Faculty Bibliography

Herpes simplex virus 1 (HSV-1) uses latency in peripheral ganglia to persist in its human host, however, recurrent reactivation from this reservoir can cause debilitating and potentially life-threatening disease. Most studies of latency use live-animal infection models, but these are complex, multilayered systems and can be difficult to manipulate. Infection of cultured primary neurons provides a powerful alternative, yielding important insights into host signaling pathways controlling latency. However, small animal models do not recapitulate all aspects of HSV-1 infection in humans and are limited in terms of the available molecular tools. To address this, we have developed a latency model based on human neurons differentiated in culture from an NIH-approved embryonic stem cell line. The resulting neurons are highly permissive for replication of wild-type HSV-1, but establish a non-productive infection state resembling latency when infected at low viral doses in the presence of the antivirals acyclovir and interferon-alpha. In this state, viral replication and expression of a late viral gene marker are not detected but there is an accumulation of the viral latency-associated transcript (LAT) RNA. After a six-day establishment period, antivirals can be removed and the infected cultures maintained for several weeks. Subsequent treatment with sodium butyrate induces reactivation and production of new infectious virus. Human neurons derived from stem cells provide the appropriate species context to study this exclusively human virus with the potential for more extensive manipulation of the progenitors and access to a wide range of preexisting molecular tools.

Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1alpha (Hif1alpha)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.

PURPOSE: Maximum dose to the left anterior descending artery (LADmax) is an important physical constraint to reduce the risk of cardiovascular toxicity. We generated a simple algorithm to guide the positioning of the tangent fields to reliably maintain LADmax <10 Gy. METHODS AND MATERIALS: Dosimetric plans from 146 consecutive women treated prone to the left breast enrolled in prospective protocols of accelerated whole breast radiation therapy, with a concomitant daily boost to the tumor bed (40.5 Gy/15 fraction to the whole breast and 48 Gy to the tumor bed), provided the training set for algorithm development. Scatter plots and correlation coefficients were used to describe the bivariate relationships between LADmax and several parameters: distance from the tumor cavity to the tangent field edge, cavity size, breast separation, field size, and distance from the tangent field. A logistic sigmoid curve was used to model the relationship of LADmax and the distance from the tangent field. Furthermore, we tested this prediction model on a validation data set of 53 consecutive similar patients. RESULTS: A lack of linear relationships between LADmax and distance from cavity to LAD (-0.47), cavity size (-0.18), breast separation (-0.02), or field size (-0.28) was observed. In contrast, distance from the tangent field was highly negatively correlated to LADmax (-0.84) and was used in the models to predict LADmax. From a logistic sigmoid model we selected a cut-point of 2.46 mm (95% confidence interval, 2.19-2.74 mm) greater than which LADmax is <10 Gy (95% confidence interval, 9.30-10.72 Gy) and LADmean is <3.3 Gy. CONCLUSIONS: Placing the edge of the tangents at least 2.5 mm from the closest point of the contoured LAD is likely to assure LADmax is <10 Gy and LADmean is <3.3 Gy in patients treated with prone accelerated breast radiation therapy.

BACKGROUND: Previous reports have proposed an association between traumatic brain injury (TBI) and subsequent glioblastoma (GBM) formation. METHODS: We used literature searches and radiographic evidence from two patients to assess the possibility of a link between TBI and GBM. RESULTS: Epidemiological studies are equivocal on a possible link between brain trauma and increased risk of malignant glioma formation. We present two case reports of patients with GBM arising at the site of prior brain injury. CONCLUSION: The hypothesis that TBI may predispose to gliomagenesis is disputed by several large-scale epidemiological studies, but supported by some. Radiographic evidence from two cases presented here suggest that GBM formed at the site of brain injury. We propose a putative pathogenesis model that connects post-traumatic inflammation, stem and progenitor cell transformation, and gliomagenesis.

Background. Extraosseous Ewing's sarcoma in the spinal epidural space is a rare malignancy, especially in adults. Case Presentation. A 40-year-old male presented with back pain and urinary hesitancy. MRI revealed a thoracic extradural mass with no osseous involvement. He underwent surgery for gross total resection of the mass, which was diagnosed as Ewing's sarcoma. He was subsequently treated with chemoradiotherapy. He remains disease-free 1 year after surgery. Review of the literature indicated only 45 previously reported cases of spinal epidural extraosseous Ewing's sarcoma in adults. Conclusions. Extraosseous Ewing's sarcoma in the spinal epidural space is a rare clinical entity that should be included in the differential for spinal epidural masses. Its treatment is multidisciplinary but frequently requires surgical intervention due to compressive neurologic symptoms. Gross total resection appears to correlate with improved outcomes.

Mutations in genes encoding Isocitrate Dehydrogenase (IDH) isoforms are found in80%of low-grade gliomas (LGGs). Sequencing of LGGs has revealed branching cancer genetics; mutant IDH1 astrocytomas contain p53 and ATRX loss of function mutations, while IDH1-mutated oligodendrogliomas have a different set of mutations that includes chr 1p/19q co-deletion. The IDH mutation is a gain-of-function change at its catalytic core that results in the production of (R)-2-hydroxyglutarate, an oncometabolite, which causes characteristic DNA and histone hypermethylation changes that may contribute to tumorigenesis. Mouse models have thus far failed to demonstrate the role of IDH1 mutations in LGG formation. To test the hypothesis that mutant IDH1 is a driver of gliomagenesis, we use human embryonic stem cell (hESC)-derived neural stem cells (NSCs) to overexpress mutant IDH1 protein in combination with p53 and ATRX knockdown. We have generated twelve NSC lines that harbor combinations of mutant IDH1, wt IDH1 or an empty vector, in combination with ATRX and/or p53 knockdown. Our preliminary data indicate that mutantIDH1 does not alter the proliferative capacity of NSCs, as shown by cell cycle analysis and Ki67 staining, but paradoxically increases their apoptotic rate (15.8% vs 5.9% n = 4), a phenotype that is exacerbated by ATRX knockdown, as detected by annexin V and TUNEL staining (17.7% vs 2.6% n = 3). shRNA-mediated knockdown of p53 salvages the pro-apoptotic phenotype of mutant IDH1 and ATRX NSCs. Furthermore, initial observations suggest that mutant IDH1 biases NSCs toward glial fates, as evidenced by upregulation of the CD44 cell surface marker. We are currently testing the effects of IDH1 mutation on i) NSC differentiation to astrocytic and neuronal lineages, ii) NSC metabolism via metabolomics profiling and iii) in vivo tumorigenesis. We propose that mutant IDH1 alters the differentiation program of human NSCs toward glial rather than neuronal fates