Patient-derived Diffuse Intrinsic Pontine Glioma Research Articles

IMMU-32. NON-INFLAMMATORY EFFECT OF DIPG ON TUMOR-ASSOCIATED MICROGLIA

Diffuse intrinsic pontine glioma (DIPG) is a diffusely infiltrating high-grade glioma that has a peak incidence from ages 6-8 and only a 9-12 month median survival. Like adult gliomas, DIPG exhibits a significant expansion of myeloid cells, with microglia/macrophages accounting for ~30% of the tumor cell mass (Caretti & Monje, 2014). In adult glioma, these glioma-associated macrophages have been implicated as supporting angiogenesis and tumor cell invasion, proliferation, and survival (Glass & Synowitz, 2014). However, genomic and epigenomic characterizations of adult and pediatric high-grade gliomas revealed striking differences in the molecular nature of these tumors (Sturm et al., 2014), and traditional chemotherapeutic approaches to adult glioblastoma are not effective in DIPG, suggesting these are unique diseases with distinct pathophysiology. Our data demonstrates that tumor-associated macrophages from primary DIPG samples exhibit gene expression signatures that are associated with pro-tumorigenic functions, but are also significantly less inflammatory than tumor-associated macrophages isolated from adult glioblastoma samples. Moreover, the cytokine secretion profiles of patient-derived DIPG cell cultures are dramatically less inflammatory when compared to patient-derived adult high-grade glioma cell cultures, and are more similar to the profile of human neural precursor cell cultures. This suggests that DIPG is less immunogenic than adult GBM, which may allow the tumor cells to evade detection and infiltrate healthy tissue. We additionally show that DIPG cells express CD200, a ligand that inhibits myeloid cell activation. Knockdown of DIPG expression of CD200 increases primary microglial cell phagocytosis in direct co-culture experiments, and also inhibits DIPG progression in in vivo xenograft models. Thus, CD200 may represent a mechanism by which DIPG evades normal immune responses to tumor growth. By understanding the DIPG-microglial interaction in molecular detail, we will gain insight into the pathophysiology of DIPG that could direct future targeted therapies.

Patient-derived DIPG cells preserve stem-like characteristics and generate orthotopic tumors.

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor, with a median survival of less than one year. Due to enormous difficulties in the acquisition of DIPG specimens and the sophisticated technique required to perform brainstem orthotopic injection, only a handful of DIPG pre-clinical models are available. In this study, we successfully established eight patient-derived DIPG cell lines, mostly derived from treatment-naïve surgery or biopsy specimens. These patient-derived cell lines can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities. In addition, these cells retained genomic hallmarks identical to original human DIPG tumors. Notably, expression of several neural stem cell lineage markers was observed in DIPG cell lines. Moreover, three out of eight cell lines can form orthotopic tumors in mouse brainstem by stereotactic injection and these tumors faithfully represented the characteristics of human DIPG by magnetic resonance imaging (MRI) and histopathological staining. Taken together, we established DIPG pre-clinical models resembling human DIPG and they provided a valuable resource for future biological and therapeutic studies.

DIPG-35. A SIRNA APPROACH FOR TARGETING IMMUNOSUPPRESSIVE IDO1 IN PEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMA

OBJECTIVE/GOALS: The purpose of our project is to delineate the role of indoleamine 2,3 dioxygenase 1 (IDO1) in pediatric diffuse intrinsic pontine glioma (DIPG), and develop therapeutic potential of IDO1 inhibition through small inhibitory (si)RNA oligonucleotides and spherical nucleic acids (SNAs). Our specific aims are: (1) confirm the gene expression, protein levels and enzymatic activity of IDO1 in different human DIPG cell lines; (2) generate and characterize siRNA oligonucleotides specifically targeting human IDO1 for an in vitro targeted approach to validate knockdown of IDO1 expression; and (3) generate and characterize gold nanoparticles for further targeted approach to inhibit IDO1. METHODS/STUDY: Unique patient-derived DIPG cell lines were grown in culture, stimulated with increasing concentrations of IFN-γ, and analyzed by levels of mRNA, protein, enzymatic activity and cell growth. IDO1 small inhibitory (si)RNA were created to target IDO1 specifically, and transfected into cells. SNA generation is currently underway. RESULTS: ANTICIPATED RESULTS: IDO1 is expressed in different human pediatric DIPG cells at the mRNA and protein levels. We created siRNA that specifically targets IDO1 and preliminary results show overall decrease of IDO1 at the mRNA and protein level. Generation of SNA is ongoing for targeted approach and improved blood brain barrier penetration & stability, with anticipated results that siIDO1-SNAs inhibit IDO1 expression and reduce tumorigenesis within brain tumor cells. DISCUSSION/SIGNIFICANCE OF IMPACT: The overall poor prognosis of children with DIPG combined with the lack of effective therapies, emphasizes the importance of understanding the IDO1 pathway and the potential effects of IDO1 inhibition for therapeutically-modulating these devastating tumors. Our research confirms IDO1 presence in DIPG, and the use of nanotechnology and development of siRNA-SNAs for IDO1 inhibition has the capability of direct translatability of therapeutically-valuable data.

EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16INK4A. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.

Diffuse Intrinsic Pontine Glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNAseq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated efficacy in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat with histone demethylase inhibitor GSKJ4 revealed synergy. Together, these data suggest a promising therapeutic strategy for DIPG.

BT-02 * FUNCTIONALLY-DEFINED THERAPEUTIC TARGETS IN DIFFUSE INTRINSIC PONTINE GLIOMA

Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal childhood cancer. Here, we performed a chemical screen in patient-derived DIPG cell cultures along with RNAseq expression analysis and integrated computational modeling to identify potentially effective therapeutic strategies. Panobinostat, among the more promising agents identified, demonstrated efficacy in pontine orthotopic xenograft models of both H3K27M and histone WT DIPG. These data suggest the potential utility of specific drug combinations and provides evidence of in vivo treatment efficacy of the multi-histone deacetylase inhibitor panobinostat.

Cell migration in paediatric glioma; characterisation and potential therapeutic targeting.

Background:Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed.Methods:Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays.Results:All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging.Conclusions:Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.