In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-β (infants) subgroups. Neuronal maturation is greater in SHH-β than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG) wherein conditionalNeuroD2-controlledRESTtransgene expression in lineage-committedPtch1+/−cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context–specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 atPtch1. Expression ofArrb1, which encodes β-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committedRESTTGcells compared with wild-type proliferating CGNPs. Lineage-committedRESTTGcells also had decreased GLI1 activity and increased histone H3K9 methylation at thePtch1locus, which correlated with premature silencing ofPtch1. These cells also had decreased expression ofPten, which encodes a negative regulator of the kinase AKT. Expression ofPTCH1andGLI1were less, andARRB1was somewhat greater, in patient SHH-β than SHH-α MBs, whereas that ofPTENwas similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling,PTCH1silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.
Background Diffuse midline gliomas (DMG), including brainstem diffuse intrinsic pontine glioma (DIPG), are incurable pediatric high-grade gliomas (pHGG). Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, rendering them therapeutically sensitive to small-molecule inhibition of chromatin modifiers. Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) is clinically relevant but has not been carefully investigated in pHGG or DIPG. Methods Patient-derived DIPG cell lines, orthotopic mouse models, and pHGG datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Immune cell cytotoxicity was assessed in DIPG cells pretreated with LSD1 inhibitors, and informatics platforms were used to determine immune infiltration of pHGG. Results Selective cytotoxicity and an immunogenic gene signature were established in DIPG cell lines using clinically relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit of this LSD1-dependent gene signature. Pretreatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. Catalytic LSD1 inhibitors induced tumor regression and augmented NK cell infusion in vivo to reduce tumor burden. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival, while CD8 T cells are negatively prognostic. Catalytic LSD1 inhibitors are nonperturbing to NK cells, while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene signature in DIPG cells. Conclusions LSD1 inhibition using catalytic inhibitors is selectively cytotoxic and promotes an immune gene signature that increases NK cell killing in vitro and in vivo, representing a therapeutic opportunity for pHGG. Key Points 1. LSD1 inhibition using several clinically relevant compounds is selectively cytotoxic in DIPG and shows in vivo efficacy as a single agent. 2. An LSD1-controlled gene signature predicts survival in pHGG patients and is seen in neural tissue from LSD1 inhibitor–treated mice. 3. LSD1 inhibition enhances NK cell cytotoxicity against DIPG in vivo and in vitro with correlative genetic biomarkers.
In order to assess the biodistribution, homing, and persistence of adoptively transferred natural killer (NK) cell immunotherapies, there is a need for imaging methodology suitable for use in preclinical studies with relevance to clinical translation. Amongst the available approaches, (19)F-MRI is very appealing for in vivo imaging due to the absence of background signal, enabling clear detection of (19)F labeled cells in vivo. Here we describe a methodology for in vivo imaging of adoptively transferred NK cells labeled with (19)F nano-emulsion, using clinically translatable technology of (19)F/(1)H magnetic resonance imaging.
Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive glial tumor that occurs in children. The extremely poor median and 5-year survival in children afflicted with DIPG highlights the need for novel biology-driven therapeutics. Here, we have implicated the chromatin remodeler and regulator of brain development called RE1 Silencing Transcription Factor (REST), in DIPG pathology. We show that REST protein is aberrantly elevated in at least 21% of DIPG tumors compared to normal controls. Its knockdown in DIPG cell lines diminished cell growth and decreased their tumorigenicity in mouse intracranial models. DIPGs are vascularized tumors and interestingly, REST loss in DIPG cells also caused a substantial decline in tumor vasculature as measured by a decrease in CD31 and VEGFR2 staining. These observations were validated in vitro, where a significant decline in tube formation by human umbilical vein endothelial cells (HUVEC) was seen following REST-loss in DIPG cells. Mechanistically, REST controlled the secretion of a pro-angiogenic molecule and ligand for VEGFR2 called Gremlin-1 (GREM-1), and was associated with enhanced AKT activation. Importantly, the decline in tube formation caused by REST loss could be rescued by addition of recombinant GREM-1, which also caused AKT activation in HUVECs and human brain microvascular endothelial cells (HBMECs). In summary, our study is the first to demonstrate autocrine and paracrine functions for REST in DIPG development. It also provides the foundation for future investigations on anti-angiogenic therapies targeting GREM-1 in combination with drugs that target REST-associated chromatin remodeling activities.
Medulloblastoma (MB) is a malignant pediatric brain tumor characterized by poor neuronal differentiation. The RE1 Silencing Transcription Factor (REST), a repressor of neurogenesis, is overexpressed in human MBs. The elevated expression of REST in a subset of patients with desmoplastic MB is associated with poor prognosis. To understand the underlying mechanisms, we generated a novel knock-in mouse expressing human REST transgene in a conditional manner in granule cell progenitors (GCPs), the cells of origin of sonic hedgehog (Shh) driven MB. Mice with constitutive activation of sonic hedgehog signaling and REST elevation (REST KI /Ptc +/-) in their GCPs developed tumors with a significant decrease in latency and increase in penetrance. We observed that 100% of REST KI /Ptc +/-animals developed heterogeneously proliferative and undifferentiated tumors with a striking increase in neural stem cell marker expression compared to tumor-bearing Ptc +/-animals. Mechanistic studies revealed direct transcriptional repression of Ptc by REST, which caused a total loss of Ptc expression in Ptc haploinsufficient GCPs. REST elevation in Ptc +/-GCPs also promoted significant Gli-1 dependent upregulation of the brain specific isoforms of Akt, and activation of AKT signaling. Consistent with this, 100% of REST KI /Ptc +/-animals developed highly vascularized, infiltrative tumors with a propensity for leptomeningeal dissemination. Thus, our studies suggest that REST elevation and constitutive Shh activation cooperatively drive an aggressive disease course by deregulating proliferation and driving the expansion of a highly undifferentiated cell population, with potential to promote rapid tumor growth and tumor dissemination.
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