Project description:Treatment-resistance of lethal high-grade brain tumors including H3K27M diffuse midline gliomas is thought to arise in part from transcriptional and electrophysiological heterogeneity. These phenotypes are readily studied in isolation using single-cell RNA-seq and whole-cell patch clamping, respectively, but their simultaneous capture is now possible by aspirating a cell's contents into a patch pipet after electrophysiology recordings using a method called 'patch-seq'. Here, we adapt this method to characterize the gene expression programs and functional responses of patient-derived glioma xenografts to neuronal firing at single-cell resolution.
Project description:The blood-brain barrier (BBB) plays important roles in brain tumor pathogenesis and treatment response, yet our understanding of its function and heterogeneity within or across brain tumor types remains poorly characterized. Here we analyze the neurovascular unit (NVU) of pediatric high-grade glioma (pHGG) and diffuse midline glioma (DMG) using patient derived xenografts and natively forming glioma mouse models. We show tumor-associated vascular differences between these glioma subtypes, and parallels between PDX and mouse model systems, with DMG models maintaining a more normal vascular architecture, BBB function and endothelial transcriptional program relative to pHGG models. Unlike prior work in angiogenic brain tumors, we find that expression of secreted Wnt antagonists do not alter the tumor-associated vascular phenotype in DMG tumor models. Together, these findings highlight vascular heterogeneity between pHGG and DMG and differences in their response to alterations in developmental BBB signals that may participate in driving these pathological differences.
Project description:Diffuse midline glioma (DMG) is a uniformly fatal pediatric, adolescent, and young adult cancer diagnosed in the midline structures of the brain. PI3K/Akt signaling is an overarching contributor to the poor outcomes of DMG patients due to their dependance on insulin and recurring mutations and amplifications in PI3K/Akt genes. Paxalisib (GDC-0084) is a potent PI3K/Akt inhibitor developed to penetrate the brain’s protective blood-brain barrier (BBB). We performed paxalisib regimen optimization by assessment of blood glucose levels, analysis in vivo pharmacokinetics/pharmacodynamics properties, and employed DMG patient derived xenograft (PDX) mouse models to determine preclinical efficacy. To identify novel combination strategies, we conducted high-resolution quantitative phosphoproteomics of DMG cells treated with paxalisib. Elevated blood glucose levels promoting hyperglycemia was seen in mice using paxalisib 10 mg/kg/day (~mouse equivalent human maximum tolerated dose- NCT03696355). Whereas 5 mg/kg/day, or 5mg/kg/b.i.d. (twice daily) non-significantly increased blood glucose levels compared to controls. Pharmacokinetic analysis of mouse tissues showed 5 mg/kg/b.i.d. increased paxalisib acumination in the brainstem, suppressing PI3K/Akt signaling to significantly extend the survival of DMG PDX models compared to all other regimens; a survival benefit amplified when combined with the anti-glycemic therapy metformin. Phosphoproteomic profiling identified increased Protein kinase C (PKC) signaling following paxalisib treatment. The combination of paxalisib and enzastaurin (PKC inhibitor) synergistically extended the survival of PDX models. Our optimized dosing regimen increased the preclinical benefits of paxalisib, with the combination of paxalisib with metformin, or paxalisib with enzastaurin, heralding rationally designed combination strategies for the treatment of DMG
Project description:This article highlights the radiation induced cytotoxic effect of the BCL2 inhibitor, venetoclax, in diffuse midline gliomas (DMG). 1. RNA-seq: We performed bulk RNA sequencing of DMG cells exposed to 6Gy radiation and found the genes upregulated after radiation compared to unirradiated controls. 2. DNA Repair shRNA screen: We also performed a DNA Repair shRNA screen and identified the genes which are responsible for radioresistance in DMG.
Project description:We generated a syngeneic H3K27M diffuse midline glioma (DMG) mouse model and performed total RNA seq on H3K27MPP cells and control cell lines H3wtMPP and Normal Neural stem cells. Aditionally we generate a Dox Inducible Mat2A Knockdown in that human DIPG cell line DIPG04 and performed RNA sequencing on cells treated with 2ug/ml Doxycyclin and no treatment