The DIPG / DMG Collaborative has funded $15,576,509 in DIPG/DMG research.
Interested in applying for a grant from the DIPG / DMG Collaborative? Learn more.
Children's National Health System and Children's Research Institute - $125,000
Comprehensive Molecular-Based Cross-Species Comparison of DIPG Biology
Comprehensive Molecular-Based Cross-Species Comparison of DIPG Biology
Scientific Merit: Despite recent advances in understanding some of the molecular mechanism of DIPG, there remains a great deal of unknown with respect to the disease pathogenesis. Mutations in histone 3 have been identified as driver mutations in up to 80% of all DIPGs. However, the exact molecular and cellular events that follow histone mutation are not well understood. Our collaborative
team of researchers have intends to use the resources of four well established DIPG laboratories to:
i) generate comprehensive molecular profiles of a large cohort of DIPG specimens,
ii) generate comprehensive cross-species comparative analysis of molecular events leading to tumorigenesis, and
iii) demonstrate the power of cross-species genomic studies for identifying and matching DIPG subgroup-specific driver mutations.
Disease Impact: Combined cross-species analysis of human DIPG specimens, DIPG primary cells and DIPG murine models. The immediate outcomes of the proposed project will be:
1. Generation of comprehensive molecular profile of a large cohort (n = 48) DIPG specimens.
2. Identification of molecular mechanism of epigenetic alterations governed by histone mutation.
3. Comprehensive cross-species comparative study of DIPG.
Our results will have immediate impact on DIPG by providing new paths for developing targeted treatments.
Innovation: Our collaborative proposal is innovative by taking advantage of the following unique opportunities:
1. A large collaborative group encompassing four established DIPG research laboratories.
2. Access to novel preliminary (proteomics, RNA profiling, exome, SNP copy number) data
supported by Collaborative.
3. Generation of new molecular data from specimens obtained through Collaborative (the research team is the first to have access to these rare biospecimens).
4. Utilization of a DIPG specific SILAC protein Atlas.
5. Incorporation of investigation into DNA structure with molecular data.
6. Comparative analysis of matched mouse and human tumors with the aim of deciphering genomic events driving DIPG.
Feasibility: PIs of the proposed project have generated preliminary data through previous Collaborative funding support. PIs have been granted access to additional specimens from Collaborative biobanks and their proposal has been approved by the Collaborative Scientific Advisory Committee. The cross-species approach is novel and provides a hypothesis driven aspect to the
Expertise: Dr. Nazarian (PI) at CNHS has led efforts in studying the protein profiling of DIPGs and generation of the first DIPG protein Atlas. Dr. Hawkins (Co-PI) at Hospital for Sick Children (SickKids) is an expert in pathology and studying the molecular basis of pediatric brain tumors. The Centre for Applied Genomics at SickKids is a pioneering center in generating genomic profiles of pediatric brain tumors. This center houses an Illumina HISeq 2500 platform which will be used for the proposed study. Dr. Becher (Co-PI) is a pediatric neuro-oncologist who is primarily laboratory based with expertise in the development and use of genetic mouse models of DIPG as preclinical tools
Dr. Drissi (Co-PI) has a strong background in telomere/telomerase biology and in DNA damage. He is PI on several funded studies on pediatric brain tumors.
Boston Children's Hospital - $110,000
Enabling epigenetic therapies by exploring DIPG chromatin sensitivities
Background: Recently, driver mutations in histone H3 have been found to correlate with particularly malignant forms of DIPG. These mutations change lysine 27 of H3 to methionine (K27M), abrogating methylation and acetylation at this site, both of which are epigenetic marks that influence chromatin architecture and are important for regulating the expression of numerous genes. Accordingly, molecules that inhibit histone deacetylases (HDACs) or the H3K27me3 demethylase inhibit DIPG growth in vitro and in tumor models in vivo. Despite this clear chromatin-dependent basis for DIPG pathology, a systematic survey of chromatin regulators important for the survival and proliferation of H3K27M mutant DIPGs has not been conducted. We hypothesize that additional chromatin regulators interact with the histone H3 mutations in DIPG to support proliferation and tumorigenesis, and that these regulators will be promising targets for developing novel therapeutics.
Research Design and Methods: We will apply multiple screening approaches to comprehensively query chromatin regulators for their effects on DIPG cell survival and proliferation. Recently, patient-derived cell lines expressing wild-type or mutant histone H3 have become available, making large-scale in vitro screens possible for the first time. We will first screen a specialized small-molecule collection that targets both conserved and specific domains found in chromatin regulators, focusing on molecules that preferentially inhibit the growth of H3-mutant DIPG cell lines, but not H3 wild-type glioblastoma cells. To obtain a more comprehensive view of epigenetic regulatory mechanisms important for DIPG growth, we will apply an shRNA-based screening platform that we developed and validated in ongoing work on chromatin regulators in acute myelogenous leukemia (AML). Cell lines harboring wild-type or mutant alleles of H3 will be transduced with a lentiviral library containing bar-coded shRNAs targeting 500 annotated chromatin regulators. After allowing these cells to propagate, we will sequence shRNAs in the population to to identify those that preferentially “drop out” of the H3-mutant population, which will theoretically identify genes that are essential for proliferation of H3-mutant cells. In parallel, CRIPSR technology will be used to target chromatin regulators in a similar screening platform, both to minimize identification of genes resulting from off-target effects of shRNAs, and to extend the coverage of the select chromatin regulators in the initial screening phase. Highpriority targets will be selected based on their identification in multiple screens, and will be confirmed by targeted knock-down or knock-out strategies. The effects of these candidates on cell viability, proliferation, differentiation, and apoptosis will be determined in follow-up experiments to more completely characterize the functional roles of these chromatin regulators in H3-mutant DIPG. Future work will focus on understanding the epigenetic processes influenced by these genes in DIPG, and on assessing the importance of these target genes for DIPG tumor growth in vivo.
Innovation: This work harnesses the unique opportunities provided by the new DIPG cell-culture models to analyze chromatin sensitivities in a systematic and comprehensive manner. As epigenetic regulators commonly considered to be easily druggable, we hypothesize that focusing on these regulators will increase the likelihood of identifying strong candidates for small-molecule therapeutics. Our use of multiple, parallel screening approaches on both wild-type and mutant cell lines is expected to uncover high-confidence, genetically-focused targets in an efficient and high-throughput manner. By including a focused chemical screen, we hope to expedite the development of novel therapeutics, as future work will on identified hits will focus on compound optimization rather than de novo compound development. The genetic screens will use the technology we have recently developed and validated for AML screening, and will represent one of the first comprehensive assessments of chromatin-dependent mechanisms in pediatric glioma. The outcome of this work is expected to be highly clinically significant, as it will identify novel components to target for DIPG therapeutics, some of which may already have small-molecule inhibitors available, thus shortening the time to clinical applications.
UCL Institute of Child Health - $200,000
T-cell based immunotherapy for DIPG.
Diffuse Intrinsic Pontine Glioma (DIPG) is a rare but incurable cancer of childhood with a pressing need for novel therapeutic strategies. Cell-based immunotherapy provides a radically new approach. Here, patient derived T-cells are grafted with the required specificity so that they can bear their destructive potential on tumour cells while leaving healthy tissue unharmed. This can be achieved using Chimeric Antigen Receptors (CARs) which are fusion proteins between the antigen binding domain of a monoclonal antibody and T-cell signalling components. Ex vivo engineering of T-cells to express CARs allows the generation of T-cells with any desired non-MHC restricted specificities. Upon recognition of the cognate antigen CAR Tcells can lyse tumour cells, proliferate, recruit other immune cells and persist to provide longlasting anti-tumour immunity. Adoptive immunotherapy with CAR T-cells has resulted in unprecedented complete and sustained clinical responses in patients with chemotherapy resistant B-cell malignancies. Pre-clinical data demonstrates that CAR T-cells can home to intracranial tumours and mount an anti-tumour response. CAR T-cell therapy studies for adult gliomas have already been initiated. We believe we can build on this experience and develop CAR T-cell therapy as an effective treatment approach for DIPG.
However, unlike B-cell malignancies with facile targeting of CD19, targets for DIPG are not well established. The aim of this proposal is to identify and validate target antigens for CAR T-cell therapy for DIPG. We (Hawkins, Toronto) have a unique set of expression data from this rare disease. This data will be analysed to identify genes encoding proteins which may serve as DIPG-specific CAR targets. We anticipate that it may not be possible to simply target a single antigen. However, the investigator team brings together the unique expertise in DIPG genetics and biology (Hawkins/Brudno SickKids) with technology and know-how in CAR T-cell therapy development (Straathof/Pule, UCL) to get around this problem. Briefly, T-cells can now be engineered to be activated only upon recognition of an antigen over a threshold expression level or upon recognition of a combination of antigens. Expression of candidate antigens will be studied extensively by immunohistochemistry on DIPG and normal tissue arrays. From this information the optimal approach will be identified to achieve discrimination by CAR engineered T-cells between DIPG and normal (brain) tissue. Then, binders specific for validated DIPG targets will be generated to construct CARs as T-cell immunotherapeutics for DIPG.
This project will form the foundation from which CAR T-cell therapy for DIPG can be developed. Once appropriate target antigens have been identified we anticipate being able to leverage further funding required for in vitro and in vivo functional testing of CARs for DIPG therapy. UCL has an established CAR T-cell programme which includes GD2-targeted CAR T-cell therapy for neuroblastoma and EGFRvIII-targeted CAR T-cell therapy for adult glioma. For clinical translation of this approach we will take advantage of this existing CAR development pipeline which will ensure rapid acquisition of further pre-clinical data including small animal models to demonstrate safety and efficacy of T-cell based therapy for DIPG.
Our overall aim is to make this promising treatment strategy available to children with DIPG and potentially high-grade gliomas in the near future.
Dana Farber Cancer Institute - $100,000
Discovering novel epigenetic dependencies in Diffuse Intrinsic Pontine Glioma
Scientific Merit Recent genetic analyses have revealed specific and unique K27M mutations in the histone 3.3 side chain in more than 90% of DIPG cases. Post-translational modifications of this histone 3.3 side chain are normally used as a “histone code” for fine-tuning gene expression across the whole genome. I hypothesize that K27M mutations induce an aberrant epigenetic and transcriptional programs, rendering tumor cells uniquely sensitive to further epigenetic perturbations. I further hypothesize that these tumor-specific alterations of the DIPG epigenome are sustained by epigenetic modifiers and master transcription factors, and as such, are potentially targetable. I expect that I will reveal key contributors that underlie DIPG epigenetic programs by applying two new technologies: 1) The CRISPR/Cas9 system, which allows the knock-out of epigenetic modifiers to identify novel tumor vulnerabilities and dependencies; and 2) Large-scale chromatin profiling, which permits a genome-wide look at DNA activation/repression states, in combination with RNA-seq to identify master transcriptional regulators.
Disease Impact Applying these new technologies to patient-derived tumor samples will shed unprecedented light on unique tumor vulnerabilities and dependencies that are not identifiable by genetic studies alone. The proposed research will provide an unparalleled view of the epigenetically mediated networks underlying DIPG biology and reveal novel tumor vulnerabilities that could rapidly enter pre-clinical and clinical trials - ultimately leading to a cure.
Innovation The proposed study is the first ever to systematically study epigenetic dependencies in DIPG and map the DIPG epigenome in a cell-, location- and differentiation-specific context. By using cutting-edge technologies and computational analysis available at our institutions and at the Broad Institute, the two distinct but complementary approaches will provide a novel view of the molecular pathways driving DIPGs.
Feasibility Both research methods proposed have already been established in collaboration with the Broad Institute. Drs. Filbin and Suva have privileged access to the Genomic Pertubation Platform, where pilot studies on CRISPR/Cas9 screens for various pediatric cancer cell lines have already been successfully completed. A close collaboration with computational scientists is also already in place. The novel findings contributed by this study will provide a rational basis for renewed attempts at improving clinical care of DIPG patients.
Expertise Dr. Suva and Dr. Filbin have a unique and complementary combination of expertise that makes them the ideal investigators to complete the proposed research. Dr. Filbin is a pediatric neurooncologist at Dana-Farber Cancer Institute and research fellow in Dr. Suva’s laboratory at Massachusetts General Hospital (MGH) and the Broad Institute. Her previous work includes the discovery of a novel combinatorial targeted treatment for glioblastoma, which led directly to clinical trials in adults and pediatric patients with high-grade gliomas. Dr. Suva is a faculty scientist at MGH and the Broad Institute and has led ground-breaking research on the underlying epigenomic and transcriptional networks in adult glioblastoma.
VU University Medical Center - $92,950
Liquid Biopsies for the Monitoring of Diffuse Intrinsic Pontine Glioma.
Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive tumor of the brain stem with the worst prognosis of any newly diagnosed childhood cancer. Until recently, the development of novel therapies was hampered by the limited understanding of the underlying genetic and biochemical abnormalities associated with this disease. However, in the past years a number of recurrent mutations has been identified through the collection of tumor tissue by reintroduction of stereotactic biopsies and rapid autopsy protocols. Since these novel mutations may serve as diagnostic- or prognostic factors or as putative targets for therapy, it will become increasingly more important to ascertain the presence of these mutations, both at the time of diagnosis and during therapy. However, the sampling of DIPG tumors through stereotactic biopsies is still a difficult and potentially hazardous neurosurgical procedure. We here propose to set up and optimize blood-based diagnostic tests for the detection of DIPG-related biomarkers. In the past years we have gained extensive experience in the detection of tumor related mutations in blood (in glioma, lung-, breast-, colon- and prostate cancer), based on the observation that tumor cells transfer (mutant) mRNA into blood platelets. Platelet isolation is simple and part of routine blood counts, and - as such - cost effective. Furthermore, it is minimally invasive, allowing for frequent assessment of the mutational status in a longitudinal fashion, without additional burden for the patient. We have recently shown that platelet derived mRNA can be used to distinguish cancer patients from healthy controls, with a sensitivity of 85-90% and a specificity of >95%. Importantly, preliminary results indicate that such blood-based assays are also suitable to detect mutations in DIPG. We here propose to establish robust blood-based assays to monitor common mutations in DIPG that may be used for the stratification of patients that do not get biopsied, and to monitor disease progression/therapy resistance in time. Gene expression profiling will be based on RNAseq experiments (aim 1), using platelet derived mRNA. Since such experiments are routinely performed in our laboratory for other types of cancer, this part may be accomplished within 6 months after start of this project. Monitoring of disease progression will be done by ultra deep amplicon sequencing (aim 2), and a specially designed method to detect H3F3A and HIST1H3B mutations, based on restriction digestion and PCR amplification (aim 3). Although we frequently perform amplicon sequencing in our laboratory, and we have been able to detect H3F3A mutations in the blood of DIPG patients, we will need to optimize amplification procedures to meet our performance criteria. Therefore, we expect to perform this part within 8 months after the start of this project. The restriction-based technique, however, is a novel method that will need extensive optimization, and thus may take up to 1 year. Nevertheless, this will be an important part of this project, as it may result in a relatively cheap diagnostic test that can easily be implemented in any laboratory. We estimate that in total we will need 92,950.- dollar for the execution of these projects. Most of this amount will be spent on next generation sequencing techniques, as personnel costs are covered by the VU University Medical Center.
Children's Cancer Institute - $145,341
Fenretinide, a novel agent targeting RTK/PI3K/AKT network for the treatment of Diffuse Intrinsic Pontine Glioma.
Diffuse Intrinsic Pontine Gliomas (DIPG) are the most aggressive of all childhood cancers. They are a type of brain tumour that peak in incidence at 5-7 years of age and are the most common form of malignant glioma to affect children. There are absolutely no effective treatments and current therapeutic strategies are palliative only. Due to their location within the brainstem the tumours cannot be removed surgically, they do not respond to chemotherapy, and radiotherapy only slows their growth temporarily. Novel and innovative treatment approaches are therefore urgently needed. In collaboration with Prof Monje (Stanford University, USA) and Dr Montero-Carcaboso (Saint John of God Foundation, Spain) we have established 7 neurosphere-forming DIPG cells and used these to perform the first ever high-throughput drug screen (HTS) of 3,500 bioactive pharmaceutical compounds. This screen has identified fenretinide as having significant activity against the DIPG cultures. Fenretinide is a synthetic retinoid that is well characterized, well tolerated in children and has been tested in the treatment of adult and paediatric cancers. It has never previously been assessed for the treatment of DIPG. The exact mechanism of action of fenretinide-induced cell death isn’t completely understood. However it has been suggested that its cytotoxic effects might be initiated by retinoic acid receptor (RAR) dependent and independent mechanisms. Furthermore it has been found to cause accumulation of reactive oxygen species (ROS) and lipid second messengers resulting in cell death through apoptosis. Our preliminary experiments have identified that fenretinide significantly inhibits proliferation of DIPG neurospheres and induces apoptosis. Furthermore we have found that ponatinib a multi-RTK inhibitor significantly enhances the cytotoxicity of fenretinide. Surprisingly we have made the novel discovery that fenretinide inhibits phosphorylation of receptor tyrosine kinases (RTK) including PDGFRa, RET and downstream targets of the Phosphoinositide 3 kinase (PI3K)/AKT pathway. Combination treatment with ponatinib, leads to enhanced suppression of this tumorigenic pathway. These findings indicate that DIPG cells are sensitive to pharmacological inhibition of RTK/PI3K/AKT pathway and that fenretinide represents an exciting new therapeutic strategy for DIPG. We seek here to build upon these initial findings, and to develop the preclinical evidence required to urgently translate these novel discoveries to clinical trial to directly benefit children with DIPG. We are currently investigating the mechanism of action of fenretinide and intend to evaluate whether its efficacy can be further enhanced with other anticancer drugs and RTK/PI3K/AKT inhibitors. Our aims are to assess the efficacy of fenretinide as a single agent and with the two most advantageous combinations in two animal models of DIPG. In doing so, we aim to develop the quantum of preclinical data required to rapidly translate this therapy from the bench to the bedside. Our team has all the necessary expertise that will ensure the success of the proposed project and ultimately the implementation of the discoveries into the clinic. Dr Maria Tsoli is a senior postdoctoral researcher with particular expertise in DIPG cell culture and xenograft models, cancer cell biology, drug discovery, computer simulations and has been involved together with Dr Ziegler in the co-supervision of a PhD student who has provided the preliminary data described in this project. Dr Ziegler has preclinical expertise in paediatric malignant brain tumours and his clinical focus on early phase clinical trials will facilitate translation of positive results to the bedside. Drs Monje and Montero-Carcaboso will continue to assist in providing DIPG cell cultures, and two DIPG animal models for performing the in vivo validations. The support of Dr Ziegler and Prof Haber at the Children’s Cancer Institute provides an invaluable environment to ensure the success of this novel research program, and with the ultimate goal of improving outcomes for children diagnosed with DIPG.
Cincinnati Children's Hospital Medical Center - $200,000
Intra-tumoral heterogeneity in diffuse intrinsic pontine and midline high-grade gliomas
Background: Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor that affects approximately 150 children in North America each year.1 Though radiation therapy can prolong survival by 2-3 months, adjuvant chemotherapy has not improved outcome. The median survival is less than 12 months and has remained unchanged for the last 30 years.3 Knowledge of the pathogenesis of DIPG has been historically limited by lack of tissue availability, as diagnostic biopsy has not been routinely performed due to safety concerns.2 More recently, however, tissue procurement through autopsy programs3-5 and slowly growing acceptance of the safety of DIPG biopsy6-9 has provided the tissue necessary to gain critical knowledge of the genetic and epigenetic landscape of DIPG and has enabled development of in vitro and in vivo models through which novel therapies may be thoroughly tested.10,11
Since 2012, high-throughput sequencing studies have yielded unprecedented insight into the biologic basis of pediatric high-grade glioma (HGG) and DIPG.12-18 Midline, non-brainstem HGGs harbor a genomic profile akin to DIPG (e.g. frequent H3K27M mutation) and carry a similarly dismal prognosis.16,19 Mutations of chromatin remodeling genes H3F3A, HIST1H3B, and HIST1H3C are present in ~80% of thalamic HGGs19 and 70-96% of DIPGs.4-9 More recently discovered ACVR1 mutations are found in ~25% of DIPGs. In addition, mutations in other canonical cancer pathways, including (RTK)-RAS-PI3K, TP53, and RB1, are commonly found in HGG and DIPG.15,20 Unfortunately, our vastly improved understanding of tumor biology has not yet translated into a therapeutic breakthrough for children with HGG and DIPG. One critical factor that will significantly impact guiding appropriate therapy is histologic and molecular intra-tumoral heterogeneity. Although such heterogeneity has been well-documented in adult HGG21-23, there is little understanding of intra-tumoral variation in pediatric HGG and DIPG. Studies of DIPG at autopsy, including our own investigations with correlative post-mortem MRI, have revealed distant tumor metastases to the periventricular and frontal brain regions.5,24 Interestingly, we have observed some histologic variability between primary and distant tumor. Preliminary WES of primary, contiguous, and metastatic sites from 8 patients in our cohort also revealed relevant genomic heterogeneity (Figure 2). Although histone mutations, if present in the primary site, were detected at all disseminated sites, several interesting variations were seen among other clinically relevant mutations. Specifically, in one patient (patient 3) with six regions sequenced (all containing H3.3K27M), we detected regional variations in TP53 mutations. The primary tumour site (primary pons) contained a TP53 p.Arg248Gln alteration (allele frequency, AF = 0.46). Only 1 of 5 contiguous and metastatic sites (leptomeningeal spread) contained this alteration at an AF of 0.10. These 5 sites also harbored a TP53 p.Arg.275His alteration, which was not detected in the primary pons. In this same patient, a PDGFRA p.Glu229Lys alteration and ~8 copy amplification was detected in the right posterior pons, but not the primary pons or any other contiguous and metastatic sites. The leptomeningeal component did exhibit a PDGFRA copy number gain of ~4 copies. We hypothesize that genomic heterogeneity exists within the primary tumor and between primary and distant disease and that better understanding of clonal evolution of DIPG and midline HGG will guide the rational design of molecularly targeted therapies for these lethal brain tumors. Specific Aim 1: To define spatial intra-tumoral heterogeneity using WES on contiguous, non-contiguous, and distant metastatic sites in DIPG and midline HGG obtained at autopsy Specific Aim 2: To demonstrate patterns of branched clonal evolution of DIPG and midline HGG using high-depth WES on contiguous, non-contiguous, and distant metastatic sites Specific Aim 3: To define temporal intra-tumoral heterogeneity using WES on matched biopsy and autopsy tumor specimens from patients with DIPG and midline HGG
Methods/Budget: We request $200,000 to perform WES on matched primary and metastatic (n=10) and matched biopsy and autopsy (n=3) specimens from patients with DIPG or midline HGG who underwent surgical biopsy and/or autopsy at Cincinnati Children’s Hospital Medical Center (CCHMC) or The Hospital for Sick Children. A portion of funding will go toward salary support for the study PIs and for bioinformatics analyses. Complete clinical annotation is available for all patients. Clinical Significance: Characterization of spatial and temporal heterogeneity and establishment of clonal evolutionary patterns in DIPG and midline HGG will have significant clinical impacts. First, this work will define the utility of primary tumor biopsy, which may misrepresent targetable genomic lesions across all disease locations, and a potential role for re-biopsy at progression. Additionally, exploration of early somatic events using high-depth sequencing and phylogenetic analyses has potential to define true disease “drivers” common across disease compartments that, if molecularly targeted, may hold more potent therapeutic potential.
The Hospital for Sick Children - $190,905
Mitochondrial DNA alterations and their potential as a novel therapeutic target in DIPG
Brain tumors are the leading cause of cancer-related death in childhood. Diffuse intrinsic pontine glioma (DIPG) arises in the pons of the brainstem and is universally fatal, comprising nearly 15% of all pediatric brain tumors.1 Despite international endeavors to improve outcome, DIPGs show poor response to conventional radiation and chemotherapeutic strategies used in adults.2 Only within the last decade have studies really begun to describe differences between adult and pediatric diseases, underscoring the need for better targeted therapies. Most recently, our group made a major breakthrough in the understanding of DIPG biology by identifying three molecular subgroups: MYCN, Silent and H3K27M.3 The MYCN subgroup has no recurrent mutations but is characterized by chromothripsis on chromosome 2p leading to amplification of MYCN and ID2. The Silent subgroup is featured by a lower mutation rate and fewer copy number alterations than the other two subgroups. The H3K27M subgroup is the largest and harbors a K27M mutation in either H3F3A (H3.3) or the HIST1H3 family (H3.1). These findings highlight the potential importance of epigenetic dysregulation in DIPG pathogenesis. However, H3K27M tumors harbor additional alterations while MYCN and Silent subgroup tumors have relatively fewer recurrent genetic alterations, strongly suggesting that H3K27M alone is likely insufficient to drive malignant transformation. Additional mechanisms, including those beyond the traditional epigenetic and genetic modifications within the nuclear genome (nDNA), are likely to be required. Unlike normal cells that rely primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate the energy needed for cellular processes, cancer cells depend on aerobic glycolysis to support their uncontrolled growth even in the presence of ample oxygen, a phenomenon termed “the Warburg effect.”4 Abnormal OXPHOS and aerobic metabolism as a result of mitochondrial dysfunction are considered robust metabolic hallmarks of many cancer entities. Numerous somatic mutations in the mitochondrial genome (mtDNA) as well as mtDNA copy number changes have been increasingly observed across a broad spectrum of primary malignancies, including adult brain tumors.5 Mounting evidence has demonstrated that mtDNA sequence and content variations are associated with neoplastic transformation, tumor progression and metastasis, chemo/radioresistance, and disease prognosis.6 Due to decreased expression of mtDNA-encoded polypeptides and compromised function of respiratory enzyme complexes, either qualitative or quantitative alterations in mtDNA could elicit a decline in mitochondrial respiratory activity and cause persistent defects in the OXPHOS system accompanied by generation of excessive reactive oxygen species (ROS). This in turn further damages mtDNA, accelerates its mutational rate, and eventually establishes a vicious cycle amplifying mitochondrial dysfunction and oxidative stress. This scenario has been proposed to positively contribute to cancer initiation and/or progression.5,6 Despite a huge explosion of DIPG genomic and transcriptomic data and tremendous efforts in characterizing the biological significance of recurrent mutations in nDNA, to date, no study has investigated mtDNA mutations and copy number changes in DIPG or their potential as therapeutic targets. We hypothesize that mtDNA alterations and consequent impairment of mitochondrial and OXPHOS functions by themselves or in a cooperative fashion with nDNA mutations are important for the initiation and/or progression of DIPG. Work proposed in this study aims to screen pathogenic “driver” mutations in the entire mitochondrial genome of DIPG tumors by harnessing the advantage of targeted next-generation sequencing (NGS) technology and to elucidate the molecular mechanisms underlying their involvement in DIPG carcinogenesis (Aim 1). The potential to exploit altered mtDNA copy number as a novel therapeutic target in DIPG will be evaluated in Aim 2. This preclinical work will determine if manipulating mtDNA copy number or stimulating mitochondrial biogenesis is capable of rescuing the metabolic function of defective mitochondria in DIPG and thus reverse the malignant phenotype. Targeted whole mitochondrial genome sequencing may lead to the identification of a set of frequent mtDNA mutations that may play a primary and causative role in DIPG development. Our innovative approach of targeting aberrant mtDNA content in combination with conventional therapies will provide clues for designing novel therapeutic strategies. Our group is a leader in the field of DIPG and being part of the largest pediatric neuro-oncology team in Canada and active contributors in the DIPG collaborative, we are ideally situated to translate discoveries made through this project into novel early diagnostic tools and more effective therapeutic trials for this devastating disease.
Children's National Medical Center - $200,000
Evaluating TORC1/2 kinase inhibition in murine and nonhuman primate (NHP) models
Scientific Merit: New therapies are desperately needed for children with DIPG. Our group participated in recent collaborative efforts that led to identification of panobinostat as an active agent in DIPG. However, additional agents are likely required in combination with panobinostat to improve long-term survival for these children. We identified mutations activating the mTOR signaling pathway in DIPGs. Our preliminary data shows that targeting mTOR using a dual TORC 1/2 inhibitor, MLN0128, suppresses DIPG growth and increases apoptosis. Our strong collaborative team will use existing available preclinical resources, including four primary DIPG lines, xenograft and allograft DIPG models, and a non-human primate model, to test DIPG response to MLN0128 and evaluate the CNS pharmacokinetics of this agent. Combinational (radiation, panobinostat) therapies will be used to assess additional impact. Our approach is based on solid preliminary data that will be further expanded using three strongly collaborating laboratories for establishing mTOR as a potential target for treating DIPGs. The clinical need, widespread activation of mTOR in DIPG, and availability of a potent drug that penetrates into the central nervous system argue for further studies of MLN0128 in DIPG. Disease Impact: The proposed project aims to explore development of a new therapeutic for DIPG. In the short-term, the investigation of MLN0128 as a therapeutic agent in DIPG will provide the pre-clinical data to support a clinical trial of this agent, potentially in conjunction with radiation/chemotherapy. Once we have the data generated by our studies, we aim to perform a clinical trial of this agent based upon a sound biological and pharmacokinetic rationale. Innovation: 1. First study to investigate MLN0128 in DIPG. 2. Use of nonhuman primate (NHP) model to study drug pharmacokinetics. a. Unique ability to concurrently sample plasma and CSF penetration at multiple time points in primates. 3. Comprehensive in vitro and in vivo analysis of MLN0128 and combinatory treatments. 4. Identification of molecular mechanism of sensitivity/insensitivity to MLN0128 treatment. 5. Potential for near future clinical assessment of MLN0128 for treating children with DIPG. Feasibility: PIs of the proposed project have generated preliminary data indicating feasibility of the proposed project. Upregulation of mTOR is a known path by which cells develop resistance to panobinostat, therefore we propose to test the TORC1/2 kinase inhibitor MLN0128 in DIPG. MLN0128 has been shown by the pediatric preclinical testing program (PPTP) to have significant activity against the D456 brain tumor xenografts. We have demonstrated MLN0128’s activity against atypical tertoid/rhaboid brain tumor orthotopic xenografts, indicating that the agent does penetrate thebrain. Proposed molecular, in vitro and in vivo studies are routine in designated laboratories. MLN0128 is currently being tested in adult GBM. MLN0128 is available through CTEP, meaning that the drug could potentially be available for pediatric clinical trials. As such we envision our proposed study will result in robust molecular justification for using this compound in treating DIPGs. Expertise: Dr. Nazarian (PI) at CNHS has extensive expertise studying the molecular profiling (mRNA, methylation, proteomics) of DIPGs. CNHS houses cutting-edge genomic, proteomic and preclinical testing murine facilities. Dr. Raabe (Co-PI) a pediatric oncologist at Johns Hopkins University School has extensive experience in cell culture, molecular biology, immunochemical analysis and xenografting. Dr. Raabe’s laboratory had developed a primary DIPG line (JHH DIPG1) extensively used for preclinical testing across many laboratories. Dr. Katherine Warren (Co-PI) is the head of Pediatric Neuro-Oncology Section at NCI. Dr. Warren has extensive pharmacologic expertise, and leads preclinical and clinical efforts in developing new therapeutic strategies for the treatment of children with tumors of the central nervous system.
European DIPG Registry - $445,870.12
European DIPG Registry.
Childhood Oncology Group (DCOG), a National Paediatric Haematology[Oncology Society (NaPHOS) member of SIOPE. DCOG is mandated by the Executive Committee of the SIOPE DIPG Network to act as a legal entity on its behalf in matters concerning the DIPG Registry. The Parties acknowledge and agree that DCOG has no individual decision making authority with respect to the allocation of the funds in relation to the SIOPE DIPG Registry. DCOG shall have no responsibility or liability with respect to the distribution of the funding received under this agreement, except for any damages resulting from DCOGs gross negligent or intentional acts.
The responsibilities of DCOG include:
• conclude on behalf of the DIPG Registry written agreements with regard to Funds received from third parties for the DIPG Registry
• receive, administer and allocate all Funds for the DIPG Registry in accordance with the reasonable instructions of the Executive Committee.
• conclude written agreements with third parties providing services to the DIPG Registry, including but not limited to such agreement with the DIPG Imaging Repository
• conclude on behalf of the DIPG Registry written agreements with scientific advisors based on the model agreement attached to the Bylaws as Annex 2.
• invoice third parties on behalf of and as instructed by Executive Committee where appropriate
• pay invoices received from DIPG Network coordinators, third party service providers and other third parties in relation with any agreements executed by or for the DIPG Network in relation to the DIPG Registry as approved by the Executive Committee;
• DCOG shall ensure transparent book[keeping in relation to the DIPG Registry’s financial administration.
• any responsibilities agreed between the Executive Committee and DCOG, pursuant to the decision making procedures of the DIPG Network.
Institute of Cancer Research - $99,470
The Evolutionary Dynamics of Diffuse Intrinsic Pontine Glioma.
Diffuse intrinsic pontine glioma are high grade glial tumours arising in the brainstem with a median survival of 9-12 months, and a distinct biology compared to similar looking tumours arising in the cerebral hemispheres in children and adults. A major challenge to improve outcomes for these tumours is their extensive intratumoral heterogeneity, reflected by differing cellular morphologies and genomic imbalances present within an individual sample. We sought to define the subclonal diversity of DIPG with a view to better understanding the evolutionary dynamics underlying this variation.
In the first year of this grant, generously funded by the Abbie’s Army and the DIPG Collaborative, we have made substantial progress in ascertaining the subclonal architecture of DIPG using next-generating sequencing of single, multi-region and longitudinal samples. We have additionally developed methodology to isolate DIPG subclones in vitro in order to determine how distinct genotypes link to function within the tumour mass. As highlighted in the original application, we are now requesting a second year of funding to more fully explore the interactions between subclonal populations of DIPG cells, and to assess the possibilities of disrupting communication between subclones as a novel therapeutic strategy.
Stanford University Medical Center - $200,000
Targeting neuronal activity-regulated DIPG infiltration
DIPG is characteristically infiltrative (i.e. diffuse and intrinsic), and this infiltrative/invasive behavior is destructive both in the brainstem and in other areas of the central nervous system to which DIPG spreads during the course of the disease. In work previously funded by the Cure Starts Now and the DIPG Collaborative, we discovered that neuronal activity promotes DIPG cell invasion through activity-regulated secreted factors that includes an endogenous antagonist to the Nogo receptor (NgR). NgR signaling is a key mechanism that restricts the plasticity and regeneration of normal brain cells, and blocking the Nogo pathway results in increased motility of cells and cellular processes such as axonal outgrowth. In the normal brain, this signaling pathway plays a role during early brain development and may continue to play a role in ongoing brain plasticity. DIPG cells express NgR and in preliminary studies we have found that blocking NgR signaling with recombinant antagonist or by deleting the NgR gene via CRISPR gene editing from patient-derived DIPG cells dramatically increases DIPG invasion in vitro. In the proposed studies, we will expand these observations to a larger number of patient-derived DIPG cell cultures to determine how universal this mechanism may be, and will assess the importance of NgR signaling to DIPG invasion in vivo using genetic models. If NgR signaling proves to be an important mechanism controlling DIPG invasion, then stimulating the NgR receptor may be an innovative therapeutic strategy to control infiltration of DIPG cells throughout the brainstem and prevent spread more diffusely to the cerebrum and spinal cord.
The regulation of the NgR signaling by neuronal activity represents one of the many ways that experience shapes brain development and plasticity. The effects on DIPG invasion represents yet another way that DIPG cells hijack normal mechanisms of brain development to promote disease progression. Ultimately, by understanding the ways DIPG subverts mechanisms of childhood brain development and adaptability, we hope to develop effective and tumor-specific strategies to disrupt the ability of the tumor cells to use these crucial signals in the tumor microenvironment.
International DIPG Registry - $699,935
Establishment of an International Diffuse Intrinsic Pontine Glioma (DIPG) Registry
Diffuse intrinsic pontine gliomas (DIPG) are the most common brainstem tumors in children, representing approximately 75-80% of all pediatric brainstem tumors. 1 Approximately 200-300 patients are diagnosed with DIPG in all of North America and Europe. 1,2 DIPG accounts for 10-15% of all new pediatric brain tumor diagnoses and is the leading cause of brain tumor-related death in children.1 The median age at diagnosis is 6 to 7 years 2-4 and prognosis for patients with DIPGs remains dismal with a median survival of less than 1 year. Although radiotherapy does improve neurological function and survival by 2-3 months, no effective chemotherapeutic regimens are currently available.1,2 Achieving cure for all children with DIPG remains a major goal of pediatric neuro-oncology. In this application, we propose the expansion of the International Diffuse Intrinsic Pontine Glioma Registry which is now the largest and most comprehensive collection of data including clinical, radiologic and pathologic data linked to a bioinformatics repository of molecular data from a diverse cohort of DIPG patients available to researchers throughout the world. With the generous support of a coalition of pediatric brain tumor foundations from the DIPG Collaborative, and collaborations with 106 academic medical centers in the US, Canada, Australia, New Zealand, Egypt, Brazil, Chile, China, Argentina, India, Japan, Saudi Arabia, United Arab Emirates, and Lebanon, the Registry is growing exponentially. From April 2012 to August 2018, 885 patients diagnosed with DIPG have been enrolled from 106 collaborating institutions. The specific aims include: 1) To continue recruitment of patients diagnosed with DIPG in the International DIPG Registry to greater than 1,000 patients and further expand internationally; 2) To provide a repository of integrated data set compromised of clinical, pathologic, radiologic and molecular features to the clinical research community for promotion of hypothesis generation and analysis and maintain follow up on all cases; 3) To facilitate conduct of autopsies and sharing of fresh tissue for establishment of in vitro and in vivo models by investigators to be shared with investigators around the world; 4) To expand the bioinformatics repository of existing molecular data on DIPGs that can be linked to patient information in the registry through the Links and Viva platform to include prospective data; 5) To develop a program focused on quality of life through a longitudinal study where patient and/or parent proxy-dyads who enroll on the International DIPG Registry will complete Health related quality of life measures at specified time intervals and aid in supportive management; 6) To broaden collaborations among investigators for hypothesis-driven research studies through the registry that will ultimately lead to better classification and more effective treatment of patients with DIPG. In the next year, the International DIPG Registry investigators will promote robust collaborative research projects on all aspects of DIPG, and will continue to make the International DIPG Registry data available to external investigators after review of the proposed research by Scientific Advisory Committee (SAC). Our long-term goal is to expand on the highly collaborative, international, hypothesis-driven research infrastructure to continue to support a wide spectrum of interdisciplinary and translational projects in DIPGs for all investigators. The data collected form a research continuum from basic biology to clinical practice that will ultimately address our primary goals of a) understanding the biology of DIPGs, b) developing more effective therapies and c) developing innovative approaches to diagnosis, response assessment and multidisciplinary treatment and follow-up that will improve patient outcome in addition to maximizing quality of life.
Stanford University - $50,000
Tumor Associated Microglia in Diffuse Intrinsic Pontine Glioma
Diffuse high-grade gliomas are a class of central nervous system tumors that carry a dismal prognosis across all ages. In children, about half of these tumors arise within the ventral pons as diffuse intrinsic pontine glioma (DIPG), which has a peak incidence from ages 6-8 and only a 9-12 month median survival. The consistent timing and location of this tumor suggests that specific developmental cues in the microenvironment of the mid-childhood ventral pons may be dysregulated. Microglia, the resident macrophages of the brain, represent both a component of the normal developmental microenvironment and also a potential contributor to pathologic microenvironments such as DIPG. Tissue macrophages are critical in the development of other organs (Pollard, 2009), and recent studies suggest that microglial trophic factors play a role in development and learning (Ueno et al., 2013; Parkhurst et al., 2013). In adult gliomas, macrophages can make up 30-50% of the tumor mass and are implicated as supporting angiogenesis and tumor cell invasion, proliferation, and survival (Glass & Synowitz, 2014). In contrast to adult gliomas, however, strikingly little is known about how microglia and macrophages contribute to the pediatric high-grade glioma microenvironment. Given the possible role of microglia in normal brain development, an intriguing hypothesis is that these pediatric gliomas hijack normal supportive roles of microglia to promote tumor growth and invasion. Understanding this interaction in molecular detail will provide insight not only into the pathophysiology of DIPG and possible adjuvant therapeutic targets, but also into the possible roles of microglia in normal brain development.
This research proposal seeks to elucidate the mechanisms through which microglia interact with DIPG. Previous work demonstrated that inhibition of colony stimulating factor-1 signaling in macrophages in a murine glioblastoma model can block glioma progression (Pyonteck et al., 2013). Moreover, recent evidence suggests that adult glioblastomas secrete the soluble factor periostin, which attracts macrophages and directs them to promote tumor growth (Zhou et al., 2015). In primary DIPG tissue samples, there is a high degree of microglial staining (Figure 1, Caretti et al., 2014), which suggests that these cells play an important role in shaping the DIPG microenvironment. Determining how this microglial role differs in DIPG compared to adult gliomas will provide a better understanding of the role of microglia in the unique midchildhood pontine microenvironment.
We hypothesize that pontine microglia promote DIPG growth through secreted growth factors, and we propose to test this hypothesis through the following aims:
1) Demonstrate how pontine microglia affect DIPG behavior at baseline and in various states of activation.
2) Identify the molecular signals that underlie microglial
effects on DIPG proliferation.
3) Determine what pathways are activated in DIPG by microglial-secreted factors.
University of California, San Francisco - $100,000
Single-cell profiling and targeting of developmentally regulated signaling programs in DIPG.
Background: As in other malignant gliomas, there is evidence that DIPG is organized as an aberrant developmental hierarchy originating from rare, treatment-resistant tumor-initiating cells (TICs), also referred to as “cancer stem cells”. Although TICs are potentially important drug targets, they are a challenge to study directly because these cells comprise a minor subset of the tumor, and prospective identification (i.e. by surface markers) is imperfect. Moreover, prospective identification of TICs is controversial, with various groups in the glioma field advocating different markers (i.e. CD133, Nestin, Sox2, integrin 6, etc.), and variability from patient-to-patient. Over 75% of DIPG tumors harbor mutations in histone H3.3 that cause global dysregulation of epigenetic marks, including H3K27me3 and CpG methylation. These epigenetic changes manifest as uncontrolled growth of cells with molecular features of pontine gliogenesis. However, the inducible and constitutively active signaling programs downstream of these epigenetic changes – and their coordination with other markers of glial differentiation – are not well understood.
Hypothesis: We hypothesize that developmentally distinct subpopulations of DIPG tumor cells rely on distinct signaling networks to maintain cell survival, and that more effective therapies can be achieved by precisely targeting these signaling programs.
Goals: To characterize developmentally regulated signaling pathways in DIPG, and to test whether targeted disruption of these pathways improves survival in xenograft models.
Design and methods: The technology enabling this study is a next-generation flow cytometry platform (‘mass cytometry’) that enables the simultaneous measurement of up to 32 antibody-based markers at the single-cell level. Mass cytometry presents opportunities to clarify phenotypic identification of developmentally distinct subsets in DIPG, including TICs, and at the same time make direct observations intracellular signaling behavior. Specific Aim 1 uses mass cytometry to map the “developmental trajectory” of pediatric DIPG by tracking the phenotypic changes along the path from primitive TICs to their more differentiated progeny. We will use phospho-specific antibodies to profile the constitutively active and inducible growth-factor signaling pathways in tumor cells along the developmental trajectory, at the single-cell level. Specific Aim 2 takes a chemical genetics approach to further elucidate the developmentally regulated signaling programs in DIPG. We will use a library of 368 well-characterized pharmacologic inhibitors to disrupt growth factor signaling and pro-survival pathways in DIPG. The results of this screen will complement information from Aim 1, to help us pinpoint the developmentally regulated signaling mechanisms in DIPG. In the final stage of this Aim, we will test promising drug candidates for the ability to improve survival in vivo using DIPG orthotopic xenografts.
Innovation: The epigenetic programs enforced by histone H3 mutations appear to lock cells in an oncogenic state, but the consequences at the level of cellular signaling and differentiation are essentially unexplored. We propose to employ mass cytometry, a powerful new technology that allows us, for the first time in pediatric glioma, to simultaneously characterize cellular phenotype and signaling behavior at the single-cell level,
Clinical significance: There is an urgent need for new therapies for DIPG. This study will lead to improved understanding of the relationship between intratumor heterogeneity and oncogenic signaling pathways in DIPG, and will test putative targeted therapies that leverage those new insights.