SUMMARYRetinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of the RB1 gene. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated RB1’s role in genome stability and considered nongenetic mechanisms of cancer pathway deregulation. Here we show that the retinoblastoma genome is stable, but multiple cancer pathways can be epigenetically deregulated. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumor cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumor cell death in vitro and in vivo. Thus, RB1 inactivation may allow preneoplastic cells to acquire multiple hallmarks of cancer through epigenetic mechanisms, resulting directly or indirectly from RB1 loss. These data provide novel targets for chemotherapeutic interventions of retinoblastoma.

SUMMARY It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro.

G protein-coupled receptors (GPCRs) are major transducers of external stimuli and key therapeutic targets in many pathological conditions. When activated by different ligands, one receptor can elicit multiple signaling cascades that are mediated by G proteins or β-arrestin, a process defined as functional selectivity or ligand bias. However, the dynamic mechanisms underlying β-arrestin signaling remain unknown. Here, by studying the cannabinoid 1 receptor (CB1R), we identify ligand-specific endocytic dwell times, that is, the time during which receptors are clustered into clathrin pits together with β-arrestins before endocytosis, as the mechanism controlling β-arrestin signaling. Agonists inducing short endocytic dwell times produce little or no β-arrestin signaling, whereas those eliciting prolonged dwell times induce robust signaling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased β-arrestin signaling. These studies reveal how receptor activation translates into β-arrestin signaling and identify a mechanism to control this pathway.

Retinoblastoma is a rare childhood cancer of the retina that begins in utero and is diagnosed in the first years of life. The goals of retinoblastoma treatment are ocular salvage, vision preservation, and reduction of short-and long-term side effects without risking mortality due to tumor dissemination. To identify better chemotherapeutic combinations for the treatment of retinoblastoma, several groups have developed genetic mouse models and orthotopic xenograft models of human retinoblastoma for preclinical testing. Previous studies have implicated the MDMX protein in the suppression of the p53 pathway in retinoblastoma and shown that the MDM2/MDMX antagonist, nutlin-3a, can efficiently induce p53-mediated cell death in retinoblastoma cell lines. However, nutlin-3a cannot be administered systemically to treat retinoblastoma, because it has poor penetration across the blood-ocular barrier. Therefore, we developed an ocular formulation of nutlin-3a, nutlin-3aOC, and tested the pharmacokinetics and efficacy of this new formulation in genetic and human retinoblastoma orthotopic xenograft models of retinoblastoma. Here we show that nutlin-3aOC specifically and efficiently targets the p53 pathway and that the combination of nutlin-3aOC with systemic topotecan is a significantly better treatment for retinoblastoma than currently used chemotherapy in human orthotopic xenografts. Our studies provide a new standardized approach to evaluate and prioritize novel agents for incorporation into future clinical trials for retinoblastoma.

A unifying principle of sensory system organization is feature extraction by modality-specific neuronal maps in which arrays of neurons show systematically varied response properties and receptive fields. Only beginning to be understood, however, are the mechanisms by which these graded systems are established. In the peripheral auditory system, we have shown previously that the intrinsic firing features of spiral ganglion neurons are influenced by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). We now show that is but a part of a coordinated package of neurotrophin actions that also includes effects on presynaptic and postsynaptic proteins, thus encompassing the input, transmission, and output functions of the spiral ganglion neurons. Using immunocytochemical methods, we determined that proteins targeted to opposite ends of the neuron were organized and regulated in a reciprocal manner. AMPA receptor subunits GluR2 and GluR3 were enriched in base neurons compared with their apex counterparts. This distribution pattern was enhanced by exposure to BDNF but reduced by NT-3. SNAP-25 and synaptophysin were distributed and regulated in the mirror image: enriched in the apex, enhanced by NT-3 and reduced by BDNF. Moreover, we used a novel coculture to identify potential endogenous sources of neurotrophins by showing that sensory receptors from different cochlear regions were capable of altering presynaptic and postsynaptic protein levels in these neurons. From these studies, we suggest that BDNF and NT-3, which are systematically distributed in complementary gradients, are responsible for orchestrating a comprehensive set of electrophysiological specializations along the frequency contour of the cochlea.

Type I and type II spiral ganglion neurons convey auditory information from the sensory receptors in the cochlea to the CNS. The numerous type I neurons have been extensively characterized, but the small population of type II neurons with their unmyelinated axons are undetectable with most recording methods. Despite the paucity of information about the type II neurons, it is clear that they must have a significant role in sound processing because they innervate the large number of outer hair cells that are critical for maintaining normal responses to stimuli. To elucidate the function of type II neurons, we have developed an approach for studying their electrophysiological features in vitro.Type II neurons obtained from postnatal day 6 -7 mice displayed distinctly different firing properties than type I neurons. They showed slower accommodation, lower action potential thresholds, and more prolonged responses to depolarizing current injection than the type I neurons. These differences were most evident in neurons from the basal, high-frequency region of the cochlea. The basal type I neurons displayed uniformly fast firing features, whereas the basal type II neurons showed particularly slow accommodation and responses to depolarization. Interestingly, neurons from the apical, low-frequency region of the cochlea showed the opposite trend. These data suggest that the type I and type II neurons have specialized electrophysiological characteristics tailored to their different roles in auditory signal processing. In particular, the type II neuron properties are consistent with cells in other sensory systems that receive convergent synaptic input for high-sensitivity stimulus detection.

Inherent in the design of the mammalian auditory system is the precision necessary to transduce complex sounds and transmit the resulting electrical signals to higher neural centers. Unique specializations in the organ of Corti are required to make this conversion, such that mechanical and electrical properties of hair cell receptors are tailored to their specific role in signal coding. Electrophysiological and immunocytochemical characterizations have shown that this principle also applies to neurons of the spiral ganglion, as evidenced by distinctly different firing features and synaptic protein distributions of neurons that innervate high- and low-frequency regions of the cochlea. However, understanding the fine structure of how these properties are distributed along the cochlear partition and within the type I and type II classes of spiral ganglion neurons is necessary to appreciate their functional significance fully. To address this issue, we assessed the localization of the postsynaptic AMPA receptor subunits GluR2 and GluR3 and the presynaptic protein synaptophysin by using immunocytochemical labeling in both postnatal and adult tissue. We report that these presynaptic and postsynaptic proteins are distributed oppositely in relation to the tonotopic map and that they are equally distributed in each neuronal class, thus having an overall gradation from one end of the cochlea to the other. For synaptophysin, an additional layer of heterogeneity was superimposed orthogonal to the tonotopic axis. The highest anti-synaptophysin antibody levels were observed within neurons located close to the scala tympani compared with those located close to the scala vestibuli. Furthermore, we noted that the protein distribution patterns observed in postnatal preparations were largely retained in adult tissue sections, indicating that these features characterize spiral ganglion neurons in the fully developed ear.

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