Michael A. Dyer

Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas

To identify somatic mutations in pediatric diffuse intrinsic pontine glioma (DIPG), we performed whole-genome sequencing of DNA from seven DIPGs and matched germline tissue and targeted sequencing of an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). We found that 78% of DIPGs and 22% of non-BS-PGs contained a mutation in H3F3A, encoding histone H3.3, or in the related HIST1H3B, encoding histone H3.1, that caused a p.Lys27Met amino acid substitution in each protein. An additional 14% of non-BS-PGs had somatic mutations in H3F3A causing a p.Gly34Arg alteration.

Neuroblastoma: developmental biology, cancer genomics and immunotherapy.

Neuroblastoma is a solid tumour that arises from the developing sympathetic nervous system. Over the past decade, our understanding of this disease has advanced tremendously. The future challenge is to apply the knowledge gained to developing risk-based therapies and, ultimately, improving outcome. In this Review we discuss the key discoveries in the developmental biology, molecular genetics and immunology of neuroblastoma, as well as new translational tools for bringing these promising scientific advances into the clinic.

The Effects of Transdermal Nicotine on Cognition in Nonsmokers with Schizophrenia and Nonpsychiatric Controls

Abundant evidence indicates that the neuronal nicotinic acetylcholine receptor (nAChR) system is integral to regulation of attentional processes and is dysregulated in schizophrenia. Nicotinic agonists may have potential for the treatment of cognitive impairment in this disease. This study investigated the effects of transdermal nicotine on attention in individuals with schizophrenia (n=28) and healthy controls (n=32). All participants were nonsmokers in order to eliminate confounding effects of nicotine withdrawal and reinstatement that may occur in the study of smokers. Subjects received 14 mg transdermal nicotine and identical placebo in a randomized, placebo-controlled, crossover design. A cognitive battery was conducted before and 3 h after each patch application. The primary outcome measure was performance on the Continuous Performance Test Identical Pairs (CPT-IP) Version. Nicotine significantly improved the performance on the CPT-IP as measured by hit reaction time, hit reaction time standard deviation and random errors in both groups. In addition, nicotine reduced commission errors on the CPT-IP and improved the performance on a Card Stroop task to a greater extent in those with schizophrenia vs controls. In summary, nicotine improved attentional performance in both groups and was associated with greater improvements in inhibition of impulsive responses in subjects with schizophrenia. These results confirm previous findings that a single dose of nicotine improves attention and suggest that nicotine may specifically improve response inhibition in nonsmokers with schizophrenia.

Association of Age at Diagnosis and Genetic Mutations in Patients With Neuroblastoma

CONTEXT Neuroblastoma is diagnosed over a wide age range from birth through young adulthood, and older age at diagnosis is associated with a decline in survivability. OBJECTIVE To identify genetic mutations that are associated with age at diagnosis in patients with metastatic neuroblastoma. DESIGN, SETTING, AND PATIENTS Whole genome sequencing was performed on DNA from diagnostic tumors and their matched germlines from 40 patients with metastatic neuroblastoma obtained between 1987 and 2009. Age groups at diagnosis included infants (0-<18 months), children (18 months-<12 years), and adolescents and young adults (≥12 years). To confirm the findings from this discovery cohort, validation testing using tumors from an additional 64 patients obtained between 1985 and 2009 also was performed. Formalin-fixed, paraffin-embedded tumor tissue was used for immunohistochemistry and fluorescence in situ hybridization. Telomere lengths were analyzed using whole genome sequencing data, quantitative polymerase chain reaction, and fluorescent in situ hybridization. MAIN OUTCOME MEASURE Somatic recurrent mutations in tumors from patients with neuroblastoma correlated with the age at diagnosis and telomere length. RESULTS In the discovery cohort (n = 40), mutations in the ATRX gene were identified in 100% (95% CI, 50%-100%) of tumors from patients in the adolescent and young adult group (5 of 5), in 17% (95% CI, 7%-36%) of tumors from children (5 of 29), and 0% (95% CI, 0%-40%) of tumors from infants (0 of 6). In the validation cohort (n = 64), mutations in the ATRX gene were identified in 33% (95% CI, 17%-54%) of tumors from patients in the adolescent and young adult group (9 of 27), in 16% (95% CI, 6%-35%) of tumors from children (4 of 25), and in 0% (95% CI, 0%-24%) of tumors from infants (0 of 12). In both cohorts (N = 104), mutations in the ATRX gene were identified in 44% (95% CI, 28%-62%) of tumors from patients in the adolescent and young adult group (14 of 32), in 17% (95% CI, 9%-29%) of tumors from children (9 of 54), and in 0% (95% CI, 0%-17%) of tumors from infants (0 of 18). ATRX mutations were associated with an absence of the ATRX protein in the nucleus and with long telomeres. CONCLUSION ATRX mutations were associated with age at diagnosis in children and young adults with stage 4 neuroblastoma. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00588068.

Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

Rb regulates proliferation and rod photoreceptor development in the mouse retina.

The retinoblastoma protein (Rb) regulates proliferation, cell fate specification and differentiation in the developing central nervous system (CNS), but the role of Rb in the developing mouse retina has not been studied, because Rb-deficient embryos die before the retinas are fully formed. We combined several genetic approaches to explore the role of Rb in the mouse retina. During postnatal development, Rb is expressed in proliferating retinal progenitor cells and differentiating rod photoreceptors. In the absence of Rb, progenitor cells continue to divide, and rods do not mature. To determine whether Rb functions in these processes in a cell-autonomous manner, we used a replication-incompetent retrovirus encoding Cre recombinase to inactivate the Rb1lox allele in individual retinal progenitor cells in vivo. Combined with data from studies of conditional inactivation of Rb1 using a combination of Cre transgenic mouse lines, these results show that Rb is required in a cell-autonomous manner for appropriate exit from the cell cycle of retinal progenitor cells and for rod development.

Identification and characterization of the first small molecule inhibitor of MDMX.

The p53 pathway is disrupted in virtually every human tumor. In ∼50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. To date, peptide inhibitors of MDMX have been developed, but no small molecule inhibitors have been reported. We have developed biochemical and cell-based assays for high throughput screening of chemical libraries to identify MDMX inhibitors and identified the first MDMX inhibitor SJ-172550. This compound binds reversibly to MDMX and effectively kills retinoblastoma cells in which the expression of MDMX is amplified. The effect of SJ-172550 is additive when combined with an MDM2 inhibitor. Results from a series of biochemical and structural modeling studies suggest that SJ-172550 binds the p53-binding pocket of MDMX, thereby displacing p53. This lead compound is a useful chemical scaffold for further optimization of MDMX inhibitors that may eventually be used to treat pediatric cancers and various adult tumors that overexpress MDMX or have similar genetic lesions. When combined with selective MDM2 inhibitors, SJ-172550 may also be useful for treating tumors that express wild-type p53.

Differentiated Horizontal Interneurons Clonally Expand to Form Metastatic Retinoblastoma in Mice

During neurogenesis, the progression from a progenitor cell to a differentiated neuron is believed to be unidirectional and irreversible. The Rb family of proteins (Rb, p107, and p130) regulates cell-cycle exit and differentiation during retinogenesis. Rb and p130 are redundantly expressed in the neurons of the inner nuclear layer (INL) of the retina. We have found that in the adult Rb;p130-deficient retinae p107 compensation prevents ectopic proliferation of INL neurons. However, p107 is haploinsufficient in this process. Differentiated Rb(-/-);p107(+/-);p130(-/-) horizontal interneurons re-entered the cell cycle, clonally expanded, and formed metastatic retinoblastoma. Horizontal cells were not affected in Rb(+/-);p107(-/-);p130(-/-) or Rb(-/-);p107(-/-);p130(+/-), retinae suggesting that one copy of Rb or p130 was sufficient to prevent horizontal proliferation. We hereby report that differentiated neurons can proliferate and form cancer while maintaining their differentiated state including neurites and synaptic connections.

Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations

UNLABELLED Ewing sarcoma is a primary bone tumor initiated by EWSR1-ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples. SIGNIFICANCE Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers.

The First Knockout Mouse Model of Retinoblastoma

The retinoblastoma susceptibility gene (RB1) was the first tumor suppressor gene identified in humans (Friend, et al., 1986) and the first tumor suppressor gene knocked out by targeted deletion in mice (Jacks, et al., Clarke, et al., Lee, et al., 1992). Children with a germline mutation in one of their RB1 alleles are likely to experience bilateral multifocal retinoblastoma; however, mice with a similar disruption of Rb1 do not develop retinoblastoma. The absence of a knock-out mouse model of retinoblastoma has slowed the progress toward developing new therapies and identifying secondary genetic lesions that occur after disruption of the Rb signaling pathway. Several advances have been made, over the past several years, in our understanding of the regulation of proliferation during retinal development (Zhang, et al., 2004; Dyer J, 2004; Dyer, Cepko, 2001) and we have built upon these earlier studies to generate the first nonchimeric knock-out mouse model of retinoblastoma. These mice are being used as a preclinical model to test new therapies for retinoblastoma and to elucidate the downstream genetic events that occur after inactivation of Rb1 or its related family members.