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.