Development of Chromosome 1q+ Specific Treatment for Highest Risk Pediatric Posterior Fossa Ependymoma.

PURPOSE: There are no effective treatment strategies for children with highest-risk posterior fossa group A ependymoma (PFA). Chromosome 1q gains (1q+) are present in approximately 25% of newly diagnosed PFA tumors, and this number doubles at recurrence. Seventy percent of children with chromosome 1q+ PFA will die because of the tumor, highlighting the urgent need to develop new therapeutic strategies for this population. EXPERIMENTAL DESIGN: In this study, we utilize 1q+ PFA in vitro and in vivo models to test the efficacy of combination radiation and chemotherapy in a preclinical setting. RESULTS: 5-fluorouracil (5FU) enhances radiotherapy in 1q+ PFA cell lines. Specifically, 5FU increases p53 activity mediated by the extra copy of UCK2 located on chromosome 1q in 1q+ PFA. Experimental downregulation of UCK2 resulted in decreased 5FU sensitivity in 1q+ PFA cells. In in vitro studies, a combination of 5FU, retinoid tretinoin (ATRA), and radiation provided the greatest reduction in cellular proliferation and greatest increase in markers of apoptosis in 1q+ PFA cell lines compared with other treatment arms. Similarly, in vivo experiments demonstrated significant enhancement of survival in mice treated with combination radiation and 5FU and ATRA. CONCLUSIONS: These results are the first to identify a chromosome 1q+ specific therapy approach in 1q+ PFA. Existing phase I studies have already established single-agent pediatric safety and dosages of 5FU and ATRA, allowing for expedited clinical application as phase II trials for children with high-risk PFA.

Comprehensive Assessment of Blood-Brain Barrier Opening and Sterile Inflammatory Response: Unraveling the Therapeutic Window.

Microbubbles (MBs) combined with focused ultrasound (FUS) have emerged as a promising noninvasive technique to permeabilize the blood-brain barrier (BBB) for drug delivery to the brain. However, the safety and biological consequences of BBB opening remain incompletely understood. This study investigates the effects of varying microbubble volume doses (MVD) and ultrasound mechanical indices (MI) on BBB opening and the sterile inflammatory response (SIR) using high-resolution ultra-high field MRI-guided FUS in mouse brains. The results demonstrate that both MVD and MI significantly influence the extent of BBB opening, with higher doses and mechanical indices leading to increased permeability. Moreover, RNA sequencing reveals upregulated inflammatory pathways and immune cell infiltration after BBB opening, suggesting the presence and extent of SIR. Gene set enrichment analysis identifies 12 gene sets associated with inflammatory responses that are upregulated at higher doses of MVD or MI. A therapeutic window is established between significant BBB opening and the onset of SIR, providing operating regimes for avoiding each three classes of increasing damage from stimulation of the NFκB pathway via TNFL signaling to apoptosis. This study contributes to the optimization and standardization of BBB opening parameters for safe and effective drug delivery to the brain and sheds light on the underlying molecular mechanisms of the sterile inflammatory response. Significance Statement: The significance of this study lies in its comprehensive investigation of microbubble-facilitated focused ultrasound for blood-brain barrier (BBB) opening. By systematically exploring various combinations of microbubble volume doses and ultrasound mechanical indices, the study reveals their direct impact on the extent of BBB permeability and the induction of sterile inflammatory response (SIR). The establishment of a therapeutic window between significant BBB opening and the onset of SIR provides critical insights for safe and targeted drug delivery to the brain. These findings advance our understanding of the biological consequences of BBB opening and contribute to optimizing parameters for clinical applications, thus minimizing potential health risks, and maximizing the therapeutic potential of this technique.

MRI-guided focused ultrasound blood-brain barrier opening increases drug delivery and efficacy in a diffuse midline glioma mouse model.

Background: Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB opening, allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Methods: Mice were orthotopically injected with a patient-derived diffuse midline glioma (DMG) cell line, BT245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa peak negative pressure, 1 Hz pulse repetition frequency, 10-ms pulse length, 3 min treatment time)/(25 µL/kg, i.v.) targeting to the tumor location. Results: In animals receiving panobinostat (10 mg/kg, i.p.) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, without significant increase of the drug in the forebrain. In mice receiving 3 weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes (P = .01). Furthermore, we showed the first survival benefit from FUS/MB improved delivery increasing the mean survival from 21 to 31 days (P < .0001). Conclusions: Our study demonstrates that FUS-mediated BBB disruption can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.

MRI-Guided Focused Ultrasound Blood-Brain Barrier Opening Increases Drug Delivery and Efficacy in a Diffuse Midline Glioma Mouse Model.

Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB disruption (BBBD), allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Mice were orthotopically injected with a patient-derived DMG cell line, BT-245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa PNP, 1 Hz PRF, 10 ms PL, 3 min treatment time) / (25 µL/kg, IV) targeting to the tumor location. In animals receiving panobinostat (10 mg/kg, IP) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, with only insignificant increase of the drug in the forebrain. In mice receiving three weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes by MRI ( p = 0.01). Furthermore, FUS/MB improved the mean survival from 21 to 31 days ( p < 0.0001). Our study demonstrates that FUS-mediated BBBD can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival. One Sentence Summary: FUS and microbubbles can increase the delivery of panobinostat to a patient-derived xenograft (PDX) orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.

Senescence Induced by BMI1 Inhibition Is a Therapeutic Vulnerability in H3K27M-Mutant DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor of childhood characterized by histone mutations at lysine 27, which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. Using a combined RNAi and chemical screen targeting epigenomic regulators, we identify the polycomb repressive complex 1 (PRC1) component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy is enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 decreases cell self-renewal and attenuates tumor growth due to induction of senescence. Prolonged BMI1 inhibition induces a senescence-associated secretory phenotype, which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to enhance tumor cell killing in vivo.

Establishment of patient-derived orthotopic xenograft model of 1q+ posterior fossa group A ependymoma.

BACKGROUND: Treatment for pediatric posterior fossa group A (PFA) ependymoma with gain of chromosome 1q (1q+) has not improved over the past decade owing partially to lack of clinically relevant models. We described the first 2 1q+ PFA cell lines, which have significantly enhanced our understanding of PFA tumor biology and provided a tool to identify specific 1q+ PFA therapies. However, cell lines do not accurately replicate the tumor microenvironment. Our present goal is to establish patient-derived xenograft (PDX) mouse models. METHODS: Disaggregated tumors from 2 1q+ PFA patients were injected into the flanks of NSG mice. Flank tumors were then transplanted into the fourth ventricle or lateral ventricle of NSG mice. Characterization of intracranial tumors was performed using imaging, histology, and bioinformatics. RESULTS: MAF-811_XC and MAF-928_XC established intracranially within the fourth ventricle and retained histological, methylomic, and transcriptomic features of primary patient tumors. We tested the feasibility of treating PDX mice with fractionated radiation or chemotherapy. Mice tolerated radiation despite significant tumor burden, and follow-up imaging confirmed radiation can reduce tumor size. Treatment with fluorouracil reduced tumor size but did not appear to prolong survival. CONCLUSIONS: MAF-811_XC and MAF-928_XC are novel, authentic, and reliable models for studying 1q+ PFA in vivo. Given the successful response to radiation, these models will be advantageous for testing clinically relevant combination therapies to develop future clinical trials for this high-risk subgroup of pediatric ependymoma.

Quantitative NMR-Based Metabolomics on Tissue Biomarkers and Its Translation into In Vivo Magnetic Resonance Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is an established analytical platform for analyzing metabolic profiles of cells, tissues, and body fluids. There are several advantages in introducing an NMR-based study design into metabolomics studies, including a fast and comprehensive detection, characterization, and quantification of dozens of endogenous metabolites in a single NMR spectrum. Quantitative proton 1H-NMR is the most useful NMR-based platform for metabolomics. The frozen tissues can be analyzed noninvasively using a high-resolution magic angle spinning (HR-MAS) 1H-NMR spectroscopy; or several extraction techniques can be applied to detect additional metabolites using a conventional liquid-based NMR technique. In this chapter, we report on tissue collection, handling, extraction methods, and 1H-NMR acquisition protocols developed in the past decades for a precise and quantitative NMR-metabolomics approach. The NMR acquisition protocols (both HR-MAS and conventional 1H-NMR spectroscopy) and spectral analysis steps are also presented. Since NMR can be applied "in vivo" using horizontal bore MRI scanners, several in vivo sequences for localized 1H-MRS (magnetic resonance spectroscopy) are presented which can be directly applied for noninvasive detection of brain metabolites.