasculature that relieves the hypoxia. Schwann cells then use the blood vessels as "tracks" to cross the bridge taking regrowing axons with them. Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair. This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue.

Development of an HTS-Compatible Assay for the Discovery of Ulk1 Inhibitors.

A rapidly accumulating body of work suggests the autophagy pathway is an attractive therapeutic target for neurodegenerative diseases and cancer. To validate autophagy as an anticancer strategy and to assess if systemic inhibition of the pathway will have deleterious effects on normal tissues and physiology, highly selective autophagy inhibitors are needed. While several inducers and inhibitors of autophagy are known, all are nonspecific and none target the enzymes that execute the pathway. A central upstream regulator of the autophagy pathway is the serine/threonine kinase Ulk1 (UNC-51-like kinase-1). Selective molecular probes that function as Ulk1-specific inhibitors are needed to improve our understanding of the autophagy pathway. To identify inhibitors of Ulk1 kinase activity, we developed an HTS-compatible, homogeneous biochemical assay using AlphaScreen technology. This novel assay design uses purified stress-activated Ulk1 and monitors phosphorylation of its full-length native substrate, Atg13. This assay was optimized and validated in a 384-well format by screening the Sigma LOPAC library. Here we report that the Ulk1 AlphaScreen assay is robust and reproducible, with a Z' factor value of 0.83 ± 0.02 and a signal to background ratio of 20 ± 1.2. Thus, this assay can be used to screen large chemical libraries to discover novel inhibitors of Ulk1.

Development of highly selective casein kinase 1δ/1ε (CK1δ/ε) inhibitors with potent antiproliferative properties.

The development of a series of potent and highly selective casein kinase 1δ/ε (CK1δ/ε) inhibitors is described. Starting from a purine scaffold inhibitor (SR-653234) identified by high throughput screening, we developed a series of potent and highly kinase selective inhibitors, including SR-2890 and SR-3029, which have IC50 ≤ 50 nM versus CK1δ. The two lead compounds have ≤100 nM EC50 values in MTT assays against the human A375 melanoma cell line and have physical, in vitro and in vivo PK properties suitable for use in proof of principle animal xenograft studies against human cancer cell lines.

A central role for the ERK-signaling pathway in controlling Schwann cell plasticity and peripheral nerve regeneration in vivo.

Following damage to peripheral nerves, a remarkable process of clearance and regeneration takes place. Axons downstream of the injury degenerate, while the nerve is remodeled to direct axonal regrowth. Schwann cells are important for this regenerative process. "Sensing" damaged axons, they dedifferentiate to a progenitor-like state, in which they aid nerve regeneration. Here, we demonstrate that activation of an inducible Raf-kinase transgene in myelinated Schwann cells is sufficient to control this plasticity by inducing severe demyelination in the absence of axonal damage, with the period of demyelination/ataxia determined by the duration of Raf activation. Remarkably, activation of Raf-kinase also induces much of the inflammatory response important for nerve repair, including breakdown of the blood-nerve barrier and the influx of inflammatory cells. This reversible in vivo model identifies a central role for ERK signaling in Schwann cells in orchestrating nerve repair and is a powerful system for studying peripheral neuropathies and cancer.

NF1 loss disrupts Schwann cell-axonal interactions: a novel role for semaphorin 4F.

Neurofibromatosis type 1 (NF1) patients develop neurofibromas, tumors of Schwann cell origin, as a result of loss of the Ras-GAP neurofibromin. In normal nerves, Schwann cells are found tightly associated with axons, while loss of axonal contact is a frequent and important early event in neurofibroma development. However, the molecular basis of this physical interaction or how it is disrupted in cancer remains unclear. Here we show that loss of neurofibromin in Schwann cells is sufficient to disrupt Schwann cell/axonal interactions via up-regulation of the Ras/Raf/ERK signaling pathway. Importantly, we identify down-regulation of semaphorin 4F (Sema4F) as the molecular mechanism responsible for the Ras-mediated loss of interactions. In heterotypic cocultures, Sema4F knockdown induced Schwann cell proliferation by relieving axonal contact-inhibitory signals, providing a mechanism through which loss of axonal contact contributes to tumorigenesis. Importantly, Sema4F levels were strongly reduced in a panel of human neurofibromas, confirming the relevance of these findings to the human disease. This work identifies a novel role for the guidance-molecules semaphorins in the mediation of Schwann cell/axonal interactions, and provides a molecular mechanism by which heterotypic cell-cell contacts control cell proliferation and suppress tumorigenesis. Finally, it provides a new approach for the development of therapies for NF1.