REST upregulates gremlin to modulate diffuse intrinsic pontine glioma vasculature

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

Pharmacologic inhibition of lysine-specific demethylase 1 as a therapeutic and immune-sensitization strategy in pediatric high-grade glioma

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

MECHANISMS OF IMMUNE RESISTANCE IN PEDIATRIC POSTERIOR FOSSA TUMORS

Posterior fossa tumors (PFT) such as medulloblastoma (MB) and atypical teratoid rhabdoid tumor (ATRT) are aggressive and malignant pediatric brain tumors. Tumor recurrence is common with no available cure. The current standard-of-care can cause significant morbidities including a sharply elevated risk for secondary malignancies, ototoxicity and neurophysiological sequelae. Here, we offer a new therapeutic approach for children with recurrent/refractory PFT, which harnesses the killing capacity of natural killer (NK) cells. This is predicated on our ability to deliver ex vivo expanded and activated clinical grade NK cells to the tumor microenvironment through an ommaya reservoir. In pre-clinical studies we observed that many MBs and ATRT were susceptible to NK cells in vitro and in vivo. These observations provided support for a first in humans Phase I trial (NCT02271711) to examine the safety and feasibility of infusing NK cells into the fourth ventricle of patients with recurrent/refractory PFTs. Our in vitro studies also identified resistance to NK-mediated lysis in a subset of MBs and ATRTs. This resistance was caused by the epigenetic upregulation of the growth and differentiation factor-1 (GDF-1), a member of the TGF-beta family, in ATRTs and the elevated expression of the programmed death-ligand 1 (PD-L1) in MBs. Their genetic and pharmacological manipulation countered tumor cell resistance to cytolysis by NK cells. Mis-regulation of these molecules was also observed in patient samples, setting the stage for their development as biomarkers to predict tumor response, and providing justification for subsequent clinical trials combining NK cells with epigenetic modifiers