PDE4A1 signaling:

Intracellular cAMP is measurable by FRET.
Areas of decreased cAMP appear yellow (asterisk,
increased 535/470) emission ratio.

We have shown that the cAMP pathway is an important regulator of brain tumorigenesis. Polymorphisms in adenylate cyclase 8 confer sex-specific risk for low grade astrocytomas, targeted inhibition of the cAMP specific phosphodiesterase 4 (PDE4) family has potent ani-brain tumor activity in medulloblastoma, glioblastoma and low grade astrocytoma models, and overexpression of one isoform of PDE4, PDE4A1, has tumor promoting effects on models of low and high grade astrocytoma and medulloblastoma. PDE4A1 is localized o the trans-golgi providing an opportunity to identify the critical mediators of cAMP effects on tumor growth as components of this subdomain in cAMP signaling. This mechanistic project utilizes site-directed mutagenesis, FRET imaging of cAMP signaling and subcellular fractionation techniques.

CXCR4 in brain tumors:

We have shown that tumor cell expression of CXCR4 is required for maintenance of a stem cell-like population and in vivo tumorigenesis in glioblastoma and medulloblastoma models. This activity requires surface localization of CXCR4 and appears to involve repression of trimethylation of histone H3 lysine 27 at the promoters of stem cell genes. This results in the loss of stem cell gene expression and stem cell activity. How CXCR4 regulates H3K27me3 is not known. This project utilizes primary brain tumor isolates, genetic and pharmacological manipulation of the CXCR4 and H3K27 mehtylation/demthylation pathways, flow cytometry and chromatin immunoprecipitation (ChIP) followed by sequencing or PCR.

Surface CXCR4 correlates with expression of stem cell markers. Primary murine medulloblastoma tumor tissue was dissociated into a single cell suspension and stained for surface CXCR4 protein expression and expression of the stem cell epitope CD15 using flow cytometry. Left panel, y-axis is CXCR4 signal, x-axis is CD15 signal. Bottom left quadrant represents background IgG gating. Cells can clearly be separated into two populations, a CXCR4hi major population (upper box) and a CXCR4lo minor population (lower box, above IgG background). Right panel, CXCR4hi and CXCR4lo cells were computationally separated and analyzed for CD15 surface expression; CXCR4lo cells express less CD15 on the cell surface

Sexual dimorphism:

We have shown that cell intrinsic sexual dimorphism in the p53 and Rb pathways results in sex differences in thresholds for transformation and responses to common therapeutics in a model of mesenchymal glioblastoma. This serves as a model for the significant sex differences in incidence and outcome for glioblastoma in patients. Current projects use primary brain tumor specimens, calling-card technology and ChIP-seq/PCR to map sex specific transcriptional regulation of oncogene/tumor suppressor pathways, genetic and pharmacological manipulation of the p53 and Rb pathways to identify mediators of sex effects on tumor biology, and sex-specific compound library screening and RNAi screens for discovery of novel aspects of sex differences in brain tumor biology.

Super-enhancer usage in female and male mes-GBMs. In collaboration with Dr. Robi Mitra we used transposon ‘Calling Cards’ to map the binding of Brd4. Super-enhancers are often defined as loci that bind large amounts of either Brd4 or Med1. We observed substantially non-overlapping super-enhancer usage in male versus female mes-GBM cells. A typical locus, Akap6, is shown in B) female and C) male mes-GBM cells. Each symbol represents an independent insertion of a transposon Calling Card that marks Brd4 binding. The height of the symbol represents the number of reads supporting that insertion. These data indicate that male mes-GBMs bind ~9 fold more Brd-4 at Akap6 than do female mes-GBMs. Akap6 functions to regulate cAMP-dependent protein kinase localization and activity. We have previously demonstrated sexual dimorphism in the cAMP pathway.

Legacy program:

The legacy program is a large cross-departmental collaboration to use advanced imaging, deep sequencing, pathology and cell biological approaches to map tumor heterogeneity in three-dimensions. It utilizes patient autopsy donations of recurrent tumors in situ in the brain. The goal of the project is to deeply understand individual tumors as complex multi-clonal and multi-domain tissues, to map treatment effects on tumor evolution and discover mechanisms of resistance.

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